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Pulmonary vascular thrombosis and thrombotic arteriopathy are common pathological findings in pulmonary arterial hypertension. Thrombin may thus play an important role in the pathogenesis and pathophysiology of pulmonary arterial hypertension.The present study aimed to elucidate the contractile effect of thrombin in the pulmonary artery and clarify its underlying mechanisms.The changes in cytosolic Ca²(+) concentrations ([Ca²(+)](i)), 20-kD myosin light chain (MLC20) phosphorylation, and contraction were monitored in the isolated porcine pulmonary artery. The production of reactive oxygen species (ROS) was evaluated by fluorescence imaging.In the presence of extracellular Ca²(+), thrombin induced a sustained contraction accompanied by an increase in [Ca²(+)](i) and the phosphorylation of MLC20. In the absence of extracellular Ca²(+), thrombin induced a contraction without either [Ca²(+)](i) elevation or MLC20 phosphorylation. This Ca²(+)- and MLC20 phosphorylation-independent contraction was mimicked by hydrogen peroxide and inhibited by N-acetyl cysteine. Fluorescence imaging revealed thrombin to induce the production of ROS. A Rho-kinase inhibitor, Y27632, inhibited not only the thrombin-induced Ca²(+)- and MLC20 phosphorylation-dependent contraction, but also the Ca²(+)- and MLC20 phosphorylation-independent contraction and the ROS production. These effects of thrombin were mimicked by a proteinase-activated receptor 1 (PAR₁)-activating peptide.This study elucidated the Ca²(+)- and MLC20 phosphorylation-independent ROS-mediated noncanonical mechanism as well as Ca²(+)- and MLC20 phosphorylation-dependent canonical mechanism that are involved in the thrombin-induced PAR₁-mediated pulmonary vasoconstriction. Rho-kinase was suggested to play multiple roles in the development of thrombin-induced pulmonary vasoconstriction.

Background and purpose: Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery. Experimental approach: The cytosolic Ca2+ concentrations ([Ca2+]i), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery. Key results: Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR1) antagonist, while it was mimicked by PAR1-activating peptide (PAR1AP), but not PAR4AP. The thrombin-induced contraction was associated with a small elevation of [Ca2+]i and an increase in MLC20 phosphorylation. Thrombin and PAR1AP induced a greater increase in tension for a given [Ca2+]i elevation than that obtained with high K+-depolarization. They also induced a contraction at a fixed Ca2+ concentration in α-toxin-permeabilized preparations. Conclusions and implications: The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR1 and thereby increasing the sensitivity of the myofilament to Ca2+. This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.

Background/Aims: The effects of oxidative stress on the vascular responsiveness to the agonists of proteinase-activated receptors (PARs) were investigated. Methods: Serum-free incubation was utilized to impose oxidative stress to isolated rat aortas. Spontaneously hypertensive rats (SHR) were investigated as a model of in vivo oxidative stress. Results: Thrombin, trypsin, PAR1-activating peptide (PAR1-AP), PAR2-AP and PAR4-AP induced little or no effect in the aortas of female Wistar-Kyoto rats (WKY). Serum-free incubation induced endothelium-dependent relaxant responses to PAR2 agonists, but not PAR1 or PAR4 agonists, in a manner sensitive to diphenyleneiodonium or ascorbic acid. In male aortas, trypsin and PAR2-AP induced a transient endothelium-dependent relaxation without serum-free incubation. The acetylcholine-induced endothelium-dependent relaxation and the sodium nitroprusside-induced endothelium-independent relaxation remained unchanged. Immunoblot analyses revealed the upregulation of PAR2 in endothelial cells, which was abolished by either diphenyleneiodonium or ascorbic acid. Aortas of female SHR expressed a higher level of PAR2 than WKY and responded to trypsin without serum-free incubation. Treatment with ascorbic acid attenuated the trypsin-induced relaxation and the PAR2 expression in SHR. Conclusion: This study provides the first evidence that oxidative stress upregulates PAR2 in endothelial cells, thereby enhancing the endothelium-dependent relaxant response to PAR2 agonists in rat aortas.

Background and purpose: The mechanism for the development of post-haemorrhagic cerebral vasospasm after subarachnoid haemorrhage (SAH) still remains unknown. Experimental approach: We investigated the role of thrombin and its receptor PAR1 in the development of hyper-contractility of the basilar artery in a rabbit double haemorrhage model, which received two injections of autologous blood into the cisterna magna. Key results: In the basilar artery isolated from the control rabbits, thrombin, only at 10 units ml−1, induced a transient endothelium-dependent relaxation and a slight smooth muscle contraction. In SAH, the contractile response to thrombin was markedly enhanced, while the endothelium-dependent relaxant effect of thrombin remained unchanged. The enhancement of the contractile responses was also observed in the absence of endothelium and thrombin induced an enhanced contraction at concentrations higher than 0.3 units ml−1. The contractile response to PAR1-activating peptide was also enhanced after SAH. However, the contractile responses to high K+ and endothelin-1, and the myofilament Ca2+-sensitivity remained unchanged after SAH. An immunoblot analysis suggested the up-regulation of PAR1 in the smooth muscle of the basilar artery. The heparinization of blood before injection prevented the enhancement of the contractile responses to thrombin and PAR1-activating peptide. Conclusions and implications: The present study demonstrated, for the first time, that the contractile response of the basilar artery to thrombin was markedly enhanced after SAH. Mechanistically, our findings suggested that the activation of thrombin following hemorrhage up-regulated the expression of PAR1, thereby inducing the hyper-responsiveness to thrombin. British Journal of Pharmacology (2007) 152, 1131–1139; doi:10.1038/sj.bjp.0707435; published online 3 September 2007

Background and purpose: The intravesical administration of dimethyl sulphoxide (DMSO) is used to alleviate the symptoms of interstitial cystitis. We investigated the relaxant effect of DMSO and its underlying mechanism in the detrusor muscle. Experimental approach: The effects of DMSO on contraction, on Ca2+ sensitivity of myofilaments, and on myosin light chain (MLC) phosphorylation were investigated in both intact and α-toxin-permeabilized strips of rabbit detrusor muscle. Key results: In fura-PE3-loaded strips, DMSO (>1%) induced a significant relaxation during sustained contractions induced by 60 mM K+-depolarization or 10 μM carbachol, while having no effect on the [Ca2+]i level. DMSO decreased the level of MLC phosphorylation during the contractions induced by 60 mM K+ and 10 μM carbachol. DMSO also inhibited both the contraction and MLC phosphorylation induced by calyculin-A in intact strips. In the α-toxin-permeabilized preparations, DMSO relaxed the Ca2+-induced contraction and also inhibited the tension development induced by a stepwise increment of Ca2+ concentrations. Such a relaxant effect of DMSO was enhanced in the presence of phosphate. Conclusions and implications: DMSO relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of myofilaments. Inhibition of the kinase activities involved in myosin phosphorylation may play a major role in DMSO-induced Ca2+ desensitization. Inhibition of the cross-bridge cycling at the step of phosphate release may also contribute to the relaxant effect of DMSO. Such relaxant effects of DMSO could be linked to the therapeutic effect of DMSO in interstitial cystitis. British Journal of Pharmacology (2007) 151, 1014–1024; doi:10.1038/sj.bjp.0707317

Objective— Plasmin is a key enzyme in fibrinolysis. We attempted to determine the possible role of plasmin in the regulation of vascular tone, while also investigating the mechanism of plasmin-induced vasorelaxation. Methods and Results— In porcine coronary artery, plasmin induced an endothelium-dependent relaxation. This relaxing effect was mostly abolished by a proteinase inhibitor, a plasmin inhibitor, or a nitric oxide (NO) synthase inhibitor. The preceding stimulation with plasmin significantly inhibited the subsequent relaxation induced by thrombin but not that induced by proteinase-activated receptor-1–activating peptide. The relaxation induced by trypsin and substance P remained unaffected by the preceding plasmin stimulation. The pretreatment with plasmin, thrombin, or trypsin significantly attenuated the plasmin-induced relaxation. In porcine coronary artery endothelial cells (PCAECs) and human umbilical vein endothelial cells (HUVECs), plasmin induced a transient elevation in the cytosolic Ca 2+ concentrations ([Ca 2+ ] i ). The preceding stimulation with plasmin inhibited the subsequent [Ca 2+ ] i elevation induced by thrombin but not that induced by trypsin. In PCAECs, plasmin concentration-dependently induced NO production. Conclusions— The present study demonstrated, for the first time, that plasmin induced an endothelium-dependent NO-mediated relaxation in the porcine coronary artery, while also showing plasmin to specifically inactivate the thrombin receptor.

Aims The contractile mechanisms of prostatic smooth muscle have been extensively investigated at the receptor level. However, the intracellular mechanisms have not yet been fully elucidated, especially in human tissue. In the present study, we examined the functional role of RhoA/Rho kinase (ROCK), one of the major intracellular molecules involved in smooth muscle contraction, in the contraction of the human prostate. Methods Ring preparations made of cultured human prostatic stromal cells (CHPSCs) or fresh human prostatic tissue was used for an isometric tension study. Gene transfer using baculovirus vector and α-toxin permeabilized preparations were also used. Results RhoA, ROCK I and ROCK II proteins were all expressed in CHPSCs and fresh human prostatic tissue. In CHPSCs ring preparations, the contraction induced by endothelin (ET)-1 was enhanced by over-expression of RhoA and inhibited by ROCK inhibitor. In α-toxin permeabilized preparations, ET-1 or GTP-γS induced an additional contraction at a constant [Ca2+]i, that was inhibited by ROCK inhibitor. In fresh human prostatic tissue, norepinephrine (NE)-induced contraction was inhibited by ROCK inhibitor at a constant [Ca2+]i in α-toxin permeabilized preparations. Conclusions These results suggested that RhoA/ROCK-mediated Ca2+ sensitization is likely involved in the contraction of the human prostate. The antagonisms of this pathway may thus be useful as an alternative target in the treatment of benign prostatic hyperplasia (BPH). Neurourol. Urodynam. 26:547–551, 2007. © 2007 Wiley-Liss, Inc.

Background and purpose: Although vascular smooth muscle cells are known to express the Na+–Ca2+ exchanger (NCX), its functional role has remained unclear, mainly because of its relatively low expression. We thus investigated the involvement of NCX in the mechanism for the forskolin-induced vaso-relaxation, using wild type (WT) and transgenic (TG) mice that specifically over-express NCX1.3 in smooth muscle. Experimental approach: We examined the relaxing effect of forskolin during the pre-contraction induced by 100 nM U46619, a thromboxane A2 analogue in the mouse isolated thoracic aorta. We also measured the intracellular Ca2+ concentration ([Ca2+]i) in fura-PE3-loaded aortic strips. Key results: The forskolin-induced decreases in [Ca2+]i and tension were much greater in aortas from TG mice than in those from WT mice. In a low Na+ solution, forskolin-induced decreases in [Ca2+]i and tension were greatly inhibited in both groups of aortas. In WT aortas, the presence of 100 nM SEA0400, an NCX inhibitor, had only a little effect on the forskolin-induced decreases in [Ca2+]i, but inhibited the forskolin-induced relaxation. However, in TG aortas, the presence of SEA0400 greatly inhibited the forskolin-induced decreases in [Ca2+]i and tension. Conclusions and Implications: The NCX was involved in the forskolin-induced reduction of [Ca2+]i and tension in the mouse thoracic aorta. Measurement of [Ca2+]i and tension in aortas of the TG mouse is thus considered to be a useful tool for evaluating the role of NCX in vascular tissue. British Journal of Pharmacology (2007) 150, 434–444. doi:10.1038/sj.bjp.0707119

Vascular smooth muscle cells (VSMCs) are not terminally differentiated and, owing to their remarkable plasticity, can change to a dedifferentiated state in response to vascular injury. Our understanding of the contractility of VSMCs is mainly based on the data obtained from normal adult animals. However, to obtain a better understanding of the abnormal contractility seen in the vascular diseases such as hypertension and vasospasm superimposed on atherosclerosis, it is important to also know the contractility of proliferating dedifferentiated VSMCs. To this end, we studied the contractility of cultured VSMCs that undergo dedifferentiation similar to that induced by vascular injury. There are only a few reports in which the contractility of cultured VSMCs has been extensively studied. We established a method to investigate the contractility of the cultured VSMCs and determined that their contraction is dramatically changed to be more dependent on the Rho-Rho kinase system but less dependent on the PKC-CPI-17 (protein kinase C-potentiated protein phosphatase 1 inhibitory protein)-mediated pathway. In this review, we focus on the contractility of the cultured VSMCs as a model of the proliferating dedifferentiated VSMCs.

We have previously reported that the contractile response to thrombin and trypsin was enhanced in the pregnant rat myometrium. We herein determined whether or not sex hormones contribute to this enhancement and the expression of protease-activated receptors (PARs). The nonpregnant rats received daily injections of either 17beta-estradiol or progesterone, and then the contractile response of the myometrium was examined ex vivo. Treatment with either 17beta-estradiol or progesterone had almost no significant enhancing effect on the high K(+)- or oxytocin-induced contraction. On the other hand, both 17beta-estradiol and progesterone dose-dependently enhanced the contractile response to trypsin. A maximal enhancement was obtained at 25 and 40 mg kg weight(-1) day(-1) for 17beta-estradiol and progesterone, respectively. The extent of the enhancement of the trypsin-induced contraction seen in the sex hormone-treated rats in the present study was comparable to that reported in the pregnant rats. However, the contractile response to thrombin and PAR1/PAR2-AP, SFLLRNP was not enhanced either by progesterone or 17beta-estradiol. PAR2-AP and PAR4-AP failed to induce contraction under any conditions. PAR1 mRNA was scarcely detected in the control myometrium by an RT-PCR analysis, while it slightly increased only in the progesterone-treated rats. Neither PAR2 nor PAR4 mRNA was detected. We thus conclude that the responsiveness to trypsin, but not thrombin, is controlled by sex hormones. A novel type of receptor, other than PAR1, PAR2 or PAR4, is suggested to mediate the trypsin-induced contraction as in the case of the pregnant rat myometrium.

Objective— Protease-activated receptor-1 (PAR1) mediates the thrombin-induced proliferation and hypertrophy of vascular smooth muscle cells. A role of Rac1 in the regulation of PAR1 expression was investigated. Methods and Results— Treatment with simvastatin, a hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, for 24 hours attenuated the transient [Ca 2+ ] i elevation induced by thrombin. Immunofluorescence staining revealed that simvastatin decreased the surface expression of PAR1 in a manner dependent on protein geranylgeranylation. Introduction of a Rac1/Cdc42 inhibitory fragment but not a RhoA inhibitory fragment using a cell-penetrating peptide also attenuated the response to thrombin and decreased the surface expression of PAR1. Finally, downregulation of Rac1, but not RhoA, using an RNA interference technique attenuated the thrombin-induced [Ca 2+ ] i elevation. However, the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged. Conclusions— Here, we provide for the first time 3 lines of evidence that Rac1 plays a critical role in maintaining the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. Rac1 is suggested to regulate the constitutive trafficking of PAR1 and thereby regulate the surface expression of PAR1.

The contractile mechanism of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was investigated in the guinea-pig Taenia coli, by simultaneously monitoring the changes in the cytosolic Ca2+ concentration ([Ca2+]i) and force. fMLP induced a significant elevation of [Ca2+]i and force at concentrations higher than 10 nM. The maximal response was obtained at a concentration of higher than 1 μM. fMLP (10 μM) augmented the force development induced by a stepwise increment of the extracellular Ca2+ concentration during 60 mM K+ depolarization, while it had no effect on the [Ca2+]i elevation, and thus produced a greater force for a given elevation of [Ca2+]i than 60 mM K+ depolarization. The removal of extracellular Ca2+ completely abolished the fMLP-induced contraction. The fMLP-induced [Ca2+]i elevation was inhibited substantially but not completely by 10 μM diltiazem, partly by 10 μM SK&F 96365, and completely by their combination. Y27632, a specific inhibitor of rho-kinase, had no significant effect on the fMLP-induced [Ca2+]i elevation and force development. Chenodeoxycholic acid, a formyl peptide receptor antagonist, specifically abolished the fMLP-induced contraction but not high K+- or carbachol-induced contractions. A dual lipoxygenase/cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, a nonselective leukotriene receptor antagonist, and a selective type 1 cysteinyl-containing leukotriene receptor antagonist specifically reduced the fMLP-induced contraction. We suggest that the low-affinity-type fMLP receptor and lipoxygenase metabolites of arachidonic acid are involved in the fMLP-induced contraction in the guinea-pig T. coli. This contraction mainly depends on the [Ca2+]i elevation due to Ca2+ influx and the enhancement of Ca2+ sensitivity in the contractile apparatus. Keywords: Ca2+ sensitivity, fMLP, gastrointestinal smooth muscle Introduction N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a synthetic analogue of bacterial chemotactic peptide (Schiffmann et al., 1975a, 1975b; Marasco et al., 1984), exerts a potent chemoattractant effect toward neutrophils and monocytes. In addition to this effect, fMLP has been shown to possess spasmogenic properties in smooth muscle tissues including airway, blood vessels, intestinal tract, and renal pelvis (Hamel et al., 1984; Armour et al., 1986; Boukili et al., 1986; Shore et al., 1987; Crowell et al., 1989; Maggi et al., 1992; Minamino et al., 1996). The mechanism for the fMLP-induced contraction appeared to vary with type of smooth muscle tissue. Many studies have suggested an indirect contractile mechanism in the human bronchus, guinea-pig lung parenchymal strips, human coronary artery, and rabbit pulmonary artery (Hamel et al., 1984; Armour et al., 1986; Shore et al., 1987; Crowell et al., 1989; Keitoku et al., 1997). The cyclooxygenase and lipoxygenase metabolites of arachidonic acid, such as thromboxane A2 and leukotriene C4, were suggested to mediate the contractile response of fMLP, based on the observation that the inhibitors of cyclooxygenase (Hamel et al., 1984; Shore et al., 1987; Crowell et al., 1989; Keitoku et al., 1997), lipoxygenase (Shore et al., 1987) and thromboxane synthase (Shore et al., 1987), or leukotriene receptor antagonist (Armour et al., 1986; Shore et al., 1987) inhibited the fMLP-induced contraction. The fMLP-activated neutrophils have also been shown to induce endothelium-dependent contraction in the human umbilical vein and canine coronary artery (Minamino et al., 1996; Kerr et al., 1998). On the other hand, the direct contractile effect of fMLP has only been suggested in the guinea-pig ileum (Marasco et al., 1982). Furthermore, the cellular mechanism for fMLP-induced contraction remains to be elucidated, especially in terms of intracellular Ca2+ signal transduction. The cellular effect of fMLP is mediated by formyl peptide receptors. Three genes for humans (formyl peptide receptor (FPR), FPRL1, and FPRL2) and eight genes for mice (FPR1, FPR-rs1, 2, 3, 4, 5, 6, and 7) have been identified as FPR gene family (Bao et al., 1992; Gao et al., 1998; Wang & Ye, 2002). This receptor family belongs to a superfamily of a seven-transmembrane, G-protein-coupled receptor (Prossnitz & Ye, 1997). Among these receptors, FPR and FPRL1 in humans and FPR1 and FPR-rs1 in mice could serve as functional receptors for fMLP. The cellular distribution of the high-affinity FPR is not restricted to phagocytic leukocytes as originally proposed, but it is expressed on a broad spectrum of tissues and cells, including neuromuscular, vascular, endocrine, and immune systems (Lacy et al., 1995; McCoy et al., 1995; Sozzani et al., 1995; Becker et al., 1998; Panaro & Mitolo, 1999). FPRL1 is also expressed in a variety of cell types including phagocytic leukocytes, lymphocytes, hepatocytes, epithelial cells, neuroblastoma cells, astrocytoma cells, and microvascular endothelial cells (Gronert et al., 1998; Le et al., 2000). However, the receptor responsible for the fMLP-induced contractile responses remains to be identified. In the present study, we investigated the mechanism of the fMLP-induced contraction in the guinea-pig Taenia coli, by simultaneously monitoring the cytosolic Ca2+ concentrations ([Ca2+]i) and force development in the fura-PE3-loaded strips. The cellular mechanism of the fMLP-induced contraction was investigated in terms of Ca2+ signaling, by examining the effect of the removal of the extracellular Ca2+, two Ca2+ entry blockers, and a rho-kinase inhibitor. The receptor mediating the fMLP-induced contraction in the guinea-pig T. coli was evaluated by examining the effects of various receptor antagonists including chenodeoxycholic acid, the formyl peptide receptor antagonist, on the fMLP-induced contraction. We also investigated the involvement of the lipoxygenase metabolites of arachidonic acid in the fMLP-induced contraction.

RhoA plays a critical role in regulating NO production in cultured endothelial cells. To determine its role in in situ endothelial cells, we investigated the effects of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors and a RhoA-binding domain of Rho-kinase (RB) on vascular contractility in the isolated rabbit mesenteric artery. Ex vivo treatment of the strips with 3×10 −5 mol/L simvastatin and fluvastatin for ≈24 to 30 hours significantly attenuated the contractile response to phenylephrine and high K + in the presence of endothelium. The addition of N ω -nitro- l -arginine methyl ester and the removal of endothelium abolished the attenuation of the contractile response. The cotreatment with geranylgeranyl pyrophosphate prevented the statin-induced attenuation of the contractile response, whereas geranylgeranyl transferase inhibitor mimicked the effect of simvastatin. Treatment with simvastatin enhanced the bradykinin-induced endothelium-dependent relaxation in the mesenteric artery, whereas it had no effect on the bradykinin-induced [Ca 2+ ] i elevation in endothelial cells of the aortic valves. Introduction of RB to the strips using a cell-penetrating peptide of Tat protein (TATHA-RB) attenuated the contractile responses in a NO-dependent manner. However, a Rac1/Cdc42-binding fragment of p21-activated protein kinase, RB without Tat peptide or TATHA-protein A had no effect. The in vivo treatment of rabbit with simvastatin and TATHA-RB attenuated the contractility in a NO-dependent manner. Simvastatin and TATHA-RB significantly upregulated eNOS in the rabbit mesenteric artery. The present study provides the first evidence that RhoA plays a physiological role in suppressing NO production in in situ endothelial cells.

Vascular smooth muscle cells (VSMCs) have a remarkable degree of plasticity and in response to vascular injury, they can change to a dedifferentiated state that can be typically seen in cell cultures. Recently, Y27632, a Rho kinase inhibitor, has been reported to preferentially correct hypertension in a hypertensive rat model. We thus tested the hypothesis that the contraction of the cultured VSMCs might be more dependent on the function of RhoA than the VSMCs in fresh tissue. For this purpose, a tissue-like ring preparation was made using the cultured porcine coronary artery SMCs (CASMCs) and collagen gel (reconstituted ring: R-ring). The R-ring developed an isometric tension on stimulation by high external K + or various receptor agonists. The phorbol ester (a protein kinase C (PKC) activator)-induced contraction of the intact R-ring was greatly inhibited, while the GTPγS (an activator of RhoA)-induced and Ca 2+ -independent contraction of permeabilized R-ring was greatly enhanced, in comparison to the fresh coronary artery ring. An immunoblot analysis showed the expression levels of RhoA and myosin phosphatase subunits (MYPT1 and PP1cδ) to be up-regulated, while the levels of CPI-17 (PKC-potentiated protein phosphatase-1 inhibitory protein), h1-calponin and PKC isoforms were downregulated in cultured CASMCs. The knock down of RhoA by RNA interference decreased the contractility of the cultured CASMCs. It is concluded that the contractility of the cultured VSMCs thus appears to be much more dependent on the function of RhoA than VSMCs in fresh tissue. The expression level of RhoA thus plays a crucial role in regulating the contractility of cultured VSMCs.

The role of Ca2+ sensitization in the contraction of human bladder urinary smooth muscle (UBSM) was investigated.Simultaneous measurements of intracellular Ca2+ concentration ([Ca2+]i) and tension in fura-2 loaded intact strips and receptor coupled strips permeabilized with alpha-toxin were applied. Protein expressions was confirmed by Western blot analysis.In intact fura-2 loaded strips 1 microM carbachol (CCh) induced a greater contraction and a lower [Ca2+]i elevation than that induced by 60 mM K depolarization. In alpha-toxin permeabilized strips 1 microM CCh induced contraction at constant [Ca2+]i and produced a leftward shift in the [Ca2+]i-tension relationship. RhoA, Rho-associated kinase (ROCK) I, ROCK II and CPI-17 proteins were expressed in human UBSM. In intact fura-2 loaded strips the application of 3 microM Y-27632, a ROCK inhibitor, or 3 microM bisindolylmaleimide I (GF109203X), a protein kinase C inhibitor, during the sustained phase of contraction induced by 1 microM CCh induced relaxation without changing [Ca2+]i. In alpha-toxin permeabilized strips the application of 3 microM Y-27632 or 3 microM GF109203X during the sustained contraction induced by 0.3 microM Ca plus 10 microM guanosine triphosphate and 1 microM CCh induced relaxation at constant [Ca2+]i.These results indicate that in human UBSM CCh induces contraction, not only by increasing [Ca2+]i, but also by increasing the Ca2+ sensitivity of the contractile apparatus in a ROCK and protein kinase C dependent manner. Antagonism of Ca2+ sensitization pathways may represent an alternative target in the treatment of overactive bladder.

Theophylline, a nonselective phosphodiesterase inhibitor, has long been regarded as a major bronchodilator in the treatment of human asthma. Using front-surface fluorometry with fura-2 and α-toxin permeabilization, the effects of theophylline on intracellular Ca2+ concentration ([Ca2+]i), tension development and Ca2+ sensitivity of the contractile apparatus were investigated in porcine tracheal smooth muscle strips. Application of theophylline induced a relaxation without a significant decrease in [Ca2+]i when strips were precontracted by 40 mM K+ depolarization, while theophylline significantly decreased both [Ca2+]i and tension induced by carbachol. The effects of theophylline on the increases in [Ca2+]i and tension induced by carbachol were significantly inhibited by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ channels. In the absence of extracellular Ca2+, theophylline significantly attenuated carbachol-induced transient increases in tension development, while it did not affect carbachol-induced transient increase in [Ca2+]i. The [Ca2+]i–force relationship, which was determined by cumulative applications of extracellular Ca2+ (0–5 mM) during 40 mM K+ depolarization, was significantly shifted to the right by theophylline. In α-toxin permeabilized strips, theophylline significantly increased the EC50 value of [Ca2+]i for contraction and enhanced the effect of cAMP, but not of cGMP. These results indicate that theophylline induces relaxation of the porcine tracheal smooth muscle through an activation of BK channels, and a resultant decrease in [Ca2+]i and an attenuation of Ca2+ sensitivity, presumably through the action of cAMP. British Journal of Pharmacology (2003) 140, 939–947. doi:10.1038/sj.bjp.0705508

We examined the role of the modulation of Ca2+ sensitivity for regulating the contractility of corpus cavernosum smooth muscle.We applied simultaneous measurements of intracellular Ca2+ concentration and tension in fura-PE3 loaded intact strips and receptor coupled permeabilization by alpha-toxin.In intact fura-PE3 loaded strips the tension induced by 10 microM. phenylephrine was significantly greater than that produced by depolarization with 118 mM. K+, although the extent of intracellular Ca2+ concentration elevations was similar. During sustained contraction induced by 10 microM. phenylephrine the application of 10 microM. Y-27632 (a Rho kinase inhibitor) induced relaxation with a slight decrease in intracellular Ca2+ concentration, while the application of 3 microM. GF109203X (a protein kinase C inhibitor) induced relaxation without changing intracellular Ca2+ concentration. In alpha-toxin permeabilized strips 10 microM. phenylephrine induced a larger increase in force at a constant intracellular Ca2+ concentration and produced a leftward shift in the intracellular Ca2+ concentration-tension relationship, a response that was partially inhibited by pretreatment with Y-27632 or GF109203X.These results indicate that in rabbit corpus cavernosum smooth muscle phenylephrine induces contraction not only by increasing intracellular Ca2+ concentration, but also by increasing Ca2+ sensitivity of the contractile apparatus in a Rho kinase and protein kinase C dependent manner. Antagonism of Ca2+ sensitization pathways in the corpus cavernosum smooth muscle represents an alternate target for the treatment of erectile dysfunction.

Thyroid hormone has a broad effect on cardiovascular system. 3,3′,5-triiodo- l -thyronine (T3), a biologically active form of thyroid hormone, increases cardiac contractility. T3 causes arterial relaxation and reduction of systemic vascular resistance, resulting in an increase in cardiac output. However, the molecular mechanisms of vascular relaxation by T3 are incompletely characterized. We studied the effect of T3 on the angiotensin (Ang) II type 1 receptor (AT 1 R) expression in vascular smooth muscle cells. T3 dose-dependently decreased expression levels of AT 1 R mRNA, with a peak at 6 hours of stimulation. Binding assay using [ 125 I]Sar 1 -Ile 8 -Ang II revealed that AT 1 R number was decreased by stimulation with T3 without changing the affinity to Ang II. T3 reduced calcium response of vascular smooth muscle cells to Ang II by 26%. AT 1 R promoter activity measured by luciferase assay was reduced by 50% after 9 hours of T3 administration. mRNA stability was also decreased by T3. Real-time quantitative reverse transcription–polymerase chain reaction and Western blot analysis revealed that AT 1 R mRNA and protein were downregulated in the aorta of T3-treated rats. These results suggest that T3 downregulates AT 1 R expression both at transcriptional and posttranscriptional levels, and attenuates biological function of Ang II. Our results suggest that downregulation of AT 1 R gene expression may play an important role for T3-induced vascular relaxation.

Thrombin is one of the key molecules involved in the development of vascular diseases. Thrombin does not only serve as a coagulation factor, but it also exerts cellular effects by activating protease (proteinase)-activated receptors (PARs), a family of seven-transmembrane G protein-coupled receptors. This study focused on the role of PARs in the vascular system. Among the four members so far identified, PAR-1 and PAR-2 were found to play an important role in the vascular system, while the functional roles of PAR-3 and PAR-4 appear to be mostly limited to platelets. The endothelial cells play a primary role in mediating the vascular effects of PARs under physiological conditions, while PARs of the smooth muscle cells can be induced under pathological conditions, and therefore play a more pathophysiological role. PAR-1 and PAR-2 mediate various vascular effects including regulation of vascular tone, proliferation and hypertrophy of smooth muscle and angiogenesis. Since proteases are activated under pathological conditions such as hemorrhage, tissue damage, and inflammation, PARs are suggested to play a critical role in the development of functional and structural abnormality in the vascular lesion. Understanding the functional role of PARs in the vascular system can thus help in the development of new strategies for the prevention and therapy of vascular diseases.

Using fura-2 fluorometry of [Ca2+]i in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca2+]i elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. The response to PAR1-AP (SFLLRNP) was significantly (P

1. This study was designed to investigate the mechanisms for the contractions induced by tachykinins (substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)) in the rabbit corpus cavernosum strips, using fura-PE3 fluorimetry and alpha-toxin permeabilization. 2. Tachykinins induced contractions in the rabbit corpus cavernosum in a concentration-dependent manner. The potency order was SP>NKA>NKB. 3. The tachykinin-induced contractions were enhanced by phosphoramidon (PPAD), an endopeptidase inhibitor, but not by N(omega)-nitro-L-arginine methylester (L-NAME). 4. The NK(1) receptor selective antagonist, SR 140333 significantly inhibited the tachykinin-induced contractions. Although the NK(2) receptor selective antagonist, SR 48968 alone did not influence the effects of tachykinins, it potentiated the inhibitory effect of SR 140333. The NK(3) receptor selective antagonist, SR142801 had no effect. 5. In the rabbit corpus cavernosum, tachykinins induced sustained increases in [Ca(2+)](i) and tension in normal PSS, while only small transient increases in [Ca(2+)](i) and tension were observed in Ca(2+)-free solution. 6. In alpha-toxin permeabilized preparations, tachykinins induced an additional force development at a constant [Ca(2+)](i). 7. These results indicated that in the rabbit corpus cavernosum: (1) Tachykinins induced contractions by increasing both the [Ca(2+)](i) and myofilament Ca(2+) sensitivity; (2) The tachykinin-induced [Ca(2+)](i) elevations were mainly due to the Ca(2+) influx; (3) Tachykinin-induced contractions were mainly mediated through the activation of NK(1) receptor expressed in the rabbit corpus cavernosum smooth muscle, and affected by the endopeptidase activity and (4) Tachykinins may thus play a role in controlling the corpus cavernosum tone.

1. The mechanism of trypsin-induced contraction in the rat myometrium was investigated using front-surface fluorimetry on fura-PE3-loaded strips. The expression of protease-activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT - PCR). 2. In non-pregnant rats, 10 microM trypsin developed a force of up to 30.5 +/- 5.1% of that obtained during the 40 mM K(+)-depolarization-induced contraction. In pregnant rats, the maximal level of the cytosolic Ca(2+) concentration and tension obtained with 3 microM trypsin was 143.2 +/- 6.0% and 63.2 +/- 7.9%, respectively. The depletion of the extracellular Ca2+ abolished the trypsin-induced contraction. 3. Trypsin-induced contraction was abolished by the pre-treatment of a serine protease inhibitor. PAR1-activating peptide (PAR1-AP) caused a potent contraction of the myometrium, while neither PAR2-AP nor PAR4-AP induced any contraction. 4. RT - PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium. 5. Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the pre-stimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1-AP remained after the pre-stimulation with thrombin and trypsin. 6. In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

1. To determine the mechanism of bradykinin-induced production of endothelium-derived contracting factors, we monitored the changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)) in in situ endothelial cells in porcine aortic valvular strips and the changes in [Ca(2+)](i) of smooth muscle cells and force in porcine interlobar renal arterial strips using front-surface fluorometry of fura-2. 2. In the presence of N(omega)-nitro-L-arginine methyl ester, bradykinin caused an endothelium-dependent transient elevation of [Ca(2+)](i) and contraction in smooth muscle in the interlobar renal artery. This contraction was completely inhibited by a prostaglandin H(2)/thromboxane A(2) receptor antagonist. 3. In the absence of extracellular Ca(2+), bradykinin failed to induce contraction. However, replenishing extracellular Ca(2+) to 0.75 mM and higher induced an instantaneous contraction. However, replenishing Ca(2+) per se did not induce any contraction in the absence of bradykinin. Pretreatment with either 10(-5) M 1-(beta-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365) or 0.2 mM Ni(2+) abolished the contraction induced by bradykinin in the presence of extracellular Ca(2+). 4. Treatment with 10(-5) M indomethacin completely inhibited the contractile response induced by Ca(2+) replenishment, regardless of the timing of its application, before or after the application of bradykinin. 5. In endothelial cells in the valvular strips, bradykinin caused a transient [Ca(2+)](i) elevation in the presence of 1.25 mM extracellular Ca(2+), but [Ca(2+)](i) returned to the resting level within 10 min. Neither 10(-5) M SKF96365 nor 0.2 mM Ni(2+) had any effect on the peak [Ca(2+)](i) elevation, but decreased [Ca(2+)](i) in the declining phase. In the absence of extracellular Ca(2+), bradykinin induced a transient [Ca(2+)](i) elevation to a level similar to that seen in the presence of 1.25 mM extracellular Ca(2+). However, [Ca(2+)](i) then rapidly returned to the prestimulation level within 5 min. Subsequent Ca(2+) replenishment to 0.75 mM and higher in the presence of bradykinin elevated [Ca(2+)](i) to significantly higher levels than the resting level seen in the media containing 1.25 mM Ca(2+). 6. In conclusion, Ca(2+) influx in the endothelial cells is essential for bradykinin to induce endothelium-dependent contraction in the porcine interlobar renal artery.

The mechanism underlying the LTC4-induced contraction of guinea-pig taenia coli was determined using the simultaneous measurements of [Ca2+]i and force in whole muscle preparations. Additional experiments were performed in receptor coupled permeabilized preparation. For comparison purposes, the contraction which was induced by a typical G-protein mediated agonist, carbachol was also characterized. LTC4 induced a contraction in the guinea-pig taenia coli in a concentration-dependent manner. The maximal response was obtained at 100 nM and the EC50 value was 5.4±1.9 nM. Both LTC4 and carbachol induced increases in [Ca2+]i and force. The maximum force induced by 100 nM LTC4 was significantly smaller than that induced by 10 μM carbachol, although an increase in [Ca2+]i produced by both agonists was similar. In the permeabilized preparations, carbachol, but not LTC4, induced an additional force development at a fixed Ca2+ concentration. LTC4 induced no increase in [Ca2+]i and force in the Ca2+-free solution, while carbachol induced transient increases in both [Ca2+]i and force in a Ca2+-free solution. Both diltiazem and SK&F 96365 significantly inhibited the LTC4− and carbachol-induced increases in [Ca2+]i and force in normal PSS. The inhibitory pattern of [Ca2+]i by these drugs was also similar. We thus conclude that LTC4 induces the contraction of the guinea-pig taenia coli mainly through Ca2+ influx via both the diltiazem-sensitive and SK&F 96365-sensitive Ca2+ channels, without affecting either the Ca2+-sensitivity or the intracellular Ca2+ release. These results indicated that the mechanism underlying the LTC4-induced contraction differs greatly from that for conventional G-protein mediated agonists, such as carbachol. British Journal of Pharmacology (2001) 133, 529–538; doi:10.1038/sj.bjp.0704122

1. An unsaturated fatty acid, leukotriene C(4) (LTC(4)), has a potent contractile effect on human airway smooth muscle, and has been implicated in the pathogenesis of human asthma. Using front-surface fluorometry with fura-PE3, the effect of LTC(4) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were investigated in porcine tracheal smooth muscle strips. 2. The application of LTC(4) induced little or no contraction despite a small and transient increase in [Ca(2+)](i). In the presence of LTC(4), however, the contractions evoked by high K(+) depolarization or a low concentration of carbachol (CCh) were markedly enhanced without inducing any changes in the [Ca(2+)](i) levels, thus indicating that LTC(4) increases the Ca(2+) responsiveness of the contractile apparatus. This LTC(4)-induced increase in Ca(2+) responsiveness could partly be reproduced in the permeabilized preparation of tracheal smooth muscle strips. 3. The LTC(4)-induced enhancement of contraction was accompanied by an increase in myosin light chain (MLC) phosphorylation and was blocked by a rho kinase inhibitor (Y-27632), but not by either a PKC inhibitor (calphostin C) or a tyrosine kinase inhibitor (genistein). 4. These results indicated that, in porcine tracheal smooth muscle, LTC(4) enhances the contraction by increasing the Ca(2+) responsiveness of the contractile apparatus in a MLC phosphorylation dependent manner, possibly through the activation of the rho-rho kinase pathway.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H(2)O(2)) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H(2)O(2) to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K(+) channel opener levcromakalim. Exogenous H(2)O(2) elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H(2)O(2) upon stimulation by ACh and that the H(2)O(2) production was markedly reduced in eNOS-KO mice. These results indicate that H(2)O(2) is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.

The mechanism of vasorelaxation induced by SR33805 was investigated by simultaneously monitoring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force, and by determining level of myosin light chain (MLC) phosphorylation in the medial strip of the porcine coronary artery. SR33805 inhibited the sustained increases in [Ca(2+)](i) and force (IC(50); 3.2+/-1.0 and 49.4+/-27.5 nM, respectively) induced by 118 mM K(+)-depolarization. There was about a 10 fold difference in the inhibitory potency between [Ca(2+)](i) and force. SR33805 completely inhibited the [Ca(2+)](i) elevation induced by a thromboxane A(2) analogue, U46619 and histamine, at concentrations (1 microM) higher than those required for the complete inhibition of K(+)-depolarization induced [Ca(2+)](i) elevation. SR33805 had no effect on the [Ca(2+)](i) elevation induced by histamine or caffeine in the absence of extracellular Ca(2+). SR33805 caused a leftward shift of the [Ca(2+)](i)-force relationship of the contraction induced by cumulative application of extracellular Ca(2+) during 118 mM K(+)-depolarization. The relationship between [Ca(2+)](i) and MLC phosphorylation also shifted to the left by SR33805, while the relationship between MLC phosphorylation and force remained unaffected. In conclusion, SR33805 caused an apparent leftward shift of the [Ca(2+)](i)-force relationship, accompanied by a greater degree of MLC phosphorylation for a given level of [Ca(2+)](i). The mechanism of this leftward shift, however, still remains to be elucidated.

Using a method employing front-surface fura-2 fluorometry to measure the cytosolic Ca2+ concentration, [Ca2+]i, the mechanism of endothelium-dependent regulation of vascular tone by thrombin was studied in porcine renal interlobar arterial strips. At concentrations lower than 3 u ml−1, thrombin evoked only early transient relaxation, while at 3 u ml−1 and higher concentrations, thrombin caused an early relaxation and a subsequent transient contraction. Both thrombin-induced relaxation and contraction were abolished by removing the endothelium. Similar biphasic responses were observed with a protease-activated receptor-1-activating peptide. Early relaxation was associated with a decrease in [Ca2+]i, while the transient contraction was not associated with a change in [Ca2+]i of smooth muscle cells. A thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10−5 M ONO-3708) completely inhibited the thrombin-induced contraction, whereas a thromboxane A2 synthase inhibitor (10−5 M OKY-046) only partly inhibited it. When the thrombin-induced contraction was inhibited by ONO-3708, either pretreatment with Nω-nitro-L-arginine methylester (L-NAME) or an increase in the amount of external K+ to 40 mM did not abolish thrombin-induced relaxation during phenylephrine-induced sustained contraction. However, the combination of pretreatment with L-NAME and an elevation of external K+ to 40 mM completely abolished the relaxation. There was no significant difference in the concentration-dependent effects of thrombin on the initial early relaxation between conditions in which the contractile components either were or were not inhibited. Thrombin is thus considered to mainly activate protease-activated receptor-1 and cause a biphasic response, early relaxation and a transient contraction, in the porcine renal interlobar artery in an endothelium-dependent manner. The thrombin-induced endothelium-dependent relaxation was mediated by nitric oxide and hyperpolarizing factors, while the contraction was mediated by TXA2 and PGH2. British Journal of Pharmacology (2000) 131, 1635–1642; doi:10.1038/sj.bjp.0703737

The effects of progesterone and 17beta-estradiol on Ca(2+) signaling in in situ endothelial cells were investigated using front-surface fluorometry of fura-2-loaded strips of porcine aortic valve. Progesterone inhibited the thapsigargin-induced sustained [Ca(2+)](i) elevation (IC(50)=33.9 microM, n=4), while 17beta-estradiol added a transient [Ca(2+)](i) elevation. Progesterone and 17beta-estradiol had no significant effect on the thapsigargin-induced [Ca(2+)](i) elevations in the absence of extracellular Ca(2+). A Mn(2+)-induced decline of fluorescent intensity at 360 nm excitation was accelerated by thapsigargin. This acceleration was completely reversed by progesterone, but not by 17beta-estradiol. Progesterone inhibited, and 17beta-estradiol enhanced the endothelin-1 (ET-1)-induced [Ca(2+)](i) elevation, while both had no effect on the ET-1-induced Ca(2+) release observed in the absence of extracellular Ca(2+) or in the pertussis toxin-treated strips. Progesterone and 17beta-estradiol thus had different effects on Ca(2+) signaling, especially on Ca(2+) influx, in endothelial cells.

Thrombin causes various cellular events by activating protease-activated receptors (PARs). Here, we showed, for the first time, that thrombin induced myometrial contraction. To determine the mechanism of thrombin-induced myometrial contraction, we simultaneously measured intracellular Ca2+ concentration ([Ca2+]i) and tension of fura-PE3-loaded rat myometrium using front-surface fluorimetry. The expression of thrombin receptor mRNA in the rat myometrium were determined by reverse transcription-polymerase chain reaction analysis (RT–PCR analysis). Thrombin (0.01–3 u ml−1) caused dose-dependent increase in [Ca2+]i and tension in the rat myometrium, and this effect was greatly enhanced in the pregnant myometrium. PAR1-activating peptide mimicked the effects of thrombin. In Ca2+-free PSS, thrombin induced no increase in [Ca2+]i and tension in the pregnant myometrium. Both diltiazem (10 μM) and SK-F 96365 (10 μM) significantly inhibited the thrombin-induced elevations of [Ca2+]i and tension, and their effects were additive. RT–PCR analysis revealed an approximately 10 fold increase in the level of thrombin receptor mRNA in the pregnant myometrium compared to that obtained in the non-pregnant myometrium. In conclusion, the contractile response to thrombin was greatly enhanced in the pregnant myometrium, mainly due to the up-regulation of thrombin receptor. We propose that initiation of a post-parturitional myometrial contraction is one of the most important physiological roles of thrombin receptor. British Journal of Pharmacology (2000) 131, 1619–1628; doi:10.1038/sj.bjp.0703729

The mechanisms of the thapsigargin (TG)-induced capacitative Ca2+ entry in in situ endothelial cells and its role in the regulation of arterial tone were investigated using front-surface fluorimetry and fura-2-loaded strips of porcine aortic valve and coronary artery. In the presence of extracellular Ca2+, TG induced an initial rapid and a subsequent sustained elevation of cytosolic Ca2+ concentration ([Ca2+]i) in valvular strips. In the absence of extracellular Ca2+, TG induced only a transient increase in [Ca2+]i. The TG-induced sustained elevation of [Ca2+]i in endothelial cells was inhibited completely by 1 mM Ni2+ and partly by 10 μM econazole and 30 μM ML-9, but not by 900 ng ml−1 pertussis toxin or 100 μM wortmannin. Therefore, cytochrome P450 and protein phosphorylation are suggested to be involved in the TG-induced Ca2+ influx in in situ endothelial cells. TG induced an endothelium-dependent large relaxation consisting of an initial and a late sustained relaxation in coronary arterial strip precontracted with U46619 (a thromboxane A2 analogue). Indomethacin alone had no effect, while indomethacin plus Nω-nitro-L-arginine (L-NOARG) markedly inhibited the sustained phase and slightly inhibited the initial phase of the TG-induced relaxation. TG induced a smaller but sustained relaxation during the 40 mM K+-induced precontraction than that seen during the U46619-induced precontraction. This relaxation was completely abolished by the pretreatment with indomethacin plus L-NOARG. In conclusion, both nitric oxide (NO) and endothelium-derived hyperpolarizing factor were suggested to mediate the TG-induced relaxation, while NO plays a major role in the sustained relaxation. The TG-induced sustained [Ca2+]i elevation in endothelial cells was thus suggested to be mainly linked to the sustained production of NO. British Journal of Pharmacology (2000) 131, 115–123; doi:10.1038/sj.bjp.0703548

To investigate the quantitative relationship between elevation in the intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) production, the changes in [Ca2+]i and NO production were determined in parallel, using fluorimetry of fura-2 and 2,3-diaminonaphthalene, respectively, in endothelial cells ex vivo of pig aortic valves. The extent of [Ca2+]i elevation was quantitatively assessed by two parameters: the level of peak [Ca2+]i elevation and the area under the [Ca2+]i curve during treatment (the integrated [Ca2+]i elevation). The amount of NO production was expressed as a percentage of that obtained with 10 μM ATP for 3 min. ATP, bradykinin, thrombin, and ionomycin were used as stimulation to induce NO production, and all these caused [Ca2+]i increases and NO production in a concentration-dependent manner. The relationships between the peak [Ca2+]i and NO production or between the integrated [Ca2+]i elevation and NO production were well described by a straight line. However, the slope value of the linear relationship in both cases varied with the type of stimulation, with thrombin giving the greatest value, followed by ATP, bradykinin and ionomycin. These data suggest that in endothelial cells ex vivo: (1) [Ca2+]i elevation regulates NO production, but (2) the peak [Ca2+]i elevation- or the integrated [Ca2+]i elevation-NO production relationships varies depending on the type of agonists. Our results thus demonstrate the presence of the agonists-dependent modulation of the relationship between [Ca2+]i elevation and NO production in endothelial cells ex vivo. British Journal of Pharmacology (2000) 130, 1140–1146; doi:10.1038/sj.bjp.0703420

Using fura-PE3 fluorimetry and α-toxin permeabilization, the characteristics of the contractile responses to neurokinin A (NKA) were determined in the pregnant rat myometrium. NKA induced contractions in rat myometrium in a concentration-dependent manner. There were no significant differences in the maximum contractions and EC50 values between the pregnant and non-pregnant myometrium, however, the contraction of only the former was greatly enhanced in the presence of phosphoramidon (PPAD), an endopeptidase inhibitor. In the pregnant myometrium, NKA induced sustained increases in [Ca2+]i and tension in normal physiological saline solution, while only small transient increases in [Ca2+]i and tension were observed in Ca2+-free solution. Both diltiazem (10 μM) and SK-F 96365 (10 μM) significantly inhibited the NKA-induced elevations of [Ca2+]i and tension. The effects were additive when these drugs were used together. NKA induced a significant leftward shift of the [Ca2+]i-tension curve obtained by changing the external Ca2+ (0–2.5 mM) during depolarization with high K+ solution. This Ca2+-sensitizing effect by NKA was also observed in the α-toxin permeabilized myometrium. These results indicated that in the pregnant rat myometrium: (1) the responsiveness to NKA increased, although it was masked by the increase in the endopeptidase activity; (2) NKA induced contractions of the myometrium by increasing both [Ca2+]i and the myofilament Ca2+ sensitivity and (3) The NKA-induced [Ca2+]i elevation was partly due to the intracellular Ca2+ release and mainly due to the Ca2+ influx, which was thought to be through both voltage dependent calcium channels and non-specification channels. British Journal of Pharmacology (2000) 130, 1165–1173; doi:10.1038/sj.bjp.0703410

The mechanism of endothelium-dependent regulation of vascular tone of bradykinin was investigated by simultaneously monitoring the changes in the cytosolic Ca2+ concentration and the force of smooth muscle in fura-2-loaded strips of the porcine renal artery with endothelium. During phenylephrine-induced sustained contraction, bradykinin (>3×10−9 M) caused endothelium-dependent triphasic changes in the force of the strips, composed of an initial relaxation, a subsequent transient contraction and a late sustained relaxation. At low concentrations (10−10–10−9 M), bradykinin caused an endothelium-dependent biphasic relaxation with no contraction. A thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10−5 M ONO-3708) completely inhibited, while a TXA2 synthase inhibitor (10−5 M OKY-046) only partially inhibited, the transient contraction induced by bradykinin. Under conditions where the bradykinin-induced contraction was inhibited by ONO-3708 during the phenylephrine-induced contraction, bradykinin induced only a transient relaxation in the presence of NΩ-nitro-L-arginine methyl ester (L-NAME). This transient relaxation was inhibited when the precontraction was initiated by phenylephrine plus 40 mM extracellular K+. The removal of L-NAME from this condition caused a partial reappearance of the initial relaxation and a complete reappearance of the sustained relaxation. In conclusion, bradykinin caused the endothelium-dependent triphasic regulation of vascular tone in the porcine renal artery. The concentrations of bradykinin required to induce a contraction was higher than that required to induce relaxation. Both TXA2 and PGH2 were involved in the bradykinin-induced contraction. The initial relaxation was mediated by nitric oxide and hyperpolarizing factors while the sustained relaxation depended on nitric oxide. British Journal of Pharmacology (2000) 129, 943–952; doi:10.1038/sj.bjp.0703141

The regulatory mechanism of thrombin receptor responsiveness in in situ endothelial cells was investigated by evaluating elevations of cytosolic Ca2+ concentration ([Ca2+]i) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the second thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused [Ca2+]i elevation, which was smaller than that seen without preceding stimulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of responsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in thrombin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by staurosporine-sensitive kinase, and restored the responsiveness through dephosphorylation by type 1 phosphatase. The mechanism of regulation of thrombin receptor activity in in situ endothelial cells differed from those reported in cultured endothelial cells. We suggest that the cell-specific regulatory mechanism may be altered by culture conditions.

1. Using front-surface fluorometry of fura-2-loaded strips, and measuring the transmembrane 45Ca2+ fluxes of ring preparations of the rabbit femoral artery, the mechanism underlying a sustained decrease in the cytosolic Ca2+ concentration ([Ca2+]i) induced by angiotensin II (AT-II) was investigated. 2. The application of AT-II during steady-state 118 mM K(+)-induced contractions caused a sustained decrease in [Ca2+]i following a rapid and transient increase in [Ca2+]i, while the tension was transiently enhanced. 3. When the intracellular Ca2+ stores were depleted by thapsigargin, the initial rapid and transient increase in [Ca2+]i was abolished, however, neither the sustained decrease in [Ca2+]i nor the enhancement of tension were affected. 4. Depolarization with 118 mM K+ physiological salt solution containing 1.25 mM Ba2+ induced a sustained increase in both the cytosolic Ba2+ concentration ([Ba2+]i) level and tension. However, the application of 10(-6) M AT-II during sustained Ba(2+)-contractions was found to have no effect on [Ba2+]i, but it did enhance tension. 5. After thapsigargin treatment, AT-II neither decreased nor increased the enhanced Ca2+ efflux rate induced by 118 mM K(+)-depolarization, whereas AT-II did increase the enhanced 45Ca2+ influx and the 45Ca2+ net uptake induced by 118 mM K(+)-depolarization. 6. Pretreatment with calphostin-C, partially, but significantly inhibited the decrease in [Ca2+]i induced by AT-II. 7. These findings therefore suggest that AT-II stimulates Ca2+ sequestration into the thapsigargin-insensitive Ca2+ stores, and thus induces a decrease in [Ca2+]i in the high external K(+)-stimulated rabbit femoral artery.

1. To investigate the underlying mechanism for the angiotensin II-induced desensitization of the contractile response during the prolonged stimulation of the vascular smooth muscle, we determined the effects of angiotensin-II on (1) cytosolic Ca2+ concentration ([Ca2+]i) and tension using fura-2-loaded medial strips of the rabbit femoral artery, (2) 45Ca2+ influx in ring preparations, and (3) Ca(2+)-sensitivity of the contractile apparatus in alpha-toxin permeabilized preparations. 2. In the presence of extracellular Ca2+, high concentrations of angiotensin-II elicited biphasic increases in [Ca2+]i and tension, which consisted of initial transient and subsequent lower and sustained phases. 3. The 45Ca2+ influx initially increased after the application of 10(-6) M angiotensin-II, and thereafter gradually decreased. At 20 min after the application, there was a discrepancy between the level of [Ca2+]i and the extent of 45Ca2+ influx. 4. The relationships between [Ca2+]i and tension suggested that the angiotensin-II-induced increase in the Ca(2+)-sensitivity of the contractile apparatus was maintained during the desensitization of smooth muscle contraction. 5. When 10(-6) M angiotensin-II was applied during the sustained phase of contraction induced by 118 mm K(+)-depolarization, at 10 min after the application, the [Ca2+]i levels were significantly lower and the tension levels were significantly higher than those prior to the application of angiotensin-II. 6. In conclusion, the decrease in [Ca2+]i, which is partially due to the inhibition of the Ca2+ influx, is mainly responsible for the desensitization evoked by high concentrations of angiotensin-II, and angiotensin-II seems to activate additional mechanisms which inhibit Ca2+ signaling during prolonged stimulation.

1. To elucidate the role of thapsigargin-induced Ca2+ entry in endothelial cells in the regulation of vascular tone, changes in Ca2+ and force of smooth muscle were simultaneously monitored in fura-2-loaded strips of porcine renal artery. 2. During phenylephrine-induced sustained contraction, thapsigargin caused an endothelium-dependent triphasic response; an initial relaxation, a subsequent transient contraction, and a sustained relaxation. The initial relaxation and the contraction were associated with a decrease and an increase in [Ca2+]i, respectively. There was no apparent [Ca2+]i decrease during the sustained relaxation. Thapsigargin-induced responses were observed at 10-8 M and higher concentrations, with the maximum response observed at 10-6 M. 3. The transient contraction was inhibited by a cyclo-oxygenase inhibitor (10-5 M indomethacin), a thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10-5 M ONO-3708), and a TXA2 synthase inhibitor (10-5 M OKY-046). 4. During the phenylephrine-induced contraction in the presence of indomethacin, thapsigargin caused an initial, but not a sustained relaxation, in the presence of Nomega-nitro-L-arginine methylester (L-NAME). During the contraction induced by phenylephrine plus 40 mM K+-depolarization in the presence of indomethacin, thapsigargin induced both a transient and a sustained relaxation. However, these relaxations were completely abolished in the presence of L-NAME. 5. Thapsigargin caused a large Ca2+ elevation in cultured endothelial cells of the renal artery. The concentration-response relation was thus similar to that for force development in the arterial strips. 6. In conclusion, thapsigargin-induced Ca2+ entry in endothelial cells led to triphasic changes in the tone of the porcine renal artery. The endothelium-dependent contraction was mediated mainly by TXA2. Nitric oxide and hyperpolarizing factor are both involved in the initial relaxation. However, a sustained relaxation was observed which mainly depended on nitric oxide.

A neuropeptide, galanin, regulates the reproductive process and directly induces myometrial contraction. The aim of this study was to determine the mechanism of galanin-induced myometrial contraction. For this purpose, we simultaneously measured intracellular Ca2+ concentration ([Ca2+]i) and tension using fura-PE3-fluorometry and the rat longitudinal myometrium. The effect of galanin on the Ca2+ sensitivity of the contractile apparatus was examined in beta-escin permeabilized strips. The expression of galanin and the galanin receptors mRNAs in the rat myometrium were determined by reverse transcription polymerase chain reaction (RT-PCR). Galanin (10-300 nM) induced phasic contraction with or without oscillation in the pregnant rat myometrium in a concentration-dependent manner. The maximal response was obtained at 100 nM. There was no significant difference either in the maximal responses or EC50 values for galanin-induced myometrial contractions among myometriums from non-pregnant and pregnant (day 4, day 11, day 20, day 22) rats. In the day 20 and 22 pregnant myometriums, assigning the levels of [Ca2+]i and tension at 40 mM K+-depolarization to be 100%, galanin increased the [Ca2+]i and tension to 126.9+/-2.9% and 116.3+/-2.7%, respectively. Diltiazem (10 microM) inhibited the galanin-induced elevation of [Ca2+]i and tension to 71.9+/-2.4% and 16.2+/-0.7%, respectively. Ni2+, by itself, decreased the basal [Ca2+]i to -50.2+/-3.9% without affecting resting tension. After Ni2+ treatment, galanin-induced increases in [Ca2+]i and tension were -19.6+/-3.4% and 0.9+/-0.1%, respectively. In myometrium treated with diltiazem, no oscillation in [Ca2+]i and tension was observed. In Ca2+-free solution with 0.1 mM EGTA, galanin increased [Ca2+]i from -40.2+/-2.7% to -18.0+/-2.6% and induced transient contraction (3.6+/-0.8%). In beta-escin permeabilized myometrium, galanin enhanced the contraction induced by 0.3 microM Ca2+ in the presence of GTP. In the presence of GDPbetaS (1 mM) instead of GTP, galanin failed to increase the Ca2+ sensitivity of the contractile apparatus. RT-PCR revealed that galanin mRNA was hardly expressed in the non-pregnant rat myometrium and increased to reach a maximal level at mid pregnancy (day 11), but decreased to the same level as in the non-pregnant myometrium at term (day 22). Type 2 galanin receptor (GALR2) mRNA was found to be expressed in the rat myometrium whereas type 1 galanin receptor (GALR1) mRNA expression was not detected. In conclusion, galanin induces contraction of the rat myometrium by increasing [Ca2+]i as well as by increasing Ca2+ sensitivity of the contractile apparatus. Galanin-induced increases in [Ca2+]i are caused by both intracellular Ca2+ release and Ca2+ influx from extracellular space. The responsiveness of the rat myometrium to galanin does not change during pregnancy. The galanin mRNA is expressed in the rat myometrium and it is upregulated during mid-pregnancy. Rat myometrium expresses GALR2 but not GALR1 mRNA. By changing mRNA expression in the myometrium during pregnancy, galanin may act as a paracrine or autocrine mediator in the regulation of myometrial contractility.

To investigate the expression of beta-adrenoceptor (AR) subtypes responsible for detrusor smooth muscle relaxation.Isolated rat detrusor smooth muscle was examined by tension measurement and reverse transcription polymerase chain reaction (RT-PCR).Norepinephrine (NE), epinephrine (EP) and isoproterenol (ISO) were found to relax the detrusor muscle pre-contracted by 6 x 10(-7) M carbachol in the presence of 10(-6) M phentolamine. NE relaxed the detrusor muscle as potently as EP. This potency order (NE=EP) thus indicated the beta-ARs of the rat detrusor muscle to possibly be a beta1 subtype. However, in the presence of 10(-6) M propranolol, beta1- and beta2- but not beta3-AR antagonist, NE showed a more potent relaxation than EP. This observation indicated that the rat detrusor muscle also possesses beta3-AR. RT-PCR revealed all three subtypes of beta-AR mRNA, namely beta1-, beta2- and beta3-AR mRNA, to be expressed in rat detrusor smooth muscle cells.We concluded that beta3-ARs exist in rat detrusor smooth muscle based on both pharmacological and molecular biological studies. Based on these findings, beta-ARs of rat detrusor smooth muscle are considered to be mixed populations consisting of three subtypes which play an important role in relaxing smooth muscle in response to catecholamines.

We investigated the expression and function of alpha1-adrenoceptor subtypes in the porcine renal artery. Reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequencing indicated that the mRNAs for alpha1a- and alpha1b-adrenoceptors were expressed in the porcine renal artery. Chloroethylclonidine, an alpha1B- and alpha1D-adrenoceptor antagonist, partially inhibited the phenylephrine-induced contraction, while 3 nM BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]dec ane-7,9-dione dihydrochloride), an alpha1D-adrenoceptor antagonist, had no effect. In contrast, 5-methylurapidil, an alpha1A-adrenoceptor antagonist, induced a rightward shift of the phenylephrine concentration-response curve. The simultaneous measurement of cytosolic Ca2+ concentration ([Ca2+]i) and tension revealed that chloroethylclonidine pretreatment abolished the phenylephrine-induced increases in [Ca2+]i and tension in the Ca2+-free solution. The application of 5-methylurapidil (3 nM) to the chloroethylclonidine-pretreated strips completely inhibited the 3 microM phenylephrine-induced [Ca2+]i and tension increase in normal PSS. We concluded that both alpha1A- and alpha1B-adrenoceptors mediate the phenylephrine-induced contraction of the porcine renal artery accompanied by an increase in [Ca2+]i, and that alpha1A-adrenoceptors cause Ca2+ influx whereas alpha1B-adrenoceptors mainly mediate Ca2+ release.

Endothelins (ETs) can modulate the airway smooth muscle tone. Using simultaneous measurements of cytosolic Ca2+ concentration ([Ca2+]i) and tension as well as the reverse transcription polymerase chain reaction (RT-PCR), we examined ET systems in the porcine trachea. In the functional study, the application of ET-1, ET-3 or sarafotoxin S6c (S6c) caused increases in [Ca2+]i and tension, in a concentration-dependent manner. These ET ligands were found to increase the Ca2+ sensitivity of the myofilament of the tracheal smooth muscle cells (SMCs). The contractions induced by ET-1 (10(-7) M), an ET receptor (ET-R) non-selective agonist, were much greater than those induced by S6c, an ET(B)-R selective agonist. BQ-123 (10(-6) M), an ET(A)-R antagonist, inhibited the ET-1 induced contraction. These functional experiments suggested the presence of both functioning ET(A)- and ET(B)-Rs in tracheal SMCs. RT-PCR experiments revealed that the tracheal SMCs expressed both ET(A)-R and ET(B)-R mRNAs, while tracheal epithelial cells (EpCs) predominantly expressed ET(A)-R mRNA. The porcine tracheal SMCs and EpCs also expressed pre-pro ET-1 (ppET-1), ppET-3, and endothelin converting enzyme-1 (ECE-1) mRNAs. These results suggested that ETs induce contraction of porcine tracheal SMCs not only by increasing [Ca2+]i but also increasing the Ca2+ sensitivity of the myofilament and that ETs could potentially be the autocrine and/or paracrine transmitters to regulate the contraction in the porcine airway smooth muscle.

This study was designed to investigate the mechanism of the relaxation induced by vasoactive intestinal peptide (VIP) in medial strips of the porcine coronary artery, by determining the effect on the cytosolic Ca2+ concentration ([Ca2+]i), the [Ca2+]i-force relation and the involvement of G-protein. Front-surface fluorometry of fura-2 revealed that U46619, a thromboxane A2 analogue, and the high K+-depolarization induced increases in both the [Ca2+]i and force of the medial strips. At a steady state of contraction, the extent of an increase in [Ca2+]i induced by 100 nM U46619 was similar to that induced by 30 mM K+-depolarization. VIP concentration-dependently (1 nM–1 μM) induced transient decreases in both the [Ca2+]i and force of the medial strips precontracted with 100 nM U46619. The decreases in the [Ca2+]i and force induced by VIP during the contraction with U46619 were much greater than those with 30 mM K+-depolarization. The VIP-induced decreases in the [Ca2+]i and force were attenuated by K+ channel blockers such as tetrabutylammonium (TBA: non-selective K+ channel blocker), charybdotoxin (large conductance Ca2+-activated K+ channel blocker), and 4-aminopyridine (4-AP: voltage-dependent K+ channel blocker). However, neither glibenclamide (ATP-sensitive K+ channel blocker) nor apamin (small conductance Ca2+-activated K+ channel blocker) had any significant inhibitory effect. In the 30 mM K+-depolarized strips, pretreatment with thapsigargin, a specific Ca2+-ATPase inhibitor of the Ca2+ store sites, completely abolished the VIP-induced decrease in [Ca2+]i, but partially attenuated the VIP-induced decrease in force. VIP shifted the [Ca2+]i-force relation of the U46619-induced contractions to the right in a concentration-dependent manner. In the α-toxin-permeabilized strips, VIP decreased the force development at a constant [Ca2+]i level (pCa=6.5) in a GTP-dependent manner, which was antagonized by guanosine-5′-O-(β-thiodiphosphate) (GDPβS). We thus conclude that VIP relaxes the coronary artery via three mechanisms: (1) a decrease in [Ca2+]i by inhibiting the Ca2+ influx presumably through the membrane hyperpolarization mediated by the activation of the large conductance Ca2+-activated (charybdotoxin-sensitive) K+ channels and voltage-dependent (4-AP-sensitive) K+ channels; (2) a decrease in [Ca2+]i by sequestrating cytosolic Ca2+ into thapsigargin-sensitive Ca2+ store sites; and (3) a decrease in the Ca2+-sensitivity of the contractile apparatus through the activation of G-protein.

By use of the reverse transcription polymerase chain reaction (RT-PCR), we determined the expression of adrenomedullin (AM) mRNA in the various tissues of the pig. To evaluate the significance of the expression of AM mRNA, we also determined the effects of AM on the cytosolic Ca2+ concentration ([Ca2+]i) and tension development of the porcine smooth muscle strips obtained from the coronary artery, pulmonary vein, trachea, ileum and urinary bladder. AM mRNA was widely expressed in the porcine tissues examined, which included myocardium (left and right ventricle and right atrium), kidney, lung, endothelial cells (aorta and aortic valve), smooth muscles (aorta, main pulmonary artery, pulmonary vein, renal artery and vein, coronary artery, ileum, trachea and urinary bladder) and epithelial cells (trachea and urinary bladder). AM induced a decrease in [Ca2+]i and tension of the coronary artery, but not the pulmonary vein. AM had no effects on either the [Ca2+]i or tension of the trachea and urinary bladder strips or on the tension development of strips of ileum. These results indicated that AM has a role as an autocrine and/or paracrine regulator of the coronary arterial tone. AM probably does not have an important role in the regulation of the pulmonary venous, tracheal, ileac and urinary bladder smooth muscle tone, even though AM mRNA is expressed in these tissues; the functional significance of AM in these smooth muscles remains to be determined. British Journal of Pharmacology (1997) 120, 193–200; doi:10.1038/sj.bjp.0700881

The expression of L- and T-type Ca 2+ channels has been reported to change during various biological events, including cellular differentiation and proliferation. The present study aimed to examine whether or not the expression of L- and T-type Ca 2+ channels depends on the cell cycle in rat aortic smooth muscle cells in primary culture. Both the phase of the cell cycle and the functional expression of Ca 2+ channels were determined in the same single cell, using an immunocytochemical analysis of cell cycle-specific nuclear antigens and a whole-cell voltage-clamp method, respectively. In the G 0 (n=130) and M (n=75) phases, all cells showed only L-type Ca 2+ currents. The cells showing a T-type Ca 2+ current appeared in the G 1 phase (37%, n=85) and increased in the S phase (90%, n=21). For L-type Ca 2+ channels, the current density was significantly greater in the G 1 phase than in the G 0 and M phases. However, either the voltage-dependent properties or the dose-response relationships of Bay K 8644- and second messenger-induced modulations of L-type Ca 2+ current did not differ in the four phases of the cell cycle. These findings thus indicate that the expression of L- and T-type Ca 2+ channels depends on the cell cycle, whereas the characteristics of L-type Ca 2+ channels do not differ between the phases of the cell cycle.

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.

1. The effects of nipradilol, a beta-adrenoceptor antagonist which possesses a nitroxy group, on cytosolic Ca2+ concentration ([Ca2+]i), and on tension development were simultaneously measured by front-surface fluorometry and fura-2-loaded strips in the proximal portion of pig coronary arteries. 2. Nipradilol reduced in a concentration-dependent manner both the [Ca2+]i and tension, irrespective of whether the strips were unstimulated or exposed to either high K+ or histamine containing solutions. However, both in the case of contractions induced by high K+-depolarization and histamine stimulation, for a given [Ca2+]i elevation the tension which developed in the presence of nipradilol was smaller than that generated in its absence, so that the [Ca2+]i-tension curves during the contraction were shifted to the right. 3. In the absence of extracellular Ca2+, the [Ca2+]i elevation due to the release of Ca2+ from histamine-sensitive store was inhibited by nipradilol. Nipradilol had no effect on the [Ca2+]i elevation due to the release of Ca2+ from caffeine-sensitive stores; however, it did inhibit the caffeine-induced increase in tension. A derivative of nipradilol, which lacked a nitroxy molecule (Nip(-N)), had no effect on the [Ca2+]i and tension elevated by histamine or caffeine in the absence of extracellular Ca2+. 4. The beta-adrenoceptor agonist, isoprenaline, reduced [Ca2+]i tension when applied to steady state contractions induced by high K+, or at the peak level of tension to histamine. The reduction of [Ca2+]i and tension induced by isoprenaline was inhibited by Nip(-N) in a concentration-dependent manner and nipradilol inhibited the isoprenaline-induced relaxation with bell-shaped concentration-response curves. At lower concentrations, nipradilol acted as a beta-blocker, the IC50- value being smaller than that of Nip(-N), and at higher concentrations, it acted as a nitrovasodilator. 5. Thus, it is suggested that, at lower concentrations, nipradilol, an antianginal drug, acts as a beta-adrenoceptor antagonist. At higher concentrations, it relaxes the proximal portion of the coronary artery by directly reducing [Ca2+]i and the Ca2+-sensitivity of the myofilaments, apparently due to the presence of the nitroxy molecule.

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.

We tested the hypothesis that adrenomedullin (ADM), a recently isolated hypotensive peptide, is an autocrine transmitter in renal arterial smooth muscle cells (RASMCs). Fura-2 fluorometry revealed that ADM decreases both the cytosolic Ca 2+ concentration ([Ca 2+ ]i) and tension induced by 0.3 μM phenylephrine (PE) in porcine RASMCs. The expression of ADM mRNA in RASMCs was assessed by the reverse transcription polymerase chain reaction (RT-PCR), using the total RNA from the RASMCs and the specific primers for the porcine ADM mRNA. A band with an expected size could be detected only when the reverse transcriptase was added, thus indicating that the porcine RASMCs express ADM mRNA. These results therefore suggested that ADM plays an important role in the regulation of the renal vascular tone, not only as a circulating hormone, but also as an autocrine transmitter.

The cellular mechanism underlying the regulation of the contraction of vascular smooth muscles by resting load is unknown. To determine the effects of changes in the resting load on vascular sensitivity to high K+ and to 9,11-dideoxy-11 alpha, 9 alpha-epoxy-methanoprostaglandin F2 alpha (U-46619), the force and cytosolic calcium concentration ([Ca2+]i) of arterial strips were recorded at resting loads of 200 (optimal load), 50, and 10 mg. A decrease in the resting load elicited a small decrease in the basal [Ca2+]i level without affecting the extent of maximal [Ca2+]i elevation induced by either stimulus. Through a decrease in the resting load, the concentration-response curves for the force development of high K+ or of U-46619 shifted to the right, whereas those for [Ca2+]i did not. We conclude that the basal [Ca2+]i level and the force development, but not the agonist-induced [Ca2+]i signals, of vascular smooth muscles depend on the resting load. We response that the resting load regulates the sensitivity of vascular smooth muscles, irrespective of types of stimuli, through a [Ca2+]i-insensitive mechanism.