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(Circulation Research. 2001;88:1283.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Distinct Pathways of Ca²⁺ Sensitization in Porcine Coronary Artery

Effects of Rho-Related Kinase and Protein Kinase C Inhibition on Force and Intracellular Ca²⁺

Koji Nobe, Richard J. Paul

From the Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio.

Correspondence to Dr Richard Paul, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576. E-mail richard.paul{at}uc.edu

Abstract

Abstract—Alterations of the Ca²⁺ sensitivity of contraction have been reported for porcine coronary artery, but the mechanisms are not clearly understood. We investigated the mechanism(s) of Ca²⁺ sensitization in response to the thromboxane A₂ analogue (U46619). Our hypothesis is that different mechanisms of Ca²⁺ sensitization could be distinguished by their distinct time courses. Therefore, we measured the time course of [Ca²⁺]_i and isometric force simultaneously in an intact artery after a single addition of U46619. The initial transient phase was associated with Ca²⁺ release from the sarcoplasmic reticulum, whereas the maintained phase was associated with Ca²⁺ influx. Two distinct types of Ca²⁺ sensitization characterized these phases with either protein kinase C (PKC)-mediated or Rho-kinase–mediated mechanisms. Their effects were quite distinct on the basis of the time courses over which the sensitization was effective. PKC inhibition (1 µmol/L calphostin C) had a much greater effect in the initial phase, diminishing the size of the transient and prolonging the rise in force and the decline in [Ca²⁺]_i. There were limited effects on the sustained force. Rho-kinase inhibition (10 µmol/L Y27632), in contrast, nearly abolished the sustained force but had a lesser effect on the transient phase. Neither inhibitor had any effect on the force versus [Ca²⁺]_i relations for KCl contractures. Our evidence suggests that both PKC-mediated and Rho-kinase–mediated Ca²⁺ sensitizations are present in coronary arteries, but the latter is dominant in thromboxane A₂ receptor–mediated contraction.

Key Words: coronary arteries • Ca²⁺ sensitization • protein kinase C • Rho-kinase • U46619

Vascular smooth muscle contractility is dependent not only on intracellular Ca²⁺ concentration ([Ca²⁺]_i) but also on the Ca²⁺ sensitivity of the contractile apparatus.¹ Agonist-mediated activation is generally associated with a higher Ca²⁺ sensitivity (greater maintained isometric force per unit increase in [Ca²⁺]_i) than that observed for activation via depolarization.² Further proof of the existence of Ca²⁺ sensitivity in smooth muscle contraction was obtained in studies on permeabilized smooth muscle, in which Ca²⁺ can be maintained at fixed levels. In the presence of GTP, agonists can enhance force at constant [Ca²⁺]_i.^{3 4} Thus, our understanding of the mechanisms of receptor-coupled activation of smooth muscle contraction now include a significant component attributable to Ca²⁺ sensitization.⁵

The mechanisms proposed for Ca²⁺ sensitization generally fall into two classes. One class alters the relation between myosin regulatory light chain phosphorylation and [Ca²⁺]_i, involving the myosin light chain kinase or phosphatase cascades. Myosin regulatory light chain phosphorylation has long been recognized as a major factor regulating smooth muscle force.⁶ Another pathway of sensitization involves alteration of the Ca²⁺ affinity of other proposed regulatory proteins, such as caldesmon or calponin. These thin filament–associated proteins are generally postulated to inhibit the actin-myosin interaction, and this inhibition is proposed to be relieved by Ca²⁺.⁷

One of the initial hypotheses proposed for Ca²⁺ sensitization involved protein kinase C (PKC).⁸ Evidence includes the observation that direct activation of the PKC by phorbol ester treatment increases force⁹ and translocation of calponin.¹⁰ More recently, much attention has been given to the role of the small G protein Rho in the Ca²⁺ sensitivity of smooth muscle contraction. Activated Rho induces phosphorylation and inactivation of myosin light chain phosphatase mediated by Rho-related kinase (Rho-kinase).¹¹ Inhibition of phosphatase has long been known to elicit contraction in permeabilized smooth muscle under conditions in which Ca²⁺ is maintained below the contraction threshold.¹²

Agonist-induced coronary artery responses have important implications for cardiac function and cardiovascular disease. Ca²⁺ sensitization based on in vivo measurements is particularly striking in porcine coronary arteries with the thromboxane A₂ (TXA₂) analogue (U46619)^{2 13} ; thus, its mechanism is of considerable interest. It is possible that multiple Ca²⁺-sensitization mechanisms are in play in this vessel. Sato et al¹⁴ have reported that endothelin-1 and carbachol enhance force at constant [Ca²⁺]_i in ß-escin–permeabilized porcine coronary arteries. They reported that the Rho-mediated Ca²⁺ sensitization might be involved in the endothelin-1–induced contraction but, surprisingly, was not involved in the responses to carbachol.

The existence of multiple mechanisms of Ca²⁺ sensitization may be differentiated in view of differences in their time courses. It has long been postulated that the mechanisms underlying the initial phase of tension development may be different from that of tension maintenance.^{15 16}

The aim of the present study was to investigate the mechanism(s) of Ca²⁺ sensitization in U46619-induced contraction in porcine coronary artery. Our hypothesis is that different mechanisms of Ca²⁺ sensitization could be distinguished by their potentially distinct time courses. The evidence for the operation of these pathways of Ca²⁺ sensitization in vivo and for their clinical significance has been inferred largely from studies based on contractility alone.^{17 18} Therefore, we measured the time course of [Ca²⁺]_i and isometric force simultaneously in an intact artery in response to a single addition of U46619. We report that U46619-induced contraction is characterized by two distinct phases of Ca²⁺ sensitization, having either PKC-mediated or Rho-kinase–mediated mechanisms, with the latter dominant in the steady state. This is the first direct evidence in intact coronary arteries for an increase in Ca²⁺ sensitivity associated with Rho-kinase.

Materials and Methods

Materials
Y27632 was a gift from the Welfide Corp (Osaka, Japan). Fura 2 was purchased from Molecular Probes. All other reagents were of the highest purity and were purchased from Sigma Chemical Co. U46619 was dissolved in ethanol, and calphostin C, phorbol 12-myristate 13-acetate (PMA), calyculin A, and SQ29548 were dissolved in dimethyl sulfoxide; no effects of vehicle were noted if total vehicle was 0.03%. Physiological salt solution (PSS) contained 122 mmol/L NaCl, 4.73 mmol/L KCl, 15.0 mmol/L NaHCO₃, 1.19 mmol/L MgCl₂, 0.02 mmol/L EDTA, 1.19 mmol/L KH₂PO₄, 2.5 mmol/L CaCl₂, and 11.1 mmol/L glucose aerated with 95% O₂/5% CO₂ for a pH of 7.4 at 37°C. MOPS-buffered PSS (MOPS-PSS) contained 140 mmol/L NaCl, 4.70 mmol/L KCl, 1.20 mmol/L NaH₂PO₄, 20.0 mmol/L MOPS, 0.02 mmol/L EDTA, 1.2 mmol/L MgSO₄, 2.5 mmol/L CaCl₂, and 11.1 mmol/L glucose with a pH of 7.4 by NaOH at 37°C.

Preparation of Coronary Artery Smooth Muscle Rings
Porcine hearts obtained shortly after slaughter were rinsed of blood and placed in cold (4°C) PSS. The distal portions of the left anterior descending coronary artery were dissected and placed in ice-cold PSS. Arteries were then cleaned of fat and connective tissue and cut into 5-mm segments. The arterial wall thickness was between 300 and 500 µmol/L. The segments were everted to expose the luminal side for fluorescence measurements and deendothelialized by rolling gently on filter paper.

Measurement of [Ca²⁺]_i and Isometric Force
[Ca²⁺]_i was measured with the fluorescent dye fura 2-AM as previously described.¹³ The arterial rings were mounted isometrically on a stainless-steel bracket. Arteries were then incubated for 3 hours at 20°C in a well-stirred MOPS-PSS containing 12.5 µmol/L fura 2-AM, 0.005% pluronic F-127, and 2 mg/mL BSA. After incubation, the tissues were rinsed in PSS for 20 minutes to remove any residual dye. Arterial segments were attached to a movable post connected to a Kent force transducer. Resting tension was adjusted to 30 to 40 mN. This value was chosen on the basis of prior experiments to set a tissue length in the optimal range for maximum tension development. Isometric force was expressed as mN/mm²; cross-sectional area was approximated as 2xwet weight/circumference.

The mounted artery was placed into a cuvette, and this assembly was placed a water-jacketed holder maintained at 37°C in a PTI Delta Scan-1 (Photon Technology International) spectrofluorometer. The cuvette was aligned such that the artery was configured for front face measurements. Fluorescence was excited at 340 and 380 nm, and emission was measured at 510 nm. The fluorescence intensity at 340-nm excitation was divided by that measured at 380 nm, and this ratio (R_340/380) was used in calibration of absolute [Ca²⁺]_i, according to Grynkiewicz et al.¹⁹ Ca²⁺ calibrations are dependent on a number of assumptions, including the value for the K_d (224 nmol/L). Although this is always a factor in interpretation of fura 2 data, the relative values (eg, when [Ca²⁺]_i is expressed in terms of the maximum) are valid. Details of various Ca²⁺ calibrations and assumptions in intact porcine coronary artery have been reported.¹³

Analysis of Data
Values given are mean±SEM; n values are the number of hearts from which arteries were taken. Significance was determined by standard ANOVA with the Bonferroni method used for multiple comparisons.

Results

U46619-Induced [Ca²⁺]_i and Isometric Force Responses in Coronary Artery
Resting [Ca²⁺]_i and force averaged 121.0±5.5 nmol/L and 2.00±0.12 mN/mm², respectively (n=29). U46619 (100 nmol/L) increased both [Ca²⁺]_i and force (Figure 1A). [Ca²⁺]_i increased rapidly, and maximal values were attained within 1 minute, averaging 1115.7±10.3 nmol/L (n=7). [Ca²⁺]_i then decreased, almost as rapidly, to a low but suprabasal steady-state level, averaging 201.6±13.5 nmol/L (n=7). Force developed with a much slower time course. The maximal response (14.93±0.48 mN/mm², n=7) occurred within 5 minutes of stimulation, and >90% of the maximal response was maintained for at least 15 minutes. When [Ca²⁺]_i reached its peak level, force was only 19.7% (4.86±0.55 mN/mm², n=7) of maximum. The relation between the peak responses of [Ca²⁺]_i and force for U46619 (1 to 300 nmol/L) is shown in Figure 1B. Values of EC₅₀ for [Ca²⁺]_i and force were 15.48 and 19.99 nmol/L, respectively. The TXA₂ receptor antagonist SQ29548 (1 µmol/L, 10-minute pretreatment) abolished the responses to U46619 without affecting baseline (Figure 1B, inset).

View larger version (26K): [in this window] [in a new window]   Figure 1. U46619-induced changes in [Ca2+]i and isometric force. Fura 2–loaded tissue was stimulated with U46619 at 37°C for 3 minutes. Fluorescence was excited at 340 and 380 nm, and emission intensities at 510 nm were recorded. A, Typical recording of simultaneous measurements of isometric force () and [Ca2+]i (•), calculated as described in Materials and Methods. B, Concentration-maximal response relations. Inset, Tissues were preincubated in the presence or absence of the TXA2 receptor antagonist (1 µmol/L SQ29548 [SQ]) for 10 minutes, and then 100 nmol/L U46619 (U4) was added. [Ca2+]i (dark bars) and isometric force (light bars) are presented as a percentage of the U46619-induced responses (% of max responses). *P<0.05 vs U46619 responses. C, Data from panel B replotted as isometric force vs [Ca2+]i relations. In the transient phase of the U46619-induced responses, the relation between the maximal [Ca2+]i value (a) and the isometric force developed at that time (b) as shown in panel A is denoted as the a-b relation (diamonds). The relation between the maximal sustained force (c) and its corresponding [Ca2+]i value (d) is designated the c-d relation (squares). The relation between the maximal isometric force (c) and the max [Ca2+]i value (a) is designated as the a-c relation (circles).

Force versus [Ca²⁺]_i relations were analyzed (Figure 1C) by using the 4 parameters indicated in Figure 1A. Point a is the maximal [Ca²⁺]_i response corresponding to a particular concentration of U46619. Point b is the value of force attained at the time of the peak [Ca²⁺]_i. Point c is the maximal force, and point d is the [Ca²⁺]_i level occurring at that time. The transient phase of the force versus [Ca²⁺]_i relation is delineated by the a-b relation; the sustained phase, by the c-d relation; and the relation between maximal increase in force and that in [Ca²⁺]_i, by the a-c relation. Strong significant correlations between force and [Ca²⁺]_i were observed for the a-b, c-d, and a-c relations; r² values were 0.908, 0.726, and 0.998, respectively. The Ca²⁺ sensitivity of the sustained phase (the slope of the c-d relation) was markedly greater than that of the transient phase (the slope of the a-b relation). The slope of the relation between the maximal force and [Ca²⁺]_i responses (a-c) was intermediate.

Source of Ca²⁺ Induced by U46619 Stimulation
To elucidate the source of the [Ca²⁺]_i increase for U46619-induced responses, we used a pharmacological approach inhibiting the sarcoplasmic reticulum Ca²⁺-ATPase with cyclopiazonic acid (CPA) and the plasmalemmal L-type Ca²⁺ channels with nifedipine (Figure 2). CPA (10 µmol/L) induced transient increases in [Ca²⁺]_i and force. The peak levels were 371.0±44.1 nmol/L and 3.72±0.74 mN/mm², respectively (n=5). These transients returned to baseline within 10 minutes. In the continued presence of CPA, the [Ca²⁺]_i transient to U46619 was significantly inhibited to 36.4±1.8% of control (n=5). The force transient was also slightly but not significantly reduced (15.1±1.8% to 6.4±5.9%, n=5). The responses in the sustained phase were not significantly inhibited; >75% of control responses remained in the presence of CPA.

View larger version (38K): [in this window] [in a new window]   Figure 2. Characterization of the Ca2+ sources in U46619-induced responses: effects of CPA (A) or nifedipine (B) on the U46619-induced [Ca2+]i and force responses. Coronary arteries were preincubated in the presence or absence of the sarcoplasmic reticulum Ca2+-ATPase inhibitor (10 µmol/L CPA) (A) or a Ca2+ channel blocker (10 µmol/L nifedipine) (B) for 10 minutes, and then 100 nmol/L U46619 was added. [Ca2+]i (darkly hatched bars) and isometric force (lightly hatched bars) were measured simultaneously as described in Materials and Methods. Values are expressed in terms of the maximal [Ca2+]i response, which occurred in the transient phases (Trans), and in terms of the maximal force, which occurred in the sustained phases (Sust). Maximal responses for CPA or nifedipine alone are designated (Max). Data, as a percentage of each maximal response, are given as mean±SEM (n5). *P<0.05 vs U46619 responses.

In contrast (Figure 2B), preincubation with nifedipine (10 µmol/L) for 10 minutes reduced the sustained force (53.1±6.3% of control, n=6). Nifedipine caused a small decrease in the transient [Ca²⁺]_i (100% to 91.1%, P<0.02). The sustained [Ca²⁺]_i was substantially reduced (19.6% to 5.6%), but the precision at these low levels of [Ca²⁺]_i was such that statistical significance was not achieved (P=0.12). Our results are consistent with the classic picture for smooth muscle in which intracellular Ca²⁺ stores underlie the transient responses and in which transmembrane Ca²⁺ influx underlies the sustained component of force.

PKC and U46619 Responses
Activation of PKC is associated with receptor-mediated stimulation in many cell types mediated by diacylglycerol formation. We investigated the role of PKC in U46619-induced responses by using the PKC inhibitor calphostin C. Figure 3 shows typical [Ca²⁺]_i and force responses (Figure 3A) and summarized data (Figure 3B). Calphostin C (1 µmol/L) had no effects on either resting [Ca²⁺]_i or force. Neither the [Ca²⁺]_i transient (1089.0±21.3 nmol/L, n=6) nor the sustained levels (216.8±56.2 nmol/L, n=6) in response to U46619 (100 nmol/L) were altered by calphostin C. In contrast, force development differed from control. The response was biphasic with a small peak (2.39±0.17 mN/mm², n=6) shortly after stimulation in most cases. This initial peak in force coincided with the [Ca²⁺]_i peak. After the small peak, force again increased to a sustained maximal level (14.95±0.18 mN/mm², n=6). This maximal level was not different from control, but the half-time for force development was significantly greater than control (97.8±3.2 versus 39.2±7.3 seconds, respectively; n=4). Calphostin C also prolonged the relaxation from the peak [Ca²⁺]_i; the half-time was increased to 61.8 seconds compared with a control value of 25.8 seconds. Calphostin C affected the relation between force and [Ca²⁺]_i during the transient phase (a-b), but the relations in both the sustained phase (c-d relation) and maximal responses (a-c relation) were unaffected (Figure 3C).

View larger version (49K): [in this window] [in a new window]   Figure 3. Effects of calphostin C on the U46619-induced [Ca2+]i and isometric force responses in coronary arteries. Coronary arteries were preincubated in the presence or absence of the PKC inhibitor (1 µmol/L calphostin C) for 10 minutes, and then U46619 was added. A, Typical responses of [Ca2+]i (•) and isometric force (). B, Summarized data for [Ca2+]i (darkly hatched bars) and force (lightly hatched bars). Values are expressed in terms of the maximal [Ca2+]i and force responses. Maximal responses to calphostin C alone are indicated (Max). Data are given as mean±SEM (n6). C, U46619 concentration (1 to 300 nmol/L) vs response data plotted as isometric force versus [Ca2+]i relations in the presence (closed symbols, solid lines) or absence (open symbols, broken lines) of calphostin C. The transient (a-b), maximum (a-c), and sustained (c-d) relations are as defined in Figure 1.

To further investigate the role of PKC, we used the PKC activator PMA. PMA (3 µmol/L) induced a transient increase in isometric force (Figure 4). The maximal value (10.56±0.95 mN/mm², n=8) was detected within 2 minutes. After attaining the peak, force slowly decreased. [Ca²⁺]_i was not significantly changed from baseline, averaging <1.5% of the U46619 response. Based on standard ANOVA for n=8, changes in [Ca²⁺]_i of 5.0 nmol/L, or 0.6% of the U46619 peak, would be detectable. In control experiments, after a similar preincubation with calphostin C, the responses to PMA were measured. As shown in Figure 4B, the isometric force responses were blocked by calphostin C, with no change in [Ca²⁺]_i.

View larger version (45K): [in this window] [in a new window]   Figure 4. PMA-induced changes in [Ca2+]i and isometric force in coronary arteries. A, Typical responses of [Ca2+]i (•), calculated as described in Materials and Methods, and force () to 100 nmol/L U46619 or 3 µmol/L PMA. B, Average (n=5) values for [Ca2+]i (darkly hatched bars) and force (lightly hatched bars) after treatment with 100 nmol/L U46619, 3 µmol/L PMA, or 1 µmol/L calphostin C, with subsequent addition of 3 µmol/L PMA from experiments as shown in panel A. Values are expressed in terms of the maximal [Ca2+]i and force responses.

Rho-Kinase and U46619 Responses
We investigated the role of Rho-kinase by using the inhibitor Y27632. Y27632 (1 µmol/L) decreased the force baseline; typical responses are shown in Figure 5A, and the summarized data are in the inset. After 10 minutes, the resting force decreased from 4.41±0.65 to 0.47±0.14 mN/mm² (n=5). No effects on [Ca²⁺]_i were observed.

View larger version (47K): [in this window] [in a new window]   Figure 5. Effects of Y27632 on U46619-induced [Ca2+]i and isometric force responses in porcine coronary artery. Arteries were preincubated in the presence or absence of the Rho-kinase inhibitor (10 µmol/L Y27632) for 10 minutes, and then U46619 was added. A, Typical responses of [Ca2+]i (•) and force (). Inset, Averaged data (mean±SEM, n=5) for [Ca2+]i (darkly hatched bars) and force (lightly hatched bars). Values are expressed in terms of the maximal [Ca2+]i or force responses. Maximal responses to Y27632 alone are indicated (Max). Sus indicates sustained; Trns, transient. *P<0.05 and #P<0.05 vs U46619 responses and nontreated resting levels, respectively. B, U46619 (1 to 300 nmol/L) response data plotted as force vs [Ca2+]i relations in the presence (closed symbols, solid lines) or absence (open symbols, broken lines) of 10 µmol/L Y27632 (Y). The transient (a-b), maximum (a-c), and sustained (c-d) relations are as defined in Figure 1. C, Expansion of panel B to highlight relations at low [Ca2+]i levels. Data showing the effects of 10 µmol/L Y27632 at high concentrations of U46619 (1 and 3 µmol/L) are also plotted.

Rho-kinase inhibition nearly abolished the contraction. U46619 (100 nmol/L) elicited only small transient (15.6%) and sustained (12.6%) increases in force in the presence of Y27632 (Figure 5A, inset). [Ca²⁺]_i increased transiently, but the maximal level (771.5±31.3 nmol/L, n=5) was decreased compared with control (1169.7±16.8 nmol/L, n=5). The sustained phase of the [Ca²⁺]_i increase was not different from control. Y27632 did not have a major effect on the relation between force and [Ca²⁺]_i for the transients (a-b), but the sustained phase (c-d) and maximal responses (a-c relation) were markedly depressed, largely reflecting the inhibition of force (Figure 5B). This can be better seen in the expanded axes (Figure 5C). To further delineate the effects of Y27632 on force and [Ca²⁺]_i, we added data points at high concentrations of U46619 (1 to 3 µmol/L). These concentrations induce larger [Ca²⁺]_i increases, but high doses are difficult to reverse on washout. The sustained phases of the contraction in response to 1 µmol/L and 3 µmol/L U46619 were inhibited by 10 µmol/L Y27632, and these points fitted well within the c-d relation, derived from lower concentrations (1 to 300 nmol/L). Our major point is that Y27632 inhibits the steady-state force with little change in [Ca²⁺]_i.

We also investigated the phosphatase inhibitor calyculin A. Calyculin A (1 µmol/L) increased force with no change in [Ca²⁺]_i (data not shown). This force developed slowly, and the maximal value was detected within 2.5 minutes after treatment (7.91±0.98 mN/mm², n=5). Over 90% of the maximal response was maintained after 10 minutes of the stimulation.

KCl-Induced [Ca²⁺]_i and Isometric Force
To compare with receptor-mediated activation, activation by depolarization with KCl, attributed to Ca²⁺ influx through L-type Ca²⁺ channels, was also investigated. KCl (80 mmol/L) quickly increased both [Ca²⁺]_i and force (Figure 6A). Maximal [Ca²⁺]_i values (1129.4±15.0 nmol/L, n=7) were detected within 1 minute. After a peak was attained, [Ca²⁺]_i slightly decreased but was still maintained at >75% of the maximum after 5 minutes. Similarly, the maximal increase in force was detected within 2 minutes (15.04±0.27 mN/mm², n=7), and >90% of the maximal response was maintained at 5 minutes. At the peak [Ca²⁺]_i, the corresponding force was 82.2±4.6% of the maximal force (12.97±0.61 mN/mm², n=7). Moreover, when force attained its maximum, [Ca²⁺]_i was 88.6±3.1% (1018.4±34.2 nmol/L, n=7) of its peak value. The maximal responses of [Ca²⁺]_i and force were a function of the KCl concentration (Figure 6B). Significant increases were detected at 30 mmol/L KCl, and the maximal responses were attained at 80 mmol/L. EC₅₀ values of [Ca²⁺]_i and force were 31.7 and 39.7 mmol/L, respectively. The relations between force and [Ca²⁺]_i were analyzed (Figure 7C) by using the 4 parameters previously described for U46619 contractions in Figure 1A. They showed strong correlations between force and [Ca²⁺]_i; for a-b, c-d, and a-c relations, r² values were 0.992, 0.997, and 0.986, respectively. Moreover, there were no differences in the relations among these phases.

View larger version (44K): [in this window] [in a new window]   Figure 6. KCl-induced changes in [Ca2+]i and isometric force development. A, [Ca2+]i (•), calculated as described in Materials and Methods, and force () from a typical recording of arteries stimulated with 80 mmol/L KCl. B, KCl concentration–maximal response relations. C and D, KCl (0 to 80 mmol/L) data plotted as force vs [Ca2+]i relations in the presence (closed symbols, solid lines) or absence (open symbols, broken lines) of 1 µmol/L calphostin (C) or 10 µmol/L Y27632 (D). The transient (a-b), maximum (a-c), and sustained (c-d) relations are as defined in Figure 1.

View larger version (25K): [in this window] [in a new window]   Figure 7. Effects of calphostin C (Cal-C) or Y27632 on the sustained phase of [Ca2+]i and isometric force for U46619 (A) or KCl (B) stimulation in porcine coronary arteries. Arteries were preincubated in the presence or absence (circles) of the PKC inhibitor (1 µmol/L calphostin C, triangles) or the Rho-kinase inhibitor (10 µmol/L Y27632, squares) for 10 minutes, and then 1 to 300 nmol/L U46619 (A) or 10 to 100 mmol/L KCl (B) was cumulatively added. Data are plotted as force vs [Ca2+]i relations in the sustained phase (c-d relation), as shown in Figure 6A. Inset A, Expanded axes to highlight relations in lower [Ca2+]i ranges. CC indicates calphostin C; Cont, control. Data are given as mean±SEM (n4).

Effects of PKC and Rho-Kinase Inhibitors on KCl-Induced Responses
Pretreatment with calphostin C (1 µmol/L) for 10 minutes did not affect either resting or KCl-induced [Ca²⁺]_i and force responses (Figure 6C). Similarly, little effects of the Rho-kinase inhibitor Y27632 were observed. Baseline values were slightly decreased, and the KCl-induced increases of [Ca²⁺]_i and force were not affected; [Ca²⁺]_i versus isometric force relations were unchanged (Figure 6D). Figure 7 shows a graphic comparison of the effects of PKC or Rho-kinase inhibition on the relations between force and [Ca²⁺]_i for U46619 (Figure 7A) and KCl stimulation (Figure 7B) in the sustained phase (c-d). Inhibition of Rho-kinase can be seen as the major player for receptor-mediated but not for KCl stimulation.

Discussion

U46619 increases coronary artery [Ca²⁺]_i and force (Figure 1A) largely via activation of TXA₂ receptors, inasmuch as its effects were blocked by the antagonist SQ29548 (Figure 1B). In general, the Ca²⁺ sensitization–associated U46619 is often greater than other agonists in other vascular tissues. Our results indicate that the responses to U46619 involve both PKC-mediated and Rho-kinase–mediated changes in Ca²⁺ sensitivity. However, their effects, based on the time courses over which the sensitization was effective, were quite distinct. PKC inhibition had a much greater effect on the initial phase of contraction. Its major effects were to diminish the size of the initial transient and to prolong the duration of force development and the decline in [Ca²⁺]_i. However, there were limited effects on the magnitude of the sustained force. Rho-kinase inhibition on the other hand, nearly abolished the sustained force but had a lesser effect on the transient phase. Although Ca²⁺ sensitization due to activation of the Rho-kinase pathway has been implicated by use of permeabilized preparations¹⁴ or contractility measurements,¹⁸ it is possible that Rho-kinase inhibition also modifies [Ca²⁺]_i. Our data provide the first evidence that Rho-kinase indeed changes Ca²⁺ sensitivity in vivo in the intact coronary artery.

These phases differed not only in their sensitivity to kinase inhibitors but also in their source of Ca²⁺ for the increase in [Ca²⁺]_i. Because the transient increase in [Ca²⁺]_i to U46619 was reduced by CPA to <40% of control, it appears that Ca²⁺ was provided largely by the sarcoplasmic reticulum stores (Figure 2A). The sustained phases of the [Ca²⁺]_i increase and isometric force were reduced by the Ca²⁺ channel inhibitor nifedipine (Figure 2B) or Ca²⁺-free PSS (data not shown), indicating a dependence of the sustained phase on extracellular Ca²⁺ influx. These results suggested the possibility of different isometric force versus [Ca²⁺]_i relations in each phase of the U46619 stimulation.

In fact, whether [Ca²⁺]_i is elevated in the steady state has important ramifications. In 10% of responses, [Ca²⁺]_i appeared to return to baseline. But in the majority of cases, [Ca²⁺]_i remained elevated. We analyzed the 17 controls for the experiments shown in Figures 2A, 2B, and 3C. When expressed as a percentage of baseline, [Ca²⁺]_i in the sustained phase averaged 242.8±38.8%. When expressed as a percentage of the maximum [Ca²⁺]_i response to 100 nmol/L U46619, the increase above baseline was 12.6±3.4%. Both were highly statistically significant. A total of 40 control U46619 responses yielded a sustained [Ca²⁺]_i of 202.5±22.2% of baseline and 10.2±2.3% of maximal response. Thus, an elevated [Ca²⁺]_i is associated with the sustained force.

Receptor-mediated activation is associated with the production of inositol triphosphate from phosphatidylinositol biphosphate and with the hydrolysis of phosphatidylinositol biphosphate by phospholipase C, with the important second messenger diacylglycerol. Inhibition of PKC by calphostin C pretreatment caused a significant delay in the force response to U46619 (Figure 3). Because the [Ca²⁺]_i transient was largely unaffected, the suppression of force development suggests the loss of a PKC-mediated Ca²⁺ sensitization. Because the maximal force and increase in [Ca²⁺]_i were not affected in the steady state, either force is saturated or calphostin C inhibition is effective only in the transient phase. The decrease in the slope of the force-[Ca²⁺]_i relation in the presence of calphostin C (Figure 3C) suggests that the latter may be the case. This is further supported by evidence from the experiments in which PKC was directly activated by PMA. A transient increase in isometric force was observed without a statistically significant increase in [Ca²⁺]_i (Figure 4). We cannot rule out changes in [Ca²⁺]_i of <10%, but our point is that with or without a small change in [Ca²⁺]_i, the increase in force in response to PMA is characterized by a very high Ca²⁺ sensitivity.

The sustained phase of the U46619-induced contraction was significantly reduced by nifedipine (Figure 2B). Similarly, pretreatment with 5 mmol/L EGTA and Ca²⁺-free PSS reduced the maintained force to <20% of control (data not shown). Thus, extracellular Ca²⁺ and transmembrane influx are necessary for the maintenance of force. The relation between isometric force and [Ca²⁺]_i indicates a much higher Ca²⁺ sensitivity than in the transient phase of the U46619 response. Because our data suggested that PKC sensitization was not likely a major player in the sustained phase, we investigated the potential role of Rho-kinase, postulated to be involved in Ca²⁺ sensitization.¹¹

The Rho-kinase inhibitor Y27632 was an impressive inhibitor of contractility, nearly completely suppressing the sustained phase of contraction. It also partially reduced the resting levels and the U46619-induced transient phases of both force and [Ca²⁺]_i. The mechanism for the reduction of the transient increase in [Ca²⁺]_i is not known. However, the sustained phase of the increase in [Ca²⁺]_i was not statistically different from that of the control. These effects of Y27632 are consistent with the loss of Rho-kinase–mediated Ca²⁺ sensitivity. Calyculin A, an inhibitor of myosin light chain phosphatase, also induces an increase in force with minimal changes in [Ca²⁺]_i, similar to that previously reported for okadaic acid.²⁰ That phosphatase inhibition can lead to an increase in force without increasing [Ca²⁺]_i supports a mechanism consistent with the hypothesis of Rho-kinase modulation of phosphatase activity and, consequently, contractility.

Our hypothesis for two different pathways modulating Ca²⁺ sensitivity is specific to receptor-mediated activation, inasmuch as it requires G-protein activation of Rho-kinase and diacylglycerol activation of PKC. Moreover, our demonstration of their presence is dependent on a pharmacological approach and limitations of the specificity of agents. To confirm that these sensitization mechanisms are specifically coupled to receptor-mediated stimulation and to control for specificity, we performed similar measurements on KCl-induced responses.

For KCl contractures, the relations between isometric force and [Ca²⁺]_i did not differ between transient and sustained phases (Figure 6). Importantly, the increases in [Ca²⁺]_i and force were not inhibited either by calphostin C or Y27632, nor were any changes in Ca²⁺ sensitivity observed (Figure 6). The Ca²⁺ sensitivity measured for KCl contractures is also of interest compared with that observed for the different phases in receptor-mediated contractions. The Ca²⁺ sensitivity for the sustained phase for U46619 was 10-fold greater than that observed for the responses to KCl. On the other hand, that of the transient PKC-modulated phase was 5-fold less. This largely reflects the much more rapid increase in [Ca²⁺]_i than in force. Some caution must be exerted in interpreting the slope of force versus Ca²⁺ in the transient phase as a Ca²⁺ sensitivity that can be readily compared with that in the steady state. Force development lags that of actomyosin activation because of the presence of any series elasticity. In smooth muscle, the series elastic component and slow contraction velocities can exacerbate the differences between the measured force and the level of activation of the smooth muscle. The latter is what is generally inferred from isometric force measurements in terms of Ca²⁺ sensitivity. However, this inference is valid in the steady state. Another potential caveat to interpretation of transient data are that although isometric force represents a tissue average, [Ca²⁺]_i is dependent on the depth of light penetration and reflected for the fluorometric measurements. Our simultaneous measurements of force and [Ca²⁺]_i for the KCl measurements set limits on these potential artifacts. The Ca²⁺ sensitivity of the steady state was <22% greater than that of the transient phase.

Independent of the exact meaning of Ca²⁺ sensitivity in transient phases, the important point is that the effects of PKC inhibition were prominent only during this initial transient. The transient phase is effective over the first 30 seconds, so there may be sufficient time for its effects on force to be of physiological relevance. Force is not necessarily the only outcome of activation of the PKC pathway; eg, contractile speed may also be affected. Although the physiological significance of the PKC sensitization is not clear, our data show unambiguously that it is present in porcine coronary artery.

In conclusion, our data show that Ca²⁺ sensitivity of smooth muscle demonstrated in permeabilized fibers¹¹ is a major factor in receptor-mediated responses to U46619 in vivo. Moreover, two distinct types of Ca²⁺ sensitization were observed. The transient phase of contraction to U46619 was associated with Ca²⁺ release from the sarcoplasmic reticulum and PKC-mediated Ca²⁺ sensitization. In the sustained phase, Ca²⁺ influx from extracellular space is central and involves Rho-kinase–mediated Ca²⁺ sensitization. Although both pathways have been postulated to play a role in coronary vasospasm,^{17 18} our data indicate that Rho-kinase is the dominant factor in thromboxane receptor–mediated contraction.

Acknowledgments

This study was supported in part by National Institutes of Health Grants HL-54829 and HL-61974. We appreciate the generous gift of the Rho-kinase inhibitor Y27632 from the Welfide Corporation.

Footnotes

Original received January 16, 2001; revision received April 24, 2001; accepted April 24, 2001.

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