Ca_movelment平滑肌钙离子运动综述
Calcium Movements,Distribution,and Functions in
Smooth Muscle
HIDEAKI KARAKI a ,HIROSHI OZAKI,MASATOSHI HORI,MINORI MITSUI-SAITO,KEN-ICHI AMANO,KEN-ICHI HARADA,
SHIGEKI MIYAMOTO,HIROSHI NAKAZAWA,KYUNG-JONG WON AND KOICHI SATO Department of Veterinary Pharmacology,Graduate School of Agriculture and Life Sciences,The University of Tokyo,Bunkyo-ku,Tokyo,
Japan
I.Introduction...........................................................................158II.Calcium movements.. (159)
A.Calcium movements predicted from muscle contraction.................................159B.Measurements of radioactive calcium fluxes . (159)
1.Slowly exchanging calcium https://www.wendangku.net/doc/a96427075.html,nthanum-inaccessible fraction ..................................................1603.Suggested calcium movements in smooth muscle....................................161C.Measurements of cytosolic free calcium level (162)
1.Aequorin........................................................................1622.Fluorescent indicators............................................................162D.Mechanisms of calcium mobilization.. (164)
1.Voltage-dependent calcium channels...............................................1642.Nonselective cation channel and calcium release-activated calcium channel............1653.Sodium-calcium exchange.........................................................1664.Calcium release from the sarcoplasmic reticulum ...................................1665.Calcium pumps in plasmalemma and the sarcoplasmic reticulum.....................1696.Mitochondria ....................................................................170E.Calcium distribution and function. (172)
1.Noncontractile Calcium compartment..............................................1722.Calcium sparks,waves,oscillations,and gradients ..................................1753.Role of localized calcium. (177)
III.Changes in calcium sensitivity (178)
A.Increase in calcium sensitivity .......................................................178B.Decrease in calcium sensitivity and inhibition of agonist-induced increase................181IV.Effects of pharmacological agents.. (181)
A.Activators and inhibitors of protein kinases and phosphatases (181)
1.Myosin light chain kinase.........................................................1812.A kinase ........................................................................1823.G kinase ........................................................................1834.C kinase ........................................................................1845.Tyrosine kinase..................................................................1866.Phosphatases....................................................................188B.Agents that change sarcoplasmic reticulum function .. (189)
1.Caffeine.........................................................................1892.Ryanodine.......................................................................1903.Inhibitors of sarcoplasmic reticulum calcium pump..................................191C.Stimulants. (191)
1.Membrane depolarization.........................................................1912.Receptor agonists ................................................................1923.Other constrictors................................................................1974.Summary .......................................................................197D.Relaxants.. (198)
1.Calcium channel blockers.........................................................1982.Potassium channel openers . (199)
0031-6997/97/4902-0157$03.00/0P HARMACOLOGICAL R EVIEWS
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3.Other relaxants..................................................................200
4.Summary .......................................................................205E.Agents affecting endothelial functions ................................................
2051.Calcium movements in vascular endothelium.......................................2052.Effects of fluid shear stress .......................................................2073.Relaxant effect of nitric oxide .....................................................207V.
Calcium movements,distribution and functions in smooth muscle ..........................208A.Calcium movements and distribution .................................................208B.Receptor-effector-structure interrelationship...........................................209VI.Conclusions ...........................................................................211VII.Acknowledgments......................................................................211VIII.
References (211)
I.Introduction
Contraction of smooth muscle is regulated by the cy-tosolic Ca 2?level ([Ca 2?]i )b ,and the sensitivity to Ca 2?of the contractile elements in response to changes in the environment surrounding the cell.The first sequence of events in regulation includes the binding of endogenous substances,such as neurotransmitters and hormones,to their specific receptors.This activates various types of guanosine 5?-triphosphate (GTP)binding proteins,which are coupled to different ion channels and en-zymes,and modulate their activities.These enzymes include both phospholipase C,which metabolizes phos-phatidylinositol and produces inositol 1,4,5-trisphos-phate (IP 3)and diacylglycerol,and adenylate cyclase,which metabolizes adenosine 5?-triphosphate (ATP)to produce cyclic adenosine 3?,5?-monophosphate (cyclic AMP).Some receptors,such as that for the atrial natri-uretic peptide,are directly coupled to guanylate cyclase,
which metabolizes GTP to produce cyclic guanosine 3?,5?-monophosphate (cyclic GMP).
The second regulatory sequence includes changes in [Ca 2?]i .Calcium influx is the major pathway to increase [Ca 2?]i .This mechanism includes voltage-dependent L -type Ca 2?channels,nonselective cation channels,the Ca 2?-release activated Ca 2?influx pathway,and the reverse mode of the Na ?/Ca 2?exchanger.Calcium re-lease from the sarcoplasmic reticulum (SR)also in-creases [Ca 2?]i .A decrease in [Ca 2?]i is mediated by Ca 2?sequestration by the SR,and extrusion by mem-brane Ca 2?pumps and Na ?/Ca 2?exchanger.Second messengers such as IP 3,diacylglycerol,cyclic AMP,and cyclic GMP alter [Ca 2?]i by affecting these mechanisms.Distribution of Ca 2?in the cytoplasm is not uniform.Calcium ion in the cytosolic compartments regulates contractile elements,whereas Ca 2?in the subplasmale-mmal compartments regulates Ca 2?-dependent mecha-nisms in the plasmalemma (ion channels,ion pumps,and enzymes).Calcium concentrations in these compart-ments are regulated independently.
The third regulatory sequence includes changes in myosin light chain kinase activity.This enzyme is acti-vated by Ca 2?and calmodulin and phosphorylates my-osin regulatory light chain (MLC).Phosphorylated my-osin interacts with actin to induce contraction.Phosphorylated MLC is dephosphorylated by MLC phos-phatase.The amount of phosphorylated MLC is there-fore dependent on the balance between MLC kinase and MLC phosphatase.However,during continuous stimu-lation,[Ca 2?]i ,the amount of phosphorylated MLC and shortening velocity gradually decrease,whereas isomet-ric force increases monotonically.This indicates that nonphosphorylated myosin is also involved in the main-tenance of contraction.Agonists and second messengers modify the MLC kinase/MLC phosphatase ratio inde-pendently of [Ca 2?]i .This mechanism,known as Ca 2?sensitivity of MLC phosphorylation,changes contractile force even in the presence of a constant level of [Ca 2?]i .Both cyclic AMP and cyclic GMP change the MLC ki-nase/MLC phosphatase balance and induce relaxation.
a Address correspondence to Hideaki Karaki.
b Abbreviations:[Ca 2?]i ,cytosoli
c Ca 2?level;GTP,guanosine 5?-triphosphate;ATP,adenosine 5?-triphosphate;IP 3,inositol 1,4,5-triphosphate;cyclic AMP,cyclic adenosine 3?,5?-monophosphate;cy-clic GMP,cyclic guanosine 3?,5?-monophosphate;SR,sarcoplasmic reticulum;MLC,myosin light chain;ADP,adenosine 5?-diphosphate;PSS,physiological salt solution;CRAC,Ca 2?release-activate
d Ca 2?channel;CICR,Ca 2?-induced Ca 2?release;IICR,IP 3-induced Ca 2?release;GTP ?S,guanosin
e 5?-O -(3-thiotriphosphate);GDP ?S,guanosine-5?-O -thiodiphosphate;SHR,spontaneously hypertensive rat;WKY,Wistar Kyoto rat;SERCA,sarcoplasmic reticulum Ca 2?-ATPase;STOC,spontaneous transient outward current;MBED,9-methyl-7-bromoeudistomin;RNA,ribonucleic acid;H-7,1-(5-iso-quinolinesulfonyl)-2-methylpiperazine dihydrochloride;M L -9,1-(5-chloronaphthalene-1-sulfonyl)-1H -hexahydro-1,4-diazepine;CGRP,calcitonin gene-related peptide;TPA,12-O -tetradecanoylphorbol-13-acetate;PDBu,phorbol 12,13-dibutyrate;DPB,12-deoxyphorbol 13-isobutyrate;DPBA,12-deoxyphorbol 13-isobutyrate 20-acetate;PDGF,platelet-derived growth factor;SD-3212,semotiadil fumarate (S )-(?)-enantiomer;KB-2796,1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)piperazine dihydrochloride;TMB-8,8-(N ,N -dieth-ylamino)octyl-3,4,5-trimethoxybenzoate;KT-362,5-[3-([2-(3,4-di-methoxyphenyl)-ethyl]amino)-1-oxopropyl]-2,3,4,5-tetrahydro-15-benzothiazepine fumarate;LP-805,8-tert -butyl-6,7-dihydropyrrolo-[3,2-e ]-5-methylpyrazolo-[1,5a]-pyrimidine-3-carbonitrile;SKF 96365,1-[3-(4-methoxyphenyl)propoxyl]-1-(4-methoxyphenyl)ethyl-1H -imidasole Hcl;fura-2/AM,acetoxymethyl ester o
f fura-2;CGRP,calcitonin gene-related peptide.
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All of these mechanisms are supported by energy sup-plied mainly from oxidative phosphorylation and partly from aerobic glycolysis.Oxidative phosphorylation sup-plies ATP mainly to contractile elements,whereas aer-obic glycolysis supplies ATP mainly to membrane ion pumps.Although smooth muscle develops approxi-mately double the force per cross-sectional area of skel-etal muscle,it consumes 100-to 500-fold lower ATP than does skeletal muscle.This difference is explained by the lower ATPase activity of the smooth muscle myosin mol-ecule.
Within the past decade,considerable progress has been made in the understanding of Ca 2?movements and distribution in smooth muscle cells.Simultaneous measurements of [Ca 2?]i and contraction in intact smooth muscle cells and tissues using various types of intracellular Ca 2?indicators have allowed analysis of Ca 2?sensitivity of contractile elements (see Karaki,1989a,1990,1991).Permeabilization of the cell mem-brane enabled the measurement of contraction in the presence of the constant concentrations of Ca 2?,ATP,and other substances in the cell.Calcium-imaging tech-niques have revealed uneven distribution of Ca 2?in the cell and localized increases in the form of Ca 2?sparks and https://www.wendangku.net/doc/a96427075.html,parison of the increase in [Ca 2?]i and contraction suggested the roles of localized Ca 2?in reg-ulation of different mechanisms located in different parts inside the cell.
This review article is focused on topics related to mechanisms regulating [Ca 2?]i and physiological roles of Ca 2?in smooth muscle.Effects of pharmacological agents on movements and distribution of Ca 2?will also be discussed.Readers should refer to review articles by Abdel-Latif (1986)and Nishizuka (1995)on the receptor-linked signal transduction,by McDonald et al.(1994),Kuriyama et al.(1995)and Knot et al.(1996)on ion channels,by Murphy (1994),Somlyo and Somlyo (1994),and Strauss and Murphy (1996)on regulation of con-tractile elements,and by Ishida et al.(1994),Paul (1995),and Hellstrand (1996)on energy supply.
II.Calcium Movements
A.Calcium Movements Predicted from Muscle Contraction
Before directly measuring [Ca 2?]i using the intracel-lular Ca 2?indicators,contraction was considered to be a good indicator of [Ca 2?]i in smooth muscle,because Ca 2?was believed to be the only regulator of contrac-tion.In vascular smooth muscle,two types of stimulants are widely used to identify the changes in [Ca 2?]i :high K ?-induced membrane depolarization and activation of the ?-adrenoceptor by norepinephrine or phenylephrine (Weiss,1977;Karaki,1987).Both of these stimuli in-duced sustained contractions,but with different charac-teristics.High K ?-induced sustained contraction was totally abolished by removing external Ca 2?and,also,
by agents blocking the Ca 2?channels,including cinna-rizine (Godfraind and Kaba,1969),?-diethylaminoethyl diphenylpropyl acetate (SKF525A)(Kalsner et al.,1970),verapamil (Peiper et al.,1971),and La 3?(Good-man and Weiss,1971a,b;Van Breemen et al.,1972).From these results,it was proposed that high K ?in-creases transmembrane Ca 2?influx,increases [Ca 2?]i and induces contraction.In contrast,norepinephrine-induced contraction was resistant to removal of external Ca 2?.It induced a transient contraction followed by a small sustained contraction in the absence of external Ca 2?.Calcium channel blockers and La 3?also inhibited the sustained phase more strongly than the transient phase.However,a part of the norepinephrine-induced sustained contraction was not inhibited by La 3?or Ca 2?channel blockers at the concentrations needed to com-pletely inhibit high K ?-induced contraction.These re-sults suggest that the norepinephrine-induced transient contraction is due to Ca 2?release from intracellular storage site (Hiraoka et al.,1968).The mechanism of the norepinephrine-induced sustained contraction was con-troversial.It was suggested that this contraction is due mainly to transmembrane Ca 2?influx because it is strongly inhibited in the absence of external Ca 2?(Som-lyo and Somlyo,1968;Hudgins and Weiss,1968;Hiraoka et al.,1968;Weiss,1977).Another possibility was that this contraction is due to Ca 2?release from storage sites because both the transient and sustained phases were less sensitive to Ca 2?channel blockers than was the high K ?-induced sustained contraction (Bohr,1963;Van Breemen et al.,1972).To further examine the mechanisms to increase [Ca 2?]i ,it was necessary to directly measure [Ca 2?]i .
B.Measurements of Radioactive Calcium Fluxes The amount of Ca 2?bound outside the cell membrane (approximately 1mmol/kg of wet tissue)is much greater than the amount of free Ca 2?in the cytoplasm (approx-imately 10nm to 1?M )and/or the amount of Ca 2?entering the cell during a contractile stimulation (500pmol of membrane-bound Ca 2?/cm 2of cell membrane compared to 0.3pmol of Ca 2?influx/cm 2of cell mem-brane)(Bolton,1979).Since it was not possible to dis-criminate between Ca 2?bound to the membrane surface and Ca 2?in the cytoplasm using radioactive 45Ca 2?,it was difficult to detect changes in transmembrane Ca 2?influx in smooth muscle.Thus,various stimulants did not change total 45Ca 2?uptake in different types of smooth muscle preparations (see Lullman,1970;Weiss,1974,1977).
1.Slowly exchanging calcium fraction.To remove that 45
Ca 2?present in the extracellular space,Briggs (1962)incubated rabbit aortic strips with solutions containing 45
Ca 2?for 30–60min followed by a 10-to 15-min wash-out period with identical non-radioactive https://www.wendangku.net/doc/a96427075.html,-ing this method,it is possible to remove rapidly exchang-ing Ca 2?and measure the slowly exchanging Ca 2?
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fraction.It was found that high K ?,epinephrine and norepinephrine increased the amount of 45Ca 2?remain-ing after the washout period (Briggs,1962;Seidel and Bohr,1971).Ouabain-induced contractions in the rabbit aorta were also shown to be accompanied by an in-creased 45Ca 2?uptake (Briggs and Shibata,1966).This method was also applied to intestinal smooth muscle of the guinea pig taenia coli by Urakawa and Holland (1964),and it was found that various stimulants,includ-ing high K ?,Ba 2?,carbachol and histamine,increased 45
Ca 2?uptake (for references see Karaki and Urakawa,1972).Thus,the amount of Ca 2?in the slowly exchang-ing fraction appears to correlate with contraction.How-ever,the time course of the increase in 45Ca 2?was slower than that of contraction,and the total amount of 45
Ca 2?increased to as much as 500?mol/kg in 30min.Furthermore,the decrease in 45Ca 2?following removal of stimulant was much slower than the decrease in mus-cle tension (Karaki and Urakawa,1972).These results suggest that this method measures 45Ca 2?in a cellular fraction in which Ca 2?gradually accumulates during contraction.Since the amount of 45Ca 2?in this fraction is larger than that in the intracellular space fraction (measured with the lanthanum method as described later),a part of this fraction may exist in the membrane surface.Neither the precise location nor the physiologi-cal role of this Ca 2?fraction has been defined.
https://www.wendangku.net/doc/a96427075.html,nthanum-inaccessible fraction.Due to their higher charge density,La 3?ions were predicted to have greater affinity than Ca 2?for any accessible anionic group that binds Ca 2?(Lettvin et al.,1964).Based upon anatomical evidence indicating that La 3?is restricted to the extracellular compartment (Laszlo et al.,1952),it was found that La 3?replaced 45Ca 2?at superficial membrane sites and prevented 45Ca 2?uptake to less accessible Ca 2?sites in smooth muscle preparations (Weiss and Goodman,1969;Goodman and Weiss,1971a,b;Weiss,1974).Van Breemen et al.(1972)attempted to remove only the extracellular 45Ca 2?by washing the tissue in a physiological salt solution (PSS)containing 2–10m M LaCl 3after completion of 45Ca 2?uptake and before tissue 45Ca 2?analysis.With this “lanthanum method,”they showed that during contraction of rabbit aorta with a high K ?solution,Ca 2?uptake was in-creased from the resting level of approximately 50?mol/kg of wet tissue to 150?mol/kg of wet tissue.They also found that replacement of Na ?in PSS by Li ?increased both 45Ca 2?uptake and muscle tension.However,there was no change in 45Ca 2?uptake during contractions induced by 10?M norepinephrine.Norepi-nephrine increased 45Ca 2?uptake only when muscle strips were preincubated with Ca 2?-free PSS (Deth and Van Breemen,1974)or in muscles depolarized by high K ?(Karaki and Weiss,1979,1980a,b).These results suggest that 45Ca 2?uptake increased only under “non-physiological”conditions and appeared to support the ideas that 1)both phases of norepinephrine-induced con-traction in the rabbit aorta are due mainly to Ca 2?release (Van Breemen et al.,1972;Bohr,1973;Cavero and Spedding,1983)and 2)access of extracellular Ca 2?is essential for refilling the intracellular release site (Deth and Van Breemen,1977).
To improve the lanthanum method by minimizing loss of 45Ca 2?during washout with La 3?solution,Godfraind (1976)employed a high concentration (50?M )of LaCl 3and found that norepinephrine increased the rate of 45
Ca 2?uptake without changing the total amount of 45
Ca 2?uptake in the rat aorta.Karaki and Weiss (1979)also modified this method for the same purpose by using a combination of high LaCl 3concentration and de-creased temperature.They found that norepinephrine increased the total amount of 45Ca 2?uptake in the rab-bit aorta only when it was depolarized.Van Breemen et al.(1981)also used decreased temperature to inhibit the loss of 45Ca 2?.Furthermore,they used EGTA instead of LaCl 3to remove the extracellular 45Ca 2?.With this method,they found that high K ?and norepinephrine increased the rate of 45Ca 2?uptake in the rabbit aorta (Meisheri et al.,1981;Van Breemen et al.,1981).
Norepinephrine also transiently increased the rate of 45
Ca 2?efflux (Godfraind,1976;Deth and Van Breemen,1977).In addition,norepinephrine decreased that Ca 2?concentration at “high affinity Ca 2?binding sites”with-out changing the Ca 2?concentration at “low affinity Ca 2?sites”(Karaki and Weiss,1979,1980a,b,c).These results provide support for the view that norepinephrine releases Ca 2?from cellular storage sites.
With the lanthanum method,increases in total 45Ca 2?uptake could be detected only under nonphysiological conditions such as stimulation with high K ?.Karaki and Weiss (1981b,1987)and Karaki et al.(1982)found that inhibition of mitochondrial function with antimycin A,oligomycin,potassium cyanide (KCN)and hypoxia abol-ished the high K ?-induced increase in 45Ca 2?uptake with little effect on contraction.Their finding indicates that the high K ?-induced increase in 45Ca 2?uptake is not associated with contraction and represents an incre-mental uptake of Ca 2?into mitochondria rather than as cytosolic free Ca 2?.This suggestion is consistent with the fact that the high K ?-induced increase in 45Ca 2?uptake (100to 300?mol/kg wet tissue;Van Breemen et al.,1972;Karaki and Weiss,1979)is much higher than the amount of Ca 2?necessary to induce contraction in permeabilized smooth muscle fibers (0.3to 3?M ;Endo et al.,1977).Thus,high K ?-induced depolarization,in-creased Ca 2?influx,and accumulation of mitochondrial Ca 2?constitute a sequential process,and the final step in this sequence can be specifically prevented by mito-chondrial inhibitors.Thorens and Haeusler (1979)found that papaverine inhibited 45Ca 2?uptake at a concentra-tion 10times lower than that needed to inhibit high K ?-induced contraction in the rabbit aorta.Since papav-erine is a potent inhibitor of mitochondrial function
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(Tsuda et al.,1977),this result also provides support for the sequence of events outlined above.
In the presence of high K ?,large amounts of Ca 2?entered the cell and were accumulated in mitochondria.Conversely,norepinephrine alone did not increase Ca 2?in mitochondria.However,norepinephrine can also in-crease Ca 2?influx because norepinephrine increased mitochondrial Ca 2?uptake in the presence of high K ?(Karaki and Weiss,1979,1981b;Meisheri et al.,1981).This result also suggests that high K ?may augment mitochondrial Ca 2?accumulation.Another alternative possibility is that high K ?may inhibit membrane Ca 2?extrusion to increase [Ca 2?]i to a level high enough to stimulate mitochondrial uptake of Ca 2?at sites of low Ca 2?affinity.However,this is not likely because inhi-bition of mitochondrial Ca 2?uptake did not change the sustained level of the high K ?-induced contraction (Karaki et al.,1982).Since Ca 2?at 1?M induces maxi-mum contractile responses in permeabilized smooth muscle,norepinephrine and high K ?may increase [Ca 2?]i to this level.Such a small increase may not be detectable by the lanthanum method because the resting level of Ca 2?uptake is as much as 50to 300?mol/kg wet tissue (Van Breemen et al.,1972;Karaki and Weiss,1979).
The effects of Ca 2?channel blockers on 45Ca 2?uptake in the rabbit aorta are also of interest.The same con-centrations of methoxyverapamil inhibited both high K ?-induced 45Ca 2?uptake and contraction (Meisheri et al.,1981).Similar results were obtained with nisoldipine (Van Breemen et al.,1985),verapamil (Karaki et al.,1984),and diltiazem (Van Breemen et al.,1981,1984;Cauvin et al.,1984a,b).These results indicate that the high K ?-induced contraction results from Ca 2?influx through the pathway sensitive to Ca 2?channel blockers.In contrast to this,methoxyverapamil at concentrations that almost completely inhibit the high K ?-induced changes had almost no inhibitory effects on that portion of the 45Ca 2?uptake and the accompanying contraction obtained with a high concentration of norepinephrine (10?M ).Higher concentrations of methoxyverapamil in-hibited the norepinephrine-stimulated 45Ca 2?uptake with little inhibitory effect on contraction.Nisoldipine (Van Breemen et al.,1985)and diltiazem (Cauvin et al.,1984b;Van Breemen et al.,1984)had similar selective inhibitory effects on 45Ca 2?uptake.These results sug-gest that a portion of the contraction induced by a high concentration (10?M )of norepinephrine in rabbit aorta is due to Ca 2?influx through a pathway less sensitive to Ca 2?channel blockers and that another portion of the contraction is not dependent on the increase in Ca 2?influx.Contractions which are not dependent on Ca 2?influx have been found to be due to both an activation of nonselective cation channels and an increase in Ca 2?sensitivity,as discussed in sections II.D.and III.A.It should also be noted that norepinephrine has con-centration-dependent dual effects on 45Ca 2?influx.
Compared to 45Ca 2?uptake and contraction stimulated by high K ?,the 45Ca 2?uptake and contraction elicited with higher concentrations of norepinephrine are less sensitive to inhibition by Ca 2?channel blockers,and those stimulated by lower concentrations of norepineph-rine are more sensitive to Ca 2?channel blockers than are those stimulated by high K ?(Van Breemen et al.,1981,1984).Furthermore,the 45Ca 2?influx pathway in resistance vessels stimulated by higher concentrations of norepinephrine is more sensitive to Ca 2?channel blockers than is the corresponding pathway in the aorta.Mechanisms of these differences are explained by acti-vation of different Ca 2?entry pathways,as is discussed in subsequent sections.
3.Suggested calcium movements in smooth muscle.Based on these observations,Bolton (1979)and Van Breemen et al.(1979),independently,suggested that the mechanisms of the increase in [Ca 2?]i in smooth muscle can be explained by two different Ca 2?influx pathways:receptor-linked and voltage-dependent Ca 2?channels (fig.1).High K ?induces membrane depolarization which,in turn,opens the voltage-dependent Ca 2?chan-nel.This channel is inhibited by agents blocking Ca 2?channels including verapamil,nifedipine and La 3?.In contrast,norepinephrine releases Ca 2?from storage sites to induce initial transient contractions and subse-quently opens the receptor-linked Ca 2?channel that is controlled by receptors for contractile agonists.In
the
F I
G .1.Calcium movements predicted mainly from contraction.High K ?depolarizes the membrane,opens the voltage-dependent Ca 2?channel,increases Ca 2?influx,and elicits sustained contrac-tion (1).Because the voltage-dependent Ca 2?channel is inhibited by Ca 2?channel blockers,contractions elicited by high K ?are inhibited by this type of blocker.In contrast,norepinephrine elicits Ca 2?release from the SR and initiates contraction (2).Because the amount of Ca 2?stored in the SR is limited,contraction due to Ca 2?release is transient.Ca 2?channel blockers do not inhibit Ca 2?re-lease.Norepinephrine also opens the receptor-linked Ca 2?channel,increases Ca 2?influx,and elicits sustained contraction (3).Calcium channel blockers only weakly inhibit the receptor-linked Ca 2?chan-nel.Thus,norepinephrine-induced contraction is less sensitive to Ca 2?channel blockers than is high K ?-induced contraction.This schema can now be revised as shown in figure 7.
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aorta,this channel is less sensitive to Ca 2?channel blockers than is the voltage-dependent Ca 2?channel.Opening of either of these channels results in a contin-uous Ca 2?influx to induce sustained contraction.Exis-tence of two types of Ca 2?channels seemed to be indi-cated by the findings in rabbit aorta that both the rates and total amounts of 45Ca 2?uptakes,stimulated by maximally effective concentrations of both high K ?and norepinephrine,are additive when the two agents were present at the same time (Karaki and Weiss,1979,1980a,b;Meisheri et al.,1981).As discussed later,how-ever,it now appears that high K ?and norepinephrine open the same L -type Ca 2?channel and that norepi-nephrine may also open a receptor-regulated nonselec-tive cation channel which conducts Na ?,K ?,and Ca 2?.High K ?and norepinephrine showed an additive effect on 45Ca 2?uptake not only because norepinephrine acti-vated both the L -type Ca 2?channel and nonselective cation channel but also because high K ?activated mi-tochondrial Ca 2?uptake.Furthermore,changes in Ca 2?sensitivity of contractile elements were not considered at the time.
C.Measurements of Cytosolic Free Calcium Level 1.Aequorin.Aequorin is a Ca 2?binding protein first extracted from the jelly fish,Aequorea aequorea ,by Shi-momura et al.(1962).This protein emits light at 465nm in the presence of Ca 2?.Ridgway and Ashley (1967)injected this photoprotein into barnacle single muscle fibers and measured [Ca 2?]i by monitoring changes in aequorin light.This method was applied to a single smooth muscle cell by Fay et al.(1979).Morgan and Morgan (1982,1984a,b)loaded the 21-kDa photoprotein into smooth muscle cells of ferret portal vein by tran-siently increasing the membrane permeability using a high concentration of EGTA,and measured [Ca 2?]i and contraction in isolated smooth muscle strips.They found that high K ?induced a sustained increase in [Ca 2?]i during sustained contraction,and both increases were inhibited by a decrease in extracellular Ca 2?concentra-tions (Morgan and Morgan,1982,1984a,b;De Feo and Morgan,1985).This supports the view that the high K ?-induced contraction is due to an increase in [Ca 2?]i resulting from activation of Ca 2?influx.In contrast,stimulation of the ?-adrenoceptors by phenylephrine in-duced a rapid rise of [Ca 2?]i to a maximum from which it decreased rapidly to a lower level and then declined more slowly,staying only slightly above basal [Ca 2?]i .At the same time,muscle tension rapidly increased to a maximum level and remained elevated as long as stim-ulation continued.During the phenylephrine-induced sustained contraction,removal of external Ca 2?de-creased [Ca 2?]i to a level lower than basal [Ca 2?]i and partially inhibited the contraction.From these results,it was postulated that the contractions induced by phen-ylephrine and high K ?are due to elevation of [Ca 2?]i above baseline,and that phenylephrine may increase the effectiveness of Ca 2?on the contractile apparatus (Morgan and Morgan,1984b).Receptor agonists pro-duced a larger force at a given [Ca 2?]i than did high K ?during the period of force maintenance also in ferret aorta (Suematsu et al.,1991b),rabbit aorta (Takuwa and Rasmussen,1987),guinea pig aorta (Jiang et al.,1994),swine carotid artery (Rembold and Murphy,1988a;Rembold,1990)and canine and bovine trachea (Gerthohoffer et al.,1989;Takuwa et al.,1987).
Although the agonist-induced sustained phase of the aequorin signal was believed to represent average [Ca 2?]i ,interpretation of the initial large transient in-crease in the aequorin signal was difficult.Measuring the light intensity of the aequorin signal,the peak level of the initial transient phase was 10to 20times higher than that of the sustained level (Abe et al.,1995).Ae-quorin has three Ca 2?binding sites in its molecule and occupation of at least two binding sites by Ca 2?results in radiation.Thus,the amount of radiation is propor-tional to 2.5th power of the Ca 2?concentration (Blinks et al.,1978).Calculating the Ca 2?concentration from light intensity by logarithmic transform,the agonist-induced transient phase of [Ca 2?]i is still 2.5to 3.3times higher than that of the sustained level.This result is different from that obtained with a fluorescent Ca 2?indicator,fura-2,which indicated that the peak levels of the ago-nist-induced transient and the sustained phases were almost identical (Abe et al.,1995).Furthermore,the agonist-induced initial increase in [Ca 2?]i was much larger than the sustained increase or the increase in-duced by high K ?.Even so,the initial transient contrac-tion was much smaller than that expected from the increase in [Ca 2?]i .Another interesting finding is that the initial transient increase in aequorin signal was rapidly desensitized by repeated applications of agonist although contractions did not change (Rembold and Murphy,1988b;Abe et al.,1995).The most likely expla-nation for the initial transient aequorin signal is that it represents the local increases in [Ca 2?]i ,as discussed later (see section II.E.1.).
2.Fluorescent indicators.A new fluorescent Ca 2?in-dicator,quin2,was synthesized by Tsien (1980).This was soon followed by the second generation of indicators including fura-2and indo-1(Grynkiewicz et al.,1985).These indicators are not membrane-permeable.To in-crease permeability,an acetoxymethyl radical is at-tached to these indicators.After loading smooth muscle cells with the acetoxymethyl esters of these indicators,the acetoxymethyl moiety is cleaved by endogenous es-terases and the indicator is trapped in the cell.
Measurements of [Ca 2?]i by the fluorescent indicators in smooth muscle tissues are much more difficult than in single cells.Abe and Karaki (1989)reported that,when 5?M acetoxymethyl ester of fura-2(fura-2/AM)was added to PSS,most of fura-2/AM was precipitated,and only 1?M was detected in the https://www.wendangku.net/doc/a96427075.html,ing this solu-tion,smooth muscle strips were not loaded with fura-2/
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AM,although platelets and single smooth muscle cells took up fura-2/AM.Centrifugation of this solution at 10,000?g for 2min decreased the effective concentra-tion of fura-2/AM to approximately 70%and there was no detectable fura-2/AM in the supernatant after a cen-trifugation at 50,000?g for 20min.This result indi-cates that fura-2/AM is insoluble in PSS,that only a small amount disperses as particles of various sizes,and that most of the particles are so large they are not able to enter the extracellular matrix of the smooth muscle tissues.To solubilize fura-2/AM,it is necessary to add small amounts of detergent and apply strong ultrasonic https://www.wendangku.net/doc/a96427075.html,ing this procedure,smooth muscle tissues can be loaded with fura-2/AM.
Using fura-2as an indicator,Ozaki et al.(1987c),in vascular tissue,and Himpens et al.(1988),in intestinal tissue,succeeded in obtaining simultaneous measure-ments of [Ca 2?]i and contraction.They found that [Ca 2?]i measured with fura-2showed better correlation with contraction than did [Ca 2?]i measured with ae-quorin.In rat aorta,both high K ?and norepinephrine induced the sustained increases in [Ca 2?]i during sus-tained contraction (Ozaki et al.,1987c;Sato et al.,1988a).In guinea pig ileum and taenia coli,high K ?elicited the sustained increases in [Ca 2?]i and sustained contractions,whereas carbachol elicited the transient increases in [Ca 2?]i and transient contractions (Him-pens et al.,1988;Ozaki et al.,1988;Mitsui and Karaki,1990).
Scanlon et al.(1987)and Malgaroli et al.(1987)re-ported a method to calculate Ca 2?concentrations from fura-2fluorescence in various types of animal tissues.However,it is difficult to obtain reliable values because of various limitations of fluorescent Ca 2?indicators (see Karaki,1989a).Among these,the most serious problem is that the change in dissociation constant (K d )of fura-2for Ca 2?.The K d value measured in vitro is different from that in cytoplasm mainly because fura-2binds to cytosolic proteins,changes K d ,and changes its fluores-cent characteristics (Konishi et al.,1988;Abe and Karaki,1989;Mitsui and Karaki,1990;Groden et al.,1991;Hochstrate and Juse,1991).Furthermore,endog-enous fluorescence,the intensity of which is also regu-lated by [Ca 2?]i (Ozaki et al.,1988),interferes with the fura-2fluorescence.Furthermore,fura-2leaks out of the cell relatively rapidly (Mitsui et al.,1993).Despite these difficulties,it was suggested that resting [Ca 2?]i is 100to 200n M and that high K ?and receptor agonists in-crease [Ca 2?]i to 300to 1500n M in vascular (Sato et al.,1988a)and intestinal smooth muscle (Himpens et al.,1988;Ito et al.,1988;Yagi et al.,1988;Mitsui and Karaki,1990).These results support the suggestion that smooth muscle contractility is primarily regulated by changes in [Ca 2?]i .
However,dissociation was observed between [Ca 2?]i and contraction in muscles stimulated with different agonists.In rat aorta,the maximum effective concentra-tion of norepinephrine induced a smaller increase in [Ca 2?]i than did the maximum effective concentration of KCl even though the norepinephrine-induced contrac-tion was larger than that induced by high K ?(Sato et al.,1988a;Karaki et al.,1988a),although the dissocia-tion was much smaller than that measured with ae-quorin.Similar results were obtained with other ago-nists including endothelin-1(Sakata et al.,1989;Kodama et al.,1994;Sudjarwo et al.,1995;Karaki and Matsuda,1996),prostaglandin F 2?(Ozaki et al.,1990c;Balwierczak,1991),serotonin (Thorin-Trescases et al.,1990),carbachol (Ozaki et al.,1990b;Himpens and Casteels,1990),clonidine (Takayanagi and Onozuka,1990),thromboxane analog (Himpens et al.,1990),pilo-carpine (Takayanagi and Ohtsuki,1990;Takayanagi et al.,1990),acetylcholine (Sato et al.,1994a)and neuro-kinin A (Sato et al.,1994b).These results support the view that agonists can increase Ca 2?sensitivity of con-tractile elements (see section III.).In guinea pig ileum (Matthijs et al.,1990;Himpens and Casteels,1990),in contrast,the Ca 2?sensitivity of the contractile elements was decreased during the sustained response to high K ?,whereas no changes were observed during pro-longed stimulation with substance P.Some relaxants showed different types of dissociation.Relaxants which increase cyclic AMP and cyclic GMP relaxed smooth muscle stimulated by high K ?or receptor agonists with a smaller inhibitory effect on [Ca 2?]i ,suggesting that both of these cyclic nucleotides decrease Ca 2?sensitivity of contractile elements (see sections III.and IV.A.2.and 3.).
Because of various problems related to [Ca 2?]i mea-surements using intracellular indicators,however,ob-served dissociation between [Ca 2?]i and contraction may be due to artifacts.These include uneven distribution of indicator in the cell,interference of the Ca 2?signal by endogenous fluorescent substances,and heterogeneous cell population in sample cells and tissues.Uneven dis-tribution of Ca 2?in the cell may also affect the relation-ship between contraction and average [Ca 2?]i in the cell.To confirm the changes in Ca 2?sensitivity,therefore,it is necessary to measure the [Ca 2?]i -force relationship using a completely different method.Permeabilized smooth muscle preparations are generally used for this purpose (Endo et al.,1977;Pfitzer,1996)and the effects of agonists and cyclic nucleotides on Ca 2?sensitivity are confirmed using this method.In the muscle permeabil-ized with ?-toxin or ?-escin,however,Kerrick and Hoar (1994)reported the possibility that the adenosine 5?-diphosphate (ADP)/ATP ratio within the cell is changed and the cells are not freely permeable to Ca 2?-ethyl-eneglycoltetraacetic acid.Care must be taken to make sure that the concentrations of intracellular ADP,ATP,and Ca 2?are held constant.Differences between the aequorin signal and the fura-2signal may be due to characteristics of aequorin including:1)insensitivity at low Ca 2?concentrations and resulting difficulty in de-
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tection of [Ca 2?]i changes near the resting level,2)non-linear response that results in an exaggerated effect in producing light if localized high concentrations of Ca 2?exist,and 3)possible inhomogenous distribution of ae-quorin in the cell (Karaki,1989a;Somlyo and Himpens,1989).
D.Mechanisms of Calcium Mobilization
1.Voltage-dependent calcium channels.There are six subtypes of voltage-dependent Ca 2?channels:L -,N-,P-,Q-,R-,and T-type.In smooth muscle,only the L -type Ca 2?channel is considered to be a major Ca 2?influx pathway (Vogalis et al.,1991;Ganitkevich and Isenberg,1991;Kuriyama et al.,1995;Knot et al.,1996;Hofmann and Klugbauer,1996).This channel is activated by membrane depolarization and inhibited by Ca 2?chan-nel blockers (see Godfraind et al.,1986).Agonists open this channel by depolarizing the cell membrane through activation of the nonselective cation channel (Pacaud and Bolton,1991),inhibition of the K ?channel and/or activation of the Cl ?channel (Kremer et al.,1989;Pac-aud et al.,1991;Miyoshi and Nakaya,1991;Iijima et al.,1991).Furthermore,agonists may open the L -type Ca 2?channels directly or indirectly through GTP-binding pro-teins in the absence of membrane depolarization (Nelson et al.,1988;Worley et al.,1991;Welling et al.,1992a,b,1993;Tomasic et al.,1992;Kamishima et al.,1992).The L -type Ca 2?channel is rapidly desensitized dur-ing sustained depolarization.However,high K ?-induced depolarization induces a sustained increase in [Ca 2?]i and a sustained contraction.Electrophysiological stud-ies showed that depolarization increased Ca 2?current,reaching a peak at about 10ms and then decreasing to a very low level.This small inward current is termed the noninactivating current,which is responsible for the sustained increases in [Ca 2?]i (Imaizumi et al.,1991;Fleischmann et al.,1994;Nakayama et al.,1996).
In rat aorta,a Ca 2?channel blocker,verapamil,in-hibited both the increase in [Ca 2?]i and the accompany-ing contraction induced by high K ?in a concentration-dependent manner.As shown in fig.2,higher concentrations of verapamil completely inhibited both the increase in [Ca 2?]i and the contraction induced by high K ?(Sato et al.,1988a;Karaki et al.,1991).Vera-pamil also inhibited the norepinephrine-induced in-crease in [Ca 2?]i in a concentration-dependent manner.Similar results were obtained with other Ca 2?channel blockers in other types of smooth muscle stimulated with other agonists,suggesting that the effects of vera-pamil are not due to nonselective inhibitory effects (see section IV.D.1.).These results do not support the idea that agonists open the receptor-linked Ca 2?channel,which is resistant to Ca 2?channel blockers (fig.1).Norepinephrine and other agonists seem to open the same verapamil-sensitive,L -type Ca 2?channel as does high K ?,and this channel may be the major Ca 2?influx pathway in smooth muscle.
The L -type Ca 2?channel activity is regulated also by the SR.Depletion of SR Ca 2?by ryanodine in rat femo-ral artery increased [Ca 2?]i and muscle tone,both of which were inhibited by verapamil (Kojima et al.,1994).In rat aorta (Sekiguchi et al.,1996),inhibition of the SR Ca 2?pump by cyclopiazonic acid depolarized the mem-brane and increased [Ca 2?]i .In guinea pig ileum (Uyama et al.,1993),cyclopiazonic acid also increased [Ca 2?]i and muscle tone both of which were inhibited by verapamil.Depletion of SR Ca 2?may inhibit the Ca 2?-activated K ?channel,depolarize the membrane and open the L -type Ca 2?channel.Agonists that release Ca 2?from the SR may have similar effects.
Calcium entry through the L -type Ca 2?channel is important to maintain the basal tone of smooth muscle (Rubart et al.,1966),especially in the arteries of spon-taneously hypertensive rats (Sada et al.,1990;Sasaki et al.,1993;Asano et al.,1993,1995b).Stretching vascular tissues activates the L -type Ca 2?channels and
increases
F I
G .2.Changes in [Ca 2?]i and contraction induced by high K ?and norepinephrine in the rat aorta without endothelium.Changes in [Ca 2?]i and contraction were measured simultaneously in the tissues loaded with a fluorescent Ca 2?indicator,fura-2.(A and B):Effects of 72.7?m KCl and 1?m norepinephrine,respectively.Ad-dition of a stimulant increased both [Ca 2?]i and muscle tension.Addition of 10m M verapamil almost completely inhibited [Ca 2?]i stimulated by high K ?or norepinephrine.High K ?-induced contrac-tion was also strongly inhibited (A).However,norepinephrine-in-duced contraction was only partially inhibited (B).Decrease in ex-ternal Ca 2?by 4?M ethyleneglycoltetraacetc acid (EGTA)decreased [Ca 2?]i below the resting level and further inhibited the norepineph-rine-induced contraction.However,a small portion of the contraction was resistant to EGTA (B).(C):Effects of norepinephrine in the presence of verapamil.Ten minutes after the addition of 10m M verapamil,1?M norepinephrine was added,which elicited a tran-sient increase in [Ca 2?]i followed by a small sustained increase.These changes were followed by rapid increase in muscle tension followed by sustained contraction that was smaller than that ob-served in the absence of verapamil in (B).(D):Effects of norepineph-rine in the presence of EGTA.Five minutes after the addition of 4m M EGTA,1?M norepinephrine was added.Norepinephrine elicited only a small transient increase in [Ca 2?]i ,accompanied by a rapid increase in muscle tension followed by a small sustained contraction that was smaller than that observed in the presence of verapamil in (C).(Modified from Ozaki et al.,1990c and Karaki et al.,1991).
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basal tone in coronary artery and basilar artery (Na-kayama and Tanaka,1989,1993).
The L -type Ca 2?channel is activated by the ?-adreno-ceptor in the cells isolated from tracheal (Welling et al.,1992a,b),rabbit ear artery (Benham and Tsien,1988),guinea pig taenia coli (Muraki et al.,1993),rat aorta (Neveu et al.,1994)and rabbit portal vein (Xiong et al.,1994).Although opening of the L -type Ca 2?channels increase [Ca 2?]i ,at least in a part of the smooth muscle cell,stimulation of the ?-adrenoceptors induce relax-ation but not contraction.This discrepancy may be ex-plained by the increase in cyclic AMP and also by the presence of a noncontractile Ca 2?compartment in the cell (see sections III.B.and IV.A.2.).
2.Nonselective cation channel and calcium release-activated calcium channel.Although the larger part of the agonist-induced Ca 2?increase was inhibited by Ca 2?channel blockers,a part of the increase was not.Verapamil did not completely inhibit the norepineph-rine-induced increase in [Ca 2?]i at concentrations which completely inhibited the high K ?-induced increase in [Ca 2?]i (Karaki et al.,1988a).Similar results were ob-tained with other Ca 2?channel blockers in other types of smooth muscles stimulated with other agonists (Sakata et al.,1989;Ozaki et al.,1990c;Sakata and Karaki,1992;Hori et al.,1992).In the presence of ve-rapamil,norepinephrine elicited a transient increase in [Ca 2?]i followed by a small sustained increase in the rat aorta (fig.2).Since the transient increase in [Ca 2?]i was inhibited by inhibitors of SR function such as ryanodine and thapsigargin,this increase may result from Ca 2?release from the SR by a mechanism that is insensitive to verapamil.In contrast,the small sustained increase in [Ca 2?]i ,which was insensitive to verapamil,was in-hibited by micromolar concentrations of La 3?(Harada et al.,1994,1996).Since the Ca 2?channel blockers are believed to selectively inhibit the L -type Ca 2?channel (see review by Godfraind et al.,1986;Catterall,1993;Kuriyama et al.,1995),and since La 3?inhibits both the
L -type and non-L -type Ca 2?
channels (Weiss,1974,1977,1996;Ruegg et al.,1989;Hescheler and Schultz,1993;Krautwurst et al.,1994;but see Inoue and Chen,1993),these results suggest that the norepinephrine-induced increase in [Ca 2?]i is due to Ca 2?influx through both the L -type and non-L -type Ca 2?channels.Enoki et al.(1995a,b)also showed that endothelin-1-induced Ca 2?influx,which was insensitive to Ca 2?channel blockers,was inhibited by a putative inhibitor of nonselective cation channel,mefenamic acid.Electrophysiological studies have also shown that receptor agonists activate the L -type Ca 2?channel and also the nonselective cation channel which is permeable to Ca 2?(Nelson et al.,1988;Kuriyama et al.,1995;Knot et al.,1996).In cultured A10smooth muscle cells,it was suggested that receptors are directly coupled to the non-L -type Ca 2?entry path-ways (Simpson et al.,1990).
In some vascular smooth muscles,Ca 2?influx through the non-L -type Ca 2?influx pathway does not seem to induce contraction.In rat aorta,the ATP-in-duced sustained increase in [Ca 2?]i ,which is due to Ca 2?influx,was only slightly inhibited by verapamil (Kitajima et al.,1994).Electrophysiological studies showed that ATP opens a nonselective cation channel which permits Ca 2?entry;this may be the mechanism of Ca 2?influx induced by ATP (Benham and Tsien,1987;Benham,1992).In single patch-clamped smooth muscle cells of rat portal vein (Pacaud et al.,1994),ATP-in-duced Ca 2?influx through nonselective cation channels activated the Ca 2?-induced Ca 2?release from the SR.However,ATP induced much smaller contractions than predicted from the increase in [Ca 2?]i (Kitajima et al.,1993,1996a).This dissociation may be explained by the presence of a noncontractile Ca 2?compartment in the cell (see section II.E.1.).
Another Ca 2?influx pathway which is not inhibited by Ca 2?channel blockers is the Ca 2?release-activated Ca 2?channel (CRAC)or capacitative Ca 2?entry path-way (Putney,1990).In smooth muscle,Casteels and Droogmans (1981)first suggested a possibility that there is a coupling between the peripheral SR and the surface membrane,allowing a one way rapid inward movement of Ca 2?.Cauvin et al.(1983,1984b)reported that lower concentrations of norepinephrine had less ability to release intracellular Ca 2?,that norepineph-rine did not release intracellular Ca 2?in the resistance arteries,and that Ca 2?channel blockers inhibited Ca 2?influx only in the resistance arteries.Their results sug-gest that Ca 2?release opens a Ca 2?influx pathway which is not sensitive to Ca 2?channel blockers.In cul-tured vascular A10cells,inhibition of the SR Ca 2?pump by thapsigargin mobilized an IP 3-sensitive SR Ca 2?pool and activated Ca 2?entry through a nicardipine-insen-sitive pathway (Xuan et al.,1992).In A7r5cells (Byron and Taylor,1995),arginine-vasopressin increased [Ca 2?]i by two different pathways,one of which is acti-vated by depletion of SR Ca 2?.In rabbit inferior vena cava,inhibition of SR Ca 2?accumulation by caffeine,ryanodine,and thapsigargin increased the steady-state [Ca 2?]i (Chen and Van Breemen,1993).In rat aorta,depletion of a Ca 2?store by ryanodine and caffeine increased [Ca 2?]i and muscle tone,both of which were insensitive to nicardipine (Hisayama et al.,1990).In bovine and porcine coronary arteries,ryanodine in-creased [Ca 2?]i (Wagner-Mann et al.,1992).In rat ileum (Ohta et al.,1995),the application of Ca 2?after expo-sure to a Ca 2?-free solution caused a small contraction and a rise in [Ca 2?]i ,both of which were potentiated when the muscle was challenged with carbachol or caf-feine before the addition of Ca 2?.Inhibition of SR Ca 2?pump by cyclopiazonic acid increased the Ca 2?-induced responses.Increases in [Ca 2?]i and contraction were inhibited by Cd 2?,Ba 2?,Ni 2?,or La 3?,but not by me-thoxyverapamil and nifedipine (Ohta et al.,1995).These
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results suggest the existence of CRAC in smooth muscle,and that an increase in [Ca 2?]i due to this mechanism is coupled to contraction.In ferret portal vein (Abe et al.,1996)and urinary bladder,however,the increases in [Ca 2?]i due to CRAC does not seem to induce contrac-tions (see section II.E.1.).
3.Sodium-calcium exchange.Bohr (1964)and Reuter et al.(1973)originally reported the contraction in rabbit aorta under conditions which implicate a Na ?/Ca 2?ex-change mechanism (Na ?pump inhibition or Na ?-free solution),although some of these effects were found to be evoked by the release of endogenous catecholamines pos-sibly due to Ca 2?influx into adrenergic nerves (Karaki and Urakawa,1977;Bonaccorsi et al.,1977;Karaki et al.,1978;Rembold et al.,1992).Experiments using a membrane-enriched microsomal fraction and smooth muscle cells revealed the presence of Na ?-dependent Ca 2?influx and efflux in smooth muscle of swine stom-ach (Raeymaekers et al.,1985),bovine trachea,porcine aorta and bovine aorta (Slaughter et al.,1987,1989)and rat aorta (Nabel et al.,1988).Lowering external Na ?concentration or increasing [Na ?]i elevated [Ca 2?]i in guinea pig taenia coli (Pritchard and Ashley,1986,1987),rat aorta (Matlib et al.,1986),swine carotid ar-tery (Rembold et al.,1992),human mesangial cells (Mene et al.,1990),cultured vascular smooth muscle (Batlle et al.,1991),the A10cells (Gillespie et al.,1992a),and the A7r5cells (Vigne et al.,1988;Bova et al.,1990;Gillespie et al.,1992b;Borin et al.,1994).The molecular structure of the Na ?/Ca 2?exchanger was also clarified (Nicoll and Philipson,1991).
Calcium influx mediated by Na ?/Ca 2?exchange in-duces contraction in some types of smooth muscle.In guinea pig aorta,ouabain and K ?-free solution induced sustained contraction with an increase in 45Ca 2?influx (Ozaki et al.,1978;Ozaki and Urakawa,1979,1981a)and an increase in [Ca 2?]i measured with fura-2(Iwamoto et al.,1992).In this preparation,Na ?-free solution alone induced sustained contraction,which was enhanced after loading with Na ?by pretreatment with ouabain (Ozaki and Urakawa,1981b).Slodzinski et al.(1995)reported that inhibition of Na ?/Ca 2?exchange by antisense in cultured arterial myocytes increased rest-ing [Ca 2?]i and inhibited the ouabain-induced augmen-tation of the agonist-induced increase in [Ca 2?]i .In rab-bit aorta,Khoyi et al.(1991)found that the 45Ca 2?uptake increased in the absence of external Na ?.
Na ?/Ca 2?exchange may be important for Ca 2?extru-sion because,in the membrane fraction of bovine aortic smooth muscle,the Na ?/Ca 2?exchanger has 3–6-fold transporting capacity than that of sarcolemmal Ca 2?-ATPase (Slaughter et al.,1989).Furthermore,co-local-ization of the Na ?/Ca 2?exchanger,Na ?-K ?pump,and a marker of the SR,calsequestrin,has been defined by high resolution,three dimensional microscope (Moore et al.,1993),suggesting a linkage between Na ?/Ca 2?ex-change and Ca 2?release from the SR.In A7r5cells,
ouabain increased both [Na ?]i and [Ca 2?]i ,and greatly augmented the release of Ca 2?from the SR evoked by thapsigargin,vasopressin and serotonin (Borin et al.,1994).Ouabain increased membrane-bound Ca 2?mea-sured with chlortetracycline,and this increase was in-hibited by thapsigargin or caffeine.These results sup-port the existence of functional linkage between Na ?/Ca 2?exchange and the SR.Ouabain may increase SR Ca 2?by increasing [Na ?]i and indirectly increasing [Ca 2?]i via Na ?/Ca 2?exchange across the sarcolemma.Most of Ca 2?that enters the cytoplasm is then stored in the SR,and this extra Ca 2?in SR can be mobilized so that the subsequent vasoconstrictor-evoked transient increases in [Ca 2?]i are amplified.
In contrast to the above results,others reported that Na ?/Ca 2?exchange plays little role in cellular Ca 2?homeostasis (Droogmans and Casteels,1979;Aaronson and Van Breemen,1981;Mulvany et al.,1984).Na ?-depletion alone did not increase muscle tone in rat aorta and mesenteric artery,whereas contractions induced by high K ?,serotonin and arginine-vasopressin were aug-mented by low Na ?solution (Bova et al.,1990).Also,in guinea pig coronary myocytes,removal of extracellular Na ?induced large increases in [Ca 2?]i only in Na ?-loaded cells,although either Na ?removal alone or Na ?loading alone did not change [Ca 2?]i (Ganitkevich and Isenberg,1993a).These results support the suggestion that Na ?/Ca 2?exchange is of minor importance for the increase in [Ca 2?]i as long as [Na ?]i is kept at physio-logical level.Aaronson and Benham (1989)reported that,in guinea pig urethra,although Na ?/Ca 2?ex-change can modulate [Ca 2?]i when [Na ?]i and mem-brane potential are at or near their physiological levels,[Ca 2?]i is regulated mainly by a Na ?-independent Ca 2?extrusion system.Morel and Godfraind (1984)showed that Na ?/Ca 2?exchange had a lower capacity,a lower affinity,and a slower rate than the ATP-dependent Ca 2?pump in plasmalemmal vesicles isolated from guinea pig ileum and aorta.In equine airway myocytes,the time constant for the decay in [Ca 2?]i after the stimulation of Ca 2?influx by depolarization pulse was not decreased in the absence of external Na ?(Fleischmann et al.,1996).Similar results were obtained in guinea pig coronary myocytes (Ganitkevich and Isenberg,1993a).
The inconsistent results for the physiological signifi-cance of Na ?/Ca 2?exchange may be due to differences between different species and different tissues (Ozaki and Urakawa,1981a;Petersen and Mulvany,1984).4.Calcium release from the sarcoplasmic reticulum.Measuring [Ca 2?]i in the SR in saponin-permeabilized cultured A7r5aortic smooth muscle cells using a fluo-rescent Ca 2?indicator,furaptra,Sugiyama and Gold-man (1995)found that the K d of the SR for Ca 2?was 49?M and resting SR Ca 2?was 75–130?M .In smooth muscle,Ca 2?is released from the SR (Stout and Diecke,1983;Yamamoto and Van Breemen,1986;Iino,1987;Sato et al.,1988a).There are two types of mechanisms to
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release Ca 2?from the SR in smooth muscle,Ca 2?-in-duced Ca 2?release (CICR)(Endo,1977;Ogawa,1994;Zucchi and Ronca-Testoni,1994)and IP 3-induced Ca 2?release (IICR)(Ferris and Snyder,1992;Mikoshiba,1993;Putney and Bird,1993).CICR is activated by Ca 2?(Itoh et al.,1981;Saida,1982;Iino,1989),whereas IICR is activated by IP 3(Suematsu et al.,1984;Somlyo et al.,1985;Islam et al.,1996).IICR is regulated not only by IP 3but also by Ca 2?.IICR is enhanced by Ca 2?below 300n M and,above this concentration,Ca 2?inhibited IICR (Iino,1990;Iino and Endo,1992;Iino and Tsukioka,1994).Calcium influx through the L -type Ca 2?channels also activates CICR in guinea pig aorta and urinary bladder and rat portal vein and mesenteric artery (Ito et al.,1991a;Ganitkevich and Isenberg,1992;Gregoire et al.,1993).Calcium influx mediated by the reverse-mode action of the Na ?/Ca 2?exchanger,which was undetectable by fura-2,released Ca 2?from the thapsigargin-sensitive intracellular stores including IP 3-releasable pools in cultured guinea pig ileum longi-tudinal muscle cells (Ohata et al.,1996).CICR is selec-tively activated by caffeine and selectively inhibited by ryanodine (Ito et al.,1986;Hisayama and Takayanagi,1988),whereas IICR is inhibited by heparin (Kobayashi et al.,1988;Ghosh et al.,1988;Chopra et al.,1989;Ganitkevich and Isenberg,1990;Komori and Bolton,1990).
In membrane fractions of guinea pig intestinal longi-tudinal smooth muscle,total binding sites of IP 3were 9–10-fold more numerous than those of ryanodine (Wibo and Godfraind,1994).The IP 3receptor and the ryano-dine receptor were localized primarily in the SR.How-ever,the stoichiometric ratio of the IP 3receptor to the ryanodine receptor was distinctly higher in the high density,ribonucleic acid (RNA)-rich subfractions than in the low density,RNA-poor subfractions,suggesting that the IP 3receptors were somewhat concentrated in the ribosome-coated portions of the SR.The low overall stoi-chiometric ratio of the ryanodine to the IP 3receptors might explain the existence of a Ca 2?-storage compart-ment that is devoid of CICR but has IICR.
Iino and co-workers (Iino et al.,1988;Yamazawa et al.,1992)classified Ca 2?stores into two subtypes using the permeabilized fibers of the guinea pig portal vein,pulmonary artery and taenia coli.One of these stores has both CICR and IICR (S ?),whereas the other has only the IICR mechanism (S ?).Ryanodine activated and then locked the CICR channels at open state,but had practically no effect on the IICR mechanism.Thus,after ryanodine-treatment,the Ca 2?store with the CICR (S ?)lost its capacity to hold Ca 2?.Changes in the agonist-evoked contraction of intact muscle due to the ryanodine treatment suggested that agonists release Ca 2?from the S ?store,which produces the initial phase of contrac-tions.In guinea pig taenia coli,CICR channels are present in 40%of the Ca 2?stores (Iino,1990).
In the ?-escin-permeabilized longitudinal smooth muscle of guinea pig ileum,caffeine,carbachol or IP 3produced a transient rise in tension in a Ca 2?-free solu-tion (Komori et al.,1995).The effect of either caffeine or carbachol was markedly reduced or abolished after pre-ceding application of the other stimulant.IP 3was with-out effect when applied subsequently to caffeine.The effects of carbachol and IP 3were abolished after com-bined treatment with ryanodine and caffeine,which causes functional removal of caffeine-releasable Ca 2?stores,but not after combined treatment with ryanodine and carbachol.These results suggest that caffeine,car-bachol and IP 3all act on common Ca 2?stores to release Ca 2?,possibly because this tissue has only the S ?store (with both IICR and CICR).Also,in guinea pig pulmo-nary artery (Iino,1990)and rat portal vein (Pacaud and Loirand,1995),most of the activator Ca 2?originates from the S ?store.
Cultured vascular smooth muscle appears to be devoid of ryanodine sensitive Ca 2?pools (Missiaen et al.,1990).In A7r5cells,vasopressin increased the fractional loss of 45
Ca 2?in Ca 2?-free solution which was not influenced by ryanodine.Caffeine did not stimulate the fractional loss of 45Ca 2?in this cell line.In saponin-skinned cells,IP 3released the 45Ca 2?which was not affected by ryan-odine or caffeine.These results suggest that A7r5cells have only S ?store (with only IICR).
In single myometrial cells from pregnant rats (Arnau-deau et al.,1994),oxytocin and acetylcholine evoked an initial peak in [Ca 2?]i followed by a smaller sustained rise.The transient increase in [Ca 2?]i was abolished by heparin,an inhibitor of IICR (Supattapone et al.,1988),and thapsigargin.In contrast,the transient [Ca 2?]i re-sponse induced by oxytocin was unaffected by ryano-dine.Moreover,caffeine failed to increase [Ca 2?]i but reduced the oxytocin-induced transient [Ca 2?]i re-sponse.In permeabilized fibers of pregnant rat myome-trium,caffeine did not produce contraction whereas both IP 3and the ionophore,A23187,evoked contractile re-sponses (Savineau,1988).These data show that myome-trial cells possess an IP 3-sensitive and thapsigargin-sensitive store (S ?),but do not possess ryanodine-and caffeine-sensitive stores (S ?).
In contrast to these observations,others suggested that Ca 2?stores cannot be classified into only two types.In rat vascular smooth muscle cells (Shin et al.,1991),some cells responded only to caffeine whereas other cells responded only to angiotensin II and released Ca 2?from the SR.In rat mesenteric artery smooth muscle cells (Baro and Eisner,1995),norepinephrine and caffeine produced a transient increase in [Ca 2?]i in Ca 2?free solution.In the presence of norepinephrine,caffeine or thapsigargin elevated [Ca 2?]i .However,if thapsigargin or caffeine was added first,the subsequent application of norepinephrine did not increase [Ca 2?]i .These results may suggest the existence of two types of Ca 2?stores;some stores are sensitive to both caffeine and agonist
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(S ?)whereas other stores are sensitive to caffeine and thapsigargin but not to agonist (S ?with only CICR).In permeabilized rabbit trachea smooth muscle cells (Chopra et al.,1991),Ca 2?release by IP 3was much greater than with guanosine 5?-O -(3-thiotriphosphate)(GTP ?S).Pretreatment with maximally effective IP 3abolished the GTP ?S-induced Ca 2?release,whereas pretreatment with GTP ?S reduced the IP 3-induced Ca 2?release by 25%.Ryanodine gave a large release of SR Ca 2?.After treatment with ryanodine,GTP ?S did not induce Ca 2?release,whereas the IP 3-induced Ca 2?re-lease was reduced by 76%.Pretreatment with ryanodine abolished the caffeine-induced Ca 2?release,and addi-tion of caffeine before ryanodine reduced the ryanodine-induced Ca 2?release by 64%.These results suggest that there are at least three Ca 2?pools present within air-way smooth muscle cells.The largest pool is released by IP 3or ryanodine (S ?),another is released only by IP 3(S ?),and the third by a high concentration of IP 3,ryan-odine or GTP ?S (which may be different from any of the above classifications).
Evidence also suggests a communication between dif-ferent types of Ca 2?stores.In cultured arterial myo-cytes,Tribe et al.(1994)found that IP 3and caffeine increased [Ca 2?]i by depleting different Ca 2?stores in the absence of external Ca 2?.Moreover,Ca 2?could be transferred between two stores,since prior application of caffeine,which alone evoked little or no increase in [Ca 2?]i ,significantly augmented the response to thapsi-gargin,which blocks Ca 2?sequestration in the IP 3-sen-sitive store.Conversely,a substantial caffeine-induced rise in [Ca 2?]i was observed only after the ability of the thapsigargin-sensitive Ca 2?store to sequester Ca 2?was inhibited.This suggests that the caffeine-sensitive store has a thapsigargin-insensitive Ca 2?sequestration mechanism.Chopra et al.(1991)also reported that,in permeabilized cultured rabbit trachea cells,Ca 2?moved from the GTP ?S-sensitive pool into the S ?store when this was depleted.Somlyo and co-workers have shown that norepinephrine released Ca 2?from both the junc-tional SR (Bond et al.,1984)and the central SR (Kowar-ski et al.,1985),and that the lumen of the various regions of the SR is continuous (Devine et al.,1972;Somlyo,1980)and permits the diffusion of Ca 2?from center to periphery or vice versa (Somlyo and Himpens,1989).Employing digital imaging technique,Tribe et al.(1994)and Golovina and Blaustein (1997)directly visu-alized the Ca 2?stores and found that although the SR appeared to be a continuous tubular network,Ca 2?stores in the SR were organized into small,spatially distinct compartments that functioned as discrete units and cyclopiazonic acid and caffeine with ryanodine un-loaded different spatially separated compartments.Characteristics of the SR seem to change during hy-pertension and other physiological and pathophysiolog-ical conditions.In vascular smooth muscle cells from spontaneously hypertensive rats (SHR)and Wistar Kyoto rats (WKY)(Neusser et al.,1994),thapsigargin induced a transient increase in [Ca 2?]i in Ca 2?free solution.The thapsigargin-induced peak [Ca 2?]i was not different in SHR cells and WKY cells.After depletion of the thapsigargin-sensitive Ca 2?pools,angiotensin II still increased [Ca 2?]i .In the SHR cells,the angiotensin II-induced increase in [Ca 2?]i was not significantly dif-ferent in the presence and absence of thapsigargin.In contrast,in the WKY cells,the response to angiotensin II was significantly diminished after depletion of the thapsigargin-sensitive pool.Furthermore,when angio-tensin II was added before thapsigargin,the thapsigar-gin response was diminished in the WKY cells but not in the SHR cells.These results suggest that vascular smooth muscle cells of WKY have two types of Ca 2?pools,a thapsigargin-and angiotensin II-sensitive type and an angiotensin II-sensitive type,whereas the SHR cells have a thapsigargin-sensitive type and an angio-tensin II-sensitive type.Levin et al.(1994)showed that partial outlet obstruction of the rabbit urinary bladder resulted in smooth muscle hypertrophy accompanied by a significant increase in the ability of ryanodine to in-hibit contraction induced by field stimulation.Ryano-dine binding also increased 4-fold at 5–7days postob-struction.Thus,smooth muscle hypertrophy secondary to partial outlet obstruction induced a marked increase in the role of intracellular Ca 2?in the mediation of the contractile response to field stimulation.
The function of the SR appears to change also with age.Neonatal rabbit bladder smooth muscle is not very sensitive to ryanodine,while that from mature rabbits is extremely sensitive.Gong et al.(1994)demonstrated that the number of ryanodine binding sites increased in rabbit bladder with normal maturation,suggesting that the bladder smooth muscle cell acquires an increased pool of sequestered intracellular Ca 2?for the develop-ment of normal contraction.
The SR is filled with Ca 2?mainly by Ca 2?influx.In resting rabbit aorta (Karaki et al.,1979),25to 30min was necessary to fill a norepinephrine-releasable store with Ca 2?.Almost all of the SR Ca 2?was released by single application of 1?M norepinephrine,as estimated by the norepinephrine-induced contraction in the ab-sence of external Ca 2?.Inhibition of Ca 2?influx by La 3?,Mn 2?,or Cd 2?inhibited the filling,whereas ve-rapamil,at the concentrations needed to completely in-hibit high K ?-induced contraction,did not inhibit the filling.This result suggests that resting Ca 2?influx,which is not mediated by the L -type Ca 2?channel,is responsible for SR Ca 2?filling.Since La 3?did not change the resting tone of the aorta,resting Ca 2?influx does not seem to be coupled to contraction.Calcium ion entering the cell through the resting Ca 2?influx path-way may be trapped by the SR without activating con-tractile elements (Casteels and Droogmans,1981).In A7r5cells,Blatter (1995)also showed that after releas-ing Ca 2?from the SR with vasopressin,the filling path-
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way of depleted stores involved Ca 2?entry into the bulk cytoplasmic compartment before uptake into the store.In the presence of high K ?,the SR accumulated greater amounts of Ca 2?and this process was inhibited by ve-rapamil (Karaki et al.,1979),suggesting that Ca 2?en-tering through the L -type Ca 2?channel is also taken up by the SR.In the presence of norepinephrine,however,accumulation of Ca 2?by the SR was inhibited in spite of an increase in Ca 2?influx.This inhibition may be due to opening of SR Ca 2?channel by norepinephrine.Bond et al.(1984)showed that repeated short-term applications of norepinephrine induced contractions in the absence of external Ca 2?and in the presence of La 3?in the high K ?-depolarized guinea pig portal vein,suggesting the recycling of SR Ca 2?when Ca 2?efflux was reduced by La 3?.
It is now generally accepted that Ca 2?release from the SR is responsible for only an initial portion of the agonist-induced sustained contraction (Karaki and Weiss,1984,1988)because,1)norepinephrine and other agonists induce only a transient contraction in the ab-sence of external Ca 2?,2)agonist-induced IP 3produc-tion is transient (Abdel-Latif,1986;Marmy et al.,1993;Dorn and Becker,1993),3)inhibitors of SR function by ryanodine inhibited the initial portion but not the sus-tained portion of agonist-induced contractions (Iino et al.,1988;Kanmura et al.,1988;Julou-Schaeffer and Freslon,1988),and 4)the agonist-induced increase in [Ca 2?]i was strongly inhibited by Ca 2?channel blockers (Sato et al.,1988b;Karaki et al.,1991)although these blockers did not inhibit Ca 2?filling of the SR (Karaki et al.,1979;Casteels and Droogmans,1981).However,Ashida et al.(1988)reported that ryanodine inhibited the norepinephrine-induced contraction by 52%in rat aorta and 14%in bovine tail artery without changing high K ?-induced contractions.Calcium channel blocker almost completely abolished high K ?-induced contrac-tions and reduced norepinephrine-induced contractions by 45%in the aorta and 82%in the tail artery.The inhibitory effects of ryanodine and Ca 2?channel blocker on the norepinephrine-induced contraction were https://www.wendangku.net/doc/a96427075.html,ing electron-microscopy,they also found that the tail artery has about 60%less SR than does the aorta and suggested that norepinephrine-induced sustained contraction is due to both Ca 2?influx through the L -type Ca 2?channel and Ca 2?release from the SR through the ryanodine-sensitive pathway;and that contractions in rat aorta are more dependent on Ca 2?release than in bovine tail artery.Weber et al.(1995)also reported that sustained contractions induced by submaximum concen-trations of norepinephrine were significantly inhibited by ryanodine whereas sustained contractions induced by a maximum concentration of norepinephrine were inhib-ited by a combination of Ca 2?channel blocker and ry-anodine.Furthermore,Iino et al.(1994a)reported that [Ca 2?]i oscillations induced by nerve stimulation or sub-maximum concentrations of norepinephrine were inhib-
ited by ryanodine in rat tail artery.These results sug-gest that,in some types of vascular smooth muscle,sustained contractions induced by submaximum concen-trations of norepinephrine are due to summation of con-tractions in individual cells which induce oscillatory con-tractions by release of SR Ca 2?.Graded contractions may result from differences in the threshold in individ-ual cells (Ohta et al.,1994;Suzuki et al.,1994).Since agonist-induced production of IP 3is transient,the oscil-latory release of Ca 2?may be due to activation of CICR.In contrast,a maximum concentration of norepineph-rine may induce Ca 2?influx to evoke sustained contrac-tions in all of the cells.Calcium ion and/or other diffus-ible messengers can diffuse between smooth muscle cells though gap junctions and propagate Ca 2?waves through silent cells (Christ et al.,1992;Young et al.,1996).This mechanism may also contribute to synchro-nize smooth muscle cells in the absence of synchroniza-tion of action potentials or sustained membrane depo-larization.
5.Calcium pumps in plasmalemma and the sarcoplas-mic reticulum.In smooth muscle,there are two types of Ca 2?ATPase,plasmalemmal Ca 2?ATPase and SR Ca 2?ATPase (Wuytack et al.,1982;Raeymaekers et al.,1985;Verbist et al.,1985;Raeymaekers and Wuytack,1996).The plasmalemmal Ca 2?-ATPase activity was four times higher than the (Na ??K ?)-ATPase activity in human myometrial smooth muscle (Popescu and Ig-nat,1983).Since Ca 2?extrusion through the Na ?/Ca 2?exchange mechanism would ultimately be limited by the (Na ??K ?)-ATPase activity,this result suggests that plasmalemmal Ca 2?-ATPase plays a more important role in Ca 2?extrusion than does Na ?/Ca 2?exchange.In cultured rat aortic smooth muscle cells,12-O-tetradeca-noylphorbol-13-acetate (TPA)increased the maximum Ca 2?efflux rate without changing the affinity for Ca 2?(Furukawa et al.,1988,1989).In Ca 2?-ATPase purified from bovine aortic smooth muscle,it was also shown that phorbol ester stimulated the ATPase activity which was accompanied by phosphorylation of the ATPase,suggesting that the plasmalemmal Ca 2?-pump in vas-cular smooth muscle is activated by protein kinase C (C kinase).Sodium nitroprusside and 8-bromo-cyclic GMP also stimulated the Ca 2?pump activity although fors-kolin and dibutyryl cyclic AMP were ineffective (Yoshida et al.,1991;Furukawa et al.,1988).
The SR Ca 2?-ATPase (SERCA)is derived from three distinct genes (Eggermont et al.,1989;Lytton et al.,1989;Amrani et al.,1995a);SERCA-1,which is ex-pressed in skeletal muscle,SERCA-2,which gives rise to the SERCA-2a and SERCA-2b isoforms,mainly ex-pressed in cardiac and smooth muscles,respectively,and SERCA-3expressed in smooth and non-muscle tis-sue.In human tracheal smooth muscle cells,expression of SERCA-2b isoform was greater than that of SERCA-2a isoform (Amrani et al.,1995a).The SERCA-2a,SERCA-2b,and SERCA-3are inhibited by thapsi-
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gargin (Lytton et al.,1992).Cyclopiazonic acid also in-hibits SERCA (Seidler et al.,1989;Bourreau et al.,1991;Low et al.,1992;Uyama et al.,1992,1993).
Luo et al.(1993)demonstrated that relaxation of ar-terial smooth muscle induced by nitroglycerin or atrial natriuretic peptide was inhibited by thapsigargin or cy-clopiazonic acid without affecting the increment of cyclic GMP content,suggesting that the enhanced sequestra-tion of Ca 2?by the SR may be an important mechanism by which nitric oxide-related compounds induce relax-ation.In canine trachea (McGrogan et al.,1995),relax-ant effects of sodium nitroprusside and 8-bromo-cyclic GMP were attenuated by cyclopiazonic acid.These re-sults are consistent with the finding that G kinase stim-ulates the plasmalemmal Ca 2?pump ATPase (Imai et al.,1990;Yoshida et al.,1991).In small mesenteric resistance arteries of the rat,3-morpholino-sydnonimine and sodium nitroprusside increased cyclic GMP and in-hibited the increase in [Ca 2?]i ,MLC phosphorylation and the contractile response to ATP (Andriantsitohaina et al.,1995).Thapsigargin reversed the inhibitory effect of the vasodilator agents when the contraction induced by ATP was elicited in the presence of the Ca 2?channel blocker,nitrendipine,or in Ca 2?-free medium.These results show that cyclic GMP inhibits ATP-induced con-traction partly by enhanced Ca 2?sequestration through a SR Ca 2?pump activation.In rat aorta,ryanodine,on the other hand,had no effect on the concentration-re-sponse curves for isoproterenol-induced relaxation (Hi-sayama et al.,1990).In rat thoracic aorta and bovine tail artery,Ashida et al.(1988)also showed that,although ryanodine had no effect on basal tone,it progressively increased tension when Ca 2?extrusion via Na ?/Ca 2?exchange was inhibited by low external Na ?.The smaller effects of ryanodine indicate that the SR plays a less important role in controlling [Ca 2?]i .
In canine tracheal smooth muscle (Bourreau et al.,1993),cyclopiazonic acid inhibited refilling of the stores occurring during high K ?stimulation.On the other hand,cyclopiazonic acid was less effective in inhibiting the refilling occurring during prolonged acetylcholine stimulation.At higher external Ca 2?or when BAY k8644was present in the medium,cyclopiazonic acid was ineffective in inhibiting the refilling during stimu-lation with acetylcholine.These results suggest the presence of two different pathways for external Ca 2?used to refill acetylcholine-sensitive internal stores.One involves active Ca 2?uptake via a cyclopiazonic acid-sensitive Ca 2?pump,and the other involves a cyclopia-zonic acid-insensitive pathway.
In bovine tail artery cells (Goldman et al.,1989),[Ca 2?]i was relatively uniformly distributed before acti-vation.During norepinephrine-evoked contractions,[Ca 2?]i increased,and the distribution of [Ca 2?]i became much more heterogeneous.On recovery from activation,discrete regions of elevated [Ca 2?]i were observed throughout the recovered cells.The large spatial varia-
tion of [Ca 2?]i after cell activation implies that Ca 2?was sequestered at localized sites in the cell during relax-ation.In rat mesenteric artery cells (Baro and Eisner,1995),both norepinephrine and caffeine released Ca 2?.The recovery of [Ca 2?]i during the application of caffeine was unaffected by the removal of external Na ?,suggest-ing that Na ?/Ca 2?exchange is not important in the reduction in [Ca 2?]i .The addition of an inhibitor of Ca 2?-ATPase,La 3?,did,however,greatly slow [Ca 2?]i recovery.From these and other results,they concluded that the three major factors responsible for removing Ca 2?ions from the cytoplasm are:a caffeine-and nore-pinephrine-sensitive store (43%),a caffeine-sensitive but norepinephrine-insensitive store (36%),and a sar-colemmal Ca 2?-ATPase (16%).Finally,a 5%contribu-tion remains to be accounted for.
6.Mitochondria.Mitochondrial inhibitors decrease ATP production and contraction in intestinal smooth muscle.However,neither ATP contents nor contractions were decreased by these inhibitors in vascular smooth muscle,possibly because ATP is supplied not only by mitochondria but also by glycolysis (Karaki et al.,1982;Nakagawa et al.,1985).Inhibition of oxidative phos-phorylation by nitrogen gas,dinitrophenol or sodium azide elicited a release of Ca 2?from mitochondria to induce transient contraction in rat aorta (Karaki et al.,1982),rabbit colon (Kowarski et al.,1985)and rat myo-metrium (Sakai et al.,1986).These results suggest the possible involvement of mitochondrial Ca 2?release in smooth muscle contraction.Inhibition of mitochondrial Ca 2?uptake may also elicit contraction.Takeo and Sa-kanashi (1985)estimated the mitochondrial Ca 2?up-take activity of the coronary artery to be 250nmol Ca 2?/mg protein/10min.Kowarski et al.(1985)ana-lyzed subcellular Ca 2?concentrations in rabbit main pulmonary artery smooth muscle cells by electron probe X-ray microanalysis and estimated the mitochondrial Ca 2?to be 2.2mmol/kg dry weight,and this was not changed after the muscle was exposed to norepineph-rine.In contrast,the central SR can accumulate larger amounts of Ca 2?,and norepinephrine released Ca 2?from the SR.The relative sizes of the central SR and mitochondrial Ca 2?pools in relaxed tissue were about 20:1.In rabbit portal vein,smooth muscle was loaded with Na ?for 3h in a K ?-free,ouabain-containing solu-tion,after which rapid Na ?/Ca 2?exchange was induced by Na ?-free solution (Broderick and Somlyo,1987).This procedure induced a large transient contraction accom-panied by a large increase in [Ca 2?]i which was taken up by mitochondria.
Grover and Samson (1986)compared affinity charac-teristics of the Ca 2?pumps toward Ca 2?in various subcellular organelles isolated from pig coronary artery.The K m value was 0.91?M for plasma membrane,0.58?M for endoplasmic reticulum,and as high as 7.1?M for mitochondria.45Ca 2?uptake experiments showed that high K ?depolarization increases mitochondrial Ca 2?
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uptake (see section II.B.2.).Ueno (1985)examined the mobilization of 45Ca 2?in the saponin-permeabilized smooth muscle cell of the porcine coronary artery and found the minimum [Ca 2?]i required for the ATP-depen-dent Ca 2?uptake by the SR and mitochondria was about 20n M and 1?M ,respectively.In saponin-perme-abilized primary cultured rat aortic smooth muscle cells,Yamamoto and Van Breemen (1986)reported that mito-chondrial 45Ca 2?uptake appeared only in the presence of nonphysiologically high concentrations of Ca 2?(10?M and higher).Stout (1991)also examined 45Ca 2?uptake in saponin-permeabilized rat caudal artery and found that mitochondrial Ca 2?content increased only when the free Ca 2?concentration exceeded 3.1?M .
Although these observations suggest the lack of in-volvement of mitochondria in the decrease in [Ca 2?]i in smooth muscle,Drummond and Fay (1996)reported that,in the voltage-clamped single stomach smooth muscle cells of Bufo marinus ,the rate of Ca 2?extrusion from the cytosol following depolarizing pulses was re-duced by more than 50%by cyanide or carbonyl cyanide p -trifluoromethoxy-phenylhydrazone.The inhibitor of both mitochondrial Ca 2?uniporter and ryanodine recep-tor,ruthenium red,produced a similar result while the ATP synthetase inhibitor,oligomycin,had no effect,in-dicating that the effect is not due to inhibition of Ca 2?-ATPase resulting from ATP insufficiency.This result suggests that mitochondria may play a significant role in removing Ca 2?from the cytoplasm in toad smooth muscle.
Glycolysis (glycogenolysis)is stimulated not only by inorganic phosphate and ADP,which activate phospho-fructokinase,but also by Ca 2?and calmodulin,which activate phosphorylase b kinase.Since reduced pyridine nucleotides,located both in the cytoplasm and mitochon-dria,and oxidized flavoproteins,located specifically in the inner mitochondrial membrane,are fluorescent sub-stances,it is possible to fluorometrically measure redox states in cells.As shown in fig.3,reduced pyridine nucleotides and oxidized flavoproteins increased in re-sponse to spontaneous mechanical activities in guinea pig taenia coli (Ozaki et al.,1988),indicating that large oxidation-reduction potentials are generated across the mitochondrial membrane during contractions.The amount of reduced pyridine nucleotides is closely corre-lated with force of contractions in guinea pig ileum (Shimizu et al.,1991).Interestingly,flavoprotein fluo-rescence started to increase 0.5–1s before the initiation of contraction,and this time course corresponded to the change in [Ca 2?]i .Furthermore,Ca 2?sensitivity was in the order of flavoprotein fluorescence ?pyridine nucle-otide fluorescence ?muscle contraction (fig.3).Chance (1965)has observed that Ca 2?increased the rate of respiration and electron transport of mitochondria.Fur-thermore,the intra-mitochondrial key enzymes for oxi-dative metabolism such as dehydrogenases were acti-vated by micromolar concentrations of Ca 2?(see
Balaban,1990).These findings suggest that the [Ca 2?]i directly activates three different mechanisms,cytoplas-mic glycolysis,mitochondrial oxidation of flavoproteins,and contractile elements in cytoplasm.
Rizzuto et al.(1992,1994)have developed molecularly engineered Ca 2?-sensitive photoproteins and applied this to study mitochondrial Ca 2?dynamics.In HeLa cells and bovine endothelial cells,mitochondrial Ca 2?increased rapidly upon stimulation with IP 3-generating agonists such as ATP,carbachol,and histamine.Moni-toring the level of NAD(P)H fluorescence suggested that the changes in mitochondrial Ca 2?were sufficiently large to induce a rapid activation of mitochondrial de-hydrogenases.
These observations suggest that contractile stimula-tions increase the Ca 2?concentration not only in cyto-plasm but also in the mitochondria.Calcium ion stimu-lates ATP production by mitochondria before it is triggered by energy consumption of contractile
ele-
F I
G .3.Changes in the fluorescence of reduced pyridine nucleo-tides (PNred)(A)and oxidized flavoproteins (FPox)(B)during spon-taneous contraction in guinea pig taenia coli.PNred and FPox fluo-rescence are shown by relative intensity of fluorescence taking the basal fluorescence as 100%.FPox started to increase before the initiation of contraction.(C):The effects of external Ca 2?concentra-tion on PNred fluorescence,FPox fluorescence,and tension develop-ment in the 45.4m M K ?-depolarized taenia coli.Responses induced by 10mM Ca 2?was taken as 100%.The Ca 2?sensitivity of each response was in the order of FPox ?PNred ?muscle contraction.(Modified from Ozaki et al.,1988).
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ments.Mitochondria may also serve as a Ca 2?sink under pathophysiological conditions where [Ca 2?]i in-creases above micromolar concentrations.E.Calcium Distribution and Function
1.Noncontractile calcium compartment.Most of the data obtained from simultaneous measurement of [Ca 2?]i and contraction confirm that there is a positive correlation between these two parameters and that smooth muscle contraction occurs following an increase in [Ca 2?]i .However,some small dissociations were iden-tified.In some types of smooth muscle,agonists induced larger contractions than predicted from the increase in [Ca 2?]i .This kind of dissociation may be explained by Ca 2?sensitization of contractile elements.In contrast,relaxants related to cyclic AMP and cyclic GMP decrease contractile force without decreasing [Ca 2?]i or with only a small decrease in [Ca 2?]i ,possibly by an attenuation of Ca 2?sensitivity of the contractile elements.However,some kinds of dissociations are explained neither by the changes in Ca 2?sensitivity nor by artifacts of [Ca 2?]i measurements.
a.A EQUORIN SIGNAL AND FURA -2SIGNAL .The Ca 2?signal obtained with aequorin was different from that predicted from contractile data in smooth muscle.Ago-nist-induced sustained contractions were accompanied by large and transient increases followed by only the small sustained increases in the aequorin signal (see section II.C.1.).The transient increase in the aequorin signal,which was due to both Ca 2?release and Ca 2?influx,was rapidly desensitized by repeated applications of agonist,although contractile tension did not change.When muscle strips were left unstimulated for 2.5–13h,the transient increase in the aequorin signal returned (Rembold and Murphy,1988b;Abe et al.,1995).Al-though the high K ?-induced sustained contraction was accompanied by a sustained increase in the aequorin signal due to Ca 2?influx,repeated applications of high K ?also gradually attenuated the aequorin signal with-out changing the magnitude of the contraction,and a 13-h resting period was needed for complete recovery of the aequorin signal (Abe et al.,1995).Although the changes in aequorin signals are much larger than the changes in [Ca 2?]i (see section II.C.1.),dissociation be-tween aequorin signals and contractions are evident.In contrast,the fura-2signal did not desensitize,and there was much better correlation between the fura-2signal and contraction.These results indicate that a part of the aequorin signal,stimulated either by Ca 2?release or Ca 2?influx,does not represent [Ca 2?]i regulating the contractile elements.
Karaki (1989a)suggested that the difference between the aequorin signal and the fura-2signal may arise from the inhomogeneous or focal increases in [Ca 2?]i .In swine carotid artery,Rembold and co-workers (Rembold et al.,1995;Van Riper et al.,1996;Rembold,1996)compared the aequorin signal and the fura-2signal and
found that the ratio of the aequorin signal and the fura-2signal changed depending upon the types of stimulation employed and that contraction is more closely correlated with the fura-2signal.From these results,they con-cluded that the aequorin/fura-2ratio can be used as an indicator of the focal increase in [Ca 2?]i .Using this method,they found that histamine-induced Ca 2?re-lease resulted in the focal increases in [Ca 2?]i in the absence of external Ca 2?.Histamine-induced increase in [Ca 2?]i was accompanied by increased MLC phosphory-lation and contraction.Caffeine elicited similar focal increase of [Ca 2?]i in the presence of external Ca 2?.However,caffeine elicited only a small increase in MLC phosphorylation and small contraction.A focal [Ca 2?]i increase was also observed when the external Ca 2?was restored in muscle treated with Ca 2?-free solution or when Na ?/Ca 2?exchange was inhibited by decreasing the external Na ?concentration.These changes were accompanied by neither MLC phosphorylation nor con-traction.These results suggest that increase in [Ca 2?]i is localized to a region distant from the contractile appa-ratus under these conditions.Only histamine increased MLC phosphorylation possibly because it increases Ca 2?sensitivity of MLC phosphorylation (see section III.A.).b.I NHIBITION OF SARCOPLASMIC RETICULUM CALCIUM ACCUMULATION AND ACTIVATION OF CALCIUM ENTRY .Inhi-bition of SR function is expected to increase [Ca 2?]i by three different mechanisms.The first mechanism is in-hibition of SR Ca 2?uptake and resulting increase in [Ca 2?]i near the SR.In rabbit inferior vena cava,inhi-bition of SR functions by caffeine,thapsigargin or ryan-odine increased the steady-state [Ca 2?]i (Chen et al.,1992;Chen and Van Breemen,1993).In guinea pig ureter (Maggi et al.,1995),inhibition of SR Ca 2?uptake by cyclopiazonic acid enhanced the contractions evoked by electrical stimulation or low-Na ?medium.Inhibition of SR Ca 2?uptake augmented contractions also in rab-bit aorta (Van Breemen et al.,1985),bovine coronary artery (Sturek et al.,1992)and guinea pig ureter (Maggi et al.,1995)(see section II.E.3.).In ferret portal vein (Abe et al.,1996),in contrast,inhibition of SR Ca 2?uptake by cyclopiazonic acid increased [Ca 2?]i measured with aequorin without changing contractions induced by norepinephrine or high K ?.However,depletion of SR Ca 2?by ryanodine and caffeine did not have such an effect,suggesting that the increase in [Ca 2?]i is due to inhibition of SR Ca 2?uptake but not to increased Ca 2?influx by activation of CRAC.Also,in rat urinary blad-der,Munro and Wendt (1994)measured [Ca 2?]i with fura-2and reported that cyclopiazonic acid augmented the increase in [Ca 2?]i induced by carbachol and high K ?without changing contraction.From these results,Abe et al.(1995,1996)suggested that there are two Ca 2?compartments in the smooth muscle cell,a com-partment containing contractile elements (contractile compartment)and another compartment unrelated to contractile elements (noncontractile compartment)(fig.
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4).On stimulation,Ca 2?concentration in the contractile compartment may increase to a level high enough to stimulate MLC kinase but not so high as to consume aequorin rapidly.In contrast,the Ca 2?concentration in the noncontractile compartment may increase so much that aequorin in this compartment is rapidly consumed.These two compartments may be separated by a diffu-sion barrier and,during a resting period,aequorin may slowly diffuse from the contractile compartment to the noncontractile compartment and thus restore the full aequorin signal.The noncontractile compartment may be located near the SR,and the Ca 2?concentration in this compartment may be regulated not only by Ca 2?influx but also by SR Ca 2?uptake.Calcium ion in this compartment cannot reach the contractile compartment because of a diffusion barrier and sequestration by the SR.
The second SR-mediated mechanism to increase [Ca 2?]i is to deplete SR Ca 2?and activate Ca 2?entry through CRAC (see section II.D.2.).In rat aorta,ryano-dine increased [Ca 2?]i measured with fura-2and muscle tone,both of which were insensitive to nicardipine (Hi-sayama et al.,1990).In ferret portal vein,in contrast,cyclopiazonic acid induced a sustained increase in [Ca 2?]i measured with aequorin without inducing con-traction (Abe et al.,1996).In rat mesenteric artery,
ryanodine and cyclopiazonic acid induced a sustained increase in [Ca 2?]i measured with fura-2without induc-ing contraction (Naganobu and Ito,1994;Naganobu et al.,1994).In rat urinary bladder,cyclopiazonic acid also increased [Ca 2?]i measured with fura-2without induc-ing contraction (Munro and Wendt,1994).There ap-pears to be tissue-specific differences in the coupling between CRAC and contraction.
The third SR-mediated mechanism to increase [Ca 2?]i is membrane depolarization resulted from inhibition of the Ca 2?-activated K ?channels (see section II.D.).De-pletion of SR Ca 2?by ryanodine or cyclopiazonic acid increased [Ca 2?]i and induced contraction,both of which were inhibited by verapamil in rat femoral artery (Kojima et al.,1994)and guinea pig ileum (Uyama et al.,1993).
c.S TIMULANT -DEPENDENT DISSOCIATION .In rat aorta,norepinephrine induced an initial large increase in [Ca 2?]i due to Ca 2?release followed by a sustained increase due to Ca 2?influx.Initial Ca 2?release was accompanied by a corresponding increase in IP 3forma-tion (Manolopoulos et al.,1991;Ahn et al.,1992;Pijuan et al.,1993)and transient contraction (Sato et al.,1988a;Karaki et al.,1988a).Endothelin-1acted on the ET A receptor and increased IP 3formation (Huang et al.,1990b)and [Ca 2?]i in a manner similar to norepineph-rine.However,the initial increase in [Ca 2?]i was not accompanied by contraction (Sakata et al.,1989;Ozaki et al.,1989;Huang et al.,1990a)or MLC phosphoryla-tion (Harada et al.,1994,1996).In contrast,the ET A receptor-mediated Ca 2?influx,observed several min-utes after the addition of endothelin-1,was accompanied by a large increase in MLC phosphorylation and contrac-tion (Harada et al.,1994,1996).Similar dissociation between Ca 2?release and contraction was reported in vascular smooth muscle stimulated with prostaglandin F 2?(Ozaki et al.,1990c;Dorn et al.,1992;Kurata et al.,1993).Simultaneous applications of norepinephrine and endothelin-1induced larger Ca 2?release than that in-duced by either of the agonists alone,although the mag-nitude of transient contraction was similar to that in-duced by norepinephrine alone (our unpublished observation),suggesting that endothelin-1does not have an inhibitory effect on contractile elements including an activation of MLC phosphatase.These results suggest that Ca 2?release induced by some agonists is not cou-pled to MLC phosphorylation and contraction,possibly because some agonists release Ca 2?in the direction of a contractile compartment whereas other agonists release Ca 2?in the direction of a noncontractile compartment.Hisayama et al.(1990)reported that Ca 2?release in-duced by prostaglandin F 2?,which was not accompanied by contraction,was insensitive to ryanodine whereas Ca 2?release induced by caffeine or phenylephrine,which was accompanied by transient contraction,was sensitive to ryanodine.These results suggest that there are two types of Ca 2?stores;one of which (sensitive
to
F I
G .4.Two Ca 2?compartments model (modified from Abe et al.,1996).The major Ca 2?compartment in the smooth muscle cell is the contractile compartment.In addition,there is a small Ca 2?compart-ment between plasmalemma and the SR that does not contain con-tractile elements (noncontractile compartment).Communication be-tween these two compartments is restricted,and aequorin cannot move freely between these compartments.Calcium ion in this com-partment also cannot reach the contractile compartment because of a diffusion barrier and sequestration by the SR.Inhibition of SR Ca 2?pump by cyclopiazonic acid increased [Ca 2?]i in the noncon-tractile compartment with little effect on the contractile Ca 2?com-partment.In contrast,depletion of the SR by ryanodine and caffeine inhibited the agonist-induced transient increase in [Ca 2?]i in con-tractile compartment with little effect on [Ca 2?]i in the noncontrac-tile compartment.Rates of decrease in contraction and [Ca 2?]i were affected neither by cyclopiazonic acid nor by ryanodine and caffeine.
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phenylephrine,caffeine and ryanodine)supplies Ca 2?only to the contractile compartment whereas the other (sensitive only to prostaglandin F 2?)supplies Ca 2?only to the noncontractile compartment.
d.N ONSELECTIVE CATION CHANNEL .ATP has been shown to increase Ca 2?influx through the nonselective cation channel (Benham and Tsien,1987;Benham,1992).In rat basilar artery,an agonist of the P 2purino-ceptor,ATP,induced contraction following an increase in [Ca 2?]i by both releasing Ca 2?and increasing Ca 2?influx through the non-L -type Ca 2?channel (Zhang et al.,1995).In rat aorta (Kitajima et al.,1993,1994,1996a),ATP also induced a larger increase in [Ca 2?]i than that induced by high K ?mainly by Ca 2?influx and partly by Ca 2?releas
e.The ATP-induced increase in [Ca 2?]i was accompanied by a smaller increase in MLC phosphorylation and a smaller contraction than those induced by high K ?-stimulated [Ca 2?]i .In swine carotid artery (Rembold et al.,1991),ATP also induced a larger increase in [Ca 2?]i measured with aequorin,and a smaller increase in MLC phosphorylation and contrac-tion than that induced by histamine.In mouse urinary bladder (Boland et al.,1993),ATP inhibited carbachol-induced contraction with little effect on [Ca 2?]i .ATP also inhibited norepinephrine-induced contraction in rat aorta with little inhibitory effect on [Ca 2?]i ,although the inhibition was very small and dissociation between [Ca 2?]i and contraction is not explained by this mecha-nism (Kitajima et al.,1996a).These results suggest that the increases in [Ca 2?]i (due not only to Ca 2?release but also to Ca 2?influx)elicited by some agonists do not increase MLC phosphorylation and contraction.
e.C YCLIC ADENOSINE 3?,5?-MONOPHOSPHATE .In rat aorta (Abe and Karaki,1989)and toad stomach (Wil-liams and Fay,1986),forskolin or isoproterenol de-creased [Ca 2?]i measured with fura-2and quin2,both of which preferentially detect [Ca 2?]i in bulk cytoplasm rather than the localized high Ca 2?compartments.Mor-gan and Morgan (1984a)observed that,in high K ?-depolarized strips of ferret portal vein,isoproterenol produced either no change or an increase in [Ca 2?]i measured with aequorin during smooth muscle relax-ation.Only in the presence of very high concentrations of isoproterenol (greater than 0.1?M )was a decrease in [Ca 2?]i detectable.Both papaverine and forskolin also caused relaxation of the muscle while [Ca 2?]i either did not change or increased.In bovine trachea (Takuwa et al.,1988),isoproterenol,forskolin,and vasoactive intes-tinal peptide induced the sustained increases in the resting [Ca 2?]i measured with aequorin by increasing Ca 2?influx,which was not inhibited by Ca 2?channel blockers.In A7r5cells,isoproterenol or forskolin in-creased Ca 2?currents by increasing single-channel ac-tivity in cell-attached patches (Marks et al.,1990).In bovine trachea (Felbel et al.,1988),isoproterenol in-creased [Ca 2?]i measured with fura-2,and the increase in [Ca 2?]i was inhibited by nitrendipine and me-
thoxyverapamil.Also,in bovine trachea (Tajimi et al.,1995),forskolin augmented the high K ?-induced in-crease in [Ca 2?]i ,measured with fura-2and inhibited the contraction.These results suggest that cyclic AMP increases [Ca 2?]i in a noncontractile compartment in bovine trachea.This possibility was confirmed in a more direct manner.Observing Ca 2?distribution by confocal microscopy in single airway smooth muscle cells loaded with fura-2,Yamaguchi et al.(1995)found that isopro-terenol decreased inner cytosolic [Ca 2?]i and increased peripheral [Ca 2?]i ,suggesting that there are two Ca 2?compartments in the cell and [Ca 2?]i in these compart-ments are regulated independently.Consistent with these findings,cyclic AMP stimulated K ?channels which are sensitive to [Ca 2?]i near the plasmalemma (see section II.E.3.).
f.S UBPLASMALEMMAL CALCIUM COMPARTMENT .In single smooth muscle cells of rabbit jejunum and rabbit ear artery,Benham and Bolton (1986)found that caffeine stimulated rapid discharge of transient K ?outward cur-rents.Subsequently,there were numerous reports de-scribing the role of SR Ca 2?on spontaneous transient outward currents (STOCs)in smooth muscle (e.
g.,Ohya et al.,1987;Sakai et al.,1988;Kitamura et al.,1989;Hume and LeBlanc,1989;Desilets et al.,1989;Stehno-Bittel and Sturek,1992;Suzuki et al.,1992;Uyama et al.,1993;Lee and Earm,1994;Kim et al.,1995b).Since activators of both IICR and CICR increase STOCs and agents known to deplete Ca 2?stores abolish STOCs after a possible initial increase of STOC discharge,it is now widely accepted that Ca 2?released from the SR activates the K ?channel (for reviews see Kuriyama et al.,1995;Bolton and Imaizumi,1996).However,the [Ca 2?]i in average cytoplasm increased only after STOCs were activated (Stehno-Bittel and Sturek,1992;Sturek et al.,1992;Imaizumi et al.,1996a,b),indicating that the Ca 2?needed to activate STOCs was not de-tected by fluorescent Ca 2?indicators such as fura-2and indo-1.
Membrane depolarization activates the K ?channel by increasing [Ca 2?]i .The increase in [Ca 2?]i is due not only to Ca 2?influx but also to Ca 2?release from the SR by CICR (see Bolton and Imaizumi,1996;Imaizumi et al.,1996a).However,CICR does not play an important role in inducing contraction in smooth muscle (Iino,1989).Furthermore,Imaizumi et al.(1993,1996a,b)found that,although caffeine-induced Ca 2?release re-sulted in the activation of K ?channels and contraction,Ca 2?release induced by 9-methyl-7-bromoeudistomin (MBED)activated the K ?channel without inducing con-traction.Since pretreatment with MBED did not change the subsequent caffeine-induced contraction,it seems likely that there are MBED-sensitive and MBED-insen-sitive SR.MBED may release Ca 2?toward the subplas-malemmal Ca 2?space to activate K ?channel but not toward the cytoplasm,where contractile proteins exist.
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2.Calcium sparks,waves,oscillations,and https://www.wendangku.net/doc/a96427075.html,ing digital imaging techniques and new intracellular Ca 2?indicators,it became possible to examine the two-or three-dimensional distribution of Ca 2?in the cell.Results of these experiments revealed that Ca 2?distrib-utes unevenly in the cell,supporting the suggestion by the functional studies.
a.C ALCIUM SPARKS .The spontaneous local increases in [Ca 2?]i ,called Ca 2?sparks,were first found in rat car-diac cells as measured with a laser scanning confocal microscope and the fluorescent Ca 2?indicator,fluo-3(Cheng et al.,1993).Calcium sparks appeared to result from the spontaneous opening of single SR Ca 2?-release channels (see Taylor,1994).Although the Ca 2?sparks were usually nonpropagating,some sparks triggered propagating waves of increased [Ca 2?]i when the Ca 2?content of the SR was increased.In cerebral artery sin-gle smooth muscle cell,Nelson et al.(1995)found the ryanodine-sensitive,spontaneous local increases in [Ca 2?]i (Ca 2?sparks)just under the surface membrane,and suggested that Ca 2?sparks may activate K ?chan-nels,hyperpolarizes the membrane and relaxes the mus-cle.
b.C ALCIUM WAVES AND OSCILLATIONS .In the eggs of a fresh water fish,medaka,fertilization started a wave of high [Ca 2?]i at the animal pole (where the sperm en-tered)and then traversed the egg as a shallow and narrow-wide band which vanished at the antipode some minutes later (Gilkey et al.,1978).This kind of Ca 2?wave occurs in all eggs investigated so far (Jaffe,1993).Injection of IP 3,but not Ca 2?,induced a Ca 2?wave (DeLisle and Welsh,1992;Lechleiter and Clapham,1992),and inhibition of the IP 3receptor abolished the Ca 2?wave (Miyazaki et al.,1992),suggesting that Ca 2?release originates from an IP 3-sensitive channel.Cal-cium waves and oscillations observed in non-muscle cells have been reviewed by Thomas et al.(1996).
In primary rat aortic smooth muscle cells,the spon-taneous increases in [Ca 2?]i were observed (Bobik et al.,1988;Weissberg et al.,1989).In cultured smooth muscle cells of the human internal mammary artery (Neylon et al.,1990),the thrombin-induced rise in [Ca 2?]i began in a discrete region typically located close to the end of the cell.Subsequently,this region of elevated [Ca 2?]i ex-panded until [Ca 2?]i was elevated throughout the cell.In some cells,the [Ca 2?]i rise began at both ends and collided midway.The rate of spreading of the region of elevated [Ca 2?]i traversed the length of most cells within about 5s.In confluent vascular smooth muscle cells,Simpson and Ashley (1989b)found spontaneous transients and elevations in [Ca 2?]i as well as main-tained oscillations.The oscillations had a periodicity of 6–9s and were not present in single cells.They also reported that endothelin-1but not vasopressin induced oscillations which were inhibited by nifedipine,and sug-gested that these oscillations are at least partly depen-dent upon the L -type Ca 2?channels (Simpson and Ash-
ley,1989a).Similar oscillations have been reported in cultured vascular smooth muscle cells (Wier and Blat-ter,1991;Gillespie et al.,1992c)and intestinal smooth muscle cells (Publicover et al.,1992;Komori et al.,1993,1996;Ohata et al.,1993;Iino et al.,1993;Kawanishi et al.,1994;Kohda et al.,1996).
In cultured rat aortic smooth muscle cells (Johnson et al.,1991),there were small regions in the cytoplasm in which [Ca 2?]i was elevated (hot spot).The initial rise in [Ca 2?]i ,triggered by stimulants,emanated from the hot spot and spread evenly throughout the cytoplasm.The increases in [Ca 2?]i lasted for about 60s and then re-treated back to the original hot spot.In half of the population of the cells,discrete oscillations in [Ca 2?]i occurred after the initial [Ca 2?]i peak.In rat tail artery (Iino et al.,1994a),both nerve stimulation and norepi-nephrine elicited oscillations of [Ca 2?]i that propagated within the cell in the form of waves.Since ryanodine inhibited the oscillations,SR Ca 2?release appears to be responsible for the oscillations.
In cultured guinea pig ileum longitudinal smooth muscle cells (Ohta et al.,1993),thapsigargin-sensitive spontaneous [Ca 2?]i oscillations were observed.Oscilla-tions in [Ca 2?]i were evoked in intact cultured human vascular smooth muscle cells and persisted in nominally Ca 2?-free media (Gillespie et al.,1992c).This indicated the existence of a cyclical mobilization of Ca 2?from internal stores.A7r5cells generated the spontaneous increases in [Ca 2?]i that were abolished by removal of extracellular Ca 2?or addition of nimodipine,indicating that Ca 2?entry through the L -type Ca 2?channels is required for Ca 2?spiking (Byron and Taylor,1993,but see Hughes and Schachter,1994).In this cell,neither ryanodine nor thapsigargin did affect Ca 2?spiking,in-dicating that mobilization of intracellular Ca 2?stores is not necessary for spike generation.In longitudinal mus-cle strips of the rat uterus (Kasai et al.,1994),cyclopia-zonic acid completely suppressed oxytocin-induced Ca 2?release without changing oxytocin-induced rhythmic contractions,suggesting that the Ca 2?stores are not directly involved in uterine rhythmic contractions.
In canine gastric muscle (Ozaki et al.,1992c),acetyl-choline transiently increased tissue levels of IP 3and increased the amplitudes of the plateau phase of slow waves and associated Ca 2?transients and phasic con-tractions.High K ?,ATP,ionomycin,thapsigargin,and caffeine also increased basal [Ca 2?]i .However,each of these compounds reduced the amplitude and duration of slow waves.Results suggest that generation of IP 3may provide negative-feedback control of Ca 2?influx during slow waves,possibly through activation of Ca 2?-acti-vated K ?channels,tending to reduce the amplitude of phasic contractile activity in gastric muscles.In cultured A7r5cells (Berman and Goldman,1992),there was an inverse relationship between SR Ca 2?content and evoked IP 3synthesis,suggesting that SR Ca 2?may serve as a signal that modulates sarcolemmal IP 3for-
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mation.The increase in [Ca 2?]i elicited by IP 3-induced Ca 2?release may inactivate IP 3-gated channels to de-crease Ca 2?release,and such a negative-feedback path-way may be responsible for the Ca 2?oscillation (Komori et al.,1993;Iino et al.,1993;Zholos et al.,1994;Carl et al.,1996).
Stimulations evoke an action potential in some,but not all vascular smooth muscles.Action potentials were only recorded from myogenic (resistant)vessels and in some elastic arteries (see Kuriyama et al.,1995).In these arteries,therefore,another mechanism of Ca 2?oscillation may be repetitive generation of action poten-tials followed by a transient opening of the L -type Ca 2?channels and a transient increase in [Ca 2?]i .Cyclic ap-pearance of trains of action potentials may be related to variations in [Ca 2?]i ,possibly via inactivation of Ca 2?-dependent K ?channels (Himpens et al.,1990).Liu et al.(1995)showed that cyclopiazonic acid and caffeine de-creased the pacemaker frequency in the canine colon.However,ryanodine did not affect the pacemaker fre-quency,which indicates that a ryanodine-sensitive store is not coupled to the biochemical clock.In A7r5cells (Wu et al.,1995),vasopressin caused an initial rapid rise and a delayed increase in [Ca 2?]i .However,in the presence of an inhibitor of K ?channel,tetraethylammonium chloride,vasopressin consistently triggered sustained Ca 2?oscillations which were preceded by a large peak of [Ca 2?]i .In the confluent monolayers of cultured vascu-lar smooth muscle (Missiaen et al.,1994a),cells are electrically coupled and spontaneous discharges of ac-tion potential and subsequent [Ca 2?]i oscillations were synchronized among all the cells.However,individual cells in the monolayer responded to arginine-vasopres-sin with different latencies,suggesting that agonist-induced [Ca 2?]i oscillations are asynchronous.Also in tail artery isolated from young rats (Iino et al.,1994a),relatively low concentrations of norepinephrine could induce oscillations of [Ca 2?]i propagated within the cell in the form of a wave and that there was no synchroni-zation in [Ca 2?]i oscillations between the cells.Cells responded to stimulation in an all-or-none manner,and increasing the concentration of norepinephrine in-creased the frequency of oscillation but not the peak concentration of the [Ca 2?]i transient.Since ryanodine abolished the [Ca 2?]i oscillation,the authors suggested that sustained contraction of smooth muscle is due to summation of [Ca 2?]i oscillations produced by Ca 2?re-lease from the SR and that graded responses to different levels of stimulation may be accomplished not by a graded response within each smooth muscle cell but by a graded number of cells within the vascular wall.Low concentrations of norepinephrine do not change mem-brane potential in rat tail artery (Itoh et al.,1983),and this may be the reason for asynchronous changes in [Ca 2?]i .
c.C ALCIUM GRADIENTS .Using one-and two-dimen-sional models,Kargacin and Fay (1991)suggested that
high Ca 2?concentrations can develop near the plasma-lemma in smooth muscle cells as a result of Ca 2?influx or Ca 2?release.Kargacin (1994)also suggested that the Ca 2?concentration in restricted diffusion spaces be-tween the plasmalemma and the SR may increase up to several ?M and this increase persists for 100–200ms.Goldman et al.(1989)examined the spatial distribu-tion of [Ca 2?]i in arterial myocytes and found that the intracellular [Ca 2?]i was relatively uniformly distrib-uted in resting cells.During norepinephrine-evoked con-tractions,[Ca 2?]i increased with much more heteroge-neous distribution.Upon removal of norepinephrine,discrete regions of elevated [Ca 2?]i were observed throughout the recovered cells.Similarly,activating Na ?/Ca 2?exchange elicited a rise in [Ca 2?]i with dis-crete areas of high [Ca 2?]i .In A7r5cells (Goldman et al.,1990),the distribution of apparent [Ca 2?]i was hetero-geneous;[Ca 2?]i was lowest in the nucleus and highest in the organelle-rich perinuclear region,while the sur-rounding cytoplasmic area (containing relatively few or-ganelles)had intermediate [Ca 2?]i .
Etter et al.(1994)loaded the toad stomach smooth muscle with C18-fura-2,a fura-2molecule conjugated to a lipophilic alkyl chain which inserts into cell mem-branes.They showed that Ca 2?influx increased [Ca 2?]i near the plasmalemma much earlier than [Ca 2?]i mea-sured globally by https://www.wendangku.net/doc/a96427075.html,ing FFP18,a Ca 2?indicator designed to selectively monitor near-membrane [Ca 2?]i ,Etter et al.(1996)further showed that during the mem-brane depolarization-induced Ca 2?influx near-mem-brane [Ca 2?]i rose faster and reached micromolar levels at early times when the cytoplasmic [Ca 2?]i ,recorded using fura-2,had risen to only a few hundred nanomo-lars.High speed series of digital images of [Ca 2?]i showed that near-membrane [Ca 2?]i ,reported by FFP18,rose within 20msec,peaked at 50to 100msec,and then declined.Calcium concentrations reported by fura-2rose slowly and continuously during membrane depolarization.It was also shown that Ca 2?release from the SR increased [Ca 2?]i ,measured with the Ca 2?-acti-vated K ?channel activity (see section II.E.3.),much earlier than the average cytosolic [Ca 2?]i measured with fura-2in bovine and guinea pig coronary arteries (Ste-hno-Bittel and Sturek,1992;Ganitkevich and Isenberg,1996a).Calcium concentrations in subplasmalemmal space seem to oscillate because STOCs were found to oscillate (Komori et al.,1993;Lee and Earm,1994;Kang et al.,1995).
d.N UCLEAR CALCIUM .Williams et al.(1985,1987)found that [Ca 2?]i in smooth muscle cytoplasm,nucleus and the SR are clearly different.The [Ca 2?]i in the nucleus and the SR were greater than in the cytoplasm and these gradients were abolished by Ca 2?ionophores,suggesting that difference in [Ca 2?]i is not due to arti-fact derived from different K d values in cytoplasm and nucleus.When external Ca 2?was increased above nor-mal in the absence of ionophores,cytoplasmic [Ca 2?]i
176KARAKI ET AL .
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2016康复医学笔记汇总
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3、成立花样跳绳课程教学实施小组，并编制花样跳绳训练方案。每周开设三节花样跳绳课外活动课程。 4、结合阳光体育（跳绳）、体育课、兴趣小组活动等开展跳绳活动，学校师生共同参与，人人学跳绳，人人爱跳绳。以课课外活动为起点，结合每天的阳光体育坚持每天一练，把跳绳活动的开展工作带入体育课，分年级、分阶段进行教学，确定每一位学生应达到的水平，确保体育活动可持续开展。 5、初步形成以“阳光课间，绳舞飞扬”为核心的学校体育文化特色。 三、跳绳内容 1、一分钟速度跳。要求每个班无论男女至少50%的同学能够达到140次/分80%的同学能够达到131次/分，100%的同学能够达到108次/分。 2、花样跳绳。要求以学校制定的《跳绳难度规定动作》为基础，要求每个班级60%的同学能够完成《跳绳一级难度规定动作》中的一半以上动作组合，25%的同学能够《跳绳二级难度规定动作》4套以上动作组合。5%的同学能够自创动作组合。 3.内容：并脚跳、踏步跳、车轮跳、侧打挽花、交互绳、网绳、绕“8”字跳、两人一绳、前后开合跳、左右开合跳、绳中绳、双飞、三飞等等。 4.开展以“花样跳绳”为主题的大队、中队活动和形式多样的花样跳绳比赛。一月一次进行了“1分钟速度绳”、“20人3分钟8字跳长绳”、“20人集体花样跳绳”等系列花样跳绳比赛。
立定跳远的运动生物力学分析
立定跳远的运动生物力学分析立定跳远成绩通常被作为评定学生身体素质好坏的一个重要指标，同时它也 经常作为运动员选材的一个重要依据。运动生物力学是一门理论与实践密切结合 的应用科学,?它直接为增强人民体质和提高运动技术水平服务。以运动力学原理来分析立定跳远各个阶段的动作技术,找出提高立定跳远技术的途径,寻求最佳立定跳远技术,以帮助提高立定跳远的成绩。换句话说,就是从这个角度来分析立定跳远应该怎么跳,为什么要这么做,如何提高立定跳成绩。立定跳远属于抛射点与落地点在同一水平面上的抛射运动,?根据远度公式得知,影响抛射远度的主要因素是腾起初速度,又根据动量定理,?要求练习者在预蹲后应立即摆臂,蹬地跳起,蹬地应快猛干脆利落。因此,在进行完整连贯地练习立定跳远时应注意以下一些动作技术方面的问题。 动作各阶段分析 1、预蹲预摆阶段。双腿预蹲与双臂预摆是同时进行且运动方向完全相反。当双腿下蹲时，双臂由前下方经体侧向后上方摆动，上体稍前倾。这个阶段应注意四个问题。 （1）下蹲的程度，是微蹲、半蹲或是全蹲应明确。立定跳远时下蹲程度要求是微蹲，这时，人体的肌肉初长度被拉长达到了最适宜的程度。若是半蹲或全蹲就不符合人体肌肉的工作特点，变成了有意识地放慢下蹲的速度而延长力的作用时间，这样会降低肌肉的收缩力量，不利于形成强大的肌肉收缩力即爆发力。 (2)预蹲摆后能不能停顿。立定跳远动作要求是不能停顿的，当预蹲预摆后应接着迅速完成蹲地动作的，其主要原因是：停顿是把连贯的动作变成静力性动作，而静力性动作较连贯性动作易使肌体产生疲劳。。 (3)摆臂的程度。预蹲时双臂后摆应做到自然，不能强扭使摆幅加大，蹬地时双臂前摆应尽力前上方摆起，以最大程度地提高身体重心。 (4)明确预蹲摆的次数是不是越多越有利于起跳。立定跳远要求只预蹲摆一至二次，并不需要进行多次的重复。多次的重复预蹲预摆不利于充分利用肌肉的弹性，同时由于肌肉松驰现象的存在，不利于肌肉产生最大收缩强力。 2蹬地结束后人体腾空到最高点阶段。预蹲结束应立即摆臂与蹬地跳起，蹬直双腿，上体尽量前送，人体在达到最高点时成一斜线，这时候整个人体也应该是遵循角动量守恒定律的。 3人体从最高点到安全落地阶段。人体蹬离地面后，由于上体尽量前倾，在最高点时，是成一条斜线根据角动量守恒定律，当人体在腾空后，在不改变外力矩作用时，身体某一环节若以一定大小的动力矩绕转轴向某一方向产生转动，必然导致身体其他环节以等量大小的动力矩绕转轴向相反方向发生转动。这时，若不急剧挥臂，向前屈体并做收腹举腿，必然导致人体按原来斜线状态落地。为保证安全落地，必定要使下肢向反方向发生转动，并且小腿前伸着地，保证了上肢上体与下肢转动的动量矩矢量和为零，才能顺利地落地。 为了提高立定跳远的成绩，在进行动作练习时还应注意以下一些训练方法的问题： 1从抛射原理的射程公式中我们可得知：初速度与远度是成正比的，初速度是影响远度的主要因素。因此，在训练中必须着重提高初速度以提高远度。由于
生物力学概论学习
运动训练生物力 学 学 习 笔 记
学校：:广州体育学院研究生部 专业：:运动训练 学号：:105852011400049 姓名：:张江龙 第一章生物力学概论 一．生物力学的定义 生物力学是研究生物系统机械运动特点及规律的科学。它既包括从宏观的角度对生物体整体和器官，组织的运动以及机械特征的研究，又包括从宏观和微观的角度对不同层次的生物组织结构内部的运动和变化进行研究。生物力学是一门力学与生物学科相互结合相互渗透的边缘学科。 1.运动生物力学 运动生物力学是研究体育运动中人体机械运动特点及规律的科学 2.运动训练生物力学 它利用力学原理和各种科学方法，对体育运动中人体的运动行为作定量 的描述和分析，并结合运动解刨学和运动生理学的生物原理对运动进行 综合评定，从力学和生物学的相关关系中得出人体运动的内在联系和基 本规律，从而确定不同运动项目运动行为的不同特点 运动生物力学 密切关注并研究体育运动对人体的有关器官的结构及机能的反作用，最 终以指导运动训练为宗旨 3. 运动生物力学研究的目的主要是探索不同运动项目的力学原理与规律，为科学训练提供必要的理论依据及方法，以提高竞技体育成绩和增强人类体质。 二．人体机械运动的特点 1.人体运动
2.人体的机械运动 人体的机械运动是在意识的支配下所完成的带有明确目的和一定意义的一系列动作行为。因此人体的机械运动可以说是人体高级运动形成的一种外在表现。 人体的机械运动是在外部作用力和内部肌肉张力的作用下产生的。所以要想揭示人体机械运动的规律，不仅要研究力学的因素，而且还必须探讨其生物学方面的因素。需要强调的是：对于分析一般的机械系统的运动，无须对引起该系统的运动发生变化的原动力来源加以仔细研究，提供符合要求的动力装置并非是力学研究者所要研究的对象。然而，使人体运动发生变化的原动力-------肌肉张力确是生物力学研究者必须关注的一个问题。肌肉力学是研究人体机械运动规律的基础。 物体系统作为整体相对于周围参照物体的位移运动 机械运动的表现形式 . 系统本身发生的变化 注意：人体在运动过程中既受自身生物学和生物力学因素的制约，又受到外部力学因素和运动规则的制约。因此必定可以找到客观存在的最合理的最有效的运动技术，以求到最好的运动成绩。寻求合理和有效的运动技术包括两方面的研究内容：一是提示动作技术原理，二是制定最佳运动技术方案。 三．生物力学的任务 1.研究人体结构和机能的生物力学特征 2.揭示动作技术原理，建立合理的动作技术模式 长期的运动实践 技术动作形成的两个途径 利用生物力学理论揭示
观点论述题
观点论述题训练 黑龙江省宝泉岭高级中学潘丽 该题型在2009——2011年新课程文综卷连续出现三年12分，希望同学们和积极训练、认真备考，在高考时一定不能空，只要经过训练，认真做答，都会得到比较满意的分数。观点论述题解题方法： 1、亮明观点。用词一定要确定。比如我认为……正确、我认为……错误等。正确、错误之后一定要用明确的语句把你的观点表述清楚。要对材料进行提练概括，尽量不要照抄材料原文。（关键词可以抄下来） 2、用史实来论证这个观点。史实要注意多角度分析。 思路一：政治、经济、文化、外交、社会生活。 思路二：外因、外因。 思路三：国际因素、国内因素。 思路四：与该事件有关联的多个主体（国家或组织）等。 不同的问题适用不同的思路，在审题时一定要先整出思路再写答案，千万不能想一句写一句。 史实与观点要紧密结合，要准确运用所学的知识，表述要准确，层次要清晰。 3、用理论来论证这个观点或写总结性语言。（这个结论一定要结合这道题目的内容写出，一般不要照搬政治课所学的原理，但要以政治课上所学原理为思路、为依据，用历史的语言来表达。） 以下是我整理的范文，如有不当之处敬请指教。 1、（2008年海南单科26题）（12分）根据材料与所学知识回答问题。 材料：天下已平，（汉）高祖乃令贾人不得衣丝乘车，重租税以困辱之。孝惠、高后时……市井之子孙亦不得仕宦为吏。---- 《史记》 工商杂色之流，假令术逾侪类（同辈之人）……止可厚给财物，必不可超授官秩，与朝贤君子比肩而立，同坐而食。---- 《旧唐书》 古者官民一家也，农商一事也……商籍农而立，农赖商而行，求以相辅，而非求以相病，则良法美意，何尝一日不行于天下哉。---- （宋）陈亮：《龙川集》 士之子恒为士，商之子恒为商。严氏之先，则士商相杂，（严）舜工又一人而兼之者也。然吾为舜工计，宜专力于商，而戒子孙勿为士。盖今之世，士之贱也，甚矣！ ---- （清）归玄恭：《归庄集》 评述中国古代商人社会地位的变迁。（12分） （解析）本题以商人的社会地位变迁为线索展开的，考查学生通过对抑商到“工商皆本”的社会地位的变化，进一步阐述对社会的影响，应坚持站在生产力的发展方面看问题，才能更好地对商人的社会地位和“重农抑商”的政策有一个客观公正的评价，本题是开放性的题目，要注意观点正确文字通顺，逻辑严谨。（评述题,先述后评） 评分标准：本题为开放性试题，采取分项评分办法。分项评分项目：观点、论证、表述。 第一等：11－12分。要求：观点正确，史论结合，能充分运用所给材料；对古代商人地位的变化及其原因分述完整，并作出总体性评价；文字通顺，逻辑严谨。 第二等：7－10分。要求：观点正确，基本能够运用材料说明问题；对古代商人地位的变化及其原因分述比较完整；文字通顺，有一定逻辑性。
运动生物力学
运动生物力学 运动生物力学：是生物力学的一个重要分支，是研究体育运动中人体机械规律的科学。 运动生物力学的主要任务：提高运动能力，预防运动损伤 运动生物力学的研究方法分为测量方法和分析方法，其中测量方法可以分为运动学测量、动力学测量、人体测量、肌电图测量 运动学测量的参数：（角）位移、（角）速度、（角）加速度 动力学测量的参数：主要界定在力的测量方面。 人体测量是用来测量人体环节的长度、围度及,（质量、转动惯量等） 肌电图测量是用来测量肌肉收缩时的神经支配特性。 动作结构：运动时所组成的各动作间相互联系、相互作用的方法或顺序 动作结构的特征主要表现在运动学和动力学，运动学特征指完成动作时的时间、空间和时空方面表现出来的形式或外貌上的特征；动力学的特征指决定动作形式的各种力（力矩）相互作用的形式和特点，包括力、惯性和能量特征。 运动学特征：时间特征、空间特征和时空特征 时间特征反映的是人体运动动作和时间的关系：半蹲起立和深蹲起立 空间特征是指人体完成运动动作时人体各环节随时间变化所产生的空间位置 改变状况：下肢和躯干等空间移动轨迹 时空特征指人体完成运动动作时人体位置变化的快慢情况。 动力学特征包括，力的特征、能量特征和惯性特征 能量特征：人体运动时完成的功、能和功率方面的表现形式。 惯性特征：人体运动中人的整体、环节以及运动器械的质量、转动惯量对运动 动作所具有的影响。 动作系统：大量单一动作按一定规律组成为成套的动作技术，这些成套的动作技术叫做动作系统。 人体基本运动动作形式可主要归纳为推与拉动作、鞭打动作、缓冲和蹬伸动作及扭转、摆动和相向运动等动作形式 上肢基本运动动作形式——推（铅球）、拉（单双杠）、鞭打（标枪）★人体基本运动下肢基本运动动作形式——缓冲、蹬伸、鞭打 动作形式全身基本运动动作形式——摆动、躯干扭转、相向运动 人体的运动是由运动器系的机能特征所决定的，即以关节为支点，以骨为杠杆，在肌肉力的牵拉下绕支点转动，各肢体环节运动的不同组合使人完成千变万化的动作。 生物运动链根据其结构特点可以分为开放链和闭合链。见书P28-图2-15 生物运动链中的杠杆同机械杠杆一样也分为平衡杠杆、省力杠杆和速度杠杆 人体中的三类骨杠杆：见书P30-图2-16 ★人体惯性参数是指人体整体及环节质量、质心位置、转动惯量和转动半径 人体简化模型：质点模型、刚体和多刚体模型
洋务运动的历史背景
洋务运动的历史背景 国内背 经过两次鸦片战争的失败，以及太平天国的打击， 国图志》中主张"师夷长技以制夷"，冯桂芬在《校邠庐抗议》中主张"以中国之伦常名教为原本，辅以诸国富强之术"。，吗吗吗吗吗 1840年，英国发动鸦片战争。遗憾的是，处于传统国家和农业文明体系下的中国在面对经过资产阶级革命后的现代国家和工业文明的英国的挑战时显得不堪一击。首先，在军事上，由于工业革命的发生使得英国军队的武器装备和军事思维发生了质的变化，在战场上，仍旧以大刀长矛和骑兵为主的清帝国的精锐部队尽管作战勇敢，但面对强大的炮火
则显得如此不堪一击。对外作战连连失败，特别是第二次鸦片战争，英法联军攻入北京，火烧圆明园，在政治上和心理上对清帝国造成了严重的阴影。在经济贸易领域，由于工业文明下的大机器生产，专业化、规模化的生产经营模式使得西方的工业产品和加工后的农产品无论是产品质量还是生产成本都远远优越于传统农耕文明下的小门小户的小农经济所生产出的产品，因此，中国长期以来的贸易大国的地位和国际贸易上的优势渐渐丧失，经济发展遭到了新兴经济模式的严峻挑战。 第二次鸦片战争结束后不久，因为清政府用领土，主权以及一系列经贸特权暂时填补了外国侵略者的肚子，国内的农民战争也进入低潮，因而呈现了暂时"稳定"的局面，即所谓"中外和好"的"和局"。但是在清朝统治集团中，一些头脑比较清楚的当权者，如曾国藩、李鸿章、左宗棠以及在中枢执掌大权的恭亲王奕?等人，并没有因为这种"和局"的出现而减少他们对清政府统治的危机感。曾，李，左诸人都为剿灭太平天国而建立殊勋，他们在借助外国侵略者对太平天国的"华洋会剿"中，亲眼看到了外国侵略者坚船利炮的巨大威力，从而感受到一种潜在的长远威胁。面临中国"数千年未有之变局"，他们
洋务运动的研究
洋务运动的研究 背景： 洋务运动对于清朝历史的发展以及中国资本主义的产生，乃至戊戌变法和辛亥革命的发生都有极其深远地影响。但这部分内容非常理论化，晦涩难懂。 目的意义：洋务运动是19世纪60-90年代由地主阶级洋务派掀起的“师夷长技以自强”的挽救清政府腐朽统治的自救运动。在中国近代化进程中起了非常重要的作用。研究此课题，对于我们了解中国近代化进程和中国历史上的变革，有很大的帮助和启发。 参与人员： 饶道诚、陈雅娟、林慧敏、李博文、苏秋李、叶晨翌 分工： 1、搜集整理史料：饶道诚、林慧敏 2、图片实物资料：李博文、苏秋李 3、撰写论文成果：陈亚娟、叶晨翌 4、制作幻灯演示：—————— 活动步骤： 1、搜集相关文字资料和影视图片资料 2、对史料进行整理，集中讨论研究 3、活动总结、成果展示 活动过程： 通过图书馆过刊和学生电子阅览室，查询相关史料和图片资料。询问相关老师和学者。 调查访问对象： 历史老师、有关学者 讨论问题： 1、洋务运动的内容 2、洋务运动为什么会出现在19世纪六十年代 3、洋务运动对中国近代社会发展的影响 4、比较洋务派和顽固派的异同
研究性学习活动小结：（？） 通过本次研究性学习活动，我们总结出了以下几点结论 1、历史选择了地主阶级洋务派担当起了推动近代化进程的任务，他们的探索历程曲折艰难。 2、洋务派近代化的探索，终究改变不了封建生产方式的抵制，形成了中国特有的扭曲的近代化经济形态。 洋务运动的最终失败，也就在情理之中。没有与先进生产力相适应的生产关系，焉能不败？“中体”不变，则企业官营国有的封建性质不变，引进的机器大生产被落后的封建体制所奴役，势必被扭曲，被戕害，其生命力焉能长久？洋务运动不变“中体”，并试图利用“西用”来“自强”，来“求富”，来巩固“中体”，“西用”被作为“富强之术”，被作为巩固“中体”的技术手段。在这种政策指导下，洋务企业不变的是封建体制，不变的是封建性质，变的仅仅是技术，变的仅仅是手段，是中国传统封建王朝官营国有经济在新形势下的进一步改良。这个新形势就是引进利用西方的机器大生产和科学技术，与以前传统王朝国家在自身内部挖掘提高有着明显不同。 洋务运动的失败，宣告了清王朝统治阶级开明派以“西用”嫁接“中体”实践的破产，宣告了对封建经济进行改良的破产。洋务运动的失败，昭示了古老封建经济的日暮途穷，只有彻底打破封建经济体制，改变经济运营模式，才能够挽救中国经济。
花样跳绳教案
花样跳绳教案 Company number：【WTUT-WT88Y-W8BBGB-BWYTT-19998】
水平三（五年级）花样跳绳教学设计 学校：厦门外国语学校翔安附属学校授课教师：陈内国 根据大纲的要求，本着“学生为本、健康第一”的原则，安排教学内容，在课的教学过程中注重跳跃能力的培养，课的设计富有趣味性、多样性、实践性。并将跳绳贯穿整个教学过程，让学生在轻松的教学气氛中达到活跃身心，增强体质，达到“寓教于乐，学生为本”的目的。 二、学情分析 本次课以小学五年级学生为授课对象。好动是学生的天性,他们对体育活动有广泛兴趣，喜欢学习别人的运动技巧。他们团体意识逐渐加深，除对个人的竞争有兴趣外，对团体竞争也发生浓厚兴趣，因此在教学过程中，运用集体合作的游戏，培养他们的集体荣辱感。开始注意教师和同学们对自己的态度。因此在教学中针对学生的心理生理特点，灵活的安排多样的跳法练习，留给学生一定的自主活动天地。使学生在参与活动中得到成功感。 三、教材分析 本课是以“花样跳绳”为教材，以花样跳绳一级动作为教学内容，在原有跳绳的基础上，使跳绳变得多样化，趣味化。既巩固了基础的跳法，又激发了学生跳绳的兴趣，鼓励学生多跳花样。在花样跳绳的过程中，学生通过模仿――实践――思考---创新，主动探究，在锻炼身体的同时，学会发现问题，解决问题，这种学习方式比较过去的学习方式，更主动，更积极，学生的学习兴趣更浓，思维也更活跃。如果说跳绳给学生带来的是尽情，那么创编花样跳绳则给学生送去了尽兴。 四、教学目标制定 1、认知与技能目标：通过教学，使学生懂得一种跳绳有多种玩法并使90%的同学能掌握一级动作3个基本动作。 2、体能与健康目标：通过教学，锻炼了学生的跳跃能力，培养了他们的灵敏、协调等体能素质。 3、情感目标：通过教学，培养学生体验群体活动的乐趣，表现出良好的集体意识和团结协作精并培养学生的创新精神。 五、教学过程 首先，在课的开始热身部分，先运用了蛇形跑进行热身，跟传统的跑步热身脱离。接着利用迷你绳操，让学生与绳子一同配合做绳操，活动学生的各个关节，并让学生懂得绳子的其它用途。接着让学生自己创新跳绳花样，并激发他们的学习兴趣。 在课的学习提高部分是以花样跳绳一级动作为主教材内容，教师从示范导入并设疑开始，运用了分解练习、完整练习、分组自主练习等。在练习过程中，教师巡回指导，纠正学生错误动作，让学生建立正确动作的表象。并利用学生争强好身胜的心理，及时为他们搭建舞台----各组展示探究成果，此后老师的激励评价，同学们的热烈掌声，学生的激情将更高了，自信心也将随之增强了，同时最佳创编奖、最佳合作奖、最佳表演奖也随之产生了。并为之后的游戏做铺垫，游戏保卫钓鱼岛开始将把整堂课推向最高潮，在加深他们的团队合作精神的同时也培养了他们的集体荣辱感。 最后是恢复整理部分，通知老师的带领使同学们在愉悦的音乐氛围中结束了本课。 整堂课，以“跳绳”贯穿始末，灵活运用跳绳的多样性，不断鼓励学生创新。游戏贯穿整个教学过程，在轻松的教学气氛中达到活跃身心，增强体质，“寓教于乐，学生为本”的目的。 六、教学中可能出现的问题及解决预防方 (1)问题：学生在学习中出现错误动作技术，动作不协调。 解决预防方法：在教学过程中，学生出现错误动作技术时，应该及时提醒纠正，教师或让优生做正确的动作示范，让学生观察，积极模仿，反复练习正确的动作，形成正确动作的动力定型。 （2）问题：学生在学习中不按要求进行练习，影响课堂教学秩序。
肌电论文阅读笔记
一、《肌电测量技术的应用》 1、肌电产生的机制 2、肌电测量电极类型 （1）针电极（2）表面电极（SEMG）：有线、无线遥测 3、肌电测量指标 （1）时域指标：是以时间为自变量，以肌电信号为函数，来描述肌电信号随时间变化的振幅特征，而不涉及肌电信号的频率变化的非时间自变量。 积分肌电IEMG(利用肌电图可以判定肌肉所处的不同状态、肌肉之间的协调程度、肌肉的收缩类型及强度、肌肉的疲劳程度及损伤、肌肉的素质等) 均方根振幅RMS, (反映一段时间内肌肉放电的平均水平) 最大值(映肌肉活动的最大放电能力) 时序（反映肌肉活动的最大放电能力) 时程（从肌电曲线开始偏离基线到回归基线的时间) （2）频域指标 4、肌电测量的应用 利用肌电图可以判定肌肉所处的不同状态、肌肉之间的协调程度、肌肉的收缩类型 及强度、肌肉的疲劳程度及损伤、肌肉的素质等。 5、肌肉与疲劳 肌肉疲劳判定 （1）肌电信号频率 肌肉疲劳，放电频率低 （2）肌电幅度 疲劳时，振幅增加 （3）肌电积分判定快肌纤维 疲劳时快肌纤维较多腓肠IEMG减小大 二、《肌电生物反馈的非线性机制》 1、使用数据：肌电振幅、肌电频率、近似熵分析 2、肌电与生物反馈：随着生物反馈次数的增加, 在肌电振幅明显降低、肌电频率明显上升 三、《肌电图（EMG）在运动生物力学研究中的运用》 1、数据处理：时域分析：(1)原始肌电图（EMG）：是直接记录下来的肌电结果，从EMG 振幅的大小可以直观看出肌肉活动的强弱。（2）平均振幅（MA）：反映肌电信号的强度，与肌肉参与的运动单位数目的多少及放电频率的同步化变化程度有关。(3）均方根肌电（RMS）：是运动单位放电有效值，其大小取决于肌电幅值的大小，与运动单位募集数量的多少和兴奋节律有关（4）积分肌电（iEMG）：是肌电信号经过全波整流后随时间变化的曲线下所包绕面积的总和，是全波整流信号的积分总值，它反映了一定时间内肌肉中参与活动的运动单位总放电量。iEMG 值的大小在一定程度上反映运动单位募集的数量多少和每个运动单位的放电大小，是评价肌纤维参与多少的重要指标.频率域分析是对肌电信号进行频率变化特征的分析，是将时域信号通过快速傅立叶转换（FFT）得出的频域信号，在表面肌电信号的检测与分析中具有重要的应用价值。频域分析主要指标有平均功率频率（MPF）、中位频率（MF）等，主要用于判断肌肉的疲劳情况。此外对肌电信号“小波处理”的方法. 2疲劳与肌电：肌肉疲劳对其肌电活动也会发生变化，因此可以用肌电研究肌肉疲劳的发生及机制。（1）肌电幅值的变化电信号振幅大小：肌肉疲劳加深肌电幅值增加，积分肌电（EMG）和均方根振幅（RMS）（2）肌电信号频谱变化频谱变化：疲劳加深，平均功率降低。平均功率频率（MPF）、中心频率（FC）
洋务运动失败原因论述
洋务运动失败原因论述 以慈禧为代表的顽固派无法坐视在洋务运动中权力旁落，对锐意进取的洋务派的打压实际上也极大的挫伤了洋务派的积极性，缺乏强有力的领导，在谨慎行事与设法进取中寻找平衡，难免只能迈出小步。也是洋务运动收效甚微的一个原因。 江南制造厂中也蕴含了李鸿章的私心，带有十分明显的封建派系的烙卸，成为对抗其他洋务派集团的工具，洋务派内部的互相分裂和抑制的狭隘目的局限了自强以立足于世界的眼光，李鸿章对江南制造局的遥控，只重枪炮的生产，使制造厂难以获得长足的发展，更不必说比肩他国。 只是封建政府的一个附庸，一个生产部门，服务于政府依赖于政府庇护，更受制于政府，也受到封建官场恶习的影响，后期的经营混乱，管理无方，任人唯亲，人员冗杂，领取干薪，徇私舞弊，扯皮推诿等现象难以避免，工厂效益低下，无生机可言，走向衰落。这样的工厂，缺乏竞争力，抵抗不了西方企业在华的强大竞争力，而其受官府支持垄断经营的模式，也限制了民族资本的发展。 仅仅停留在仿制的层面上，满足政府对新式武器的需求，但对技术追求的偏执，导致这家军工企业陷入了高成本的误区。江南制造总局虽然掌握了一定的西方先进武器的制造技术，但其推出与西方先进国家存在一定的延迟，并且仿制品与真品之间也存在着性能的差距，可见其技术的落后。而且以轮船为例，由于基础工业发展的落后，进
口原料的成本相较于进口成品更甚。在管理方面， 洋务运动主要在地方展开，缺乏统一规划，大量的国家资源也消耗在低水平的重复建设中封建官僚制度的另一个严重恶果,是经营管理腐败 , 生产效率低下, 生产成本高昂,大部分经费用于支付薪水和工食。江南制造总局每年的支出中,薪水、工食、修建、办公等费约占全部经费的百分之四十左右。以封建官僚主义的经营管理方法管理近代化企业，其后果是使这些企业被它窒息，受其腐蚀而相继失败。
中国近代灾荒史研究综述
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