Difference in Contractile Mechanisms between the Early and Sustained Components of Ionomycin-Induced Contraction in Rat Caudal Arterial Smooth Muscle

Abstract

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K⁺ concentration in rat caudal arterial smooth muscle involves a Ca²⁺-induced Ca²⁺ sensitization mechanism whereby Ca²⁺ entry through voltage-gated Ca²⁺ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) induced by a Ca²⁺ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca²⁺. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC₂₀) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca²⁺]_i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca²⁺]_i elevation.

INTRODUCTION

Smooth muscle contraction is regulated by phosphorylation and dephosphorylation of the 20 kDa light chain of myosin (LC₂₀) catalysed by Ca²⁺/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), respectively.^1–3) However, much attention has been focused recently on the molecular mechanisms underlying regulation of force independent of changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), referred to as Ca²⁺ sensitization.³⁾ Ca²⁺ sensitization is mediated predominantly via inhibition of MLCP, leading to an increase in LC₂₀ phosphorylation.³⁾ The monomeric guanosine triphosphatase (GTPase), RhoA, plays a major role in Ca²⁺ sensitization of smooth muscle contraction: RhoA-GTP activates Rho-associated kinase (ROCK), which subsequently phosphorylates the myosin-targeting subunit of MLCP (MYPT1), thereby inactivating the phosphatase and leading to enhanced LC₂₀ phosphorylation and smooth muscle contraction.^1,3) The activation of RhoA/ROCK is, therefore, a major downstream pathway of receptor-dependent, G protein-mediated Ca²⁺ sensitization.^3,4)

Electromechanical coupling operates through changes in membrane potential, which affects [Ca²⁺]_i. Stimulation by high [K⁺] induces depolarization of the cell membrane, which opens voltage-gated Ca²⁺ channels causing Ca²⁺ influx, increases [Ca²⁺]_i, binding of Ca²⁺ to CaM, activation of MLCK, LC₂₀ phosphorylation, and contraction.^3,5) We previously indicated that 60 mM K⁺-induced membrane depolarization evokes a rapid increase in force of de-endothelialized rat caudal arterial smooth muscle, which then declines to a steady-state level that is significantly greater than resting force.⁶⁾ Both phases of the contractile response to K⁺-induced membrane depolarization were found to be absolutely dependent on the influx of Ca²⁺ through voltage-gated Ca²⁺ channels, since they could be blocked by a Ca²⁺ channel blocker or by removal of extracellular Ca²⁺. The initial phasic contraction is attributable to an increase in [Ca²⁺]_i and the phosphorylation of LC₂₀ catalyzed by Ca²⁺/CaM-dependent MLCK. On the other hand, the sustained tonic contraction involves activation of the RhoA/ROCK pathway leading to inhibition of MLCP.⁶⁾ Moreover, we recently found that the Ca²⁺-dependent proline-rich tyrosine kinase 2 (Pyk2, also known as FAK2, CAKβ and RAFTK) lies upstream of RhoA activation in response to membrane depolarization in de-endothelialized rat caudal arterial smooth muscle, leading to MLCP inhibition and sustained contraction.^7–9) Furthermore, Pyk2 was shown to be associated with the RhoA/ROCK pathway in the spontaneous colonic smooth muscle by a similar mechanism to that seen in vascular smooth muscle.¹⁰⁾ However, the mechanism whereby Pyk2 activates the RhoA/ROCK pathways remains unclear.

In this study, the Ca²⁺ ionophore ionomycin was used to increase [Ca²⁺]_i, rather than the influx of Ca²⁺ through voltage-gated Ca²⁺ channels, to determine whether Pyk2 activation is indeed downstream of Ca²⁺ influx, and we investigated the effects of each inhibitor on ionomycin-induced contraction and LC₂₀ phosphorylation.

MATERIALS AND METHODS

Materials

Prazosin (1-[4-amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]-piperazine), DL-propranolol (1-[isopropylamino]-3-[1-naphthyloxy]-2-propanol), ML-9 (1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine) and PF-431396 (N-[2-[[[2-[(2,3-dihydro-2-oxo-1H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]-N-methyl-methanesulfonamide hydrate) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.), ionomycin from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.), dithiothreitol (DTT), sodium salicylate and Phos-tag^® Acrylamide from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), HA-1077 from MP Biomedicals (Irvine, CA, U.S.A.), and W-7 and W-5 from Toronto Research Chemicals (North York, ON, Canada). 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid was from Dojindo Laboratories (Kumamoto, Japan) and all other chemicals were of reagent grade. Stock solutions were prepared in water for prazosin, propranolol, HA-1077 and sodium salicylate, and in dimethylsulphoxide (DMSO) for ionomycin, ML-9, PF-431396, W-7 and W-5.

Force Measurements in Intact Muscle Strips

Male Sprague–Dawley rats (400–550 g) were anesthetized with 5% isoflurane and euthanized by carbon dioxide as approved by the Institutional Ethics Committee for Animal Research at Meiji Pharmaceutical University and conforming to Guidelines for Proper Conduct of Animal Experiments in Japan. De-endothelialized caudal arterial smooth muscle strips were prepared for force measurements as previously described.¹¹⁾ Caudal arteries were removed and helical strips [0.5 mm × (6–7) mm] were cut in Hepes-Tyrode (H-T) solution (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 10 mM Hepes, pH 7.4). The endothelium was removed mechanically from all segments by a rubbing procedure. Muscle strips were mounted horizontally between two hooks and immersed in a pool of H-T solution. Once mounted, tissues were stimulated with 60 mM KCl by replacing NaCl in H-T solution with equimolar KCl. The absence of vasorelaxation in response to 1 µM acetylcholine indicated successful endothelial cell disruption. After stable 60 mM K⁺-induced contractions were achieved, the strips were transferred to H-T solution including 60 µM ionomycin in the presence or absence of each inhibitor. Each inhibitor was applied for 20 min before the transfer to 60 µM ionomycin. All measurements of 60 mM K⁺- or 60 µM ionomycin-induced contraction were carried out in the presence of 1 µM prazosin and 0.1 µM propranolol to block the α₁- and β-adrenergic effects of noradrenaline, which is released from nerve terminals.⁶⁾ All buffers were pre-oxygenated with 100% O₂ at room temperature.

LC₂₀ Phosphorylation Measurements in Muscle Strips

At indicated times before or after stimulation by 60 µM ionomycin, tissues were rapidly frozen in 10% (w/v) trichloroacetic acid (TCA), 10 mM DTT in dry ice/acetone. The residual TCA was washed out with dry ice-cold 10 mM DTT/acetone and tissues were lyophilized for 16 h and stored at −80 °C until LC₂₀ extraction. Protein was extracted from freeze-dried tissues by addition of sample buffer (65 mM Tris–HCl, pH 6.8, 10% glycerol, 4% sodium dodecyl sulfate (SDS), 100 mM DTT and 0.01% bromophenol blue) by a modification of the method of Wilson et al.¹²⁾ Quantification of LC₂₀ phosphorylation was achieved by Phos-tag SDS-polyacrylamide gel electrophoresis (PAGE) (12.5% acrylamide) and Western blotting with enhanced chemiluminescence detection, as previously described.^7,8,13,14) Anti-LC₂₀ rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A., catalogue number sc-15370) was used at 1 : 3250 dilution.

Statistical Analysis

Data represent the mean ± standard error of the mean (S.E.M.). Values of n indicate the numbers of smooth muscle strips utilized. Student’s t test was used for statistical comparisons. One-way ANOVA followed by Tukey–Kramer multiple-comparisons test was used to compare three or more groups. p-Values <0.05 were considered to be statistically significant. These analyses were performed using JMP Pro16 (SAS Institute Japan, Tokyo, Japan).

RESULTS

Effects of HA-1077, Sodium Salicylate, PF-431396 and ML-9 on Ionomycin-Induced Contraction

A Ca²⁺ ionophore, ionomycin (60 µM), in the presence of extracellular Ca²⁺, elicited a slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca²⁺, which reached a maximum after approx. 15 min (Fig. 1). Because ionomycin-induced contraction is very slow and requires a long time for recovery, data for ionomycin-induced contractions with and without inhibitors were obtained from separate strips to avoid muscle damage. The maximal force of 60 µM ionomycin-induced contraction was 33.2 ± 6.5% (n = 11) of that induced by 60 mM K⁺. The average maximal force developed in response to 60 mM K⁺ was 0.32 ± 0.01 mN/mg tissue wet weight (n = 45). Pre-treatment with a ROCK inhibitor, HA-1077 (3 µM) and a Pyk2 inhibitor, sodium salicylate (10 mM) and a Pyk2/focal adhesion kinase (FAK) inhibitor, PF-431396 (3 µM), inhibited only the sustained phase (30 min), but not the early phase (4 min), of 60 µM ionomycin-induced contraction (Figs. 1, 2). On the other hand, pre-treatment with a MLCK inhibitor, ML-9 (30 µM), reduced potently both the early phase and the sustained phase of ionomycin-induced contraction (Figs. 1, 2).

Fig. 1. Effect of Pre-treatment with HA-1077, Sodium Salicylate, PF-431396 or ML-9 on Ionomycin-Induced Contractions of Rat Caudal Arterial Smooth Muscle

After stable 60 mM K⁺-induced contraction was attained, strips were pre-incubated for 20 min with each inhibitor, and then incubated with 60 µM ionomycin in the presence of each inhibitor. Ionomycin-induced contractions with and without inhibitors were obtained from separate strips. a, Typical traces showing ionomycin-induced contraction of rat caudal arterial smooth muscle in the absence or presence of each inhibitor. b, Cumulative force data expressed as a percentage of the maximal force of the phasic contraction induced by 60 mM K⁺. Values represent the mean ± S.E.M. (A) The time–courses of ionomycin-induced contraction in the absence of inhibitors (○; n = 11). (B) The time–courses of ionomycin-induced contraction in the absence (○; n = 11) or presence (●; n = 7) of 3 µM HA-1077. (C) The time–courses of ionomycin-induced contraction in the absence (○; n = 11) or presence (●; n = 5) of 10 mM sodium salicylate. (D) The time–courses of ionomycin-induced contraction in the absence (○; n = 11) or presence (●; n = 6) of 3 µM PF-431396. (E) The time–courses of ionomycin-induced contraction in the absence (○; n = 11) or presence (●; n = 6) of 30 µM ML-9.

Fig. 2. Ionomycin-Induced Contraction after 4 or 30 min of Ionomycin Stimulation in the Presence of Each Inhibitor

After stable K⁺-induced contraction was attained, strips were pre-incubated for 20 min with indicated concentrations of each inhibitor, and then incubated with 60 µM ionomycin in the presence of the indicated concentrations of each inhibitor. Ionomycin-induced contractions with and without inhibitors were obtained from separate strips. Force is expressed as a percentage of the maximal force of the phasic contraction induced by 60 mM K⁺ and was measured 4 min (A) or 30 min (B) after ionomycin addition. Values represent the mean ± S.E.M. (the data in the absence of each inhibitor (n = 11), and the data in the presence of 3 µM HA-1077 (n = 7), 10 mM sodium salicylate (n = 5), 3 µM PF-431396 (n = 6), 30 µM ML-9 (n = 6), 150 µM W-7 (n = 6) or 150 µM W-5 (n = 4)). Each value was compared with the 60 µM ionomycin-induced contraction in the absence of each inhibitor, and significance was tested using Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from 60 µM ionomycin-induced contraction at 4 or 30 min after ionomycin addition in the absence of inhibitors.

Effects of W-7 and W-5 on Ionomycin-Induced Contraction

A CaM inhibitor, W-7 (150 µM), suppressed the early phase of contraction (Figs. 2, 3). However, ionomycin-stimulated contractions rose slowly in the presence of W-7. Consequently, the sustained phase of ionomycin-induced contraction was slightly inhibited by W-7, but the difference was not significant, compared with the 60 µM ionomycin-induced contraction in the absence of W-7 (Figs. 2, 3). Ionomycin-induced contraction was not affected by W-5 (150 µM), which has only a weak CaM inhibitory effect (Figs. 2, 3).

Fig. 3. Effect of Pre-treatment with W-7 or W-5 on Ionomycin-Induced Contractions of Rat Caudal Arterial Smooth Muscle

After stable 60 mM K⁺-induced contraction was attained, strips were pre-incubated for 20 min with 150 µM W-7 or W-5, and then incubated with 60 µM ionomycin in the presence of W-7 or W-5. Ionomycin-induced contractions with and without inhibitors were obtained from separate strips. (A) a–c, Typical traces showing ionomycin-induced contraction of rat caudal arterial smooth muscle in the absence or presence of each inhibitor. A typical trace and a graph (○) for 60 µM ionomycin-induced contraction in Figs. 3Aa and 3B are the same as shown in Fig. 1(A). (B) Cumulative force data expressed as a percentage of the maximal force of the phasic contraction induced by 60 mM K⁺. Values represent the mean ± S.E.M. The time–courses of ionomycin-induced contraction in the absence of each inhibitor (○; n = 11), or the presence of 150 µM W-5 (▲; n = 4) or 150 µM W-7 (●; n = 6).

Effects of HA-1077, Sodium Salicylate, PF-431396, ML-9, W-7 and W-5 on Ionomycin-Induced LC₂₀ Phosphorylation

Mono-phosphorylation of LC₂₀ was measured following stimulation with 60 µM ionomycin (Figs. 4, 5, Supplementary Figure). LC₂₀ phosphorylation levels increased significantly in response to 60 µM ionomycin from a resting level of 0.18 ± 0.04 mol P_i/mol LC₂₀ to 0.36 ± 0.01 mol P_i/mol LC₂₀ at 4 min (Fig. 4) and 0.18 ± 0.01 mol P_i/mol LC₂₀ to 0.36 ± 0.02 mol P_i/mol LC₂₀ at 30 min (Fig. 5) after ionomycin addition.

Fig. 4. Ionomycin-Induced LC₂₀ Phosphorylation at 4 min after Ionomycin Stimulation in the Presence of Each Inhibitor

Muscle strips were pre-treated for 20 min without or with each inhibitor and treated with 60 µM ionomycin in the absence or presence of each inhibitor for 4 min at which time the tissue was frozen and LC₂₀ phosphorylation quantified as described in Materials and Methods. Blotting data for W-5 was obtained from a different gel run under the same conditions. (A) Representative western blots of LC₂₀ showing separation of the phosphorylated from the unphosphorylated species by Phos-tag^® SDS-PAGE. We observed no di-phosphorylated LC₂₀ in any experiment using intact strips. (B) Cumulative data (n = 6–10). LC₂₀ phosphorylation was expressed as mol P_i/mol LC₂₀ in the absence and presence of each inhibitor. Values represent the mean ± S.E.M. Statistical comparisons of means between groups were performed using a one-way ANOVA followed by Tukey–Kramer multiple-comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from the level of LC₂₀ phosphorylation without the stimulation. ^## p < 0.01; significantly different from the level of LC₂₀ phosphorylation with 60 µM ionomycin in the absence of inhibitors.

Fig. 5. Ionomycin-Induced LC₂₀ Phosphorylation at 30 min after Ionomycin Stimulation in the Presence of Each Inhibitor

Muscle strips were pre-treated for 30 min without or with each inhibitor and treated with 60 µM ionomycin in the absence or presence of each inhibitor for 30 min at which time the tissue was frozen and LC₂₀ phosphorylation quantified as described in Materials and Methods. Blotting data for W-5 was obtained from a different gel run under the same conditions. (A) Representative western blots of LC₂₀ showing separation of the phosphorylated from the unphosphorylated species by Phos-tag^® SDS-PAGE. We observed no di-phosphorylated LC₂₀ in any experiment using intact strips. (B) Cumulative data (n = 5–11). LC₂₀ phosphorylation was expressed as mol P_i/mol LC₂₀ in the absence and presence of each inhibitor. Values represent the mean ± S.E.M. Statistical comparisons of means between groups were performed using a one-way ANOVA followed by Tukey–Kramer multiple-comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001; significantly different from the level of LC₂₀ phosphorylation without the stimulation. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001; significantly different from the level of LC₂₀ phosphorylation with 60 µM ionomycin in the absence of inhibitors.

Ionomycin-induced LC₂₀ phosphorylation at 4 min after ionomycin addition was not significantly reduced by pre-treatment with HA-1077 (3 µM), sodium salicylate (10 mM), PF-431396 (3 µM) or W-5 (150 µM), compared with the level of LC₂₀ phosphorylation stimulated by 60 µM ionomycin in the absence of inhibitors (Fig. 4). However, ML-9 (30 µM) and W-7 (150 µM) significantly reduced the elevation of LC₂₀ phosphorylation induced by ionomycin at 4 min after ionomycin addition, compared with the level of LC₂₀ phosphorylation stimulated by 60 µM ionomycin in the absence of inhibitors (Fig. 4). On the other hand, ionomycin-induced sustained elevation of LC₂₀ phosphorylation at 30 min after ionomycin addition was significantly reduced by pre-treatment with HA-1077 (3 µM), sodium salicylate (10 mM), PF-431396 (3 µM) or ML-9 (30 µM), compared with the level of LC₂₀ phosphorylation stimulated by 60 µM ionomycin in the absence of inhibitors (Fig. 5). In particular, pre-treatment with sodium salicylate almost abolished LC₂₀ phosphorylation induced by ionomycin. W-7 (150 µM) significantly inhibited ionomycin-induced sustained elevation of LC₂₀ phosphorylation at 30 min after ionomycin addition, compared with the level of LC₂₀ phosphorylation stimulated by 60 µM ionomycin in the absence of inhibitors, but LC₂₀ phosphorylation levels were significantly increased above resting levels (Fig. 5). W-5 (150 µM) had no effect on ionomycin-induced sustained elevation of LC₂₀ phosphorylation at 30 min after ionomycin addition. Consequently, the changes in early or sustained phases of ionomycin-induced contraction (Fig. 2) and LC₂₀ phosphorylation (Figs. 4, 5) in the presence of each inhibitor were similar.

DISCUSSION

The caudal artery is a well-innervated distributing artery with a relatively simple structure and plays an important role in regulating local blood flow. The tunica media is well-developed and contains a large amount of smooth muscle, allowing for effective regulation of vasoconstriction and vasodilation. Therefore, the smooth muscle of the caudal artery is useful for elucidating the mechanisms of vasoconstriction and for developing model systems for vascular pathology. We have previously reported many useful research findings using caudal arterial smooth muscle.^{4,6–9,11,12,14–16)}

We previously reported that high [K⁺]-induced sustained contraction of rat caudal arterial smooth muscle involves the RhoA/ROCK pathway leading to phosphorylation of MYPT1,⁶⁾ and this pathway is facilitated by Pyk2, which is activated by an increase in [Ca²⁺]_i via membrane depolarization-induced Ca²⁺ influx from the extracellular space.^7–9) In this study, to determine whether Ca²⁺-dependent RhoA/ROCK activation through Pyk2 activation is indeed downstream of Ca²⁺ influx, the Ca²⁺ ionophore ionomycin was used to increase [Ca²⁺]_i, and the effect of pre-treatment with each inhibitor on ionomycin-induced contraction was assessed.

Ionomycin, in the presence of extracellular Ca²⁺, elicited slow and sustained contraction (Fig. 1). This shows that ionomycin-induced Ca²⁺ influx is slow and MCLK is gradually activated by Ca²⁺ influx, resulting in slow phosphorylation of LC₂₀ and contractile response. Therefore, pre-treatment with a highly selective MLCK inhibitor, ML-9,^17,18) inhibited both the early phase and the sustained phase of ionomycin-induced contraction (Figs. 1, 2). On the other hand, pre-treatment with a ROCK inhibitor, HA-1077,¹⁹⁾ and a Pyk2 inhibitor, sodium salicylate^20,21) and a Pyk2/FAK inhibitor, PF-431396,²²⁾ suppressed only the sustained phase (15 to 30 min), but not the early phase (within 5 min), of ionomycin-induced contraction (Figs. 1, 2). Moreover, the effects of each inhibitor on LC₂₀ phosphorylation showed similar results to those of these contractile responses (Figs. 2, 4, 5). The concentrations of each inhibitor used in this study were 3 to 10 times the IC₅₀ value for high [K⁺]-induced contractions, as used in our previous research,^6–8) suggesting that the concentrations used are appropriate. MLCK is directly activated by immediate influx of Ca²⁺ after stimulation, and the subsequent gradual increase of [Ca²⁺]_i activates Pyk2. It seems that the subsequent activation of RhoA/ROCK via activated Pyk2 causes sustained contraction. This finding is the first report showing that the sustained phase of ionomycin-induced contraction is due to inhibition of MLCP through Pyk2-mediated RhoA/ROCK pathway activation. This finding is similar to the mechanism to develop the early phase of contraction via MLCK activation induced by high [K⁺] stimulation and the mechanism of sustained phase due to inhibition of MLCP through RhoA/ROCK activation via Pyk2 activation as previously reported.^6–9)

Sodium salicylate is well known as a cyclooxygenase (COX) inhibitor. However, it was reported that sodium salicylate is rather less potent, but quite selective, as an inhibitor of Pyk2²⁰⁾ and its unique ability to induce vasodilation has been attributed to the action of Pyk2 inhibition rather than its cyclooxygenase inhibitory activity.²¹⁾ In fact, sodium salicylate was a relatively weak inhibitor of both COX-1 and COX-2 isoforms in intact cells and was inactive against COX in either broken cells or purified enzyme preparations,²³⁾ further strongly supporting that the vasodilator action of sodium salicylate is COX-independent. We previously indicated that sodium salicylate reversed 60 mM K⁺-induced rat caudal arterial smooth muscle contraction in a concentration-dependent manner with an IC₅₀ value of 2.9 ± 0.5 mM, and pre-treatment of the tissue with sodium salicylate (10 mM) eliminated the tonic component of 60 mM K⁺-induced contraction and the sustained elevation of LC₂₀ phosphorylation without affecting the phasic contractile response and the phasic K⁺-induced LC₂₀ phosphorylation.⁸⁾ Moreover, we also found sodium salicylate had no effect on the activity of purified recombinant ROCK.⁸⁾ On the other hand, another Pyk2/FAK inhibitor, PF-431396, inhibited both phasic and tonic components of the contractile response and LC₂₀ phosphorylation to 60 mM K⁺, suggesting an off-target effect of PF-431396 during the phasic contraction.⁸⁾ Furthermore, we previously reported that FAK activation does not correlate with K⁺-stimulated contraction.⁸⁾ PF-431396 probably has other inhibitory actions on contraction besides the inhibition of Pyk2, that is likely MLCK itself because skeletal muscle MLCK has been shown to be partially inhibited by PF-431396.²²⁾ Therefore, PF-431396 cannot be used at concentrations higher than those used in this study. In short, it seems that sodium salicylate has a high selectivity for Pyk2, although its effect is not strong, and the vasodilator action of sodium salicylate is likely to be COX-independent.

Pyk2, which may act upstream of RhoA/ROCK, is a non-receptor, Ca²⁺-dependent protein-tyrosine kinase that is regulated by various extracellular signals and increased [Ca²⁺]_i.^24–26) Although the mechanism of activation of Pyk2 by [Ca²⁺]_i is not known in detail, the Ca²⁺/CaM complex binds to the FERM domain of Pyk2. The FERM domain has been suggested to mediate Ca²⁺/CaM-dependent Pyk2 homodimerization (via binding of CaM to the α2-helix of the F2 subdomain) and transphosphorylation at Tyr402 to recruit Src, which binds via its SH2 domain and phosphorylates Pyk2 at Tyr579 and Tyr580 within the activation loop of the kinase domain to generate maximal Pyk2 activity.²⁷⁾ In addition, it was confirmed that RhoA activation and MYPT1 phosphorylation induced by angiotensin (Ang) II stimulation are mediated by PDZ-RhoGEF, and that its activity is activated by Pyk2 in the study using rat aortic vascular smooth muscle cells.²⁸⁾ We previously reported that the phosphorylation level of Pyk2 at Tyr402 was increased when treating caudal smooth muscle with high [K⁺] and ionomycin.^7–9) However, the mechanism of Pyk2 activation via Ca²⁺ influx due to membrane depolarization by high [K⁺] rather than receptor stimulation is still unclear. Therefore, we determined whether Pyk2 is directly activated by Ca²⁺ or whether an intermediate, such as CaM, is involved, using the CaM antagonist, W-7, but not the inactive analog, W-5.

It is well known that W-7 is a cell-permeable CaM antagonist that inhibits Ca²⁺/CaM-regulated enzyme activities with IC₅₀ values of 28 µM for Ca²⁺/CaM-dependent phosphodiesterase and 25 µM for Ca²⁺/CaM-induced activation of MLCK.^29–31) On the other hand, W-5 interacted more weakly with CaM and inhibited the activation of Ca²⁺/CaM-dependent enzymes to a lesser extent than did W-7.^29–31) Pre-treatment with W-7 (150 µM) suppressed the early phase of ionomycin-induced contraction. On the other hand, the suppression of the sustained phase was only slight but not significant, compared with the 60 µM ionomycin-induced contraction in the absence of W-7 (Figs. 2, 3). Additionally, pre-treatment with the inactive analog, W-5 (150 µM) showed no inhibitory effect in both the early and sustained phase of ionomycin-induced contraction (Figs. 2, 3). The effects of W-7 and W-5 on the increase of LC₂₀ phosphorylation induced by ionomycin were similar to their effects on the contractile response (Figs. 4, 5). Therefore, we conclude that the stimulation with ionomycin initially results in LC₂₀ phosphorylation by MLCK activated by increased Ca²⁺/CaM complex, followed by suppression of MLCP by Pyk2, which is directly activated by Ca²⁺, resulting in sustained LC₂₀ phosphorylation and contraction. The conclusion that the increase of [Ca²⁺]_i is responsible for Pyk2 activation was supported by the observation that the Ca²⁺ channel blocker nifedipine inhibited both K⁺- and ionomycin-induced contraction and Pyk2 autophosphorylation.⁸⁾ On the other hand, it was reported that W-7 (200 µM), a higher concentration than that used in our study, showed an inhibitory effect on contraction and RhoA activation induced by high [K⁺] or ionomycin stimulation in experiments using rabbit aorta, and W-5 showed no inhibitory effect.³²⁾ These studies have suggested that the Ca²⁺/CaM complex is involved upstream of RhoA activation. However, in our study, although W-7, but not W-5, strongly inhibited the early phase of ionomycin-induced contraction and LC₂₀ phosphorylation, the sustained phase of these parameters was only slightly inhibited. This slight inhibition of the sustained contraction by W-7 is thought to be due to inhibition of Ca²⁺/CaM-dependent MLCK activation by W-7.

The activity of the RhoA/ROCK pathway, which is involved in the inhibition of MLCP, is thought to be regulated by Rho guanine nucleotide exchange factor (RhoGEF) and Rho GTPase-activating protein (RhoGAP).³³⁾ RhoGEF releases GDP from RhoA and promotes GTP binding, following which GTP-bound RhoA activates ROCK.³³⁾ It was reported that RhoGEFs/RhoGAPs involved in blood pressure regulation in vascular smooth muscle cells include p115-RhoGEF, PDZ-RhoGEF, Leukemia-associated RhoGEF (LARG), p63-RhoGEF, Rho-specific GTPase activating protein (GRAF) 3 and p190-RhoGAP.³⁴⁾ Moreover, it was reported that G_12/13, which is activated by receptor stimulation, directly activates RhoA and causes contraction in rabbit aortic smooth muscle cells³⁵⁾ and that G_12/13 binds to specific RhoGEFs such as PDZ-RhoGEF³⁶⁾ and LARG³⁷⁾ in cultured cells. On the other hand, transfection experiments with cultured aortic vascular smooth muscle cells have suggested that PDZ-RhoGEF may connect activated Pyk2 to RhoA activation via phosphorylation and activation of its RhoGEF activity.²¹⁾ Moreover, it was reported that Pyk2 activated by Ang II induces PDZ-RhoGEF tyrosine phosphorylation thereby activating the Rho/ROCK cascade in vascular smooth muscle cells.³⁸⁾ Artamonov et al.³⁹⁾ showed that the activation of PDZ-RhoGEF and LARG plays an important role in the sustained contraction of vascular smooth muscle tissues stimulated with the thromboxane A₂ receptor agonist U46619 and endothelin-1. However, the involvement of RhoGEFs in the Ca²⁺-dependent RhoA/ROCK pathway via Pyk2 remains unclear. Therefore, we are currently trying to identify RhoGEFs coupled with Pyk2.

We conclude that the early phase of contraction caused by the slow increase in [Ca²⁺]_i by stimulation is due to slow MLCK activation mediated by Ca²⁺/CaM, and at the same time, that Pyk2 is involved in the sustained phase of contraction, which is due to activation of RhoGEFs via Pyk2 activated by influx of Ca²⁺, followed by activation of the RhoA/ROCK pathway. It was reported that up-regulation of the RhoA/ROCK pathway was involved in the etiology of hypertension⁴⁰⁾ and sodium salicylate lowered blood pressure in hypertensive rats at concentrations that inhibit Pyk2.²¹⁾ Therefore, Pyk2 may be a suitable therapeutic target for the treatment of cardiovascular diseases associated with hypercontractility. We are currently investigating the identification of Ca²⁺-dependent Pyk2-coupled RhoGEFs in caudal artery smooth muscle, with a view to the identification of novel therapeutic agents that control vascular smooth muscle contractility.

Acknowledgments

The authors thank Professor Michael P. Walsh (University of Calgary, Canada) for his valuable critique of this article.

Funding

This work was supported by JSPS KAKENHI Grant Number JP19K07108 to MM.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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