effects of calponin on force generation by single smooth muscle cells

Effects of calponin on force generation by single smooth muscle cells

ARIE HOROWITZ, ODILLE CLEMENT-CHOMIENNE, MICHAEL P. WALSH, TERENCE TAO, HIDEAKI KATSUYAMA, AND KATHLEEN G. MORGAN Cardiovascular Division, Beth Israel Hospital, and Program in Smooth Muscle Research, Department of Medicine, Harvard Medical School, Boston 02215; Boston Biomedical Research Institute, Boston 02114; and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111; and Smooth Muscle Research Group and Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Horowitz, Arie, Odille CluSment-Chomienne, Michael P. Walsh, Terence Tao, Hideaki Katsuyama, and Kath- leen G. Morgan. Effects of calponin on force generation by single smooth muscle cells. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1858-H1863, 1996.-Although the actin- binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+1) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca2+ -independent pathway by either phenylephrine or the E isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase CE treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175+Ala replacement had no effect on contractile force. A peptide corresponding to Leul@-Glylg4 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviat- ing the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca2+ -independent pathway downstream of protein kinase CE.

ferret; aorta; single cell force measurement; thin filament regulation

CALPONIN (Cap) is an abundant thin filament-associ- ated protein present in vascular smooth muscle (28). The in vitro properties of Cap, including its capacity to inhibit actomyosin adenosinetriphosphatase (ATPase) (31), have been well characterized (reviewed in Ref. 6); however, its in vivo role remains obscure. Despite the central role of myosin light chain (MLC) phosphoryla-

tion in initiating smooth muscle contraction, there is accumulating experimental evidence for an uncoupling of force maintenance and, under certain circumstances, MLC phosphorylation (8, 9, 14,26, 27,30). This uncoupling may be reconciled by invoking thin filament regulation in addition to MLC phosphorylation. The actin-binding and -inhibitory properties of CaP make it a likely candidate for involvement in thin filament regulation.

Three recent studies addressed the physiological role of CaP by applying the intact protein (12, 13, 23), a large amino-terminal fragment (13), or several CaP peptides (11) to skinned multicellular smooth muscle preparations of either mesenteric artery or taenia coli. The overall conclusion of these studies was that exogenously applied CaP is capable of inhibiting both force and shortening velocity to varying degrees. There were, however, considerable quantitative differences between the studies on the extent of these effects. Itoh et al. (12) observed a force reduction of -30% in both Ca2+-stimulated and MLC-thiophosphorylated muscle strips. Jaworowski et al. (13), on the other hand, observed no force reduction in Ca2+-induced contractions and only a minimal force reduction with MLC- thiophosphorylated muscle strips, but found a 25% decrease in shortening velocity in both contractions. Similarly, Obara et al. (23) found that the primary effect of exogenous CaP was to strongly suppress shortening velocity in Ca 2+-stimulated strips but, unlike Jaworowski et al. (13), they observed a threefold larger force reduction in Ca2+-induced contractions than with thiophosphorylated muscle strips.

In addition to the divergent conclusions, the above studies employed Ca2+- induced contractions and MLC thiophosphorylation, conditions that may not have been optimal for monitoring the effects of thin filament regulation. Because the myosin phosphorylation pathway is fully activated under these conditions, the effects of thin filament regulation are likely to be masked. Moreover, in the presence of adenosine-5’-0-(3- thiotriphosphate), CaP may also have been irreversibly thiophosphorylated by calmodulin-dependent protein

H1858 0363-6135/96 $5.00 Copyright o 1996 the American Physiological Society

CALPONIN AND SMOOTH MUSCLE REGULATION H1859

kinase II, losing its capacity to bind actin and inhibit the actomyosin ATPase (31,32).

It has been shown that ferret aorta smooth muscle cells can be induced to contract at low intracellular [Caz+] ([Ca’+]i> by phenylephrine (PE) in a protein kinase C (PKC)-dependent manner (3, 18, 25) without an increase in MLC phosphorylation above the basal level (14,20>. Also, we have recently shown that PKC-E is the specific isoenzyme involved in Ca2+-independent contraction of these cells, and that CaP can serve as its substrate in vitro (10). Changes in the contractile properties of these cells stimulated by either PE or PKC-E at low [Ca2+]i are therefore more likely to reflect thin filament regulatory processes.

Here we address the physiological role of CaP in smooth muscle contraction by application of either intact CaP or an actin-binding CaP peptide (LG29C) to permeabilized ferret aorta single smooth muscle cells. We found that intact CaP inhibited the maximal contractile force. In contrast, LG29C triggered contraction in a dose-dependent manner. Thes CaP plays a role in regul ating

findin -gs suggest that mooth m uscle con .trac-

tion via a Ca2+ -independent, PKC-dependent pathway.

METHODS

Materials. ATP, creatine kinase, PE, L-a-phosphatidyl+ serine, and saponin were purchased from Sigma Chemical (St. Louis, MO). 1,2-Diolein was purchased from Serdary Research Laboratories (London, Ontario, Canada). CM- Sephadex was purchased from Pharmacia (Baie d’Urfe, Que- bec, Canada). Electrophoresis reagents were purchased from Bio-Rad (Richmond, CA). Other reagents were of analytic grade or better and were purchased from Fisher Scientific (Pittsburgh, PA). The unphosphorylated wild-type CaP was expressed in Escherichia coli and purified as described (7). Two synthetic 30-residue peptides-LG29C, corresponding to the CaP Leu166-Gly1g4 sequence followed by a Cys (LQMGT NKFAS QQGMT AYGTR RHLYD PKLGC), and XLG29C, having a scrambled sequence of the same residues (MRLKY LGTPG AHTKQ LRDGA CYSGN QTFQM)-were synthesized (ABI 431A, Applied Biosystems, Foster City, CA) and purified by reverse-phase high-performance liquid chromatog- raphy (Beckman 344, Beckman Instruments, Foster City, CA). The identity and purity of the peptides were verified by sequencing (ABI 477, Applied Biosystems). The peptides were solubilized at a concentration of 10 mM in 4 M urea and 10 mM dithiothreitol (DTT). PKC+ was purified from rat brain as described (10) and stored in 20 mM Na N-2-hydroxyethyl- piperazine-W-2ethanesulfonic acid (HEPES) (pH 7.4), 1 mM ethylene glycol-bis( P-aminoethyl ether)-N,N,N’,N’-tetraace- tic acid (EGTA), 1 mM EDTA, 0.02% (wt/vol> NaN3, 0.03% (vol/vol) Triton X-100, and 25% (vol/vol) glycerol. PKC+ was purified free of other PKC isoenzymes as described (10).

CaP phosphorylation. Recombinant CaP (0.2 mg/ml) (7) was incubated for 60 min at 30°C in 25 mM tris(hydroxymeth- yl)aminomethane (Tris)=HCl (pH 7.5), 5 mM MgClz, 0.1 mM CaCIZ, 0.3 mg/ml L-cc-phosphatidyl+serine, 62 ug/ml 1,2- diolein, 0.03% (wt/vol) Triton X-100, and 1 mM ATP, in the absence and presence of PKC (0.5 ug/ml) in a volume of 20 ml [the PKC used for CaP phosphorylation was a mixture of (x, p, and y isoenzymes, purified from rat brain as described (l)]. EDTA was then added to a final concentration of 6.3 mM (1.2 mM excess over Mg2+ plus Ca2+) to quench the reaction. Parallel reactions containing [Y-“~P]ATP indicated that, in

the presence of PKC, CaP was phosphorylated to the extent of 2.25 mol Pi/m01 CaP [quantified as described (31)]. Reaction mixtures containing unlabeled ATP were applied to columns (1 X 10 cm) of CM-S ep a h d ex previously equilibrated with 20 mM TrisHCl (pH 7.5), 50 mM KCl, and 1 mM DTT. CaP was eluted with 20 mM Tris HCl (pH 7.5), 1 M KCl, and 1 mM DTT. Cap-containing fractions, identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were combined and dialyzed against two changes (10 liters each) of 20 mM TrisHCl (pH 7.5), 0.1 M KCl, and 1 mM DTT.

CaP mutagenesis. CaP in which Ser175 was mutated to Ala (CaPS175A) was engineered by polymerase chain reaction mutagenesis, expressed in E. coli, and purified as described (2%

Cell isolation method. Ferrets were anesthetized with chloroform according to procedures approved by the Institu- tional Animal Care and Use Committee. Aortas were removed and smooth muscle cells were isolated by gentle enzymatic digestion as previously described (3). Cells were used within l-3 h of isolation.

SingZe cell mounting and force measurement. The experimental procedures, equipment, and solutions were identical to those described in Ref. 10. Briefly, cells were plated on glass coverslips in a Ca2+- and Mg2+-free Hanks’ buffered saline solution (in mM: 137 NaCl, 10 HEPES, 5.5 dextrose, 5.4 KCl, 4.17 NaHC03, 0.44 KH2PO4, 0.42 Na2HP04; pH adjusted to 7.3 with NaOH). The solution was exchanged before the experiments to a pCa 7.0 or 9.0 buffer [ionic strength 0.2 M, pH 7.0 set with 3-(N-morpholino)propanesulfonic acid (MOPS), 135 mM K+, 15 mM creatine phosphate, 15 mM EGTA, 3 mM ATP, and 1 mM Mg2+ 1, and the coverslip was placed on the stage of an inverted microscope (Nikon Diaphot, Nikon Corporation, Tokyo, Japan). The cells were permeabilized by saponin (120 pg/ml) for 3 min. Experiments were carried out in 200 pl of pCa 7.0 or 9.0 buffer to which 20 U/ml of creatine kinase were added daily before the experiment. Cells were chosen for attachment to the microtools and force measurement only if their length exceeded 60 urn and if they adhered to the glass coverslip. Force measurements were carried out as previously described (2, 3, 15). Average length of the cells used in the experiments was 75 ? 6 urn (mean t SD, n = 50). Cell contractility was tested daily before the experiment by application of PE to nonpermeabilized cells in a physiological saline solution containing 1 mM Ca2+ (3). All experiments were performed at room temperature (22°C).

Cell imaging. Cells processed and permeabilized as above were incubated for 10 min in pCa 9.0 buffer containing 5 uM CaP fluorescently labeled (30) with 5-iodoacetamidofluores- cein (IAF) (Molecular Probes, Eugene, OR). The cells were fixed and imaged as previously described (24).

RESULTS

Effects of exogenous CaP on smooth muscle contraction. Single permeabilized smooth muscle cells were activated either by 10F4 M PE (Fig. 1A) or by 0.1 U/u1 PKC-E (Fig. 1B). The effect of exogenous CaP was initially studied on cells at pCa 7.0 to simulate the resting free Ca2+ concentration (4). In response to lop4 M PE, these cells produced a steady-state mean force of 89 t 9 pg (n = 7) that was reduced by 60% after the application of 5 uM CaP (Fig. 1C). At pCa 7.0, however, the cells already generate 20% of maximal force (2). Most interestingly, though, ferret aortic smooth muscle cells were found to produce force in response to PE and PKC+ even at pCa 8.6-9.0 (3, 10). Therefore, to

H1860 CALPONIN AND SMOOTH MUSCLE REGULATION

1C4 mM PE 5pM CaP 1

4 pi PKCE 5 pM CaP

C

PE (pCa 7) PE (pCa 9) PKC& (pCa 9)

Fig. 1. Effect of calponin (Cap, 5 pM) on force produced by single permeabilized ferret aorta smooth muscle cells. A-B: single force traces at pCa 9.0. A: after stimulation by lop4 M phenylephrine (PE). B: after stimulation by E subunit of protein kinase C (PKC-E) (4 pl containing 20.3 U diluted into a volume of 200 ul on coverslip, yielding a final concentration of 0.1 U/ul; 1 U is the amount of enzyme catalyzing transfer of 1 pmol Pi to E-peptide/min, e-peptide being a synthetic peptide corresponding to autoinhibitory domain of PKC-e, see Ref. 1). C: mean steady-state forces (+ SE) before (solid bars) and after (hatched bars) application of Cap. PE (pCa 7.0), n = 7; PE (pCa 9.0>, n = 7; PKC-E (pCa 9.0>, n = 5.

simplify interpretation of results, the remaining experiments were carried out at pCa 9.0. Cells activated by PE and by PKC-E at pCa 9.0 produced forces of 110 2 10 ug (mean 2 SE; n = 5) and of 105 t 12 ug (n = 5), respectively. These forces are not significantly different from each other or from the values we previously obtained under the same conditions (10).

Exogenous CaP (final concentration 5 pM) was applied to the cell bathing solution when force reached plateau. The application of CaP caused a mean fall by 45 and 43% of the steady-state force amplitude in PE- and PKC-E-stimulated cells, respectively. Typical force records from single cells are shown in Fig. 1, A and B, and the mean steady-state forces before and after the application of CaP are indicated in Fig. 1C. The mean half-time to maximum force inhibition was 3.4 t 0.8 (n = 5) and 3.1 t 0.5 min (n = 5) in cells activated by PE and PKC+, respectively. To verify CaP penetration, cells were also incubated for 10 min (a duration similar to the time required for full force reduction) in 5 pM CaP fluorescently labeled with IAF and viewed by fluorescence microscopy. The images confirmed that the exogenous CaP diffused into the entire smooth muscle

cell within this time (data not shown). The similarities between the steady-state forces obtained with PE and PKC-e and between the effects of CaP on these forces are consistent with the suggestion that both PE and PKC-E activate the same pathway in smooth muscle (10, 17).

The inhibitory effect of exogenous CaP applied after stimulation, when force had already reached a plateau, may have been minimized by the experimental conditions. In cells stimulated by PKC-E, some of the unphosphorylated exogenous CaP could have been phosphorylated by the PKC-E and ATP present in the bathing solution, thus losing its inhibitory effect; in cells stimulated by PE, the kinase pathway in the cell was activated and could have similarly phosphorylated some of the exogenous Cap. To test whether a larger inhibitory effect can be obtained when the CaP is less likely to be phosphorylated, we pretreated unstimulated cells with 5 uM exogenous CaP. Addition of 0.1 U/u1 PKC-E to these cells failed to elicit a force response (Fig. 2). As described previously, cells of the ferret aorta possess an intrinsic tone, even at pCa 9.0 (3), and the pretreatment by CaP reduced this intrinsic tone by 75 t 7 pg (n = 9).

Effects ofphosphorylated and mutant CaPs on smooth muscle contraction. Phosphorylated CaP loses its ability to bind F-actin and inhibit actomyosin ATPase activity in vitro (3 1). The site of phosphorylation in vivo was reported to be Ser 175 (32). CaP (5 uM) phosphorylated by PKC was, therefore, applied to cells activated with 0.1 U/u1 PKC -E. In marked contrast to unphosphorylated CaP, phosphorylated CaP did not have an immediate inhibitory effect on contractile force (Fig. 3). When a prolonged force recording was made, a delayed decrease in force by 40 t 10% (n = 3) was seen; the mean half-time to maximum force inhibition was 10.3 or: 0.3 min (n = 3), three times slower than in cells treated

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PKC E CaP CaP followed alone followed by CaP by PKC E

Fig. 2. Effect of post- vs. pretreatment with CaP (5 uM) on PKC-e- stimulated single smooth muscle cells. PKC+ concentration was 0.1 U/ul; pCa = 9.0. PKC-E alone, ~2. = 7; PKC-E followed by CaP, zz = 7; CaP alone and CaP followed by PKC-E, yt = 9.


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(Fig. 4, A and C). Peptide concentrations ~50 uM had no detectable contractile effect. Similarly, sham solutions containing diluent but no peptide had only a transient effect on force (similar to the initial transient seen in Fig. 4A) but produced no steady-state increase in force. The relatively high peptide concentration required to produce force is probably due to the small size and, consequently, lower F-actin affinity of LG29C compared with that of wild-type Cap. Given the relatively high concentration of the peptide required to elicit a force response, the possibility of a concentration- induced nonspecific effect was investigated by applying a peptide with a scrambled sequence of the same amino acid composition as peptide LG29C. This scrambled peptide (XLG29C) produced no sustained force response when applied to single cells at a concentration of

cap phos CaP CaPS175A 200 pM (n = 5) (Fig. 4B).

Fig. 3. Effect of phosphorylated (phos CaP) or mutant CaP DISCUSSION (CaPSl75A) on PKC-e-induced force in single smooth muscle cells. Force was measured immediately before (solid bars) or 10 min after (hatched bars) application of 5 uM phosphorylated or mutant Cap.

We have demonstrated that exogenous CaP signifi-

PKC-E concentration was 0.1 U/ul; pCa = 9.0. Cap, ~2. = 7; phos Cap, cantly reduced or, when CaP application preceded n = 3; CaPS175A, y2 = 4. stimulation by PKC-E, completely eliminated contrac-

with unphosphorylated Cap. The fact that the phosphorylated CaP did have an inhibitory effect, though delayed, suggests that the CaP was probably dephosphorylated by an end .ogenous phosphatase (5).

To further test whether the capacity of CaP to inhibit 200 pM LG29C

force depen ds on its ability to bind F-actin, we used CaPS175A. CaPS175A has a reduced affinity for actin and consequently is only a weak inhibitor of the in vitro actin-activated Mg-ATPase activity of phosphorylated smooth muscle myosin (29). Correspondingly, it also had no detectable inhibitory effect on contractile force of cells stimulated by 0.1 U/u1 PKC-E (peak force 125 ? 20 ug, n = 4) (Fig. 3). Unlike phosphorylated CaP, the site-directed mutant CaP did not produce a delayed force reduction during the time period of observation, up to 20 min. This is consistent with the above interpretation that exogenous phosphorylated CaP was probably dephosphorylated by an endogenous phosphatase in the cell.

Effects of CaPpeptides on smooth muscle contraction. Al .though the above resul .ts clearly indicate the ability of CaP to reduce force in the smooth muscl .e cell, application of exogenous CaP presumably produced supraphysiological concentrations of this protein in the intracellular space. Thus, to examine whether endogenous CaP acts in a physiologically significant manner to regulate force, we synthesized a peptide spanning residues Leul@-Gly lg4 (LG29C). This peptide contains part of the actin-binding domain, encompassing 38 residues from Ala14” to Tyr 182 (21), but does not contain the 19 residues, Ala 145-11e163, thought to be essential for actomyosin ATPase inhibition (22). Thus LG29C may act as a CaP antagonist by competing for actin binding.

Application of peptide LG29C to single permeabilized smooth muscle cells at pCa 9.0 elicited a concentration- dependent force response betw ‘een 50 an .d 200 pM with a maximum of 166 t 27 ug (n = 4) at 200 uM peptide

200 PS 5 min

200 FM XLG29C

Fig. 4. Effects of CaP peptides LG29C and XLG29C on unstimulated single cells. Single force trace of a cell treated by 200 uM LG29C (A) or by 200 uM XLG29C (B). Concentration dependence of force (C) in cells treated with 25-200 uM LG29C (n 2 4) is shown; size and direction of initial force transient varied from experiment to experiment depending on position of cell on application of reagent. pCa = 9.0.

coverslip relative to point of

H1862 CALPONIN AND SMOOTH MUSCLE REGULATION

tile force in skinned single smooth muscle cells. On the other hand, a CaP peptide consisting of the actin- binding region minus the actomyosin ATPase inhibitory domain induced contractile force when applied to unstimulated cells. These results were obtained in PE- or PKC-E-induced contractions and at low [Ca2+], conditions that should not increase MLC phosphorylation above basal levels (20).

The mechanism we hypothesize for the reduction in contractile force by exogenous CaP is based on the observation that the endogenous CaP translocates to the cell cortex on PE stimulation (24). The binding sites on the thin filaments vacated by the endogenous CaP thus become available to the exogenous CaP, which reinhibits the thin filaments. The suppression of contractile force was more pronounced when the application of exogenous CaP preceded, rather than followed, stimulation, because in the former case the CaP was less likely to be phosphorylated by either the exogenously applied PKC+ or the PE-stimulated cellular kinase pathway, losing its inhibitory effect (31). An alternative mechanism of force reduction by exogenous CaP could be competitive substrate inhibition of either endogenous or exogenous PKC-E, assuming that CaP is indeed the in vivo substrate of PKC-E (10). This mechanism cannot adequately explain, however, the results of experiments with the CaP peptide or the ability of CaP to relax the resting muscle (see CaP alone in Fig. Z), where PKC is primarily cytosolic and inactive (16).

The triggering of contraction by the F-actin-binding CaP peptide LG29C probably resulted from disturbing the conformation of the endogenous CaP on the thin filaments, interfering with its inhibitory function. Be- cause peptide LG29C itself lacks the ATPase inhibitory domain Ala 145-Ile163 (ZZ), it did not block force produc- tion by the basal population of phosphorylated myosin [estimated to be 12% of the total myosin content in vascular smooth muscle (14)].

The inhibitory effect of CaP we observed is in general agreement with previous studies that found similar inhibition of force and shortening velocity. We measured a larger force reduction, however, particularly in comparison to the effects of CaP on MLC-thiophosphorylated muscle strips; Itoh et al. (12) and Obara et al. (23) obtained a 2530% force reduction, and Jaworowski et al. (13) obtained only a -10% force reduction. The smaller effects observed by others could have resulted from the supramaximal myosin activation caused by thiophosphorylation, which may have obscured thin filament regulation by Cap. Another possible reason for the smaller effects observed in previous studies may be the larger size of the skinned preparation used (either taenia coli or mesenteric arterial strips), in which some of the inner cells may have remained impermeable to the exogenous Cap.

Our results with peptide LG29C were more pronounced than those of Itoh et al. (11), who applied a smaller 11-residue CaP peptide (Phe173-Arg1s5) con- tained within the 29-residue sequence of LG29C, to P-escin-permeabilized strips of rabbit mesenteric artery. In their study, the CaP peptide augmented the

contraction of Ca 2+-stimulated smooth muscle strips but did not trigger contraction in the presence of EGTA. Moreover, the augmentation (1.5 5 0.2-fold) was relatively small compared with that of a control nonactin- binding peptide (Gly 213-Lys225) (1.2 t 0.2 times the force in the absence of the peptide). Thus, although further confirmation is necessary, the peptide used in the present study (LG29C) may prove to be a better experimental tool as a “Cap antagonist.”

Ferret vascular smooth muscle cells were shown to contract via a Ca2+ -independent pathway (3, 15, ZS), without a detectable increase in the basal MLC phosphorylation level (20). The ability of several PKC inhibitors to block the Ca2+-independent contractions (3, 15, 18) and the semblance between the time course of contraction and of PKC translocation observed in the contracting cell (17) suggest that PKC is involved in this pathway. CaP was also found to translocate in PE-stimulated vascular smooth muscle cells, with a temporal and spatial pattern similar to PKC (24). This similarity suggests that both proteins participate in the same regulatory pathway. PKC may act either by phosphorylating CaP directly or by phosphorylating another, as yet undetermined, protein upstream of Cap. Interaction between CaP and caldesmon as part of the thin filament regulatory mechanism also has to be considered (19). The results presented here do not favor one specific alternative over the other. However, we have shown that PKC-E phosphorylates CaP purified from chicken gizzard in vitro (10). Put together with the above findings, the results presented are consistent with the hypothesis that CaP has a regulatory function in PKC-dependent smooth muscle contraction.

The authors are very grateful to Christopher A. Parker for the imaging of smooth muscle cells and to Dr. Paul T. Szymanski for phosphorylating calponin.

This work was supported in part by National Institute ofArthritis and Musculoskeletal and Skin Diseases Grant POl-AR-41637, Na- tional Heart, Lung, and Blood Institute Grants HL-42293 and HL-31704, and by a grant from the Heart and Stroke Foundation of Alberta. 0. Clement-Chomienne is the recipient of fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). M. Walsh is an AHFMR Medical Scientist.

Address for reprint requests: A. Horowitz, Cardiovascular Div., Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215.

Received 14 November 1995; accepted in final form 23 January 1996.

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effects of calponin on force generation by single smooth muscle cells

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