Regulation of the Plasma Membrane Ca2+ Pump ... - Semantic Scholar

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263,No. 17, Issue of June 15, pp. 8058-8065,1988 Printed in U.S.A.

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of the Plasma Membrane Ca2+ Pump by Cyclic Nucleotides in Cultured Vascular Smooth Muscle Cells* (Received for publication, October 23, 1987)

Ken-Ichi Furukawa, Yuko Tawada, andMunekazu Shigekawa From the Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan

We investigated the mechanisms of Ca2+ extrusion from cultured rat aortic smooth muscle cells while monitoring changes in the cytosolic Ca2+concentration ([Ca2+]i)using fura 2 fluorescence. “Caz+ efflux from these cells consisted of two major mechanisms; one was dependent on the extracellular sodium concentration (Na+,) and the other was independent of Na+o.Na+,dependent effluxincreased monotonically withincreasing [Ca2+]ibetween 0.1 and 1.0 PM, whereas Na+,-independent efflux reached a plateau at 0.6-1 PM [Ca2+liwith a half-maximum obtained at about 0.16 MM. At [Ca2+Ii below1 PM, the latter was significantly greater than the former. Unlike the Na+,-dependent mechanism, Na+,-independent 4aCa2+ efflux wasinhibited almost entirely by extracellularly added La3+or a combination of high extracellular pH (pH 8.8) and 20 mM M g + . It was also inhibited, although not completely, by compound 48/80, a calmodulin antagonist, andvanadate. These resultsstrongly suggest that Na+,-dependent and Na+,-independent “Ca2+ effluxes occur via the Na+/Ca2+exchanger and the ATP-dependent Ca” pump, respectively. Sodium nitroprusside andatrial natriureticfactor, which are agents that stimulate intracellularproduction of cGMP, and 8-BrcGMP significantly accelerated the Na+,-independent “Ca2+ efflux especially at low [Ca2+]i. Forskolin, dibutyryl CAMP, and 8-Br-CAMP, however, showed no stimulation. These results suggest thatthe plasma membrane Ca” pump is regulated by cGMP but not by cAMP in intact vascularsmooth musclecells.

evidence, however, that significant Ca2+influx or efflux across the cell surface membrane occurs under a variety of conditions, which appear able to induce, either directly or indirectly, contraction or relaxation of vascular smooth muscle (see Refs. 3-5 for review). Ca2+extrusion from smooth muscle cells, like that in other cell types (6-8), most likely involves both the Na+/Ca2+exchanger and theATP-dependent Ca2+pump. Existence of the latter system was unequivocally demonstrated in the plasma membrane of vascular (9) and gastric smooth muscle cells (10). Evidence for existence of the former system in thesame membrane also has been obtained (11, 12). However, the magnitudes of activities of these systems and their relative importances in producing Ca2+extrusion from intact smooth muscle cells remain uncertain. The contractile state of vascular smooth muscle is affected by drugs as well as many physiological vasoactive substances. Relaxation can be induced by agents such as theendotheliumderived relaxing factor, atrial natriuretic factor (ANF), and nitrovasodilators, which increase cGMP levels within vascular smooth muscle cells, and agents such as p-adrenergic hormones and forskolin, which increase intracellular cAMP levels (13-16). Available evidence obtained so far indicates that there is agood parallelism between smooth muscle relaxation and accumulation of these cyclic nucleotides (17-19). The mechanisms by which these nucleotides induce relaxation, however, have been controversial. These nucleotides have beensuggested to cause relaxation by decreasing [Ca2+]i through activation of various ion transport systems including Ca2+flux mechanisms across the plasma membrane and the sarcoplasmic reticulum or by inhibiting directly the activity The contractile state of vascular smooth muscle cells is of the contractile system (4, 16, 20-22). Using a plasma dependent on availability of Ca2+for activation of their con- membrane Ca2+pump ATPase preparation partially purified tractile systems (1). Regulation of Ca2+ movement within from bovine aortic smooth muscle and the membrane reconthese cells is not well understood because activities of Ca2+ stitution technique, we have recently shown that the plasma transport systems in membranes of these cells have not been membrane Ca2+pump is a target for cGMP-dependent reguquantitatively assessed. Recent ultrastructural studies using lation and have provided evidence suggesting that activation a technique of electron probe microanalysis (2) suggest that of the Ca2+pump occurs through cGMP-dependentphosphounder physiological conditions, uptake by and release of Ca” rylation of the Ca2+pump ATPase itself (23). from the sarcoplasmic reticulum plays a major role in control In the present study, we first quantitatively characterized of [Ca2+]>in vascular smooth muscle cells. There is ample Ca2+ extrusion systems inintactsmooth muscle cells by measuring ‘%a2+ efflux from cells as a function of [Ca2+],in * This work was supported by Research Grant 62A-1 for Cardio- the presence and absence of added extracellular Na+ and then vascular Diseases from the Ministry of Health and Welfare and Grant-in-Aid 62780240 for Scientific Research from the Ministry of studied the effect of cyclic nucleotides on the activity that Education, Science, and Culture. The costs of publication of this presumably corresponds to that for the plasma membrane article were defrayed in part by the payment of page charges. This Ca2+pump in order to establish the physiological importance article must therefore be hereby marked “aduertisement” in accord- of cyclic nucleotide-dependent regulation of the plasma memance with 18 U.S.C. Section 1734 solelyto indicate this fact. brane Ca2+pump. The abbreviations used are: [Ca2+],,intracellular Ca2+concentration; [ M e ] , , extracellular M e concentration; Na+,, extracellular EXPERIMENTALPROCEDURES Na+; pH;, intracellular pH; pH,, extracellular pH; BSS, balanced salt Cell Culture-Vascular smooth muscle cells were isolated from rat solution; ANF, atrial natriuretic factor; diS-C8(5), 3,3’-dipropylthiodicarbocyanine iodide; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth-thoracic aorta (250-300 g, Wistar-Kyoto male rat) by enzymatic dispersion as described by Chamley et al. (24). The resulting cells anesulfonic acid.

8058

Cyclic Nucleotide Regulationof Sarcolemmal Ca2+Pump weregrown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 pg/ml streptomycin and were passaged twice a weekby harvesting with trypsin plus EDTA and seeding. Cells from the same stock at passage levels of 5-10 were seeded into 35-mm culture dishes for 45Ca2+ efflux measurements or onto sterile glass cover slips laid in a 100 X 100-mm square dish for measurements of [Ca2+],,pH;, and the membrane potential. Measurement of [Caz+];-[Ca2+]; was monitored by using fluorescent Ca2+ indicator fura 2. Smooth muscle cells attached to glass cover slips were loaded with fura 2 by incubating them with 4 pM fura 2-acetoxymethylester for 40 min a t 37 "C in balanced salt solution (BSS; 146 mM NaCl, 4 mM KCl, 2 mM MgCL, 0.5 mM CaC12,10 mM glucose, 0.1% bovine serum albumin, and 10 mM Hepes-Tris, pH 7.4). Loaded cells were washed 3 timesand thenincubated with fresh BSS for additional 20 min at 37°C to completely hydrolyze the entrapped ester. The glass cover slip was fixed to a holder which was inserted into a cuvette diagonally. The fluorescence signal was monitored a t 510 nm with excitation wavelengths alternating between 350 and 380 nm using a Hitachi MPF-4 spectrofluorometer equipped with a built-in stirrer. A high pass cut-off filter (cut off below 430 nm) was placed in front of the emission monochrometer to reduce the background signal. After each measurement, Mn2+was added to a cuvetteto a final concentrationof 10 mM to evaluate fura 2leakage from cells. To estimate autofluorescence from cells a t 350 and 380 nm, 10 p~ ionomycin was then added to completely quench the fura 2 fluorescence. The [Ca"]; was calculated as described by Grynkiewicz et al. (25) after correction for fluorescence from extracellular fura 2 and autofluorescence determined as described above. In Fig. 1, A and B, we plotted calculated values of [Ca2+],obtained a t appropriate times before and after the ATP stimulation as a function of time, instead of presenting raw records of Ca2+transient. efflux from cells cultured in Measurement of 45Cu2'Effl~x-"~Ca~+ a 35-mm dish was measured essentially as described by Smith et ul. (26). After cells were rinsed 3 times a t 37°C with BSS, they were incubated in 1ml of BSS containing 10 pCi of "Ca2+ for 4 hat 37 "C. Immediately afterward, cells were rinsed 10 times with the efflux media containing no CaCL (Ca2+-freeBSS) or no CaC12and no NaCl (Ca2+-and Na+-free BSS) for 1 min. In the Ca2+-and Na+-free BSS, NaCl was replaced by 146 mM choline chloride. Time courses of 45Ca2+ efflux were followed thereafter by replacing the efflux media (1.5 ml of Ca2+-freeBSS or Caz+-and Na'-free BSS) either every 5 s or every 10 s. To replace the efflux media, we removed the reaction medium by quickly turning the dish upside down and collected the liquid by pouring the contents into a funnel and then added temperatureequilibrated new medium to the dish with an automatic dispenser. We repeated this procedure every 5 or 10 s. One min after the start of "Caz+ efflux measurement, the efflux media used for replacement were switched to ones containing either ATP or angiotensin I1 to induce a transient increase in [Ca2+Ii.The amount of "Ca" lost from cells during each time interval was measured by liquid scintillation counting. In some experiments, '%az+ efflux was measured from cells loaded with fura 2. "Caz+ efflux profiles from these cells were not different from cells not loaded with fura 2. Therefore, 45Caefflux was usually measured from cells not loaded with fura 2. potential Measurement of Membrane Potential-Membrane changes were monitored with a potential-sensitive dye diS-C3(5)(27) under conditions similar to those used for measurement of 45Ca2+ efflux. Cultured smooth muscle cells attached to glass cover slips were preincubated with 0.5 p~ diS-C3(5) a t 37 'C for 5 min in BSS before assaying fluorescence. Fluorescence measurements were performed with excitation a t 620 nm and emission a t 680 nm, respectively. Fluorescence signal was calibrated by changing extracellular K+ concentrations in the presence of 0.5-1.0 p~ valinomycin, which enabled us to determine the extracellular K+ concentration for which there was no change in fluorescence. The membrane potential was estimated using Nernst's equation by assuming that it equals the equilibrium potential for K+ in the presence of valinomycin. The intracellular K' concentration was assumed to be 156 mM (28). Measurement of pH;-The fluorescent pH indicator dye, 2',7'bis(carboxyethy1)carboxyfluoresceinwas used to monitor changes in pHi pH;. 2',7'-Bis(carboxyethy1)carboxyfluorescein loading and measurement in cultured vascular smooth muscle cells attached to glass cover slips were performed according to Kurtz and Golchini (29). Cellular Nu' Content-The cellular Na' content of cultured aortic smooth muscle cells were determined by a Hitachi atomic absorption spectrometer as described previously (30).

8059

Cellular Protein Content-Cellswere solubilized in 1% sodium aliquot was taken dodecyl sulfate and 10 mM Na2B407, and then an for protein determination. Protein was measured by the modified Lowry method (31) with bovine serum albumin as a standard. Materials-The sources of materials used in this workwere as follows: Fura 2, fura 2-acetoxymethylester, and 2',7'-bis(carboxyethy1)carboxyfluorescein from Dojin Chemicals; diS-C3(5) from Nippon Kankoh-Shikiso Kenkyusho; ATP, dibutyryl CAMP, 8-Br-cAMP, 8Br-cGMP, and bovine serum albumin (fatty acid-free) from Sigma; angiotensin I1 from Peptide Institute Inc.; sodium nitroprusside from Wako Pure Chemical Industries; ionomycin, valinomycin, and forskolin from Behring Diagnostics; 3-isobutyl-1-methylxanthinefrom Aldrich; Dulbecco's modified Eagle's medium and antibiotics from Flow Laboratories; fetal calf serum from M. A. Bioproducts; 45CaC12 from Du Pont-New England Nuclear. All other chemicals wereof analytical grade. RESULTS

Characterization of Ca2+ ExtrusionSystemsinAortic Smooth Muscle Cells-The excitable cells are known to have two major mechanisms for extrusion of Ca2+from cells (6-8). One is dependent on Na+o,coupled with Na+ influx, a n d is catalyzed by theNa+/Ca2+exchanger. T h e o t h e ris independent of Na", and may be catalyzed by the plasma membrane Ca2+pump. In t h e following, we characterize Ca2+extrusion systemsinculturedaorticsmoothmusclecells to obtain information about the relative magnitude and functional significance of these systems. Smooth muscle cells were stimulated with various concentrationsof either ATP or angiotens i n I1 to raise [Ca2+], transiently,and the resultant unidirectional effluxes of Ca2+were measured. The ATP-induced Ca2+ mobilization in cultured aortic smooth muscle cells appears to occur through binding to a purinergic receptor located on t h e cell surface as investigated in detailin our previous study

(32). Figs. 1, A and B, shows typical records of ATP-induced intracellular Ca2+ transient and increased 4sCa2+effluxes measured in the presence and absence of Na+,. CaCl, was always omitted from the extracellular medium toinhibit the calcium-calciumexchangeacross the plasmamembrane. When the physiological concentration of Na+ (146 mM) was present inthe medium, 30 PM ATP raised [Ca2+Iifrom a basal level of 74 k 16 nM (mean f S.D., n = 3) t o a peak level of 495 f 30 nM ( n = 3) in 10 s, followed by a relatively slow decline toward the basal level (Fig. lA).45Ca2+efflux changed in parallel with [Ca2+],. The amount of 45Ca2+label lost from cells during a 5-s period was 0.081 f 0.027 nmol/mg ( n = 3) at t h e basal [Ca"], whereas itwas 0.590 f 0.020 nmol/mg ( n = 3) at near the peak [Ca2+Ii.When 146 mM Na+, was replaced bythesameconcentration of choline+,theATP-induced increase in [Ca2+], became significantly greater (compare Fig. 1,A and B ) ;the peak [Ca2+],, which was reached in15 s, was about 1.4-fold that obtained in the presence of Na+,. In addition, the Ca2+ transient appeared to decay at a slower rate than in the presenceof Na+,. In spite of the greater Ca2+ response, the highest rate of 4sCa2+efflux measured at near the peak [Ca2+jiwas 0.357 f 0.016 nmol/mg/5 s ( n = 3) in the absence of Na+a,which was significantly lower than that obtained in the presence of 146 mM Na', (see above). We examined [Ca2+Ii dependenceof the 4sCa2+efflux rate that was induced with measured at near the peak [Ca2+], various concentrations of ATP (2-30 p ~ (Fig. ) 2). At each concentration of ATP, the peak [Ca2+]; was always reached in about 10 and 15 s i n the presence and absence of Na+,,, respectively (cf. Fig. 1, A and B ) . An important finding was that changes in [Ca2+], around the peaks of Ca2+transients were relatively small. We estimated the mean peak [Caz+], by taking the mean of the maximum and minimum values of

8060

Cyclic Nucleotide Regulation of Sarcolemmal ea2+ Pump

A

I:‘K

- 0.6

Oca,

I 7.0 6.5 -log[Ca*’l, (MI

SECONDS

6.0

FIG.2. [Ca”], dependence of “CaZ+ efflux measured in the presence or absence of Na+..IntracellularCa2+transient and“Ta2+ efflux weremeasured in Ca2+-freeBSS (0)or Na+- and Ca2+-free BSS (0)after stimulation of cells with various concentrations of ATP. In the figure, the “Ca2+ efflux rate was plotted as a function of the mean peak [Ca2+Ii.Both parameters were estimated as described in the text. The brohn line indicates [Ca2+Iidependence of the Na+,dependent component of “Ca” efflux which was estimated as the difference between the values obtainedin the presence and absence of Na+,.

from cultured aorticsmooth muscle cells consists of two components, one being Na+,-dependent and the other being Na+,-independent. The Na+,-dependent component, which was estimated by subtracting “Ca” efflux in the absence of Na+, from that in the presence of Na+,, increased monotonically with an increase in [Ca2+]iand did not show a plateau at [Ca2+]ibelow 1p ~ In. addition, at these [Ca2+Ii,the Na+,dependent “Ca2+ efflux was significantly smaller than the SECONDS Na+,-independent one (Fig. 2). It should be mentioned that efflux were essentially the same FIG. 1. Effect of Na+.on intracellular Ca2+mobilization and [Ca2+]i-dependencesof 45Ca2+ unidirectional “Ca2+ efflux in vascular smooth muscle cells. whether cells were stimulated with angiotensin I1 (0.01-0.3 A , intracellular Ca2+transient and “Ca2+ efflux were measured after p ~ or) ATP (2-30 p M ) ( d a t a not shown). stimulation of cells with 30 p~ ATP in Caz+-freeBSS containing 146 Fig. 3 shows the effect of La3+on ‘5Ca2+efflux from smooth mM NaCl as described under “Experimental Procedures.”For measurement of “Ca2+ efflux, the amount of ‘%a2+lost into the medium muscle cells. In this experiment, cells were stimulated with 1 was measured every 5 s after ATP stimulation.B, experiments of the p~ angiotensin 11, instead of ATP, to avoid precipitation of type shownin A were repeated usingNa+- and Ca2+-freeBSS in place ATP with La3+. The Na+,-independent “Ca” efflux was of Ca2+-freeBSS. In these and subsequent figures, the results are inhibited by La3+in a dose-dependent manner; La3+at 1mM shown as the mean f S.D. of triplicate determinations. decreased the activity to about 10% of the control, whereas 65 p~ La3+produced a half-maximal inhibition. In contrast, the Na+,-dependent “Ca2+ efflux was accelerated by La3+at [Caz+Iimeasured during the periods between 5 and15 s in the concentrations between 10 and 200 p~ (cf. Fig. 3). The presence of Na+, and between 10 and 20 s in the absence of concentration of La3+ producing the maximum stimulation Na+,, respectively. The ‘5Ca2+efflux rates were estimated was about 20 p ~ which , accelerated the Na+,-dependent from the amounts of 45Ca2+label lost from cells during these ‘Ta2+efflux by60%. On the other hand, higher La3’ inhibited same periods. The values of [Ca2+]ioccurring during these the Na+,-dependent 45Ca2+efflux and the half-maximal inhiperiods deviated only by 5-9% from the respective value of bition occurred at 3 mM La3+.Therefore, the Na+,-independthe mean peak [Ca2+]i(see the sizes of horizontal deviation ent 45Ca2+efflux was much more susceptible to inhibition by La3+than the Na+,-dependent 45Ca2+ efflux. shown in Fig. 2). Fig. 2 shows that in the presence of Na+., In Fig. 4, we examined the effect of high pH, and high 45Ca2+efflux increased with increasing [Ca2+]iup to about 1 [Mg“l0 on &Ca2+efflux from smooth muscle cells stimulated p ~the , highest concentration measured. Replacement of Na’ with 1 p~ angiotensin 11. In the absence of Na+o, eitherhigh with choline+ decreased “Ca2’ efflux and changed its [Ca2+Ii- pH (pH 8.8) or 20 mMMg2+ inhibited “Ca2+ efflux by 15dependence. 45Ca2+efflux increased with an increase in 19%, whereas these in combination inhibited it by 80%. In [Ca2+Ii but reached a plateau at about 0.6 p~ [Ca2+]i.The contrast, the Na+,-dependent component of “Ca2+ efflux, plateau value of the 45Ca2+efflux rate measured at 0.9 p~ which was estimated as the difference between the values [Ca2+Iiwas 0.67 f 0.02 nmol/mg/lO s, which was 1.7 times obtained in the presence and absence of Na+,, wasnot affected less thanthat obtained at the similar [Ca2+]ibut in the significantly by changes in pH, and [ M P ] , (0.65 f 0.07 presence of Na+O.These results indicate that 45Ca2+efflux nmol/mg/lO s for control cells uersus 0.67 f 0.02 nmol/mg/

Cyclic Nucleotide Regulation

of Sarcolemmal Ca2+Pump

8061

independent component of 45Ca2+efflux induced by 30 p M ATP (0.82 k 0.065 nmol/mg/20 s for control cells versus 0.39 f 0.032 nmol/mg/20 s for compound 48/80-treated cells). Effects of Intracellular cGMP and CAMPon V a 2 +EffluxcGMP andcAMP have been implicated as second messengers for many agents that induce relaxation of vascular smooth muscle cells (13-16). One of the possible mechanisms for relaxation by these messengers is that they lower [Ca2+]iby accelerating Ca2+ extrusion from cells. In the experiments described below, we examined effects of agents that presumably increase intracellular levels of cGMP or cAMP on the Na+,-independent 45Ca2+efflux from aortic smooth muscle cells. Fig. 5A shows the effect of sodium nitroprusside on the Na+,-independent 45Ca2+efflux induced by ATP stimulation. In cells preincubated with 10 PM sodium nitroprusside for 3 min at 37 “C in BSS, the 45Ca2+efflux rate was found to be significantly greater than in control cells, especially at low [Ca2+Ii;at about 0.1 p~ [Ca2+Ii,the 45Ca2+efflux ratein nitroprusside-treated cells was 0.378 f 0.07 nmol/mg/lO s ( n -log [LaCl,l (M) = 3), which was about 2.4-fold that incontrol cells. Essentially similar results were obtained when cells were treated under FIG. 3. Effect of La*+ on Na+.-dependent and Na+.-independent “Ca2’ effluxes. 4sCa2+effluxes from ‘%az+-loaded cells comparable conditions with 100 nM ANF, another cGMPwere measured in Ca2+-freeBSS (0)or Ca2+-and Na+-free BSS (0) producing agent (Fig. 5 B ) or 500 p~ 8-Br-cGMP,a membrane and in the presence of various concentrations of LaCL after stimu- permeable analogue of cGMP (Fig. 5 C ) .The observed leftward lation with 1 PM angiotensin 11. The “Ca2+ efflux rate was estimated shifts in [Ca2+]idependence suggest that these agents accelfrom the amount of‘%a2+ label lost from cells during the period erate 45Ca2+efflux by increasing the affinity of the Na+obetween 5 and 15 s or 10 and 20 s after the angiotensin I1 stimulation in Ca2+-freeBSS or Ca2+-and Na+-free BSS, respectively. The broken independent Ca2+extrusion system for intracellular Ca2+. In Fig. 6 , we examined effects of incubation of cells with 10 line represents the difference between the values obtained in the ~ L Mforskoiin (Fig. 6 A ) or 0.4 mM dibutyryl cAMP (Fig. 6B) presence and absence of NG. for 3 min at 37°C in BSS on the Na+,-independent “Ca2+ efflux. Dibutyryl cAMP at 0.4 mM did not change [Caz+]i 0 Ca, 0 Nan. Oca, dependence of the Na+,-independent 45Ca2+efflux (Fig. 6B). Essentially the same results was obtained when cells were treated with 0.5 mM 8-Br-cAMP, another cAMP analog, under equivalent conditions (data not shown). Treatment of cells with 10 p~ forskolin, on the other hand, resulted in a slight increase (10-15%) in the 45Ca2+efflux rate (Fig. 6A). However, we did not consider this small increase to be significant because the experimental errorwas relatively large. The results obtained in these experiments were similar even when 1 mM 3-isobutyl-1-methylxanthinewas included in the reaction media. It should also be mentioned that the absence of stimulatory effects of cAMP analogues or forskolin is unlikely to be due to inability of these agents to raise intracellular levels of cAMP or its analogues, because in these experiments cAMP analogues or forskolin increased the peak heights of Ca2+ transient induced with 10-30 p~ ATP about 1.6-fold FIG. 4. Effects of alkaline pH and high Mg2* on “‘Ca2* ef- relative to those in control cells. The mechanism for this cells were washed with either Ca2+-freeBSS effect of cAMP is currently under study. flux. After 46Ca2+-loaded or Na+- and Ca2+-freeBSS for 2 min (see “Experimental Procedure”), Effect of Cyclic Nucleotides and Other Experimental Condithey were transferred into modified BSS whose pH and saltconcentions on Cellular Na+ Content, pHi, and Membrane Potentialtrations were shown in the figure and then incubated for 10 s. Cells In the experiments described above, “Ca2+ efflux was measwere subsequently stimulated with 1 PM angiotensin 11, and the amount of “Ca2+ label lost from cells during the following 20 s was ured after cells were pretreated with cyclic nucleotides for 3 measured. min in normal BSS and then incubated in either Ca2+-free BSS or Na+- and Ca2+-freeBSS for 2 min before stimulation with ATP orangiotensin I1 (see “Experimental Procedures”). 10 s for cells treated at pH8.8 and 20 mM MgC12) (Fig. 4). Effects of other inhibitors of the plasma membrane Ca2+ We examined the effects of the cyclic nucleotide treatment pump were also studied on 45Ca2+efflux from smooth muscle and other experimental conditions on the Na’ content (Fig. cells. Pretreatment of cells with 3 mM ammonium vanadate 7), pHi, and the membrane potential of the cells. Dibutyryl for 5 min at 37°C in BSS inhibited Na+,-independent com- cAMP (Fig. 7) or 8-Br-CAMP (datanot shown) but not 8-Brponent of 45Ca2+efflux induced by 30 p~ ATP by 75% (0.740 cGMP (Fig. 7) decreased the cellular Na+ content by about f 0.020 nmol/mg/lO s at 776 f 64 nM mean peak [Ca2+]ifor 30% when cells were incubated for 3 min with 0.5 mM of these control cells uersus 0.180 f 0.018 nmol/mg/lO s at 1040 & 85 nucleotides in normal BSS. The Na+ content also decreased nM mean peak [Ca2+Iifor vanadate-treated cells). Pretreat- by about 30% when cells were treated simply with Na+- and ment of cells with 0.5 pg/ml of compound 48/80 under the Ca2+-freeBSS for 2 min (compare the first and fourth columns comparable conditions produced 53% inhibition of the Na+,- in Fig. 7 ) ,although it was not affected significantly by similar J

1

I

8062

Cyclic Nucleotide Regulation of Sarcolemmal ea2+ Pump 0.6-

I

A

0.6-

. 7

F

-

% E

0.4X

c c

-

m8 0.2-

1

t

I

0‘

7.0

6.5

6.0

- I O ~ I C ~ “ I(M) , 0.8

B

7.0

6.5

6.0

0‘

-~og[~a~’l, (MI

6.0 7.0

6.5

-1og[~a”1 (M)

FIG. 6. Effects of forskolin ( A )and dibutyryl cAMP ( B )on Na+.-independent ‘‘CaB* efflux. The experimental conditions were the same as those described in the legend to Fig. 5 except that 10 p~ forskolin ( A ) and 0.4 mM dibutyryl cAMP (&-CAMP) ( B ) were used. 0,control cells; 0, drug-treated cells.

I

NORMAL

ON%.

. oca.

1

‘ TT

0

7.0

6.5

6.0

-log[Ca2*li IM)

FIG. 5. Effects ofsodium nitroprusside (SNP)( A ) , ANF ( B ) ,and 8-Br-cGMP (C) on Na+,-independent ‘%aB+efflux. Ca2+transient and “Ca2+ efflux were measured as described in the legend to Fig. 2 except that they were measured only in Na+- and Ca2+-freeBSS and that cells were pretreated with 10 WM sodium nitroprusside ( A ) , 100 nM ANF ( B ) , or 0.5 mM 8-Br-cGMP (C) in BSS for 3 min before the start of washing with Na+- and Ca*+-free FIG. 7. Effect of cyclic nucleotides on intracellular Na+conBSS (see “Experimental Procedures”). For the control experiment, tent. Cells were incubated with 0.5 mM 8-Br-cGMPor 0.5 mM cells were pretreated with vehicle for each drug. 0, control cells; 0, dibutyryl cAMP in BSS for 3 min. After incubation of cells further drug-treated cells. for 2 min in normal BSS orNa+- and Ca*+-freeBSS, the intracellular Na+ content was assayed as described under “Experimental Procetreatment of cells with Ca2+-freeBSS (data not shown). As dures.”

these effects of dibutyryl cAMP and the Na+- andCa2+-free medium were not additive, the cellular Na+ content became almost equal when the cells that were pretreated with or without the cAMP analogue in normal BSS were incubated further with Na+-and Caz+-free BSS for 2 min (Fig. 7). Subsequent addition of extracellular ATP or angiotensin I1

at 30 and 1p ~ respectively, , did not induce a further change in the cellular Na+ content (data notshown). The pHi of the cells (7.18 f 0.01) ( n = 3), on the other hand, was not affected significantly when cells were treated with either 0.5 mM 8-Br-cGMP or 0.5 mM dibutyryl cAMP in

Cyclic Nucleotide Regulation of Sarcolemmal Ca2+Pump

8063

when measured in the absence of added Ca2+.As shown in Fig. 2, [Ca2+Iidependences of Na+,-dependent and Na+,independent 4sCa2+effluxes were distinctly different; the latter showed a typical saturation at 0.6-1.0 phi [Ca2+]iwith a half-maximum obtained at about 0.16 PM [Ca2+]i,whereas the former did not show a sign of saturation even at 1 I.IM [Ca2+Ii,the highest concentration measured. Interestingly, the [Ca2+Iidependences of these two components of 45Ca2+efflux and therelative magnitude of these activities were remarkably similar to those obtained with internally dialyzed squid axons (6). In addition, the [Caz+]idependence of the Na+,-independent 45Ca2+efflux (Fig. 2) was very similar to thatobtained for 45Ca2+uptake by proteoliposomes reconstituted from the plasma membrane Caz+ pump ATPase partially purified from bovine aortic smooth muscle (23). Other features of Na+,-dependent and Na+,-independent 45Ca2+effluxes observed in the present study are comparable to those obtained with dialyzed squid axons (see Ref. 6 for summary of squid axon data). High La3+ inhibited both of these activities in smooth muscle cells (Fig. 3) as in squid axons. It is important to note that the Na+,-independent component of “Ca2+ efflux was approximately 50 times more sensitive to the inhibitory effect of La3+ than the Na+,dependent component (Fig. 3). Low concentrations of La3+ inhibited the Na+,-independent 45Ca2+efflux monotonically by up to 90%. In contrast, the effect of La3+ on the Na+,dependent “Ca2+ efflux was biphasic, and low La3+ (at less than 300 PM) accelerated this 45Ca2’efflux significantly, instead of inhibiting it (Fig. 3). A combination of high extracellular pH and high extracellular MgZ+ inhibited the Na+,independent component of 45Ca2+ efflux up by to80%, whereas it did not affect the Na+,-dependent component (Fig. 4). Inhibition of Ca2+transport by high pH and high M g + or DISCUSSION high Caz+from the side opposite to the ATP-binding side of In thepresent study, we have attempted to evaluate quanthe membrane appears to be a general property of the memtitatively the Ca2+ transport capability of Ca2+ extrusion brane Ca2+ pump ATPases because the erythrocyte Ca2+ systems in cultured aortic smooth muscle cells.Ca2+extrusion pump, the sarcoplasmic reticulum Ca2+pump, and the Na+,was measured using fura 2 fluorescence as an indicator of independent *Ca2+ efflux in dialyzed squid axons, which is [Ca2+]iafter stimulation of cells with ATP or angiotensin 11. presumably effected by the plasma membrane Ca2+ pump Measurement of [Caz+]iby fura 2 fluorescence was made with (36), have been shown to be inhibited by high pH and high a homogeneous population of cells which were stably subcul- M P or high Caz+(37-39). In addition to these observations, tured for 4-5 days on glass cover slips. It is thus likely that the Na+,-independent ‘%a2+ efflux from smooth muscle cells Ca2+ responses within individual cells are all alike. It is was found to be inhibited, although incomplete, when cells possible, however, that thetotal cell fura 2 fluorescence meas- were treated with either vanadateor compound 48/80, known ured may not correctly reflect the cytosolic Ca2+level. Wil- inhibitors of the Ca2+pump ATPase (40,41). The incomplete liams et al. (35) observed that Ca2+is not uniformly distributed inhibitions by these reagents may be due to inactivation of within the smooth muscle cell but is present at higher con- vanadate within the living cells or inabilityof the calmodulin centrations in both the nucleus and the sarcoplasmic reticu- antagonist to inhibit the calmodulin-independent activity of lum than in thecytoplasm. This subcellular heterogeneity of the plasma membrane Caz+pump when intracellular ATP is [Ca2+Ii,however, does not appearto affect our results seriously not very low (42). because according to theirestimation, it could cause an overAll of the features of Na+,-dependent and Na+,-independestimation of the resting cytosolic Ca2+levels by &lo% (35). ent 45Ca2+ effluxes described above strongly suggest that Caz+ In addition, the size of this overestimation would be reduced extrusion from cultured aorticsmooth muscle cells is effected further when cells are stimulated with increasing concentra- mainly by two distinct flux mechanisms, which are the Na+/ tions of the agonist, which wouldraise the cytosolic Ca2+level Ca2+ exchanger and the ATP-dependent Ca2+pump. Some and consequently increase fluorescence contribution from the uncertainties could remain, however, about the quantitative cytosolic Ca2+to total cell fluorescence. In this study, Ca2+ aspect of these &Ca2+effluxes. In this study, “Caz+ effluxes extrusion was measured in a time range where values for were measured either in Ca2+-freeBSS or in Na+- and Ca2+[Ca”]i deviated only by 5 9 % from the mean peak value with free BSS after the agonist stimulation. Thesedifferent extrawhich the 45Ca2+efflux rate was correlated. Therefore, the cellular conditions may induce changes in the intracellular observed 46Ca2+ efflux rate should have reflected [Ca2+Iifairly conditions which could affect activities of Ca2+ extrusion accurately although [Ca2+Iichanged constantly after the ag- systems indirectly. The results show that pHi changed minionist stimulation (Fig. 1). mally under the conditions used, whereas the cellular Na’ As in the giant squid axon (6) and cardiac muscle (7, 8), content decreased by 30% when cells were incubated in Na+45Ca2+efflux from cultured aortic smooth muscle cells con- and Ca2+-free BSS to measure Ca2+extrusion by the Ca2+ sisted of Na+,-dependent and Na+,-independent components pump (see “Results”). Such a decrease in the Na+ content,

normal BSS(datanot shown). Subsequent stimulation of cells with 10 PM ATP also did not produce a change in PHi. When cells were treated with either 0.5 mM 8-Br-cGMP, 0.5 mM dibutyryl CAMP, or none of these agents for 3 min in normal BSS and then incubated in Na+- and Ca2+-freeBSS for 2 min, the values of pHi were 7.16 f 0.02 (n = 3), 7.16 f 0.02 ( n = 3), and 7.15 f 0.01 ( n = 3), respectively. A t 20 s after subsequent stimulation with 10 PM ATP, these values changed to 7.10 f 0.01 (n = 3), 7.11 f 0.01 (n = 3), and 7.12 f 0.02 (n = 3), respectively. When pH and theM P concentration of the Na+- and Ca2+-freeBSS were suddenly changed to 8.8 and 20 mM as in Fig. 4, pHi increased only at a slow rate (0.054 f 0.06 pH unit in 1 min). In thisstudy, we used a potential sensitive dye diS-C3(5)to estimate membrane potentials in cultured aortic smooth muscle cells.The value obtained for the resting membrane potential in normal BSS was found to be -49 f 3 mV (n = 3) (inside negative). This estimate is ratherclose to that(-58 k 3 mV) determined recently by direct impalement with microelectrode intothe same type of cells (33). Therefore, we considered that theuse of a potentialsensitive dye in cultured aortic cells, although it is an empirical methods (34), gave a fairly accurate estimation of the membrane potential across the plasma membrane. The resting membrane potential decreased slightly to -47 f 2 mV (n = 3) and -45 f 4 mV ( n = 3) when cells were incubated for 2 min with Ca2+-freeBSS or Ca2+-freeand Na+-free BSS, respectively. Under respective conditions, treatment of cells with either 0.5 mM 8-Br-cGMP or 0.5 mM dibutyryl CAMP and/or stimulation of cells with 30 FM ATP did not produce significant changes in the membrane potential.

8064

Cyclic Nucleotide Regulation of Sarcolemmal Ca2+Pump

however, would not have affected the activity of the Ca2+ pump significantly because it was shown with sealed insideout red cell membrane vesicles that Na+ and K+ can substitute for each other in activatingthe plasma membrane Ca2+pump (43). Another factor that could modify activities of Ca2+ extrusion systems is a change in the membrane potential across the plasma membrane. Membrane hyperpolarization would accelerate Ca2+extrusion by the Na+/Ca2+exchanger (441, whereas it could exert eitherno effect or inhibitory effect on the Ca2+pump activity depending on electrogenic nature of the plasma membrane Ca2+pump, over which there is no general consensus (44,45). Thisstudy showed that theresting membrane potential in cultured aorticsmooth muscle cells in normal BSS was -49 mV (inside negative) as estimated with a potential sensitive dye (see “Results”). Incubation of cells in Ca2+-freeBSS orCa2+-and Na+-free BSS for 2 min resulted in slight depolarization (by 2 or 4 mV, respectively). In these cells, we could notdetectany significant changes inthe membrane potential when they were treated with cyclic nucleotides and/or ATP (see “Results”). Recently it has been reported that in the rabbit ear artery, extracellularly added ATP induced transient membrane depolarization which occurred within 1-2 s after the ATP stimulation (46). Such an early transient change in the membrane potential, if it existed in our system, may not have been detected with the method used in this study. At any rate, all of these findings suggest that different extracellular conditions used may not preclude estimation of the relative magnitude of intrinsic activities of the Ca2+extrusion systems. For the reasons given above, the present results can be considered to provide clear-cut evidence for the relative importance of the two Ca2+extrusion systems in cultured aortic smooth muscle cells. This information would be especially important because procedures for isolation of pure membrane fractions from vascular smooth muscle have not so far been established. The data show that the affinity of the ATPdependent Caz+ pump for Ca2+ is apparently much higher than thatof the Na+/Ca2+exchanger and that theCa’+ pump extrudes significantly more Ca2+than the Na+/Ca2+ exchanconsistent ger at [Ca2+Iibelow 1PM (Fig. 2). The data are thus with a view that the Ca2+pump may be the primary mechanism responsible for maintaining the low resting [Ca2+’]i.At present, however, we do not know the extent to which Ca2+ sequestration by the sarcoplasmic reticulum contributes to the maintenance of resting [Ca2+]i.In thedialyzed squid axon, it was shown that the activity of the Na+/Ca2+exchanger increases sharply at [Ca’+]i above 1PM and reaches a level 10 times the activity of the plasma membrane Caz+ pump at about 100 PM [Ca2+]i(6). The very similar [Caz+]idependences of the 45Ca2+effluxes obtained at low [Ca2+]ifor smooth muscle cells (Fig. 2) and for squid axons (6) suggest that the Na+/Ca2+exchanger could extrude a large amount of Ca2+ from the smooth muscle cells when [Ca”]i rises to a value much higher than 1PM. In vascular smooth muscle, a variety of agents that produce intracellular accumulation of cAMP or cGMP induce relaxation of smooth muscle as stated in the introduction. The mechanisms by which cAMP or cGMP induces relaxation, however, have been controversial. Recently, we have shown that the plasma membrane Ca2+pump ATPase is indeed a target for cGMP-dependent regulation in vascular smooth muscle (23). Furthermore, using the Ca2+pump ATPase partially purified from bovineaortic smooth muscle, we provided evidence suggesting that theCa2+pump ATPase proteinitself is phosphorylated by cGMP-dependent proteinkinase, which results in an increased Ca2+pump activity especially at low

Ca2+concentrations (23). In the present study, we examined whether this cGMP-dependentregulation of the plasma membrane Ca’+ pump is also operative in uiuo. We studied effects of agents such as sodium nitroprusside and ANF, which are known to cause intracellular cGMPaccumulation (13,14), on the Na+.-independent component of‘%a’+ efflux from cultured aortic smooth muscle cells. Pretreatment of cells with nitroprusside (Fig. 5 A ) , ANF (Fig. 5B), or8-Br-cGMP,a membrane-permeable analogue of cGMP (Fig. X), resulted in acceleration of the Na+,-independent “Ca” efflux especially at low [Ca’+]i; At 0.1 p~ [Ca’+]i, which appears to be the threshold level for contraction (47), the “Ca’+ efflux rate was morethan twice as great in cells treated with these agents. These results thus strongly suggest that the activity of the plasma membrane Ca’+ pump is regulated by cGMP in cultured vascular smooth muscle cells. In contrastto the data obtained with the cGMP-producing agents, neither forskolin nor cAMP analogues evoked significant acceleration of the Na+,-independent 46Ca2+ efflux (Fig. 6, A and B, and see “Results”). Although we did not measure the intracellular cAMP level directly, it can be inferred that levels of cAMP and its analogues were certainly elevated in cells used inthese experiments because cAMP analogues decreased the cellular Na+ content as discussed below and because under the conditions of Fig. 6, A and B, the peak heights ofCa’+ transient elicited by the ATP stimulation increased by about 1.6-fold in cells pretreated with forskolin or cAMP analogues (see “Results”). These results, therefore, strongly suggest that cAMP does not accelerate the plasma membrane Ca” pump in the intactaortic cells. This conclusion is consistent with our observation that no significant phosphorylation of the Caz+ pump ATPase protein purified from bovine aortic smooth muscle was detected in the presence of [y3’P]ATPandthecatalyticsubunit of CAMPdependent protein kinase.’ In the experiment of Fig. 7, we studied effects of intracellular cyclic nucleotides on the Na+ content of cells. When cells were incubated with dibutyryl cAMP or 8-Br-cGMP for 3 min in normal BSS, the former but not the latterdecreased the Na+ content by about 30%. 8-Br-CAMP also decreased the Na+ content of cells (see “Results”). This CAMP-induced decrease in the Na+ content is consistent with the previous proposal (48) that cAMP stimulates the activity of the Na+ pump of smooth muscle membrane. In this context, the absence of effect of 8-Br-cGMP in a dose comparable to the effective doseof dibutyryl cAMP or 8-Br-CAMP may be taken as indicating that cGMP does not stimulate the Na+ pump activity significantly. Thus, the present result does not supportthe contention that the primaryeffect of cGMPis stimulation of the Na+ pumpactivity (22). In summary, we have demonstrated that Ca2+ extrusion from cultured aortic smooth muscle cells occurs via two distinct pathways, which are presumably the ATP-dependent Ca2+pump and the Na+/Ca2+exchanger. As the Ca2+pump extrudes significantly more Ca’+ than the Na+/Ca2+exchanger at [Ca2+]i below1 p ~ it , may be concluded that the Ca2+ pump is the primary mechanism responsible for maintaining the low resting [Ca2+]iin these cells. Treatment of cells with sodium nitroprusside, ANF and 8-Br-cGMP but not forskolin, dibutyryl cAMP and 8-Br-CAMP stimulated the Ca’+ pump activity. Theseresults strongly supportour proposal that cGMP stimulationof the plasma membrane Ca2+pump activity plays an important role in the cGMP-induced relaxation of vascular smooth muscle.

* K.-I. Furukawa, Y. Tawada, and M. Shigekawa, unpublished observations.

Cyclic Nucleotide Regulation of Sarcolemmal Ca2+Pump Acknowledgments-We are grateful to Drs. TakahideWatanabe and Yukio Hirata of Institute this for their advice.

REFERENCES 1. Hartshorne, D. J., and Gorecka, A. (1980) Handb. Physiol. 11, 93-120 2. Somlyo, A. P. (1985) Circ. Res. 5 7 , 497-507 3. Bolton, T. B. (1979) Physwl. Reu. 59,606-718 4. Kuriyama, H., Ito, Y., Suzuki, H., Kitamura, K., and Itoh, T. (1982) Am. J. Physiol. 2 4 3 , H641-H662 5. Johns, A., Leijten, P., Yamamoto, H., Hwang, K., and van Breemen, C. (1987) Am. J. Cardiol. 59,18A-23A 6. Baker, P. F., and DiPolo, R. (1984) Curr. Top. Membr. Tramp. 22,195-247 7. Reuter, H., and Seitz, N. (1968) J. Physiol. (Lond.) 1 9 5 , 451470 8. Barry, W. H., Rasmussen, C. A. F., Jr., Ishida, H., and Bridge, J. H. B. (1986) J. Gen. Physwl. 88,393-411 9. Furukawa, K.-I., and Nakamura, H. (1984) J. Biochem. (Tokyo) 96,1343-1350 10. Wuytack, F., De Schutter, G., and Casteels, R. (1981) FEES Lett. 129,297-300 11. Reuter, H., Blaustein, M. P., and Haeusler, G. (1973) Philos. Trans. R. SOC.Lond. E Biol. Sci. 265,87-94 12. Morel, N., and Godfraind, T. (1984) Biochem. J. 2 1 8 , 421-427 13. Ignarro, L. J., and Kadowitz, P. J. (1985) Annu. Reu. Pharmacol. Toxicol. 25,171-191 14. Murad, F. (1986) J. Clin. Znuest. 7 8 , 1-5 15. Kramer, G. L., and Hardman, J. G. (1980) in Handb. Physwl. 11, 179-199 16. Bulbring, E., and Tomita, T. (1987) Pharmacol. Reu. 39,49-96 17. Jones, A.W., Bylund, D. B., and Forte, L. R. (1984) Am. J. Physiol. 2 4 6 , H306-H311 18. Gruetter, C. A., Gruetter, D. Y., Lyon, J. E., Kadowitz, P. J., and Ignarro, L. J. (1981) J. Pharmacol. Exp. Ther. 2 1 9 , 181-186 19. Galvas, P. E., and DiSalvo, J. (1983) J. Pharmocol. Exp. Ther. 224,373-378 20. Popescu, L. M., Panoiu, C., Hinescu, M., and Nutu, 0.(1985) Eur. J. Pharmacol. 107,393-394 21. Kobayashi, S., Kanaide, H., and Nakamura, M. (1985) Science 229,553-556 22. Rapoport, R. M., Schwartz, K., and Murad, F. (1985) Circ. Res. 57,164-170 23. Furukawa, K.-I., and Nakamura, H. (1987) J. Biochem. (Tokyo) 101,287-290 24. Chamley, J. H., Campbell, G. R., McConnell, J. D., and GroschelStewart, U. (1977) Cell Tissue Res. 177,503-522

8065

25. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260,3440-3450 26. Smith, J. B., Smith, L., Brown, E. R., Barnes, D., Sabir, M.A., Davis, J. S., and Farese, R. V. (1984) Proc. Natl. Acad. Sci. U. S. A . 81,7812-7816 27. Sims. P. J.. Waggoner. A. S., Wang, C.-H., and Hoffman, J. F. (1974) B i b c h e Z t V i3.3315-3350 28. Jones, A. W. (1982) in Vascular Smooth Muscle: Metabolic, Ionic, and Contractile Mechanisms (Crass, M. F., 111, and Barnes, C. D., eds) pp. 37-70, Academic Press, New York 29. Kurtz, I., and Golchini, K. (1987) J. Biol. Chem. 262,4516-4520 30. Tamura, H., Hopp, L., Kino, M., Tokushige, A., Searle, B.M., Khalil, F., and Aviv,A. (1986) Am. J. Physwl. 2 5 0 , C939c947 31. Dulley, J. R., and Grieve, P. A. (1975) Anal. Biochem. 6 4 , 136141 32. Tawada, Y., Furukawa, K.-I., and Shigekawa, M. (1987) J. Biochem. (Tokyo) 1 0 2 , 1499-1509 33. Blennerhassett, M. G., Kannan, M. S., and Garfield, R. E. (1987) Am. J. Physiol. 2 5 2 , C555-C569 34. Laris, P. C., and Hoffman, J. F.(1986) in Optical Methods in Cell Physiology (De Weer, P., and Salzberg, B. M., eds) pp. 199210, Wiley-Interscience, New York 35. Williams, D. A., Becker, P. L., and Fay, F. S. (1987) Science 2 3 5 , 1644-1648 36. DiPolo, R. (1978) Nature 274,390-392 37. DiPolo, R., and Beaug6, L. (1982) Biochim. Biophys. Acta 6 8 8 , 237-245 38. Kratje, R. B., Garrahan, P. J., and Rega, A. F. (1985) Biochim. Biophys. Acta 816,365-378 39. Wakabayashi, S.,Ogurusu, T., and Shigekawa, M. (1987) J. Biol. Chem. 262,9121-9129 40. Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Bbl. Chem. 2 5 6 , 395-401 41. Gietzen, K., Adamczyk-Engelmann, P., Wuthrich, A., Konstantinova, A., and Bader, H. (1983) Biochim. Bwphys. Acta 7 3 6 , 109-118 42. Rossi, J. P. F. C., Rega, A. F., and Garrahan, P. J. (1985) Biochim. Biophys. Acta 816,379-386 43. Sarkadi, J. D., Macintyre, J. D., and Gordos, G. (1978) FEES Lett. 89,78-82 44. Allen, T. J. A., and Baker, P. F. (1986) J. Physiol. (Lond.) 3 7 8 , 77-96 45. Niggli, V., Sigel, E., and Carafoli, E. (1982) J. Biol. Chem. 2 5 7 , 2350-2356 46. Benham, C.D., Bolton, T. B., Byrne, N.G., and Large, W. A. (1987) J. Physiol. ( L o n d . ) 387,473-488 47. Itoh, T., Kanmura, Y., Kuriyama, H., and Sasaguri, T. (1985) Br. J. Pharmacol. 84,393-406 48. Scheid, C. R., and Fay, F. S. (1984) Am. J. Physiol. 2 4 6 , (2431C438