©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Calponin Peptide Enhances Ca Sensitivity of Smooth Muscle Contraction without Affecting Myosin Light Chain Phosphorylation (*)

(Received for publication, January 2, 1995; and in revised form, May 22, 1995)

Takeo Itoh (1)(§) Akito Suzuki (1) Yoshimasa Watanabe (1) Terumasa Mino (2) Michiko Naka (2) Toshio Tanaka (2)

From the  (1)Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812 and the (2)Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Edobashi, Tsu, Mie 514, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In permeabilized smooth muscle, exogenously applied calponin binds to myofibrils and reduces Ca-activated tension (Itoh, T., Suzuki, S., Suzuki, A., Nakamura, F., Naka, M., and Tanaka, T.(1994) Pflügers Arch. Eur. J. Physiol. 427, 301-308). A calponin peptide (calponin Phe-Arg), which inhibits the binding of calponin to actin, blocks the action of calponin and enhances the contraction induced by submaximal Ca in permeabilized vascular smooth muscle. Unlike calmodulin, this peptide enhances the Ca-induced contraction without a corresponding increase in the level of myosin light chain phosphorylation. These results suggest that calponin decreases the sensitivity of smooth muscle to Ca at a given level of myosin light chain phosphorylation.


INTRODUCTION

Phosphorylation of myosin light chain by Ca-calmodulin-dependent myosin light chain kinase is believed to be responsible for the coupling between an increased intracellular concentration of Ca and contraction in smooth muscle(1, 2, 3, 4) . However, a dissociation of the relationship between the intracellular concentration of Ca and tension and that between Ca and myosin light chain phosphorylation in smooth muscle contraction has been reported(3, 5, 6) , and thus other mechanisms that act in concert with, or without, the participation of myosin light chain phosphorylation have also been suggested. Recently, much attention has been paid to thin filament-linked mechanisms that are mediated by the actin-binding proteins, calponin and caldesmon.

Calponin is a major component of the smooth muscle thin filament(7) , and it binds to F-actin, tropomyosin, and calmodulin. This protein inhibits the actomyosin ATPase activity of smooth muscle through its binding to actin in vitro(8, 9, 10) . Moreover, calponin is known to be an excellent substrate for protein kinase C and Ca-calmodulin-dependent protein kinase II in vitro(9, 10) , the phosphorylation of calponin by each kinase resulting in the loss of its ability to inhibit ATPase activity (9, 10) . Three tryptic phosphopeptides of smooth muscle calponin made using protein kinase C were recently isolated and designated T(1) (Gly-Lys), T(2) (Phe-Arg), and T(3) (Gly-Arg)(11) . Phosphorylation of Ser(12) or Thr(11) in the T(2) peptide accounts for over 50% of the total calponin phosphorylation, and the phosphorylation is competitively inhibited by actin, suggesting that peptide T(2) may be adjacent to the actin-binding region of calponin.

Here, we present evidence derived from experiments using T(2) peptide that calponin acts to lower the myofilament Ca sensitivity in smooth muscle contraction at a given level of myosin light chain phosphorylation.


EXPERIMENTAL PROCEDURES

Materials

Calponin was purified from chicken gizzard(8) . Actin was purified from rabbit skeletal muscle by the method previously described(14) . Calmodulin was isolated from bovine brain and purified by DEAE-cellulose chromatography and phenyl-Sepharose chromatography (15) . An antibody against myosin light chain was a gift from Dr. James T. Stull (Department of Physiology, University of Texas Southwestern Medical Center). T(1) and T(2) peptides were purchased from Peptide Institute, Inc. (Osaka, Japan).

Measurement of Phosphorylation of 20-kDa Myosin Light Chain

Phosphorylation of myosin light chain was measured by the Western blotting method using antibody against bovine tracheal myosin light chain as a first antibody and biotin-labeled goat anti-rabbit IgG as a second antibody, as described previously(16) .

Tissue Preparation

A segment of the third branch of the rabbit mesenteric artery distributing to the ileum region (diameter about 100 µm) was excised and cleaned by removal of connective tissue. Very thin smooth muscle strips (0.2-0.3-mm length, 0.05-0.07-mm width, 0.02-0.03-mm thickness) were made, transferred into a chamber of 0.05-ml volume, and mounted horizontally, the resting tension being adjusted to obtain a maximum contraction in 128 mM K(17) .

Permeabilized Smooth Muscle Strips

The muscle strip was then permeabilized by an application for 30 min of beta-escin in a relaxing solution containing 4 mM EGTA, 110 mM potassium methanesulfonate, 5.2 mM Mg(MS)(2), 5 mM ATP, 5 mM creatine phosphate, 20 mM PIPES, (^1)and 1 µM ionomycin (pH 7.1), as described previously(17) . To prevent deterioration of the Ca-induced contraction, 0.1 µM calmodulin was present throughout the experiment. Free Ca concentration was calculated as described previously(18) . All experiments were performed at room temperature (23-25 °C).

Fluorescence Measurement

Actin was labeled with N-(1-pyrenyl)iodoacetamide by the method described by Cooper et al.(19) . Pyrene-labeled F-actin (3 mM) in 20 mM Tris-HCl (pH 7.4) containing 100 mM KCl and 0.1 mM CaCl(2), was preincubated for 1 h at 25 °C. Fluorescence intensity (excited at 365 nm, emitted at 407 nm) was measured 15 min after the addition of T(2).

Assay for Binding of Calponin to F-actin

For all assays assessing the cosedimentation of T(2) with F-actin, the T(2) was added to F-actin in 20 mM Tris-HCl (pH 7.5) containing 30 mM KCl, 2 mM MgCl(2), 1 mM ATP, 1 mM dithiothreitol and 0.2 mM CaCl(2), and the reaction mixture was incubated for 30 min at 25 °C following the addition of calponin. Subsequent analysis was performed as described by Nakamura et al.(11) .

Assay of Mg-ATPase Activity

The actin-activated myosin Mg-ATPase activity was determined by the method described previously (20) under the following conditions. 0-8 µM F-actin and 2.2 µM thiophosphorylated myosin were mixed in 25 mM Tris-HCl, pH 7.5, 83 mM KCl, 10 mM MgCl(2), 1 mM CaCl(2), and 1 mM dithiothreitol to a final volume of 100 µl. The reactions were started by the addition of 1 mM [-P]ATP. After preincubation for 10 min at 25 °C, the reaction was stopped for 10 min with 10% perchloric acid. The effect of T(1) and T(2) peptides on actin-activated myosin Mg-ATPase was observed in the presence of 5 µM F-actin.


RESULTS AND DISCUSSION

Effect of Calponin on Smooth Muscle Contraction

Fig. 1A shows the inhibitory effect of exogenously applied 3 µM calponin on the contraction induced by 1 µM Ca. This action of calponin was concentration-dependent over the range 0.32-3 µM, and the maximum reduction was by 35.2 ± 18.3% of control (n = 4, p < 0.01; Student's t test). The apparent binding of calponin to the myofibril in permeabilized muscle was determined from the density ratio of calponin to actin in SDS-polyacrylamide gel electrophoresis with or without application of calponin. Under our conditions, it was in the range 0.04-0.05 in control and 0.08-0.15 after a 30-min application of calponin followed by a 30-min washout. Thus, the increase in the concentration of calponin was estimated to be by 2.1 ± 0.2 times control (n = 3, p < 0.05).


Figure 1: Effect of exogenously applied calponin and a calponin peptide (calponin Phe-Arg; T(2)) on Ca-induced contraction in permeabilized vascular smooth muscle. A, calponin (CaP) was applied during the maintained contraction induced by 1 µM Ca. Subsequent application of T(2) increased the tension to the level recorded before application of calponin. B, concentration-dependent effects of T(2) on contraction induced by 0.3 µM Ca. After a recording of the maximum Ca-induced contraction (on application of 10 µM Ca), 0.3 µM Ca was applied. Subsequently, various concentrations of T(2) (0.03-0.3 mM) were applied on the Ca-induced contraction, in ascending steps. C, effect of simultaneous application of CaP and T(2) on contraction induced by 0.3 µM Ca. Both agents were applied during the steady state contraction induced by 0.3 µM Ca followed by a washout of T(2) only; 10 µM Ca was finally applied to register the maximum Ca-induced response.



Protein kinase C phosphorylates calponin, causing dissociation of calponin from actin and thus attenuates the calponin-induced inhibition of actin-activated myosin Mg-ATPase in reconstituted smooth muscle contractile proteins(9, 10) . Calponin (3 µM) phosphorylated by protein kinase C (0.6 mol of P(i)/mol of calponin) had less inhibitory action (0.95 ± 0.05 times control, n = 3) than nonphosphorylated calponin (0.48 ± 0.16 times control, n = 3, p < 0.05) on the contraction induced by 1 µM Ca. The result was consistent with the previous findings(13) . Under these conditions, the binding of the phosphorylated calponin to the myofibril in permeabilized smooth muscle was decreased (0.67 ± 0.21 times that obtained by unphosphorylated calponin, n = 3). These results suggest that the action of exogenously applied calponin is not nonspecific and that, on application, the unphosphorylated form of calponin first binds to the myofibril and then inhibits Ca-induced contraction.

Interaction of T(2)Peptide with F-actin

Fig. 2A shows the effect of T(2) peptide (FASQQGMTAYGTR) on the fluorescence intensity of pyrene-labeled F-actin. The fluorescence intensity of actin was reduced by the addition of 0.3 mM T(2), suggesting that T(2) peptide binds to the actin filament and then causes the same conformational changes in actin as calponin does (21) .


Figure 2: A, effect of a synthetic calponin peptide, T(2), on the fluorescence intensity of pyrene-labeled F-actin. Pyrene-labeled F-actin (3 µM) in 20 mM Tris-HCl (pH 7.4) containing 100 mM KCl and 0.1 mM CaCl(2) was preincubated for 1 h at 25 °C. Fluorescence intensity (excited at 365 nm, emitted at 407 nm) was measured 15 min after the addition of T(2). The values are the mean of four determinations with S.D. B, effect of T(2) on binding of calponin or caldesmon to F-actin. T(2) (0.3 mM) or T(1) (0.3 mM) was added to F-actin (3 µM) in 20 mM Tris-HCl (pH 7.5) containing 30 mM KCl, 2 mM MgCl(2), 1 mM ATP, 1 mM dithiothreitol, and 0.2 mM CaCl(2), and the reaction mixture was incubated for 30 min at 25 °C following the addition of either calponin (1.5 µM) or caldesmon (1.5 µM). Subsequent analysis for an actin cosedimentation assay was performed as described under ``Experimental Procedures.'' Each bar represents the mean of at least four experiments, and each verticalbarabove indicates S.D. The squarebracket indicates values that are significantly different (p < 0.01; Student's t test). C, effects of T(2) peptide on actin-activated Mg-ATPase activity. The conditions for ATPase assay were as described under ``Experimental Procedures.'' Actin (2-8 µM) was added in the presence (bullet) or absence () of 0.3 mM T(2) peptide. The rates of myosin Mg-ATPase activity are given as the mean of triplicate measurements in the same assay conditions. D, effects of T(2) and T(1) peptides on actin-activated Mg-ATPase activity. Conditions for the ATPase assay were as described under ``Experimental Procedures.''



Calponin and caldesmon simultaneously bind to F-actin, but calponin uses both specific and common binding sites(22, 23) . The effect of T(2) peptide on the binding of calponin and caldesmon to F-actin was investigated using the cosedimentation method. T(2) peptide (0.3 mM) inhibited calponin binding to F-actin compared with T(1) peptide (GASQAGMTAPGTKR). Moreover, T(2) peptide had no effect on caldesmon binding to F-actin (Fig. 2B), indicating that T(2) peptide interacts with F-actin at its specific binding sites. On the other hand, T(2) peptide had little effect on actin-activated Mg-ATPase activity at the concentration of 0.3 mM (Fig. 2, C and D).

Effect of T(2) Peptide on Smooth Muscle Contraction

To investigate the role of endogenous calponin in smooth muscle contraction, we studied the effect of T(1) and T(2) peptides on Ca-induced contraction in permeabilized smooth muscle. T(2) (0.03-0.3 mM) concentration-dependently enhanced the contraction induced by 0.3 µM Ca, the maximum enhancement induced by 0.3 mM T(2) being to 1.5 ± 0.2 times control (n = 4, Fig. 1B). In contrast, T(1) (0.3 mM) had almost no effect on the Ca-induced contraction (1.2 ± 0.2 times that induced by 0.3 µM Ca alone, n = 9). Furthermore, T(2) (0.3 mM) totally prevented the inhibitory action of 3 µM calponin (Fig. 1C). These results could be interpreted to indicate that, in permeabilized vascular smooth muscle, T(2) enhances Ca-induced contraction through an inhibition of the action of endogenous calponin.

Effect of T(2) Peptide on CaSensitivity of Contraction and Myosin Light Chain Phosphorylation

To study whether or not such an inhibition of endogenous calponin in smooth muscle cells is sufficient to produce contraction without myosin light chain phosphorylation, the effect of T(2) peptide on contraction and myosin light chain phosphorylation was observed in Ca-free solution containing 4 mM EGTA. Under these conditions, T(2) (0.3 mM) evoked neither a contraction (tension = 0.02 ± 0.02 times the maximum Ca-induced contraction, n = 4) nor an increase in myosin light chain phosphorylation (0.05 ± 0.02 mol of PO(4)/mol of myosin light chain in control and 0.04 ± 0.02 mol of PO(4)/mol of myosin light chain in the presence of T(2), n = 5).

ITP is a substrate for myosin ATPase but not for myosin light chain kinase(24) . In a solution containing 5 mM Mg-ITP (with no ATP), T(2) (0.3 mM) did not produce contraction in Ca-free solution containing 4 mM EGTA (tension = 0.02 ± 0.02 times the maximum Ca contraction in ATP-containing solution, n = 4). A lack of action of T(2) on smooth muscle contraction was also found when this peptide (0.3 mM) was applied in Mg-ITP solution containing 10 µM Ca (tension = 0.03 ± 0.02 times the maximum Ca response in ATP-containing solution, n = 4). These results suggest that T(2) may enhance the contraction induced by a given level of myosin light chain phosphorylation.

To test this hypothesis, the relationship between Ca and tension and that between Ca and myosin light chain phosphorylation was determined in the presence and absence of T(2), and the effects were compared with those obtained with calmodulin (Fig. 3). Calmodulin (6 µM) enhanced both the contraction and myosin light chain phosphorylation induced by submaximal concentrations of Ca without any change in the maximum responses induced by 10 µM Ca. The enhancing action of calmodulin was apparent at concentrations over 0.3 µM Ca (Fig. 3). The Hill coefficient and ED value for Ca in producing contraction were 1.7 ± 0.1 and 0.6 ± 0.1 µM, respectively, in control (n = 4) and 2.4 ± 0.5 (p < 0.05) and 0.31 ± 0.02 µM (p < 0.05), respectively, in the presence of calmodulin (n = 4). In contrast, 0.3 mM T(2) enhanced the Ca-induced contractions without a corresponding increase in the level of myosin light chain phosphorylation; this action was apparent on the contractions induced by Ca at concentrations over 0.1 µM. T(2) had no effect on either the maximum contraction or myosin light chain phosphorylation induced by 10 µM Ca. The Hill coefficient and ED value for Ca in producing contraction in the presence of T(2) (n = 4) were 1.5 ± 0.4 (p < 0.05 against control) and 0.4 ± 0.1 µM (p < 0.05), respectively. These results indicate that T(2) peptide enhances the contraction without a change in the level of myosin light chain phosphorylation at a given concentration of Ca (0.1-1.0 µM).


Figure 3: Effects of T(2) peptide and calmodulin on the relationship between Ca and tension (A) and on that between Ca and 20-kDa myosin light chain (MLC) phosphorylation (B) in beta-escin-permeabilized vascular smooth muscle. A, increasing concentrations of Ca (0.03-10 µM) were cumulatively applied from low to high in Ca-free solution containing 4 mM EGTA. B, measurement of phosphorylation of MLC was performed as described under ``Experimental Procedures.'' The phosphorylation of MLC is expressed in mol of PO(4)/mol of MLC. In A and B: bullet, control; , 6 µM calmodulin; , 0.3 mM T(2). Results shown are each the mean of 4-5 observations with S.D. The slope of the concentration-response relationship (of Ca against tension and against MLC phosphorylation) is shown by the Hill coefficient (N) and midpoint position (pK = (-log K), where K is the dissociation constant). These parameters were obtained by fitting the data points for each curve to the following equation by a nonlinear least squares method. F/F(0) = (C/K)/(1 + (C/K)), where C represents the concentration of Ca. In A, F is the amplitude of contraction at any given concentration of Ca, and F(0) is the maximum response evoked by 10 µM Ca expressed as a relative tension of 1.0. *, values that are significantly different with a corresponding control (p < 0.05).



Effect of T(2) Peptide on the Relationship between Contraction and Myosin Light Chain Phosphorylation

Fig. 4shows the effects of calmodulin (6 µM) and T(2) (0.3 or 1 mM) on the relationship between tension and myosin light chain phosphorylation in the presence of various concentrations of Ca. Calmodulin did not seem to modify the relationship between tension and myosin light chain phosphorylation. In contrast, T(2) shifted the tension-myosin light chain phosphorylation relationship to the left. The myosin light chain phosphorylation required for the half-maximum tension in the presence of T(2) (0.13 ± 0.02 mol of PO(4)/mol of myosin light chain) was significantly less than that in the absence of T(2) (0.26 ± 0.02 mol of PO(4)/mol of myosin light chain, p < 0.05). These results suggest that, unlike calmodulin, T(2) peptide enhances the contraction evoked at a given level of myosin light chain phosphorylation over the physiological range of Ca concentrations (0.1-1.0 µM).


Figure 4: Effect of calmodulin and T(2) peptide on the relationship between tension and MLC phosphorylation in beta-escin-permeabilized smooth muscle. The curves were obtained by fitting the data points to the following equation by a nonlinear least squares method: F/F(0) = (C/K)/(1+ (C/K)). N, Hill coefficient; K, dissociation constant. C and F/F(0) represent phosphorylation of MLC (mol of PO(4)/mol of MLC) and relative tension, respectively. Results shown are each the mean of four observations with S.D. The fitted values of N and K were, respectively, 2.4 and 0.26 mol of PO(4)/mol of MLC in control (r = 0.996) and 1.6 and 0.13 mol of PO(4)/mol of MLC in the presence of T(2) (r = 0.992). bullet, control (n = 4); , 6 µM calmodulin (n = 4); , 0.3 mM T(2) (n = 4); up triangle, filled, 1 mM T(2) (n = 4); black square, 1 mM T(2) with 6 µM calmodulin (n = 4).



It has been suggested that the phosphorylation of myosin light chain does not entirely depend on the cellular concentration of Ca. In permeabilized smooth muscles, agonist with GTP and GTPS alone both enhance, at a given concentration of Ca, the phosphorylation of myosin light chain and the tension produced, possibly through an inhibition of myosin phosphatase (6, 25) . Phosphorylation of myosin light chain kinase by Ca-calmodulin-dependent protein kinase II causes an increase in the concentration of Ca-calmodulin required for the activation of myosin light chain kinase(26, 27) . These two processes increase and decrease, respectively, the Ca-sensitivity of myosin light chain phosphorylation without a change in the relationship between tension and myosin light chain phosphorylation. However, our study has raised the interesting point that, in contrast, T(2) peptide did change the tension-myosin light chain phosphorylation relationship.

In conclusion, under physiological conditions, calponin acts to lower the myofilament Ca sensitivity in smooth muscle contraction at a given level of myosin light chain phosphorylation.


FOOTNOTES

*
This work was partly supported by a grant-in-aid from the Ministry of Education of Japan and a research grant for the study of cardiovascular diseases from the Ministry of Health and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan. Tel.: 81-52-853-8149; Fax: 81-52-851-9106.

(^1)
The abbreviations used are: PIPES, 1,4-piperazinediethanesulfonic acid; GTPS, guanosine 5`-3-O-(thio)triphosphate; MLC, myosin light chain.


ACKNOWLEDGEMENTS

We thank Dr. R. J. Timms for the language editing and Dr. H. Suzuki for useful comments.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.