Mitsuo Mita

Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase.

Depolarization of the sarcolemma of smooth muscle cells activates voltage-gated Ca2+ channels, influx of Ca2+ and activation of cross-bridge cycling by phosphorylation of myosin catalysed by Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK). Agonist stimulation of smooth muscle contraction often involves other kinases in addition to MLCK. In the present study, we address the hypothesis that membrane depolarization-induced contraction of rat caudal arterial smooth muscle may involve activation of Rho-associated kinase (ROK). Addition of 60 mM K+ to de-endothelialized muscle strips in the presence of prazosin and propranolol induced a contraction that peaked rapidly and then declined to a steady level of force corresponding to approx. 30% of the peak contraction. This contractile response was abolished by the Ca2+-channel blocker nicardipine or the removal of extracellular Ca2+. An MLCK inhibitor (ML-9) inhibited both the phasic and tonic components of K+-induced contraction. On the other hand, the ROK inhibitors Y-27632 and HA-1077 abolished the tonic component of K+-induced contraction, and slightly reduced the phasic component. Phosphorylation levels of the 20-kDa light chain of myosin increased rapidly in response to 60 mM K+ and subsequently declined to a steady-state level significantly greater than the resting level. Y-27632 abolished the sustained and reduced the phasic elevation of the phosphorylation of the 20-kDa light chain of myosin, without affecting the K+-induced elevation of cytosolic free Ca2+ concentration. These results indicate that ROK activation plays an important role in the sustained phase of K+-induced contraction of rat caudal arterial smooth muscle, but has little involvement in the phasic component of K+-induced contraction. Furthermore, these results are consistent with inhibition of myosin light-chain phosphatase by ROK, which would account for the sustained elevation of myosin phosphorylation and tension in response to membrane depolarization.

Protein kinase C-independent sensitization of contractile proteins to Ca2+ in α-toxin-permeabilized smooth muscle cells from the guinea-pig stomach

Involvement of protein kinase C in receptor‐operated Ca2+ sensitization of cell shortening was investigated by use of α‐toxin‐permeabilized smooth muscle cells from the fundus of the guinea‐pig. Most of the isolated cells responded to 0.6 μm Ca2+ with a maximal shortening to approximately 65% of the resting cell length. Addition of acetylcholine (ACh) at a maximal concentration (10 μm) resulted in a marked decrease in the concentration of Ca2+ required to trigger a threshold response from 0.6 μm to 0.2 μm. The augmentation of Ca2+ sensitivity by ACh was not inhibited by specific protein kinase C inhibitors, calphostin C and K‐252b at a concentration of 1 μm. These findings suggest that protein kinase C is not involved in the muscarinic receptor‐operated augmentation of Ca2+ sensitivity.

Receptor‐coupled shortening of α‐toxin‐permeabilized single smooth muscle cells from the guinea‐pig stomach

1 Isolated single smooth muscle cells from the fundus of the guinea‐pig stomach were permeabilized by use of Staphylococcus aureus α‐toxin. Receptor‐coupled shortening of individual cells was monitored under phase contrast microscopy. 2 Most of the isolated cells responded to 0.6 μm Ca2+, but not to 0.3 μm Ca2+, with a resulting maximal shortening to approximately 65% of the resting cell length. The contractile activity of these permeabilized cells lasted for several hours and repeated shortening was readily achieved after washing out. 3 Addition of acetylcholine (ACh) at a maximal concentration (10 μm) resulted in a marked decrease in the concentration of Ca2+ required to trigger a threshold response from 0.6 μm to 0.2 μm, and 1 mm guanosine 5′‐diphosphate (GDP) blocked this decrease. Moreover, treatment with 100 μm guanosine 5′‐triphosphate (GTP) mimicked the action of ACh. 4 Addition of 100 μm inositol 1,4,5‐trisphosphate (InsP3) with 0.2 μm Ca2+ did not cause cell shortening, whereas 10 μm ACh with 0.2 μm Ca2+ did, suggesting that InsP3‐induced Ca2+ release is not involved in ACh‐operated cell shortening. 5 The present study demonstrates an α‐toxin‐permeabilized single smooth muscle cell preparation which retains its receptor function and also provides an insight into mechanisms leading to augmentation of Ca2+ sensitivity by stimulation of muscarinic receptors or GTP‐binding proteins.

A Role for the Tyrosine Kinase Pyk2 in Depolarization-induced Contraction of Vascular Smooth Muscle

Background: Depolarization-induced tonic contraction of vascular smooth muscle involves tyrosine phosphorylation. Results: Depolarization activates the Ca2+-dependent tyrosine kinase Pyk2, leading to activation of the RhoA/Rho-associated kinase pathway. Conclusion: Activation of Pyk2 is required for the sustained phase of depolarization-induced contraction. Significance: Knowledge of the mechanisms responsible for sustained contraction is crucial for identification of defects leading to disease associated with vascular contractile dysfunction. Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca2+-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K+-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K+, Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca2+, elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca2+ channel blocker nifedipine inhibited peak and sustained K+-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K+-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca2+ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.

Attenuation of store‐operated Ca2+ entry and enhanced expression of TRPC channels in caudal artery smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. Previously, we found that Ca2+ entry from the extracellular space via α1‐adrenoceptor‐activated, Ca2+‐permeable channels, but not voltage‐gated Ca2+ channels, is impaired in endothelium‐denuded caudal artery smooth muscle from Type 2 diabetic Goto‐Kakizaki (GK) rats. In the present study, we investigated the impairment of Ca2+ entry mechanisms via Ca2+‐permeable channels from the extracellular space in response to α1‐adrenoceptor stimulation (cirazoline) in endothelium‐denuded caudal artey strips isolated from GK rats.

Muscarinic receptor binding and Ca2+ influx in the all-or-none response to acetylcholine of isolated smooth muscle cells

Isolated smooth muscle cells from guinea pig taenia caecum were prepared by collagenase digestion. Isolated single smooth muscle cells showed an all-or-none response to acetylcholine (ACh) under our experimental conditions. A Scatchard plot of the specific binding of [3H]quinuclidinyl benzilate (QNB) to the cells gave a straight line, and the KD and Bmax values were calculated as 0.18 +/- 0.03 nM and 1.11 +/- 0.10 pmol/mg protein, respectively. ACh competitively inhibited the specific binding of [3H]QNB in a concentration-dependent manner. Thus, although the cells showed an all-or-none response, ACh bound to the receptor concentration dependently. The contraction of the cells in response to ACh was inhibited in a concentration-dependent manner, by nicardipine suggesting that the contraction of smooth muscle cells in response to ACh depended on the influx of extracellular Ca2+. The ACh-stimulated increase of the Ca2+ influx was very rapid and correlated well with the contraction of the cells; it decreased after reaching a maximum. The all-or-none response of the cells could be due to a rapid influx of Ca2+. These results suggest that ACh binds to its receptor in a concentration-dependent manner, and that when the binding reaches a certain threshold, a rapid influx of Ca2+ occurs and the cells show an all-or-none response, followed by an efflux of Ca2+.

Comparison of pharmacodynamics between carvedilol and metoprolol in rats with isoproterenol-induced cardiac hypertrophy: Effects of carvedilol enantiomers

A recent clinical study has shown that carvedilol has a significantly more favorable effect than metoprolol on survival rate in patients with heart failure. This may be due to actions of carvedilol such as beta(2)-adrenoceptor blockade, alpha-adrenergic receptor blockade and other properties such as anti-oxidant effects that are not yet fully understood. We compared the effects of racemic carvedilol, metoprolol and carvedilol enantiomers on cardiac hypertrophy at similar heart rate in rats with isoproterenol-induced cardiac hypertrophy. Continuous administration of isoproterenol for 2 weeks produced heart failure, which is characterized by an increased heart rate, cardiac hypertrophy and downregulation of beta-adrenoceptors. The doses of racemic carvedilol and metoprolol were adjusted to obtain a similar heart rate in rats with isoproterenol-induced cardiac hypertrophy. The reduction of left ventricular weight and improvement of cAMP production induced by carvedilol were superior to those induced by metoprolol. Although heart rate, blood pressure and cAMP production were not affected by R-carvedilol, left ventricular weight was significantly reduced as a result of alpha-adrenoceptor blockade. The improvement of cAMP production by S-carvedilol was significantly higher than that induced by coadministration of R-carvedilol and metoprolol, suggesting that beta(2)-adrenoceptor blockade partly contributed to the improvement of signal transduction in rats with isoproterenol-induced cardiac hypertrophy. This study has demonstrated that the effects of carvedilol on cAMP production and cardiac hypertrophy in rats with isoproterenol-induced cardiac hypertrophy are superior to those induced by metoprolol at a similar heart rate.

Effect of Ca2+ deprivation on short-term desensitization of isolated smooth muscle cells showing an all-or-none response to acetylcholine.

1. Short-term desensitization of the contractile response of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine was examined in the absence of external Ca2+. 2. Isolated smooth muscle cells contracted to 10(-7)-10(-6) M acetylcholine, showing an all-or-none response. 3. Desensitization was induced by incubating the cells with 10(-4) M acetylcholine for 10 min in Ca2+-free solution. 4. Desensitized cells showed an all-or-none response but required a higher concentration of acetylcholine for induction of contraction, indicating that the desensitization was due to a change in the threshold concentration. 5. Desensitization is suggested to be induced before massive influx of Ca2+ into the cells.

The change in the threshold for short-term desensitization in isolated smooth muscle cells showing an all-or-none response to acetylcholine.

1 Isolated smooth muscle cells from guinea‐pig taenia caecum were prepared by collagenase digestion. The cells showed an all‐or‐none response to acetylcholine (ACh) under our experimental conditions. 2 Desensitized cells showed an all‐or‐none response but required a higher concentration of ACh for induction of contraction, indicating that the desensitization was due to a change in the threshold concentration. 3 In [3H]‐quinuclidinyl benzilate ([3H]‐QNB) binding to the desensitized cells, KD and Bmax were not significantly different from those estimated in the control cells. The competitive inhibition curve for specific binding of [3H]‐QNB by ACh in the desensitized cells was in agreement with that of control cells. 4 The ACh‐stimulated increase of the 45Ca2+ influx was very rapid and correlated well with the contraction of the cells. The concentrations of ACh inducing the maximal 45Ca2+ influx were increased by desensitization. 5 These results indicated that although the binding of ACh to the receptor was not changed by desensitization, the threshold concentration of ACh for their contraction was raised by desensitization, and the 45Ca2+ influx accompanying the contraction was shifted to the side of high concentration of ACh. 6 These results suggest that the development of short‐term desensitization is due to an uncoupling of the receptor from the mechanism for initiation of the contraction.

Impairment of α1‐adrenoceptor‐mediated contractile activity in caudal arterial smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. In the present study, we compared the responsiveness of de‐endothelialized caudal artery smooth muscle strips, isolated from Type 2 diabetic Goto‐Kakizaki (GK) and normal Wistar rats, to α1‐adrenoceptor stimulation (cirazoline) and membrane depolarization (K+).