Enhanced Contraction of Arterial Smooth Muscle Cell in Skin Artery Is Sensitive to Hyperpolarization Mediated by BKCa Channel in Chronic Constriction Injury Model Rat.

Previous our study found that improvement of skin blood flow associated with neuropathic pain using vasodilators is useful for alleviation of neuropathic pain. In this study, we aimed to elucidate the mechanism underlying enhanced vasorelaxation induced by vasodilators, which increase cAMP and cyclic guanosine monophosphate (cGMP), in chronic constriction injury model rat. We assessed vasorelaxation effect of vasodilators by measurement of isometric contraction in isolated plantar artery from chronic constriction injury of sciatic nerve model rats. Nifedipine, a voltage-dependent Ca2+ channel inhibitor, NS1619, Ca2+-activated K+ (BKCa) channel opener, and diazoxide, an ATP-sensitive potassium channel opener, -induced vasorelaxation in ipsilateral plantar artery was enhanced compared to the these in contralateral plantar artery. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and substance P, a NK1 receptor agonist, caused vasorelaxation in both ipsilateral and contralateral artery. The vasorelaxation induced by SNP and substance P in ipsilateral artery is enhanced compared to the these in contralateral artery. Isoprenaline, a ß adrenoceptor agonist, and salbutamol, a ß2 adrenoceptor agonist, caused strong vasorelaxation in ipsilateral artery but not in contralateral artery. Iberiotoxin, a BKCa channel inhibitor, prominently suppressed the enhanced vasorelaxation induced by SNP, substance P, isoprenaline and salbutamol. In summary, the enhanced contraction of arterial smooth muscle cell in skin artery is sensitive to hyperpolarization in chronic constriction injury model rat. Furthermore, ß adrenoceptor agonist would be a good drug to improve the decreased skin blood flow because it has selective vasorelaxation to ipsilateral plantar artery.

Harmine suppresses collagen production in hepatic stellate cells by inhibiting DYRK1B.

Liver fibrosis is a major consequence of chronic liver disease, where excess extracellular matrix is deposited, due caused by the activation of hepatic stellate cells (HSCs). The suppression of collagen production in HSCs is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated effects of harmine, which is a ß-carboline alkaloid and known as an inhibitor of dual-specificity tyrosine-regulated kinases (DYRKs), on the production of collagen in HSCs. LX-2 cells, a human HSC cell line, were treated with harmine (0-10 µM) for 48 h in the presence or absence of TGF-ß1 (5 ng/ml). The expression of collagen type I α1 (COL1A1) and DYRK isoforms was investigated by Western blotting, quantitative RT-PCR, or immunofluorescence. The influence of knockdown of each DYRK isoform on the COL1A1 expression was further investigated. The expression of COL1A1 was markedly increased by treating with TGF-ß1 for 48 h in LX-2 cells. Harmine (10 µM) significantly inhibited the increased expression of COL1A1. LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, DYRK2, and DYRK4, although the expression of DYRK4 was much lower than the others. Knockdown of DYRK1B, but not DYRK1A or DYRK2, with siRNA significantly suppressed TGF-ß1-induced increase in COL1A1 expression. These results suggest that harmine suppresses COL1A1 expression via inhibiting DYRK1B in HSCs and therefore might be effective for the treatment of liver fibrosis.

Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3ß inactivation.

Differentiation-inducing factor-1 (DIF-1), a morphogen produced by the cellular slime mold Dictyostelium discoideum, is a natural product that has attracted considerable attention for its antitumor properties. Here, we report a novel inhibitory effect of DIF-1 on the activation of hepatic stellate cells (HSCs) responsible for liver fibrosis. DIF-1 drastically inhibited transdifferentiation of quiescent HSCs into myofibroblastic activated HSCs in a concentration-dependent manner, thus conferring an antifibrotic effect against in the liver. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, showed any effect on the inhibition of HSC activation by DIF-1. In contrast, TWS119, a glycogen synthase kinase 3ß (GSK3ß) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3ß (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of ß-catenin and reduced the level of non-phospho (active) ß-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/ß-catenin signaling pathway through dephosphorylation of GSK3ß. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis.

Mechanism of Membrane Depolarization Involved in α1A-Adrenoceptor-Mediated Contraction in Rat Tail and Iliac Arteries.

Our previous studies have shown that phenylephrine-induced contraction of cutaneous arteries is primarily mediated via α1A-adrenoceptors, but not α1D-adrenoceptors that generally mediate vascular contraction, and that the larger part of the contraction is induced in a voltage-dependent Ca2+ channel (VDCC)-independent manner. Here, we investigated the mechanism underlying the smaller part of the α1A-adrenoceptor-mediated contraction, i.e., VDCC-dependent one, in cutaneous arteries. Isometric contraction was measured with wire myograph in endothelium-denuded tail and iliac arterial rings isolated from male Wistar rats. LOE908 (10 µM), a cation channel blocker, partially inhibited the contraction induced by phenylephrine in tail and iliac arteries. Nifedipine (10 µM) further suppressed the phenylephrine-induced contraction that remained in the presence of LOE908 (10 µM) in iliac arteries but barely in tail arteries, suggesting that phenylephrine-induced depolarization in tail arteries is due to the activation of LOE908-sensitive cation channels. In iliac arteries, the contraction induced by A-61603, a specific α1A-adrenoceptor agonist, was also partially inhibited by LOE908 (10 µM); however, nifedipine had little effect on the A-61603-induced contraction that remained in the presence of LOE908 (10 µM), suggesting that depolarization mediated via α1A-adrenoceptors is due to the activation of LOE908-sensitive cation channels even in iliac arteries. These results suggest that membrane depolarization mediated via α1Α-adrenoceptors is caused by cation influx through LOE908-sensitive cation channels. Less contribution of VDCC to phenylephrine-induced contraction in tail arteries compared to in iliac arteries is likely due to that α1Α-adrenoceptor-mediated activation of VDCC is caused only by depolarization via cation influx through LOE908-sensitive cation channels.

Differential Contribution of Nerve-Derived Noradrenaline to High K+-Induced Contraction Depending on Type of Artery.

High K+-induced contraction of arterial smooth muscle is thought to be mediated by membrane depolarization and subsequent activation of voltage-dependent Ca2+ channels (VDCCs). In line with this, this study found that contraction induced by 80 mM K+ was almost abolished by nifedipine (1 µM), a VDCC inhibitor, in isolated rat aorta, and was markedly suppressed in the iliac artery. However, nifedipine (1 µM) only partially suppressed high K+-induced contraction in the tail artery. The contractions remaining in the arteries were further reduced by non-selective cation channel (NSCC) inhibitors, including 2-aminoethoxydiphenyl borate (2-APB) (100 µM), SK&F96365 (10 µM), and 3,4-dihydro-6,7-dimethoxy-α-phenyl-N,N-bis[2-(2,3,4-trimethoxyphenyl)ethyl]-1-isoquinolineacetamide hydrochloride (LOE908) (10 µM). In particular, sustained tonic contraction was nearly abolished. Prazosin (0.3 µM), an α1-adrenoceptor antagonist, partially inhibited high K+-induced contraction in the tail and iliac arteries, but had no effect in the aorta. Consistently, tyramine potently induced contraction in the tail and iliac arteries, but not in the aorta. Furthermore, the inhibition by prazosin and NSCC inhibitors of the high K+-induced contraction in the presence of nifedipine was comparable. These results suggest that depending on the type of artery, high K+-induced contraction is mediated by Ca2+ influx not only through VDCCs but also through NSCCs, the activation of which is due to the activation of α1-adrenoceptors by the released noradrenaline from sympathetic nerve terminals resulting from high K+ stimulation.

Caffeine Suppresses the Activation of Hepatic Stellate Cells cAMP-Independently by Antagonizing Adenosine Receptors.

During liver injury, hepatic stellate cells (HSCs) are activated by various cytokines and transdifferentiated into myofibroblast-like activated HSCs, which produce collagen, a major source of liver fibrosis. Therefore, the suppression of HSC activation is regarded as a therapeutic target for liver fibrosis. Several epidemiological reports have revealed that caffeine intake decreases the risk of liver disease. In this study, therefore, we investigated the effect of caffeine on the activation of primary HSCs isolated from mice. Caffeine suppressed the activation of HSC in a concentration-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator, had no effect on the caffeine-induced suppression of HSC activation. None of the isoform-selective inhibitors of phosphodiesterase1 to 5 affected changes in the morphology of HSC during activation, whereas CGS-15943, an adenosine receptor antagonist, inhibited them. Caffeine had no effect on intracellular cAMP level or on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. In contrast, caffeine significantly decreased the phosphorylation of Akt1. These results suggest that caffeine inhibits HSC activation by antagonizing adenosine receptors, leading to Akt1 signaling activation.

Caffeine-induced inversion of prostaglandin E2 effects on hepatic stellate cell activation.

BACKGROUND AND AIMS: Liver inflammation leads to the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. The present study aimed to investigate the effects of prostaglandin E2 (PGE2), which is biosynthesized by Kupffer cells, hepatocytes, and HSCs during inflammation, on HSC activation, including its combinatory effect with caffeine. METHODS: HSCs isolated from mice were activated by culturing in a medium supplemented with 10% fetal bovine serum for 7 days on plastic plates. The activation of HSCs was evaluated by immunofluorescence of α-smooth muscle actin in HSCs. Comprehensive gene expression analysis was performed using mRNA-sequencing to compare HSCs cultured for 1 or 7 days, with or without PGE2, caffeine, or both. RESULTS: PGE2 (1 µM) facilitated the activation of HSCs but inhibited the HSC activation in the presence of caffeine (3 mM). Comprehensive gene expression analysis revealed that HSCs treated with PGE2 in the presence of caffeine were classified in the same class as HSCs cultured for 1 day, i.e., quiescent HSCs. In contrast, PGE2 did not exhibit an inhibitory effect on HSC activation when co-treated with any isoform-specific phosphodiesterase inhibitors. Although the adenylate cyclase inhibitor 2',5'-dideoxyadenosine suppressed the elevation of intracellular cAMP level induced by PGE2 in the presence of caffeine, it had no effect on the inhibition of HSC activation by PGE2 plus caffeine. CONCLUSION: The effect of PGE2 on HSC activation is changed from facilitatory to inhibitory when combined with caffeine, suggesting that caffeine may effectively suppress liver fibrosis during inflammation.

Na+-dependent inactivation of vascular Na+/Ca2+ exchanger responsible for reduced peripheral blood flow in neuropathic pain model.

Reduced skin blood flow has been reported in neuropathic pain patients as well as various peripheral neuropathic pain model animals. We have previously shown that vasodilators, which improves reduced skin blood flow, correlatively alleviate neuropathic pain in chronic constriction injury (CCI) mice, a model of neuropathic pain from peripheral nerve injury. Here, we sought to elucidate the mechanism underlying the reduced skin blood flow in CCI rats. The skin blood flow of the ipsilateral plantar arteries was significantly reduced compared to that of the contralateral ones 4 weeks after loose ligation of the sciatic nerve. The contraction induced by noradrenaline, serotonin, and U46619, a thromboxane receptor agonist, in the isolated ipsilateral plantar arteries was significantly enhanced compared to that in the contralateral ones. KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, shifted the concentration-response curves of noradrenaline to the left in the contralateral arteries but had no effect on the ipsilateral side. There was no significant difference in concentration-response curves of noradrenaline between the ipsilateral and contralateral arteries in the presence of KB-R7943. Amiloride, a non-specific inhibitor of Na+ channels and transporters, comparably shifted concentration-response curves of noradrenaline to the left in both the contralateral and ipsilateral arteries. One hundred nM of noradrenaline induced intracellular Ca2+ elevation in the ipsilateral arteries, which was significantly larger than that induced by 300-nM noradrenaline in the contralateral arteries. These results suggest that reduced peripheral blood flow after nerve injury is due to Na+-dependent inactivation of NCX in the ipsilateral plantar arteries.

Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca2+ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd3+ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase.

Toxins affecting actin filaments and microtubules.

Actin and tubulin are the two major proteins of the cytoskeleton in eukaryotic cells and both display a common property to reversibly assemble into long and flexible polymers, actin filaments and microtubules, respectively. These proteins play important roles in a variety of cellular functions and are also involved in numbers of diseases. An emerging number of marine-derived cytotoxins have been found to bind either actin or tublin, resulting in either inhibition or enhancement of polymerization. Thus, these toxins are valuable molecular probes for solving complex mechanisms of biological processes. This chapter describes actin- and tubulin-targeting marine natural products and their modes of action, with reference to their use as research tools and their clinical applications.

Alleviation of mechanical stress-induced allodynia by improving blood flow in chronic constriction injury mice.

Reduced blood flow in the skin is observed in patients with neuropathic pain and in animal models. The aim of the present study was to elucidate the relationship between reduced skin blood flow and neuropathic pain in mice with a chronic constriction injury (CCI). Noradrenaline-induced contraction was enhanced in isolated plantar arteries ipsilateral to the CCI surgery compared to the contralateral arteries. Ten µM hydralazine, a peripheral vasodilator, at improved the enhanced contractile response in the ipsilateral arteries. The plantar blood flow in vivo was lower on the ipsilateral side of the CCI mice than on the contralateral side, and a 50% paw withdrawal threshold, as measured using the von Frey filament test, was lower on the former than on the latter side. An intraperitoneal injection (i.p.) of hydralazine (1 mg/kg) or phentolamine (5 mg/kg) improved blood flow in the skin and hyperalgesia in the ipsilateral plantar. In adrenalectomized CCI mice, plantar blood flow in the skin on the ipsilateral side was increased compared to in sham-operated mice, which was accompanied by alleviation of hyperalgesia. Moreover, the enhanced contractile response to noradrenaline was also observed in the ipsilateral plantar arteries isolated from the adrenalectomized CCI mice. Either hydralazine (1 mg/kg, i.p.) or an adrenalectomy barely affected mean arterial pressure in the CCI mice, whereas phentolamine (5 mg/kg, i.p.) lowered it. These results suggest that reduced blood flow in the skin contributes to neuropathic pain and that improving that blood flow with peripheral vasodilators, such as hydralazine, can alleviate it.

α1A-Adrenoceptors, but not α1B- or α1D-adrenoceptors, contribute to enhanced contractile response to phenylephrine in cooling conditions in the rat tail artery.

Cutaneous arteries show enhanced contraction in response to cooling, which is suggested to be mediated via α2C-adrenoceptors. We have previously shown that α1-adrenoceptors are also involved in the enhanced contraction in cooling conditions. In the present study, we aimed to identify the α1-adrenoceptor subtype involved in the response. Phenylephrine-induced contraction was enhanced by cooling to 24 °C in isolated rat tail arteries but suppressed in iliac arteries and aorta. At 37 °C, RS100329 (3 nM), an α1A-adrenoceptor antagonist, shifted the concentration-response curve of phenylephrine to the right in tail and iliac arteries, but not in aorta, while BMY7378 (10 nM), an α1D-adrenoceptor antagonist, shifted them to the right in aorta and iliac arteries, but not in tail arteries. At 24 °C, RS100329 (3 nM) shifted the concentration-response curve of phenylephrine to the right and decreased the maximum contraction in tail arteries. The inhibitory effects of RS100329 (3 nM) were more pronounced at 24 °C, compared to at 37 °C, implying larger contribution of α1A-adrenoceptors at 24 °C. In tail arteries, the maximum contraction of A-61603, an α1A-adrenoceptor agonist, was larger at 24 °C than at 37 °C. In contrast, in iliac arteries, the maximum contraction of A-61603 was smaller and its EC50 was smaller at 24 °C than at 37 °C. Under the condition where α1D-adrenoceptors were blocked, phenylephrine-induced contraction of iliac arteries was rather enhanced by cooling to 24 °C. These results suggest that α1A-adrenoceptors contribute to the enhanced contraction of cutaneous arteries in cooling conditions.

Differential contribution of calcium channels to α1-adrenoceptor-mediated contraction is responsible for diverse responses to cooling between rat tail and iliac arteries.

Our previous studies have shown that α1-adrenoceptors, in addition to α2-adrenoceptors, are involved in enhanced contraction of cutaneous blood vessels during cooling. The present study aimed to elucidate the mechanism underlying it. In tail and iliac arteries isolated from rats, isometric contraction was measured using a myograph and the phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) was quantified by western blotting. The phenylephrine-induced contraction was enhanced by cooling to 24 °C in tail arteries, but was suppressed in iliac arteries. Endothelium denudation or treatment with iberiotoxin enhanced the phenylephrine-induced contraction in tail arteries at 37 °C; however, neither affected the contraction at 24 °C. The phenylephrine-induced contraction at 37 °C was largely suppressed by nifedipine in iliac arteries, but only slightly in tail arteries. The Rho kinase inhibitor H-1152 largely suppressed the phenylephrine-induced contraction at 24 °C, but only slightly at 37 °C, in both arteries. The phosphorylation level of MYPT1 at Thr855 in tail arteries was increased by the cooling. Taken together, these results suggest the following mechanism in regard to cooling-induced enhancement of α1-adrenoceptor-mediated contraction in tail arteries: Cooling enhances the contraction of tail arteries via α1-adrenoceptor stimulation by reducing endothelium-dependent, large-conductance Ca2+-activated K+ channel-mediated relaxation and by inducing Rho kinase-mediated Ca2+ sensitization, although the latter occurs even in iliac arteries. A smaller contribution of voltage-dependent Ca2+ channels, which are largely suppressed by cooling, to α1-adrenoceptor-mediated contraction in tail arteries seems to be more crucially involved in the appearance of the enhanced contractile response to cooling.

G-protein coupled estrogen receptor-mediated non-genomic facilitatory effect of estrogen on cooling-induced reduction of skin blood flow in mice.

An enhanced vasoconstrictor activity of cutaneous arteries participates in the reduction of skin blood flow induced by cooling stimulation. Raynaud's phenomenon, which is characterized by intense cooling-induced constriction of cutaneous arteries, is more common in women during the period from menarche to menopause. We thus investigated the effect of 17ß-estradiol (E2) on cooling-induced reduction of plantar skin blood flow (PSBF) in mouse in vivo. Ovariectomized female ddY mice, anaesthetized with pentobarbital, were treated with tetrodotoxin for eliminating the sympathetic nerve tone and artificially ventilated. The PSBF was measured by laser Doppler flowmetry. Cooling air temperature around the foot from 25 to 20, 15, or 10°C decreased the PSBF in a temperature-dependent manner, which was suppressed by the specific α2C-adrenoceptor antagonist MK-912. When E2 was intravenously administered as a bolus followed by a constant infusion for 10min just before the cooling stimulation, the cooling-induced reduction of PSBF was facilitated by E2 in a dose-dependent manner. The facilitatory effect of E2 was not induced after the treatment with MK-912. Similar facilitatory effect was induced by an intravenous application of G-1, an agonist of G protein-coupled estrogen receptor (GPER, also termed GPR30). Moreover, the facilitatory effect of E2 was abolished by the GPER antagonist G15. These results suggest that acute administration of E2 leads to the facilitation of cooling-induced, α2C-adrenoceptor-mediated reduction of skin blood flow via the activation of the non-genomic estrogen receptor GPER.

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family.

By DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Calpha (PKCalpha) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCalpha activation in PCs.

RA-VII, a cyclic depsipeptide, changes the conformational structure of actin to cause G2 arrest by the inhibition of cytokinesis.

In L1210 cells, RA-VII (0.1-100 nM) caused the concentration-dependent inhibition of the proliferation and G2 arrest. Treatment of PC12 cells with 10 nM RA-VII changed cell shape round with binucleation, suggesting the inhibition of cytokinesis. The fluorescence intensity of FITC-phalloidin bound to F-actin was enhanced by RA-VII. In surface plasmon resonance experiments, the signal of F-actin was modified by RA-VII in close agreement with a concentration of FITC-phalloidin binding to F-actin. These results suggest that RA-VII causes the conformational change of F-actin and the stabilization of actin filaments to induce G2 arrest.

Functional role of Cl- channels in acidic pH-induced contraction of the aorta of spontaneously hypertensive and Wistar Kyoto rats.

pH regulates various cellular functions. Previously, we have described that acidic pH produces depolarization and contraction in isolated aorta from spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats [Br. J. Pharmacol. 118 (1996) 485]. The aim of the present study was to investigate the involvement of Cl- channels in acidic pH-induced contraction. Changing the pH of the bathing solution from 7.4 to 6.5 induced a contraction in both SHR and WKY aorta, which was 127.50+/-13.32% and 79.27+/-0.94% of the 64.8 mM KCl-induced contraction, respectively. The acidic pH-induced contraction was partially inhibited by the voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (1 microM) and nifedipine (0.1 microM). The Cl- channel inhibitors, diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (0.5 mM), 9-anthracene chloride (0.5 mM), indanyloxyacetic acid (30 microM) and niflumic acid (3 microM) also inhibited the acidic pH-induced contraction and the degree of attenuation was comparable to that of VDCC blockers. DIDS, 9-anthracene chloride and niflumic acid at concentrations used to inhibit the acidic pH-induced contraction also inhibited the 10 microM phenylephrine-induced contraction partially, without affecting the 64.8 mM KCl-induced contraction, whereas both the contractions were inhibited by indanyloxyacetic acid with equal efficacy. Indanyloxyacetic acid but not DIDS, 9-anthracene chloride or niflumic acid inhibited the 24.8 mM KCl-induced contraction. Simultaneous measurement of cytosolic Ca2+ and tension showed that niflumic acid reversed the increase in intracellular Ca2+ level and inhibited the contraction caused by acidic pH. Similarly, acidic pH depolarized the cultured vascular smooth muscle cells from SHR and the depolarization was completely reversible after the administration of niflumic acid. All these results suggest that the activation of Cl- channels is an important mechanism underlying the depolarization and contraction induced by acidic pH in SHR and WKY aortas.

Extracellular acidosis results in higher intracellular acidosis and greater contraction in spontaneously hypertensive rat aorta.

Acidic pH induces a contraction in aorta from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. The contractile response to acidic pH in SHR aorta is greater than that in WKY aorta. The purpose of this study was to investigate the correlation among extracellular pH (pH(o)), intracellular pH (pH(i)) and contraction in order to understand the exaggerated contractile response to acidic pH in SHR aorta. pH(i) measurement showed that at pH(o) 6.5, intracellular acidification was greater in SHR aorta than in WKY aorta. Decreasing pH(o) further to 6.2 in WKY aorta produced intracellular acidification close to that achieved at pH(o) 6.5 in SHR aorta, and at this level, the difference in contractile response between the two strains was also abolished. These results suggest that acidic pH(i), but not pH(o), is closely correlated with the contractile response and that the exaggerated contractile response in SHR aorta is due to a greater fall in pH(i).

Strain-specific effects of acidic pH on contractile state of aortas from Wistar and Wistar Kyoto rats.

The effects of acidosis were investigated on the resting and precontracted aortas from Wistar and Wistar Kyoto (WKY) rats. Decrease in pH from 7.4 to 6.5, having no effect on the resting tension of Wistar aorta, induced a marked contraction of WKY aorta. Acidic pH markedly relaxed the contraction to 300 nM phenylephrine in Wistar aorta, whereas in WKY aorta, it produced a biphasic response, an initial relaxation followed by potentiation of the contraction. In aortas loaded with fura 2-AM, phenylephrine caused an increase in intracellular Ca2+ ([Ca2+]i) and a contraction in both Wistar and WKY rats. pH 6.5 produced a decrease in [Ca2+]i to a near-basal level and almost abolished the phenylephrine-induced contraction in Wistar rat aorta. However, in WKY aorta, a biphasic response, an initial decline and later a recovery of [Ca2+]i level, was observed. Interestingly, at similar sustained [Ca2+]i, the contractile response to phenylephrine in WKY aorta was potentiated under acidic pH conditions. Acidic pH-induced inhibition of the contraction to phenylephrine was unaffected by iberiotoxin, 4-aminopyridine, and glibenclamide (Ca2+-activated, delayed rectifier and ATP-sensitive K+ channel inhibitors, respectively), in aortas from both Wistar and WKY. Decrease in extracellular pH was associated with a rapid fall in intracellular pH (pHi) and the intracellular acidification profile was not different in both strains. All these results show that acidic pH induces strain-specific inhibitory and excitatory effects on the contractile state of aortas from Wistar and WKY rats, respectively. The sustained and transient relaxant responses to acidic pH in Wistar and WKY aortas, respectively, are due to decrease in [Ca2+]i levels, but this decrease in [Ca2+]i is independent of the activation of K+ channels.

Functional role of ryanodine-sensitive Ca2+ stores in acidic pH-induced contraction in Wistar Kyoto rat aorta.

Acidic pH induced a contraction in the isolated aorta from Wistar Kyoto rat. The magnitude of contraction was dependent upon the degree of extracellular acidification. The maximum level of contraction observed at pH 6.5 was 84.6 +/- 3.4% of the 64.8 mM KCl-induced contraction. To investigate the role of extracellular as well as intracellular Ca(2+) in acidic pH-induced contraction (APIC), we changed the extracellular pH in the presence of EGTA. Sustained contraction induced by acidic pH in the presence of extracellular Ca(2+) was completely abolished in the presence of EGTA, while a transient but significant contraction was still observed. Ryanodine, a selective ryanodine receptor blocker and cyclopiazonic acid (CPA), an inhibitor of sarco-/endoplasmic reticulum Ca(2+) ATPase, abolished the transient contraction, when pH was decreased in Ca(2+)-free solution. On the other hand, neither xestospongin C, a selective inositol-1,4,5-trisphosphate receptor antagonist nor U-73122, a phospholipase C inhibitor showed this effect. These results suggest the involvement of Ca(2+) release from ryanodine-/CPA-sensitive store of sarcoplasmic reticulum (SR). In normal Ca(2+)-containing solution, ryanodine and CPA did not alter the maximum level of APIC. However, they significantly decreased the rate of rise of APIC. U-73122, suppressed the maximum contraction induced by acidic pH without affecting the rate of rise of APIC, while xestospongin C and U-73343, an inactive analogue of U-73122, had no effect on both parameters of APIC. From these results, it is concluded that acidic pH induces Ca(2+) release from the ryanodine-/CPA-sensitive store of SR and that release provides supportive effect on initiating rapid transient contraction, but not on the sustained contraction, which is entirely due to Ca(2+) influx.