Shigeru Hishinuma

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.

Ca2+/Calmodulin‐Mediated Regulation of the Desensitizing Process in Gq Protein‐Coupled Histamine H1 Receptor‐Mediated Ca2+ Responses in Human U373 MG Astrocytoma Cells

We investigated Ca2+/calmodulin (CaM)‐mediated regulation of the desensitizing process of the histamine H1 receptor‐mediated increase in intracellular Ca2+ concentration in human U373 MG astrocytoma cells. The desensitizing process was evaluated by measuring the histamine‐induced Ca2+ responses in cells pretreated with histamine for 15 s‐30 min under various conditions. Under normal physiological conditions, desensitization developed with three successive phases : a fast desensitization within 15 s, a transient resensitization at 45 s, and a prompt and sustained redesensitization from 1 to 30 min. Similar processes of desensitization/resensitization occurred even under hypertonic conditions, where histamine‐mediated internalization of the histamine H1 receptor is inhibited. The transient resensitization phase was selectively prevented by deprivation of extracellular Ca2+ and, even more strikingly, by the presence of W‐7 (a CaM antagonist). FK506 and cyclosporin A, Ca2+/CaM‐dependent protein phosphatase (PP2B) inhibitors, mimicked such effects. In the presence of KN‐62, a Ca2+/CaM‐dependent protein kinase II (CaM kinase II) inhibitor, the early development of desensitization disappeared, allowing a slow and simple development of desensitization. The early processes of desensitization and resensitization were unaffected by W‐5, okadaic acid, and KN‐04 (less potent inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X and chelerythrine (protein kinase C inhibitors). The high‐affinity site for histamine was converted to a lower‐affinity site by histamine treatment, which also showed a transient restoration phase at 45 s in a manner sensitive to KN‐62 and FK506. These results provide the first evidence that Ca2+/CaM plays a crucial role in determining the early phase of the desensitizing process via activation of CaM kinase II and PP2B, by regulating agonist affinity for histamine H1 receptors.

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca2+-mobilization and Ca2+-influx pathways in taenia caeci

1 We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN‐A‐343 and AHR‐602 with those of carbachol in smooth muscle of guinea‐pig taenia caeci. 2 McN‐A‐343 and AHR‐602, as well as carbachol, completely displaced the atropine‐sensitive binding of [3H]‐quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol>McN‐A‐343>4AHR‐602. 3 In the presence of 2.2 mM extracellular Ca2+, McN‐A‐343 and AHR‐602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4‐DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF‐DX 116 (M2 selective). These contractions were inhibited by an L‐type Ca2+ channel blocker, verapamil. 4 In Ca2+‐free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN‐A‐343 and AHR‐602. Application of McN‐A‐343 or AHR‐602 inhibited the carbachol‐induced contraction in Ca2+‐free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5 The EC50 value for carbachol‐induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6 The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor‐mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN‐A‐343 and AHR‐602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.

Novel regulation of muscarinic receptors and their coupling with G proteins in smooth muscle: transient resensitization during desensitizing process.

1 Muscarinic stimulation of the smooth muscle of guinea‐pig taenia caeci was produced with 10−4 m carbachol for 15 s, 30 s, 1 min, 2 min and 30 min, and the time course of developing desensitization was studied by measuring the muscle contractility and the binding characteristics of muscarinic receptors. 2 The contractile response to carbachol was analyzed using dose‐response curves. The response to 10−7 m carbachol was reduced by treatment for 15 s with 10−4 m carbachol (fast desensitization), but recovered partially after 30 s treatment and completely after 1 min treatment (resensitization). Contractility was reduced again after 2 min and 30 min treatment (re‐desensitization). 3 The affinity of carbachol for muscarinic receptors was changed by the carbachol treatment in a manner similar to the contractility. Thus, the affinity was reduced at 15 s, restored slightly at 30 s and completely at 1 and 2 min, and was reduced again at 30 min. 4 5′‐Guanylylimidodiphosphate (GppNHp), a non‐hydrolysable analogue of guanosine triphosphate (GTP) reduced the affinity of muscarinic receptors for carbachol via guanine nucleotide‐binding regulatory proteins (G proteins). A similar effect was observed in tissues desensitized by 15 s carbachol treatment. This effect disappeared after 30 s, recovered completely after 1 and 2 min, and disappeared again after 30 min carbachol treatment. 5 Neither the dissociation constant (Kd value) nor the maximal binding (Bmax) of [3H]‐quinuclidinyl benzilate ([3H]‐QNB) to muscarinic receptors were changed by the carbachol treatment. 6 These results indicate that the whole process of desensitization, resensitization and re‐desensitization are related to changes in the binding ability of muscarinic receptors, in their coupling with G proteins and in the post‐receptor steps of the signal transduction. We emphasize that the desensitizing process involves an early transient phase of resensitization that could be caused by restoration of both the affinity of carbachol for muscarinic receptors and their coupling with G proteins. This novel resensitization mechanism may have some physiological significance for cellular homoeostasis by modulating cellular responsiveness transiently or even in an oscillatory manner during the process of desensitization.

Ubiquitin/proteasome‐dependent down‐regulation following clathrin‐mediated internalization of histamine H1‐receptors in Chinese hamster ovary cells

J. Neurochem. (2010) 113, 990–1001.

Ca2+/calmodulin-mediated regulation of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells.

Abstract: We investigated the regulation by intracellular Ca2+ of agonist‐induced sequestration of Gq protein‐coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine‐induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3H]mepyramine binding sites on intact cells accessible to the hydrophilic H1‐receptor antagonist pirdonium. The changes in the pirdonium‐sensitive binding of [3H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine‐induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin‐mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W‐7 and calmidazolium. Inhibitors of protein kinase C (H‐7 and GF109203X), Ca2+/calmodulin‐dependent protein kinase II (KN‐62), or protein phosphatase 2B (FK506) did not alter the time course of H1‐receptor sequestration. These results provide the first evidence that agonist‐induced, clathrin‐mediated sequestration of Gq protein‐coupled receptors is transiently inhibited by Ca2+/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.

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+).

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase

Ca2+‐DEPENDENT DIFFERENTIAL DEVELOPMENT OF CARBACHOL‐INDUCED DESENSITIZATION TO RECEPTOR AGONISTS AND HIGH K+ IN GUINEA‐PIG TAENIA CAECI

1 Carbachol (CCh)‐induced desensitization to CCh was interrupted by a transient resensitization during its early stage, with concomitant changes at the muscarinic receptor/G‐protein level in smooth muscle of guinea‐pig taenia caeci. To assess whether such a peculiar desensitizing process may heterologously regulate smooth muscle contraction, we examined the developmental processes of CCh‐induced desensitization to histamine and high K+ and compared it with that to CCh. 2 Under Ca2+‐containing physiological conditions, treatment with 10−4 mol/L CCh for 30 min induced heterologous desensitization to histamine and high K+. The development of desensitization to histamine was interrupted by a transient resensitization at 1 min in a manner similar to that to CCh. In contrast, CCh‐induced desensitization to high K+ reached a peak at 1 min and was followed by a gradual resensitization up to a partial restoration at 30 min. 3 Under Ca2+‐free conditions containing 0.2 mmol/L EGTA, treatment with 10−4 mol/L CCh for 30 min failed to induce heterologous desensitization to either histamine or high K+, whereas the CCh treatment developed homologous desensitization to CCh in a simple time‐dependent manner without a resensitization phase. 4 These results suggest that cellular responsiveness to receptor agonists and non‐receptor‐mediated depolarizing stimulation is differentially regulated by Ca2+‐dependent heterologous desensitization in smooth muscle.

DIFFERENTIAL DEVELOPMENT OF CARBACHOL-INDUCED DESENSITIZATION IN RECEPTOR-MEDIATED Ca2+ INFLUX AND Ca2+ RELEASE PATHWAYS IN SMOOTH MUSCLE OF GUINEA-PIG TAENIA CAECI

1 We have found that development of carbachol (CCh)‐induced desensitization to receptor agonists, but not to receptor by‐passed stimulation, is transiently interrupted by a Ca2+‐dependent resensitization during the early stage in the smooth muscle of guinea‐pig taenia caeci. To further characterize the receptor‐mediated signal transduction pathways involved in this peculiar desensitization process, we examined the desensitization processes during Ca2+ influx‐ and Ca2+ release‐mediated contractions in response to activation of muscarinic receptors or histamine H1 receptors. 2 Desensitization treatment with 10−4 mol/L CCh for 30 min in the presence of extracellular Ca2+ resulted in desensitization to the muscarinic agonists McN‐A‐343 or AHR‐602, which are known to induce contraction only in the presence of extracellular Ca2+ in taenia caeci. The development of desensitization to these agonists was interrupted by a transient resensitization at 1 min. In contrast, the transient resensitization phase was lost following removal of extracellular Ca2+ during the desensitization treatment with CCh; under these conditions, the desensitization developed gradually without an apparent resensitization phase. 3 Contractions to 10−4 mol/L CCh and 10−4 mol/L histamine in the absence of extracellular Ca2+ were gradually desensitized without a resensitization phase following the CCh desensitization treatment, irrespective of the presence or absence of extracellular Ca2+ during CCh treatment, although the onset of the desensitization was delayed under Ca2+‐free conditions. 4 These results suggest that the receptor‐mediated Ca2+ influx and Ca2+ release pathways are differentially desensitized to CCh and that the transient resensitization appears to regulate the desensitization process in response to Ca2+ influx‐mediated contraction. Such differential processes of desensitization in receptor‐mediated bifurcated signalling pathways may determine cellular responsiveness to certain types of stimuli, depending on the different Ca2+ sources required for contraction.