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 β adrenoceptor agonist, and salbutamol, a β2 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, β adrenoceptor agonist would be a good drug to improve the decreased skin blood flow because it has selective vasorelaxation to ipsilateral plantar artery.
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in SLC26A4 cause a broad phenotypic spectrum, from typical Pendred syndrome to nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Identification of these mutations is ...important for accurate diagnosis, proper medical management and appropriate genetic counseling and requires updated information regarding spectrum, clinical characteristics and genotype-phenotype correlations, based on a large cohort. In 100 patients with bilateral enlarged vestibular aqueduct among 1511 Japanese hearing loss probands registered in our gene bank, goiter data were available for 79, of whom 15 had Pendred syndrome and 64 had nonsyndromic hearing loss. We clarified the mutation spectrum for the SLC26A4 mutations and also summarized hearing levels, progression, fluctuation and existence of genotype-phenotype correlation. SLC26A4 mutations were identified in 82 of the 100 patients (82.0%). Of the Pendred syndrome patients, 93% (14/15) were carriers, as were 77% (49/64) of the nonsyndromic hearing loss patients. Clinical characteristics of patients with SLC26A4 mutations were congenital, fluctuating and progressive hearing loss usually associated with vertigo and/or goiter. We found no genotype-phenotype correlations, indicating that, unlike in the case of GJB2 mutations, the phenotype cannot be predicted from the genotype. Our mutation analysis confirmed the importance of mutations in the SLC26A4 gene among hearing loss patients with enlarged vestibular aqueduct and revealed the mutation spectrum, essential information when performing genetic testing.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
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.
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.
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.
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•Prostaglandin E2 (PGE2) facilitated the activation of mouse hepatic stellate cells (HSCs) induced by fetal bovine serum.•In the presence of caffeine, PGE2 rather suppressed HSC activation.•RNA-sequencing analysis showed that HSCs treated with PGE2 plus caffeine were classified in the same class as quiescent HSCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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-bis2-(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.
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 3β (GSK3β) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3β (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of β-catenin and reduced the level of non-phospho (active) β-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/β-catenin signaling pathway through dephosphorylation of GSK3β. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis.
•A cellular slime mold product DIF-1 blocked hepatic stellate cell (HSC) activation.•An inhibitor of glycogen synthase kinase (GSK) 3β attenuated the effect of DIF-1.•The level of inactive GSK3β was significantly reduced by DIF-1.•DIF-1 reduced nuclear translocation of β-catenin and the level of active β-catenin.•DIF-1 is a possible candidate for liver fibrosis therapy to target HSC activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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