The Cl(-) channel blocker niflumic acid

The Clàchannel blocker niflumic acid releases Ca2tfrom an intracellular store in rat pulmonary artery smooth muscle cells

1Stuart F.Cruickshank,1Lynne M.Baxte r&*,1Robert M.Drummond

1Department of Physiology and Pharmacology,University of Strathclyde,27Taylor Street,Glasgow G4ONR

1The effect of the Clàchannel blockers niflumic acid(NFA),5-nitro-2-(3-phenylpropylamino)-

benzoic acid(NPPB),4,40-diisothiocyanatostilbene-2,20-disulfonic acid(DIDS),and anthracene-9-

carboxylic acid(A-9-C),on Ca2tsignalling in rat pulmonary artery smooth muscle cells was

examined.Intracellular Ca2tconcentration([Ca2t]i)was monitored with either fura-2or fluo-4,and

caffeine was used to activate the ryanodine receptor,thereby releasing Ca2tfrom the sarcoplasmic

reticulum(SR).

2NFA and NPPB significantly increased basal[Ca2t]i and attenuated the caffeine-induced increase

in[Ca2t]i.These Clàchannel blockers also increased the half-time(t1/2)to peak for the caffeine-

induced[Ca2t]i transient,and slowed the removal of Ca2tfrom the cytosol following application of

caffeine.Since DIDS and A-9-C were found to adversely affect fura-2fluorescence,fluo-4was used to

monitor intracellular Ca2tin studies involving these Clàchannel blockers.Both DIDS and A-9-C

increased basal fluo-4fluorescence,indicating an increase in intracellular Ca2t,and while DIDS had

no significant effect on the t1/2to peak for the caffeine-induced Ca2ttransient,it was significantly

increased by A-9-C.

3In the absence of extracellular Ca2t,NFA significantly increased basal[Ca2t]i,suggesting that the

release of Ca2tfrom an intracellular store was responsible for the observed effect.

4Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in

basal[Ca2t]i induced by NFA.Additionally,incubating the cells with ryanodine also prevented the

increase in basal[Ca2t]i induced by NFA.

5These data show that Clàchannel blockers have marked effects on Ca2tsignalling in pulmonary

artery smooth muscle cells.Furthermore,examination of the NFA-induced increase in[Ca2t]i

indicates that it is likely due to Ca2trelease from an intracellular store,most probably the SR.

British Journal of Pharmacology(2003)140,1442–1450.doi:10.1038/sj.bjp.0705571

Keywords:Clàchannel blocker;Ca2tsignalling;pulmonary artery;smooth muscle

Abbreviations:A-9-C,anthracene-9-carboxylic acid;CPA,cyclopiazonic acid;DIDS,4,40-diisothiocyanatostilbene-2,20-disulfonic acid;NFA,niflumic acid;NPPB,5-nitro-2-(3-phenylpropylamino)-benzoic acid

Introduction

In smooth muscle cells,the intracellular chloride(Clà) concentration is normally maintained at levels higher than its electrochemical equilibrium.It is widely accepted that this high intracellular Clàconcentration plays an important role in regulating a number of cellular functions,including intracel-lular pH,cell volume,contractility,and membrane potential (for a review see Chipperfield&Harper,2000).Many of these cellular effects of Clàare dependent upon the activities of Clàchannels.For example,in smooth muscle,agonist-induced release of Ca2tfrom intracellular stores can activate plasma membrane Clàchannels resulting in membrane depolarization. This in turn leads to activation of voltage-gated Ca2tchannels and Ca2tinflux(for review see Large&Wang,1996; Kitamura&Yamazaki,2001).Chloride channels in the plasma membrane have been extensively studied using the patch-clamp technique,in vascular(Hogg et al.,1994a,b; Yuan,1997),tracheal(Janssen&Sims,1992),gastric(Wade et al.,1996),and urogenital(Cotton et al.,1997)smooth muscle preparations.

A number of studies,examining the effect of Clàchannel blockers on the contractile activity of vascular smooth muscle, have supported the above role for Clàchannels.Specifically, the Clàchannel blocker niflumic acid(NFA)has been shown to reduce the magnitude of agonist-induced contractions in the rat aorta(Criddle et al.,1996;Lamb&Barna,1998)and pulmonary artery(Wang et al.,1997;Yuan,1997;Hyvelin et al.,1998),as well as the rabbit basilar(Uchida et al.,1990) and middle cerebral arteries(Gokina&Bevan,2000).Other Clàchannel blockers including4,40-diisothiocyanatostilbene-2,20-disulfonic acid(DIDS)and anthracene-9-carboxylic acid (A-9-C)have been shown to have similar inhibitory effects on vascular smooth muscle contraction(Lamb&Barna,1998; Gokina&Bevan,2000).However,establishing the role of Clàchannels in cell physiology is often complicated by the fact that many of these Clàchannel blockers also have nonspecific effects.For instance,NFA has been shown to block nonselective cation channels(Gogelein et al.,1990),L-type Ca2tchannels(Doughty et al.,1998),and also activate Ca2t-

*Author for correspondence:

E-mail:robert.drummond@https://www.wendangku.net/doc/f511470490.html, Advance online publication:17November

2003

British Journal of Pharmacology(2003)140,1442–1450&2003Nature Publishing Group All rights reserved0007–1188/03$25.00

https://www.wendangku.net/doc/f511470490.html,/bjp

activated Ktchannels(Greenwood&Large,1995;Toma

et al.,1996).

There is increasing evidence that Clàmovement also plays an

important role in counteracting electrogenic Ca2tfluxes across

the sarcoplasmic reticulum(SR)membrane,during contrac-

tion–relaxation cycles in cardiac(Meissner&McKinley,1982),

skeletal(Kourie et al.,1996a,b),and smooth muscle(Pollock

et al.,1998).In saponin-permeabilized gastric smooth muscle

cells from rabbit,5-nitro-2-(3-phenylpropylamino)-benzoic

acid(NPPB)and indanyloxyacetic acid were found to almost

completely block Ca2tuptake by the SR,suggesting a role for

Clàchannels in SR Ca2thandling(Pollock et al.,1998).

Despite a large number of studies examining the effect of Clà

channel blockers on the contractile and electrophysiological

properties of smooth muscle,there have been very few studies

that systematically look at the effect of these drugs on

intracellular Ca2tsignalling(Pon et al.,1993;Yuan,1997;

Hyvelin et al.,1998).Thus,the objective of the present study

was to determine the effect of four different Clàchannel

blockers on caffeine-induced Ca2tsignalling in rat pulmonary

artery smooth muscle cells(PASMCs).Caffeine was used as an

agonist since it activates the ryanodine receptors in the SR

membrane,thereby allowing the effects of Clàchannel blockers

on SR Ca2tsignalling to be examined simultaneously.Our

results show that,in rat PASMCs,Clàchannel blockers have

significant effects on[Ca2t]i signalling,while detailed study of

the effect of NFA indicates that it releases Ca2tfrom an

intracellular store,probably the SR.A preliminary account of

this work has been presented to the British Pharmacological

Society(Cruickshank&Drummond,2002).

Methods

Cell isolation

Experiments were carried out on smooth muscle cells freshly

isolated from rat pulmonary and mesenteric arteries.Male

Sprague–Dawley rats(200–300g)were killed by cervical

dislocation in accordance with current UK legislation.For

PASMC isolation,the heart and lungs were removed en bloc

and placed in a dissecting solution of the following composi-

tion(in m M):NaCl119,KCl4.7,KH2PO41.18,MgSO41.17,

glucose5.5,NaHC0325,HEPES10,pH adjusted to7á4with

NaOH.Thereafter,intrapulmonary arteries(400–800m m

outside diameter)were dissected out and PASMCs isolated

using the method previously described by Drummond&Tuft

(1999).Briefly,arterial ring segments(B1mm in length)were

placed in a dissociation solution of the following composition

(m M):NaCl128,KCl 5.4,KH2PO40.95,Na2HPO40.35,

MgSO41.17,glucose10,sucrose2.9,NaHCO34.16,HEPES

10,pH adjusted to7.3with NaOH.Papain(1.5mg mlà1)and DL-dithiothreitol(1mg mlà1)were added to the solution and the tissue was maintained at41C for60min in a refrigerator.

The solution containing the tissue was then transferred to a

water bath at371C for6min.Thereafter,the arterial rings were

transferred to fresh dissociation solution containing collage-

nase(Sigma type VIII,1.5mg mlà1)and incubated in the water

bath for a further5min at371C.Gentle trituration of the tissue

with a fire-polished Pasteur pipette yielded single smooth

muscle cells,which remained viable for up to6h.A similar

procedure was used to isolate cells from small branches of the mesenteric artery(200–500m m outside diameter),with the exception that the incubation period in papain was15min,and 10min in collagenase.

Measurement of[Ca2t]i

To measure[Ca2t]i,PASMCs were incubated with5m M fura-2 AM for40min at room temperature.Fura-2fluorescence was monitored using a PTI DeltaRam microfluorimeter(PTI, Lawrenceville,NJ,U.S.A.),coupled to a Nikon Eclipse TE200 microscope with an?40NA 1.3objective lens.Ratio measurements of fura-2were made at20Hz and converted to [Ca2t]i using the method described by Grynkiewicz et al.(1985), with an assumed Ca2t-fura-2K d of200n M.R max,R min and b were calculated as follows:R max is the fluorescence ratio in saturating Ca2t(340nm/380nm excitation);R min is the fluores-cence ratio in the absence of Ca2t(340nm/380nm excitation); and b is the fluorescence ratio for dye excited at380nm(Ca2t-free/saturating Ca2t),as described in Moore et al.(1990).Both DIDS and A-9-C were found to affect fura-2fluorescence(see Figure2);therefore,in studies using DIDS and A-9-C,fluo-4 AM(5m M)was used to monitor intracellular Ca2t.

Caffeine(20m M),which activates the ryanodine receptor to release SR Ca2t,was applied to a single cell via a pressure-ejection pipette,positioned about100m m from the cell,using a Picospritzer II(General Valve,Fairfield,NJ,U.S.A.).Cells were bathed in an extracellular solution of the following composition(in m M):NaCl150,KCl5.4,MgCl21.2,CaCl2 1.8,HEPES10,glucose10,pH adjusted to7.4with NaOH. The Ca2t-free extracellular solution had the following composition(m M):NaCl150,KCl5.4,MgCl23.0,HEPES 10,EGTA1,glucose10,and the pH adjusted to7.4with NaOH.All experiments were carried out at room temperature (20–251C).

Reagents and data analysis

Cyclopiazonic acid(CPA)and ryanodine(both Calbiochem, CN Biosciences,Nottingham,U.K.),NFA,DIDS,NPPB,and A-9-C(all from Sigma,Sigma-Aldrich Ltd.,Poole,U.K.)were prepared as stock solutions in DMSO and diluted as required. Unless otherwise stated,all drugs were bath applied.Bath concentrations of DMSO did not exceed0.2%and had no effect on Ca2tsignalling.Fura-2AM and fluo-4AM were obtained from Molecular Probes(Cambridge Bioscience, Cambridge,U.K.).

Following caffeine application,the half-time(t1/2)for recovery of[Ca2t]i to its basal level was determined.Data are presented as the mean7s.e.m.,and n?number of cells studied.For graphical clarity in the[Ca2t]i records,not all data points are shown.Statistical tests of difference were made using Student’s paired t-test,with P o0.05being considered statistically significant.

Results

Caffeine-induced changes in[Ca2t]i in PASMCs and the effect of ryanodine

The application of20m M caffeine for5s produced a transient increase in[Ca2t]i,from a basal value of8072n M to a peak

S.F.Cruickshank et al Clàchannel blockers and[Ca2t]i in smooth muscle cells1443

British Journal of Pharmacology vol140(8)

value of752744n M(n?6),in rat PASMCs.The half-time (t1/2)to peak,for the caffeine-induced increase in[Ca2t]i,was 0.570.1s after the onset of caffeine application,with[Ca2t]i returning to basal levels over the subsequent20–40s period. The t1/2for recovery of[Ca2t]i following the application of caffeine was870.6s.In order to evoke reproducible responses,a5–6min interval was allowed between caffeine applications(Figure1a).

Incubating PASMCs in50m M ryanodine for5min reduced the magnitude of the caffeine-induced[Ca2t]i transient; however,subsequent responses to caffeine were completely abolished(Figure1b).This indicates that the caffeine-induced [Ca2t]i transients in PASMCs are entirely dependent upon Ca2trelease from the SR via the ryanodine receptor.

Effect of NFA,NPPB,DIDS,and A-9-C on fura-2 excitation spectra

The excitation spectra for fura-2was recorded in the presence of either NFA,NPPB,DIDS or A-9-C,in order to determine whether any of these agents affected fura-2fluorescence. Figure2a and b show that neither NFA(50m M)nor NPPB (10m M)affected fura-2fluorescence,in either Ca2t-containing or Ca2t-free solution.However,both100m M DIDS and50m M A-9-C were found to affect fura-2fluorescence,when excited between320and380nm.This was observed in both Ca2t-containing and Ca2t-free solutions(Figure2c,d).Conse-quently,studies with DIDS and A-9-C used fluo-4to monitor intracellular Ca2t,as neither had an effect on fluo-4 fluorescence(not shown).

Effect of NFA,NPPB,DIDSor A-9-C on caffeine-induced Ca2ttransients in PASMCs

After obtaining a control[Ca2t]i transient to caffeine,a5min recovery period was allowed before the addition of NFA (50m M)to the extracellular solution.Following a2min equilibration period,NFA was found to significantly increase basal[Ca2t]i from87715to253745n M(n?6,P o0.05, Figure3a and b).In the presence of NFA,the peak[Ca2t]i attained during application of caffeine was reduced from 10177143to642754n M,showing a significant reduction in the change in[Ca2t]i(D[Ca2t]i)(P o0.05,Figure3c).NFA also increased the t1/2to peak for the caffeine-induced increase in[Ca2t]i from0.670.1to1.570.3s(P o0.05,Figure3d),and increased the t1/2for recovery of[Ca2t]i from7.271.3to 17.473.3s(P o0.05,Figure3e).

In a separate series of experiments,NPPB(10m M)was also found to significantly increase basal[Ca2t]i from10473to 16779n M(n?5,P o0.05)after a2min equilibration period (Figure4a and b).The peak[Ca2t]i attained during applica-tion of caffeine was reduced from712772to415774n M

in

Figure1Caffeine increases[Ca2t]i via a ryanodine-sensitive Ca2t

store.Caffeine(20m M)was applied to single PASMCs via a

pressure-ejection pipette positioned approximately100m m from the

cell.Reproducible[Ca2t]i transients were obtained when a5–6min

recovery period was allowed between caffeine applications(a).The

break between transients represents the recovery period during

which time recordings were not made.Cells were incubated with

ryanodine(50m M)for5min and caffeine re-applied.Subsequent

caffeine-induced[Ca2t]i transients were abolished(b).Data are

shown as mean values7s.e.m.(n?

6).

Figure2Effect of the Clàchannel blockers NFA,NPPB,DIDS,

and A-9-C on fura-2excitation spectra.Excitation spectra for1m M

fura-2in Ca2t-free and Ca2t-containing solution in the absence or

presence of NFA(50m M)(a).Excitation spectra for1m M fura-2in

Ca2t-free and Ca2t-containing solution in the absence or presence

of NPPB(10m M)(b).Excitation spectra for1m M fura-2in Ca2t-free

and Ca2t-containing solution in the absence or presence of DIDS

(100m M)(c).Excitation spectra for1m M fura-2in Ca2t-free and

Ca2t-containing solution in the absence or presence of A-9-C

(50m M)(d).Emission was measured at510nm with a10nm

bandpass filter.

1444S.F.Cruickshank et al Clàchannel blockers and[Ca2t]i in smooth muscle cells

British Journal of Pharmacology vol140(8)

the presence of NPPB,again indicating a significant reduction in D [Ca 2t]i (P o 0.05,Figure 4c).NPPB also increased the t 1/2to peak for the caffeine-induced increase in [Ca 2t]i from 0.770.1to 2.270.4s (P o 0.05,Figure 4d),and increased the t 1/2for recovery of [Ca 2t]i following stimulation from 10.572.5to 25.271.3s (P o 0.05,Figure 4e).

Since DIDS and A-9-C were found to affect the fura-2fluorescence,fluo-4was used to monitor intracellular Ca 2t.Due to uncertainties regarding the calibration of the fluo-4signal,fluo-4fluorescence was not converted to [Ca 2t]i .Following a 2min incubation period with DIDS (100m M ),fluo-4fluorescence increased by 2371%(n ?6,P o 0.05),which most likely reflects an increase in [Ca 2t]i (Figure 5a and b).DIDS did not affect the magnitude of the caffeine-induced increase in fluo-4fluorescence (Figure 5c),suggesting that it had no effect on the amount of Ca 2treleased from the SR.Furthermore,DIDS had no effect on the t 1/2to peak for the caffeine-induced increase in fluo-4fluorescence (0.870.2for control and 170.3s in the presence of DIDS)(Figure 5d),nor did it affect the t 1/2for recovery of fluo-4fluorescence (4.470.5s for control and 5.570.6s in the presence of DIDS)(Figure 5e).

After a 2min incubation period with A-9-C (500m M ),basal fluo-4fluorescence was increased by 870.2%(n ?7,P o 0.05),

which most likely reflects an increase in [Ca 2t]i (Figure 6a and b).The magnitude of the caffeine-induced increase in fluo-4fluorescence,as assessed by D F /F 0,was reduced from 0.9970.05to 0.8370.06in the presence of A-9-C (P o 0.05,Figure 6c).The t 1/2to peak for the caffeine-induced increase in fluo-4fluorescence was increased from 1.170.2s to 1.770.2s in the presence of A-9-C (Figure 6d).However,the t 1/2for recovery was not affected (6.870.8s for control and 6.270.5s in the presence of A-9-C)(Figure 6e).

Effect of reducing extracellular Cl àon [Ca 2t]i in PASMCs

To examine the effect of reducing the extracellular Cl àconcentration on Ca 2thomeostasis,PASMCs were bathed in extracellular medium in which NaCl had been replaced with equimolar sodium glutamate.In reduced Cl àextracellular medium,basal [Ca 2t]i was 80710n M ,which was not different from that observed in regular extracellular medium (78714n M ,n ?4,P 40.05).Furthermore,caffeine-induced [Ca 2t]i transients were not affected in reduced Cl àextra-cellular medium,where the peak increase in [Ca 2t]i of 525722n M was not different from that observed in regular extracellular medium (573729n M ,n ?4,P 4

0.05).

Figure 4Effect of NPPB on caffeine-induced [Ca 2t]i transients in rat PASMCs.Averaged caffeine-induced [Ca 2t]i transients from five cells,before and after the addition of 10m M NPPB (a).[Ca 2t]i was not recorded during the preincubation period (2min)with NPPB.Summary data showing:the effect of NPPB on basal [Ca 2t]i (b);the change in [Ca 2t]i produced by caffeine ([Ca 2t]i )(c);the half-time (t 1/2)to peak for the caffeine-induced [Ca 2t]i transient (d);and the half-time (t 1/2)for Ca 2tremoval following caffeine application (e).Data are shown as mean values 7s.e.m.(n ?5)and *P o

0.05.

Figure 3Effect of NFA on caffeine-induced [Ca 2t]i transients in rat PASMCs.Averaged caffeine-induced [Ca 2t]i transients from six cells,before and after the addition of 50m M NFA (a).[Ca 2t]i was not recorded during the preincubation period (2min)with NFA.Summary data showing:the effect of NFA on basal [Ca 2t]i (b);the change in [Ca 2t]i produced by caffeine ([Ca 2t]i )(c);the half-time (t 1/2)to peak for the caffeine-induced [Ca 2t]i transient (d);and the half-time (t 1/2)for Ca 2tremoval following caffeine application (e).Data are shown as mean values 7s.e.m.(n ?6)and *P o 0.05.

S.F .Cruickshank et al Cl àchannel blockers and [Ca 2t]i in smooth muscle cells 1445

British Journal of Pharmacology vol 140(8)

NFA-induced increase in basal [Ca 2t]i

In order to further characterize the NFA-induced increase in [Ca 2t]i in smooth muscle cells,the Cl àchannel blocker was applied to a single cell using a pressure ejection pipette.This enabled the kinetics of the NFA-induced increase in [Ca 2t]i to be determined,as well as allowing investigation of the source of Ca 2tbeing mobilized to produce the response.

When NFA (0.1–1000m M )was applied to single smooth muscle cells,it produced a concentration-dependent increase in [Ca 2t]i ,with the effect being half maximal at approximately 65m M (Figure 7a).Therefore,in further studies,we continued to use 50m M NFA.Following application of NFA (50m M ),[Ca 2t]i increased from 5171to 8972n M (n ?4,P o 0.05),reaching a plateau 7174s after beginning the drug applica-tion.When the application of NFA was terminated,[Ca 2t]i recovered to the original basal level.

To establish whether the increase in [Ca 2t]i was dependent upon extracellular Ca 2t,NFA was applied to cells bathed in Ca 2t-free extracellular medium.In the absence of extracellular Ca 2t,NFA was still capable of increasing [Ca 2t]i from 5373to 99710n M (n ?6,P o 0.05),reaching a plateau 58716s after beginning the drug application (Figure 7b).This finding suggested that NFA was releasing Ca 2tfrom an intracellular store.

NFA was also found to cause an increase in [Ca 2t]i in mesenteric artery smooth muscle cells.Specifically,application of NFA via a pressure-ejection pipette increased [Ca 2t]i from 8073to 12679n M (n ?8,P o 0.05).When NFA was applied to cells bathed in Ca 2t-free extracellular medium,[Ca 2t]i increased from 6474to 7875n M (n ?6,P o 0.05).

SR depletion prevents the NFA-induced increase in basal [Ca 2t]i

In order to identify the intracellular source of Ca 2t,PASMCs were treated with the Ca 2t-ATPase inhibitor CPA (10m M ),and the SR was depleted by application of caffeine.Normally,after 1min treatment with CPA,caffeine still produced an increase in [Ca 2t]i although the kinetics of the caffeine-induced [Ca 2t]i transient differed between control and CPA-treated cells (compare Figure 1a to Figure 8).A second application of caffeine in the continued presence of CPA did not produce a [Ca 2t]i transient,indicating that the SR was depleted of Ca 2t.When 50m M NFA was then applied to cells,no increase in [Ca 2t]i was observed (Figure 8).

These

Figure 5Effect of DIDS on caffeine-induced changes in fluo-4fluorescence in rat PASMCs.Averaged caffeine-induced changes in fluo-4fluorescence,recorded from six cells,before and after the addition of 100m M DIDS (a).Fluorescence was not recorded during the preincubation period (2min)with DIDS.Summary data showing the effect of DIDS on basal fluo-4fluorescence (b);the change in fluorescence produced by caffeine (D fluo-4)(c);the half-time (t 1/2)to peak for the caffeine-induced fluorescence transient (d);and the half-time (t 1/2)for Ca 2tremoval following caffeine application (e).Data are shown as mean values 7s.e.m.(n ?6)and *P o

0.05.

Figure 6Effect of A-9-C on caffeine-induced changes in fluo-4fluorescence in rat PASMCs.Averaged caffeine-induced changes in fluo-4fluorescence,recorded from seven cells,before and after the addition of 500m M A-9-C (a).Fluorescence was not recorded during the preincubation period (2min)with A-9-C.Summary data showing the effect of A-9-C on basal fluo-4fluorescence (b);the change in fluorescence produced by caffeine (D fluo-4)(c);the half-time (t 1/2)to peak for the caffeine-induced fluorescence transient (d);and the half-time (t 1/2)for Ca 2tremoval following caffeine application (e).Data are shown as mean values 7s.e.m.(n ?7)and *P o 0.05.

1446S.F .Cruickshank et al Cl àchannel blockers and [Ca 2t]i in smooth muscle cells

British Journal of Pharmacology vol 140(8)

data suggest that the NFA-induced increase in [Ca 2t]i is due to Ca 2trelease from the SR.

Further support of this notion is provided by the studies with ryanodine.Application of NFA produced reproducible increases in basal [Ca 2t]i .Cells were then incubated with 50m M ryanodine for 5min and caffeine applied to activate the ryanodine receptor.Subsequent application of NFA,in the continued presence of ryanodine,did not produce any increase in basal [Ca 2t]i .Taken together,the data from Figures 8and 9

suggest that NFA releases Ca 2tfrom a ryanodine-sensitive intracellular store,most likely the SR.

Discussion

The present study has shown that the Cl àchannel blockers NFA and NPPB cause an increase in [Ca 2t]i in rat PASMCs.While fura-2could not be used to measure [Ca 2t]i in the studies involving DIDS or A-9-C,the increase in fluo-4fluorescence observed is nevertheless indicative of them also causing an increase in intracellular Ca 2t.This increase in [Ca 2t]i was maintained for as long as the smooth muscle cells were exposed to the Cl àchannel blocker.NFA also produced an increase in [Ca 2t]i in mesenteric artery smooth muscle cells,suggesting that the effect is not unique to pulmonary vascular smooth muscle cells.Increases in basal [Ca 2t]i have previously been observed with both NFA and flufenamic acid in visceral smooth muscle cells (Nitecki et al .,1994),ST 885cells (Poronnik et al .,1992),epithelial cells (Schultheiss et al .,2000),and neurones (Shaw et al .,1995;Partridge &Valenzuela,2000).In order to establish whether Cl àchannels were having a direct influence on basal [Ca 2t]i ,experiments were carried out under conditions where extracellular NaCl was substituted with an impermeant anion in the form of sodium glutamate.Under these conditions,basal [Ca 2t]i was not affected,nor was the magnitude of the caffeine-induced [Ca 2t]i transient;thus,it seems unlikely that the effects observed in the present study are mediated by Cl àchannels per se .Normally,activation of Ca 2t-activated Cl àchannels in smooth muscle would produce membrane depolarization,leading to the opening of voltage-dependent Ca 2tchannels and Ca 2tinflux.Indeed,chloride channel blockers have been shown to cause membrane hyperpolarization and dilation of pressurized cerebral arteries (Nelson et al .,1997).Thus,the increase in [Ca 2t]i produced by the Cl àchannel blockers used in the present study was rather unexpected,and a more detailed investigation of the underlying mechanism was therefore carried out for NFA.

When NFA was applied to either pulmonary or mesenteric artery smooth muscle cells using a pressure ejection pipette,the increase in [Ca 2t]i occurred almost immediately upon drug application,and returned to basal levels after drug application was terminated.The increase in [Ca 2t]i induced by NFA was still observed when cells were bathed in Ca 2t-free extracellular medium,suggesting that NFA is capable of releasing Ca 2tfrom an intracellular store.While the magnitude of the NFA-induced increase in [Ca 2t]i in mesenteric artery smooth

muscle

Figure 7Effect of NFA on [Ca 2t]i in PASMCs.Concentration –response curve showing the magnitude of the increase in [Ca 2t]i when NFA was applied to single PASMCs using a pressure-ejection pipette.Data are shown as mean values 7s.e.m.,and each point is representative of 5–7different cells (a).PASMCs were incubated in Ca 2t-free extracellular medium (containing 1m M EGTA)for 1min prior to application of NFA (50m M ).NFA was applied to a single PASMC,using a pressure-ejection pipette positioned approximately 100m m from the cell.Data are shown as mean values 7s.e.m.(n ?

5).

Figure 8Effect of depleting the SR Ca 2tstores on NFA-induced increases in [Ca 2t]i .Cells were incubated with the SR Ca 2tATPase inhibitor CPA (10m M )for 1min prior to repeated caffeine (20m M )application.Thereafter,NFA (50m M )was applied.Data are shown as mean values 7s.e.m.(n ?5).

S.F .Cruickshank et al Cl àchannel blockers and [Ca 2t]i in smooth muscle cells 1447

British Journal of Pharmacology vol 140(8)

cells was slightly reduced in Ca 2t-free extracellular medium,this may be due to the Ca 2tstores being depleted more rapidly in this preparation.After depleting the SR of Ca 2t,by repeatedly applying caffeine in the presence of the SR pump inhibitor CPA,the increase in [Ca 2t]i induced by NFA was completely abolished in PASMCs.This suggests that the NFA-induced increase in [Ca 2t]i is most likely due to Ca 2trelease from the SR.

Further support for the involvement of the SR as the source of Ca 2twas provided by studies with ryanodine.When PASMCs were treated with ryanodine,at a concentration that completely abolished the caffeine-induced Ca 2ttransient,the NFA-induced increase in [Ca 2t]i was also prevented.Addi-tional,indirect support for an effect of Cl àchannel blockers on Ca 2tstores in smooth muscle is provided by studies where spontaneous transient outward currents (STOCs)have been studied.STOCs are due to activation of Ca 2t-activated potassium channels,that open in response to transient increases in [Ca 2t]i following spontaneous release from intracellular stores (Nelson et al .,1995).In portal vein smooth muscle cells,niflumic,flufenamic,and mefenamic acids reduced the frequency and amplitude of STOCs (Greenwood &Large,1995),while NPPB completely abolished STOCs (Kirkup et al .,1996).Both groups suggested that a possible explanation for the observed effect was that the Cl àchannel blockers were modifying intracellular release from,or seques-tration into,intracellular stores.

In addition to increasing basal [Ca 2t]i in PASMCs,NFA and NPPB also reduced the magnitude of the caffeine-induced [Ca 2t]i transient.One explanation for the effects of NFA and NPPB on the caffeine-induced [Ca 2t]i transient in PASMCs is that these Cl àchannel blockers are actually releasing Ca 2tfrom the SR,thereby reducing the amount of Ca 2tthat can subsequently be released by caffeine.A similar effect has been observed with NPPB on vasopressin-induced [Ca 2t]i transients in A7r5cells (Pon et al .,1993).However,in contrast to these studies,NFA (50m M )has been shown to have no effect on the endothelin-1-induced Ca 2toscillations in freshly isolated rat PASMCs (Hyvelin et al .,1998),or on the 5-HT-induced [Ca 2t]i transients in cultured rat PASMCs (Yuan,1997).A number of studies have shown that Cl àchannel blockers are capable of activating the ryanodine receptor from skeletal (Kawasaki &Kasai,1989;Oba,1997)and cardiac muscle

(Zahradn?kova

&Zahradn?k,1993;Hill &Sitsapesan,2002).Specifically,in bilayer studies,DIDS and 4-acetoamido-40-isothiocyanatostilbene-2,20-disulfonic acid (SITS)have been shown to activate the skeletal muscle ryanodine receptor,and lock it in an open state without decreasing the single-channel conductance (Kawasaki &Kasai,1989).NFA was found to have a dual effect on the skeletal muscle ryanodine receptor,increasing the open probability of the channel at 10m M and blocking the channel at 100m M (Oba et al .,1996).A recent study on cardiac ryanodine receptors has shown that DIDS increased single-channel conductance and open probability,and altered the voltage dependence of channel gating (Hill &Sitsapesan,2002).Such an effect of Cl àchannel blockers on the ryanodine receptor in situ could result in the release of Ca 2tfrom the SR,thereby reducing the magnitude of the caffeine-induced [Ca 2t]i transient in PASMCs.

Although DIDS was found to increase basal fluo-4fluorescence in the present study,it had no effect on either the magnitude or kinetics of the caffeine-induced fluo-4fluorescence transient.Given that DIDS is known to have a direct effect on the ryanodine receptor,as alluded to above,this result may be considered somewhat surprising.However,it is known that DIDS is membrane impermeant (Cabantchik &Rothstein,1974;Brayden et al .,1993),and,unless there is a specific transport process for getting DIDS inside the cell,it is unlikely to have any effect on the ryanodine receptor in an intact cell.DIDS is known to be capable of inhibiting the plasma membrane Ca 2tpump (Niggli et al .,1982),so this could explain the increase in basal fluo-4fluorescence observed;however,this would need to be confirmed in future studies.While A-9-C also increased basal fluo-4fluorescence,its effect was markedly reduced compared to that of DIDS.Unlike the other Cl àchannel blockers used in the present study,the ability of A-9-C to activate the ryanodine receptor does not appear to have been investigated,and,although A-9-C is known to be membrane permeable,the mechanism responsible for the increase in basal fluo-4fluorescence

is

Figure 9Effect of ryanodine on the NFA-induced increase in [Ca 2t]i .Repeated application of NFA (50m M )from a pressure-ejection pipette increased [Ca 2t]i .Cells were then incubated with ryanodine (50m M )and caffeine (20mM)was applied to activate the ryanodine receptor (not shown ).Thereafter,NFA was applied.Data are shown as mean values 7s.e.m.(n ?3).

1448S.F .Cruickshank et al Cl àchannel blockers and [Ca 2t]i in smooth muscle cells

British Journal of Pharmacology vol 140(8)

unclear at the present time.A-9-C also reduced the magnitude of the change in fluo-4fluorescence in response to caffeine,as well as increasing the t1/2to peak.The slowed release of Ca2tfrom the SR in the presence of A-9-C could account for the reduced magnitude of the fluo-4fluorescence transient,as Ca2t-removal processes would be more effective at removing Ca2tfrom the cytosol.

As with A-9-C,NFA and NPPB were also found to increase the t1/2to peak for SR Ca2trelease in PASMCs,following stimulation with caffeine.Recent hypotheses have suggested that Clàflux across the SR membrane plays an important role in counteracting any charge build-up that occurs during Ca2trelease from the SR(Pollock et al.,1998),that would otherwise inhibit SR Ca2trelease(Kargacin et al.,2001).Janssen(2002) has hypothesized that Clàchannels in the plasma membrane also increase Ca2trelease from the SR by maintaining a high SR to cytosol Clàgradient,promoting Clàand hence Ca2trelease from the SR.Inhibiting Clàchannels,with for example NFA,could prevent Clàefflux such that Ca2trelease from the SR is reduced.Thus,the increased time to peak could be due to inhibition of anion channels in both the SR and plasma membrane,resulting in impaired release of Ca2tfrom the SR. It has also been shown that certain Clàchannel blockers, including NPPB and indanyloxyacetic acid94,inhibit SR Ca2tuptake in permeabilized gastric smooth muscle cells, while they had no effect on SR Ca2tuptake in permeabilized cardiac cells(Pollock et al.,1998).It is therefore possible that the slowing of intracellular Ca2tremoval following caffeine stimulation in the present study,may in part be due to SR Ca2tuptake being inhibited.However,as indicated above, activation of the SR ryanodine receptor would also impair the ability of the SR to sequester Ca2t,thereby having the apparent effect of slowing the rate of Ca2tremoval.This notion is supported by the observation that ryanodine had a marked effect on Ca2tremoval(Figure1b),most likely because the SR was‘leaky’and therefore unable to function as a Ca2tstore.

In conclusion,we have presented evidence that four different Clàchannel blockers have marked effects on[Ca2t]i signalling in PASMC.A consistent finding was that all the four Clàchannel blockers increased basal[Ca2t]i,while a more detailed examination of the NFA-induced increase in[Ca2t]i indicated that this was due to release of Ca2tfrom a ryanodine-sensitive store.This ability of NFA to release Ca2tfrom the SR could explain why this Clàchannel blocker has been found to attenuate the magnitude of agonist or caffeine-induced contractions(e.g.Gokina&Bevan,2000),and also inhibit STOCs,which are due to the activation of Ktchannels via SR Ca2trelease(Greenwood&Large,1995).Clearly,direct monitoring of SR Ca2tin intact cells would enable a greater understanding of the effect of Clàchannel blockers on SR Ca2trelease and uptake.

We would like to thank Drs Agustin Guerrero-Hernandez and Karen McCloskey for helpful discussion and critical reading of this manu-script.This work was supported by the British Heart Foundation.

References

BRAYDEN, D.J.,KROUSE,M.E.,LAW,T.&WINE,J.J.(1993).

Stilbenes stimulate T84Clàsecretion by elevating Ca2+.Am.J.

Physiol.,264,G325–G333.

CABANTCHIK, A.I.&ROTHSTEIN, A.(1974).Membrane proteins related to anion permeability of human red blood cells.I.

Localization of disulfonic stilbene binding sites in proteins involved in permeation.J.Membr.Biol.,15,207–226. CHIPPERFIELD,A.R.&HARPER,A.A.(2000).Chloride in smooth muscle.Progr.Biophys.Molec.Biol.,74,175–221.

COTTON,K.D.,HOLLYWOOD,M.A.,MCHALE,N.G.& THORNBURY,K.D.(1997).Ca2+current and Ca2+-activated chloride current in isolated smooth muscle cells of the sheep urethra.J.Physiol.,505,121–131.

CRIDDLE, D.N.,DE MOURA,R.S.,GREENWOOD,I.A.&LARGE, W.A.(1996).Effect of niflumic acid on noradrenaline-induced contractures of the rat aorta.Br.J.Pharmacol.,118,1065–1071. CRUICKSHANK,S.F.&DRUMMOND,R.M.(2002).The effect of Clàchannel blockers on[Ca2+]i signalling in rat pulmonary artery smooth muscle cells.Br.J.Pharmacol.,137,43.

DOUGHTY,J.M.,MILLER, A.M.&LANGTON,P.(1998).Non-specificity of chloride channel blockers in rat cerebral arteries: block of the L-type calcium channel.J.Physiol.,507,433–439. DRUMMOND,R.M.&TUFT,R.A.(1999).Release of Ca2+from the sarcoplasmic reticulum increases mitochondrial[Ca2+]in rat pulmonary artery smooth muscle cells.J.Physiol.,516,139–147. GOKINA,N.I.&BEVAN,J.A.(2000).Role of intracellular Ca2+release in histamine-induced depolarization in rabbit middle cerebral artery.Am.J.Physiol.,278,H2105–H2114.

GOGELEIN,H.,DAHLEM,D.,ENGLERT,H.C.&LANG,H.J.(1990).

Flufenamic acid,mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas.FEBS Lett.,268,79–82.

GREENWOOD,I.A.&LARGE,W.A.(1995).Comparison of the effects of fenamates on Ca-activated chloride and potassium currents in rabbit portal vein smooth muscle cells.Br.J.Pharmacol.,116, 2939–2948.GRYNKIEWICZ,G.,POENIE,M.&TSIEN,R.Y.(1985).A new generation of Ca2+indicators with greatly improved fluorescence properties.J.Biol.Chem.,260,3440–3450.

HILL,A.P.&SITSAPESAN,R.(2002).DIDS modifies the conductance, gating and inactivation mechanisms of the cardiac ryanodine receptor.Biophys.J.,82,3037–3047.

HOGG,R.C.,WANG,Q.&LARGE,W.A.(1994a).Effects of Cl channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.Br.J.Pharmacol., 111,1333–1341.

HOGG,R.C.,WANG,Q.&LARGE,W.A.(1994b).Action of niflumic acid on evoked and spontaneous calcium-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.

Br.J.Pharmacol.,112,977–984.

HYVELIN,J.-M.,GUIBERT,C.,MARTHAN,R.&SAVINEAU,J.-P.

(1998).Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes.Am.J.

Physiol.,275,L269–L282.

JANSSEN,L.J.(2002).Ionic mechanisms and Ca2+regulation in airway smooth muscle contraction:do the data contradict dogma?Am.J.

Physiol.,282,L1161–L1178.

JANSSEN,L.J.&SIMS,S.M.(1992).Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes.J.Physiol.,453,197–218.

KARGACIN,G.J.,ALI,Z.,ZHANG,S.J.,POLLOCK,N.S.& KARGACIN,M.E.(2001).lodide and bromide inhibit Ca2+uptake by cardiac sarcoplasmic reticulum.Am.J.Physiol.,280, H1624–H1634.

KAWASAKI,T.&KASAI,M.(1989).Disulfonic stilbene derivatives open the Ca2+release channel of the sarcoplasmic reticulum.

J.Biochem.(Tokyo),106,401–405.

KIRKUP,A.J.,EDWARDS,G.&WESTON,A.H.(1996).Investigation of the effects of5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB)on membrane currents in rat portal vein.Br.J.Pharmacol., 117,175–183.

S.F.Cruickshank et al Clàchannel blockers and[Ca2t]i in smooth muscle cells1449

British Journal of Pharmacology vol140(8)

KITAMURA,K.&YAMAZAKI,J.(2001).Chloride channels and their functional roles in smooth muscle tone in the vasculature.Jpn.J.

Pharmacol.,85,351–357.

KOURIE,J.I.,LAVER, D.R.,AHERN,G.P.&DULHUNTY, A.F.

(1996a).A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.Am.J.Physiol., 270,C1675–C1686.

KOURIE,J.I.,LAVER, D.R.,JUNANKAR,P.R.,GAGE,P.W.& DULHUNTY,A.F.(1996b).Characteristics of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.Biophys.J.,70,202–221.

LAMB,F.S.&BARNA,T.J.(1998).Chloride ion currents contribute functionally to norepinephrine-induced vascular contraction.Am.

J.Physiol.,275,H151–H160.

LARGE,W.A.&WANG,Q.(1996).Characteristics and physiological role of the Ca2+-activated Clàconductance in smooth muscle.Am.

J.Physiol.,271,C435–C454.

MEISSNER,G.&MCKINLEY, D.(1982).Permeability of canine cardiac sarcoplasmic reticulum vesicles to K+,N a+,H+,Clà.

J.Biol.Chem.,257,7704–7711.

MOORE,E.D.,BECKER,P.L.,FOGARTY,K.E.,WILLIAMS,D.A.& FAY,F.S.(1990).Ca2+imaging in single living cells:theoretical and practical issues.Cell Calcium.,11,157–179.

NELSON,M.T.,CHENG,H.,RUBART,M.,SANTANA,L.F.,BONEV,

A.D.,KNOT,H.J.&LEDERER,W.J.(1995).Relaxation of arterial

smooth muscle by calcium sparks.Science,270,633–637. NELSON,M.T.,CONWAY,M.A.,KNOT,H.J.&BRAYDEN,J.E.(1997).

Chloride channel blockers inhibit myogenic tone in rat cerebral arteries.J.Physiol.,502,259–264.

NIGGLI,V.,SIGEL, E.&CARAFOLI, E.(1982).Inhibition of the purified and reconstituted calcium pump of erythrocytes by m M levels of DIDS and NAP-taurine.FEBSLett.,138,164–166. NITECKI,S.,FARRUGIA,G.,RICH,A.&SZURSZEWSKI,J.H.(1994).

Effects of flufenamic acid in vivo on MMC activity and in vitro on intracellular calcium in the canine lower small intestine.Gastro-enterology,170,1226.

OBA,T.,KOSHITA,M.&VANHELDEN,D.F.(1996).Modulation of frog skeletal muscle Ca2+release channel gating by anion channel blockers.Am.J.Physiol.,271,C819–C824.

OBA,T.(1997).Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca2+-release channels.Am.J.Physiol., 273,C1588–C1595.

PARTRIDGE,L.D.&VALENZUELA,C.F.(2000).Block of hippocam-pal CANchannels by flufenamate.Brain Res.,867,143–148.POLLOCK,N.S.,KARGACIN,M.E.&KARGACIN,G.J.(1998).

Chloride channel blockers inhibit Ca2+uptake by the smooth muscle sarcoplasmic reticulum.Biophys.J.,75, 1759–1766.

PON, D.J.,FLEZAR,M.,LISTER, D.L.&HEISLER,S.(1993).

Diphenylamine-2-carboxylate analogues block Clàconductances in A7r5cells by affecting cellular Ca2+homeostasis.Eur.J.

Pharmacol.,245,119–127.

PORONNIK,P.,WARD,M.C.&COOK,D.I.(1992).Intracellular Ca2+ release by flufenamic acid and other blockers of the non-selective cation channel.FEBSLett.,296,245–248.

SCHULTHEISS,G.,FRINGS,M.,HOLLINGSHAUS,G.&DIENER,M.

(2000).Multiple action sites of flufenamate on ion transport across the distal colon.Br.J.Pharmacol.,130,875–885.

SHAW,T.,LEE,R.J.&PARTRIDGE,L.D.(1995).Action of diphenylamine carboxylate derivatives,a family of non-steroidal anti-inflammatory drugs,on[Ca2+]i and Ca2+-activated channels in neurons.Neurosci.Lett.,190,121–124.

TOMA,C.,GREENWOOD,I.A.,HELLIWELL,R.M.&LARGE,W.A.

(1996).Activation of potassium currents by inhibitors of calcium-activated chloride conductance in rabbit portal vein smooth muscle cells.Br.J.Pharmacol.,118,513–520.

UCHIDA,R.,NISHIJIMA,H.,YAMAZAKI,J.,SHIMAMURA,K.& KITAMURA,K.(1990).Effects of Cl channel blockers on the contractions recorded in the rabbit basilar artery.Jpn.J.

Pharmacol.,79(Suppl I),210.

WADE,G.R.,BARBERA,J.&SIMS,S.M.(1996).Cholinergic inhibition of Ca2+current in guinea-pig gastric and tracheal smooth muscle.

J.Physiol.,491,307–319.

WANG,Q.,WANG,Y.-X.,YU,M.&KOTLIKOFF,M.I.(1997).Ca2+-activated Clàcurrents are activated by metabolic inhibition in rat pulmonary artery smooth muscle cells.Am.J.Physiol.,273, C520–C530.

YUAN,X.-J.(1997).Role of calcium-activated chloride current in regulating pulmonary vasomotor tone.Am.J.Physiol.,272, L959–L968.

ZAHRADNI KOVA, A.&ZAHRADNI K,I.(1993).Modification of cardiac Ca2+release channel gating.Pflu¨gers Arch.,425, 555–557.

(Received August5,2003

Revised September16,2003

Accepted October7,2003)

1450S.F.Cruickshank et al Clàchannel blockers and[Ca2t]i in smooth muscle cells British Journal of Pharmacology vol140(8)