RESUMEN
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.
Asunto(s)
Arterias , Sustancia P , Animales , Ratas , Isoproterenol , Constricción , Vasodilatadores , Nitroprusiato , Receptores de Neuroquinina-1 , Albuterol , Receptores AdrenérgicosRESUMEN
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.
Asunto(s)
Cadena alfa 1 del Colágeno Tipo I , Harmina , Células Estrelladas Hepáticas , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Factor de Crecimiento Transformador beta1 , Cadena alfa 1 del Colágeno Tipo I/antagonistas & inhibidores , Cadena alfa 1 del Colágeno Tipo I/biosíntesis , Harmina/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Quinasas DyrKRESUMEN
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.
Asunto(s)
Circulación Sanguínea/efectos de los fármacos , Neuralgia/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , Sodio/metabolismo , Vasodilatadores/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Amilorida/farmacología , Animales , Arterias/efectos de los fármacos , Compuestos de Boro/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Ionóforos de Calcio/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Contracción Muscular/efectos de los fármacos , Nifedipino/farmacología , Norepinefrina/farmacología , Ouabaína/farmacología , Ratas Wistar , Serotonina/farmacología , Tiourea/análogos & derivados , Tiourea/farmacología , Vasoconstrictores/farmacologíaRESUMEN
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.
Asunto(s)
Fenómenos Fisiológicos Celulares/efectos de los fármacos , Endotelina-1/farmacología , Células Estrelladas Hepáticas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Calcio/metabolismo , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Calmodulina/antagonistas & inhibidores , Calmodulina/metabolismo , Células Cultivadas , Antagonistas de los Receptores de Endotelina/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Masculino , Ratones , Miosina Tipo II/antagonistas & inhibidores , Miosina Tipo II/metabolismo , Fenilpropionatos/farmacología , Piridazinas/farmacología , ARN Mensajero/genética , Receptor de Endotelina A/metabolismo , Sulfonamidas/farmacología , Quinasas Asociadas a rho/metabolismoRESUMEN
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.
Asunto(s)
Cafeína/farmacología , Dinoprostona/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Inflamación/tratamiento farmacológico , Animales , Cafeína/administración & dosificación , Células Cultivadas , AMP Cíclico/metabolismo , Dinoprostona/administración & dosificación , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Inflamación/patología , Cirrosis Hepática/prevención & control , Masculino , Ratones , Factores de TiempoRESUMEN
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.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Células Estrelladas Hepáticas/efectos de los fármacos , Hexanonas/farmacología , Transporte Activo de Núcleo Celular , Adenina/análogos & derivados , Adenina/farmacología , Animales , Antineoplásicos/farmacología , Diferenciación Celular , Transdiferenciación Celular , Dictyostelium , Relación Dosis-Respuesta a Droga , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Ratones , Oxadiazoles/farmacología , Fosforilación , Pirimidinas/farmacología , Pirroles/farmacología , Quinoxalinas/farmacología , Transducción de Señal , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Canales de Calcio/fisiología , Arteria Ilíaca/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Cola (estructura animal)/irrigación sanguínea , Acetamidas/farmacología , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Imidazoles/farmacología , Isoquinolinas/farmacología , Masculino , Nifedipino/farmacología , Fenilefrina/farmacología , Ratas Wistar , Cola (estructura animal)/fisiología , Tetrahidronaftalenos/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
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 (1â¯mg/kg) or phentolamine (5â¯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 (1â¯mg/kg, i.p.) or an adrenalectomy barely affected mean arterial pressure in the CCI mice, whereas phentolamine (5â¯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.
Asunto(s)
Hiperalgesia/fisiopatología , Flujo Sanguíneo Regional , Estrés Mecánico , Animales , Constricción , Hiperalgesia/inducido químicamente , Hiperalgesia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Neuralgia/complicaciones , Neuralgia/fisiopatología , Umbral del Dolor/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/efectos de los fármacos , Piel/fisiopatología , Vasodilatadores/farmacologíaRESUMEN
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 (3â¯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 (10â¯nM), an α1D-adrenoceptor antagonist, shifted them to the right in aorta and iliac arteries, but not in tail arteries. At 24⯰C, RS100329 (3â¯nM) shifted the concentration-response curve of phenylephrine to the right and decreased the maximum contraction in tail arteries. The inhibitory effects of RS100329 (3â¯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.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Fenilefrina/farmacología , Receptores Adrenérgicos alfa 1/metabolismo , Vasoconstricción/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Frío , Arteria Ilíaca/efectos de los fármacos , Arteria Ilíaca/metabolismo , Arteria Ilíaca/fisiología , Masculino , Modelos Animales , Piperazinas/farmacología , Ratas , Ratas Wistar , Piel/irrigación sanguínea , Timina/farmacologíaRESUMEN
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.
Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Canales de Calcio/fisiología , Frío/efectos adversos , Receptores Adrenérgicos alfa 1/fisiología , Vasoconstricción/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Arteria Ilíaca/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio , Masculino , Modelos Animales , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Nifedipino/farmacología , Fenilefrina/farmacología , Fosforilación , Proteína Fosfatasa 1/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Cafeína/farmacología , AMP Cíclico/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Receptores Purinérgicos P1/efectos de los fármacos , Animales , Células Cultivadas , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Cirrosis Hepática/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Fosforilación , Quinazolinas/farmacología , Triazoles/farmacologíaRESUMEN
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.
Asunto(s)
Arterias/inervación , Arterias/fisiología , Contracción Muscular/fisiología , Norepinefrina/fisiología , Acetamidas/farmacología , Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Compuestos de Boro/farmacología , Calcio/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Imidazoles/farmacología , Canales Iónicos/fisiología , Isoquinolinas/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/inervación , Músculo Liso Vascular/fisiología , Nifedipino/farmacología , Potasio/farmacología , Prazosina/farmacología , Ratas WistarRESUMEN
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.
Asunto(s)
Frío/efectos adversos , Estradiol/farmacología , Estrógenos/farmacología , Receptores de Estrógenos/metabolismo , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguínea , Animales , Femenino , Ratones , Ovariectomía , Vasoconstricción/efectos de los fármacosRESUMEN
Cutaneous arteries are known to constrict in response to cooling via α2C-adrenoceptors. The involvement of α1-adrenoceptors in the cooling response has also recently been suggested by in vivo studies in mice. The present study was thus aimed to confirm it in the isolated mouse cutaneous plantar artery. Changes in vessel diameter were measured by pressurized arteriography. Myogenic constriction was induced depending on intraluminal pressure, and was nearly abolished by the Ca(2+) channel blocker nifedipine or by lowering bath temperature to 24°C. The α1-adrenoceptor agonist phenylephrine produced two-phase constriction composed of phasic and tonic components, both of which were enhanced by the cooling to 24°C. Nifedipine partly inhibited the phenylephrine constriction at 37°C, and the nifedipine-resistant constriction was further inhibited by the inositol 1,4,5-trisphosphate (IP3) receptor inhibitor xestospongin C. Although the cooling to 24°C still enhanced the phenylephrine constriction in the presence of nifedipine, the enhancement was not observed in the presence of both nifedipine and xestospongin C. In Ca(2+)-free solution, phenylephrine produced two-phase constriction at 37°C, which was abolished by 30-min treatment with thapsigargin, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). In contrast, short-term treatment with thapsigargin for 3min rather enhanced the phenylephrine constriction in Ca(2+)-free solution at 37°C; however, the enhanced constriction by the cooling to 24°C was not further enhanced by the SERCA inhibitor. These results suggest that cooling inhibits Ca(2+) re-uptake by SERCA, thereby enhancing constriction induced by Ca(2+) released via IP3 receptors in the mouse plantar artery.
Asunto(s)
Receptores Adrenérgicos alfa 1/fisiología , Piel/irrigación sanguínea , Vasoconstricción/efectos de los fármacos , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Arterias/efectos de los fármacos , Arterias/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Frío/efectos adversos , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Masculino , Ratones , Nifedipino/farmacología , Fenilefrina/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Piel/efectos de los fármacos , Tapsigargina/farmacología , Vasoconstricción/fisiología , Vasodilatadores/farmacologíaRESUMEN
An enhanced vasocontrictor activity of cutaneous vessels participates in the reduction of skin blood flow induced by cooling. The present study investigated changes in the local response to cooling in hyperglycemic conditions. Male diabetic db/db and control C57BL/6J mice, anaesthetized with pentobarbitone, were treated with tetrodotoxin for eliminating the sympathetic nerve tone and artificially ventilated. The plantar skin blood flow (PSBF) was measured by laser Doppler flowmetry. Cooling the air temperature around the foot reduced PSBF in a temperature-dependent manner in control and db/db mice. The PSBF reduction was significantly smaller in db/db mice than in control mice. Phentolamine, a non-selective α-antagonist, bunazosin, a selective α1-antagonist, MK-912, a selective α2C-antagonist, and Y-27632, a Rho-kinase inhibitor, significantly inhibited the PSBF reduction induced by cooling to 15 °C in both mice and the inhibitory effects were comparable between these mice. The cooling-induced PSBF reduction was also significantly inhibited by N(ω)-nitro-L-arginine, an inhibitor of nitric oxide synthase, in control mice; however, the inhibitory effect of N(ω)-nitro-L-arginine was not observed in db/db mice. The reduction of PSBF induced by the intraarterial administration of adrenaline was comparable between control and db/db mice both before and after the treatment with N(ω)-nitro-L-arginine. It is thus likely that the reduction of skin blood flow induced by local cooling might be partly mediated by a decrease in endothelium-derived nitric oxide production, and that an impairment of the nitric oxide production might be related to reduced vasocontrictor response to cooling in db/db mice.
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Diabetes Mellitus/fisiopatología , Óxido Nítrico/fisiología , Flujo Sanguíneo Regional/fisiología , Piel/irrigación sanguínea , Antagonistas Adrenérgicos alfa/farmacología , Amidas/farmacología , Animales , Frío , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Temperatura Cutánea/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
In addition to its regulatory effect on bone mass, calcitonin has been shown to relieve pain and alleviate peripheral circulatory disturbance in patients with Raynaud's syndrome and complex regional pain syndrome. In the present study, we investigated whether calcitonin ameliorates diminished blood flow and enhanced arterial contraction in response to noradrenaline in chronic constriction injury (CCI) of the sciatic nerve in rats. Following surgically induced CCI, laser Doppler flowmetry studies showed a significant decrease in plantar skin blood flow of the ipsilateral hind paw compared to the contralateral side. A subcutaneous bolus injection of elcatonin (20 U/kg), a synthetic derivative of eel calcitonin, significantly improved decreased skin blood flow in the ipsilateral side. In vitro analysis of plantar arteries isolated from the ipsilateral hind paw 7-13 days after the CCI procedure showed higher sensitivity to noradrenaline than the plantar arteries from the contralateral side. Elcatonin (0.1-10 nm) significantly reduced noradrenaline-induced contraction in the arteries of the ipsilateral side, whereas it had little effect on those of the contralateral side. These results suggest that calcitonin selectively ameliorates enhanced arterial contractility in CCI neuropathic rats, thus leading to its alleviating effect on peripheral circulatory disturbance.
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Arterias/fisiología , Calcitonina/fisiología , Proteínas de Peces/fisiología , Nervio Ciático/lesiones , Nervio Ciático/fisiología , Vasoconstricción/fisiología , Animales , Velocidad del Flujo Sanguíneo/fisiología , Enfermedad Crónica , Constricción Patológica/fisiopatología , Constricción Patológica/terapia , Anguilas , Miembro Posterior/irrigación sanguínea , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Piel/irrigación sanguíneaRESUMEN
An involvement of signal transduction other than phosphatidylinositol turnover in thromboxane A(2) receptor (TP receptor)-mediated vascular contraction was investigated in rat aorta. The contraction induced by U46619, a TP receptor agonist, at low concentrations (≤ 30 nM) was partially inhibited by verapamil, an inhibitor of voltage-dependent Ca(2+) channels (VDCC), and was further diminished in Ca(2+)-free solution. Twenty nanomolar of U46619 induced contraction and elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)), which were consisted of two phases; slowly developing first phase followed by quickly rising second phase. The second phase was inhibited by verapamil, and all the [Ca(2+)](i) response was abolished in Ca(2+)-free solution. The contraction and [Ca(2+)](i) elevation induced by 20 nM U46619 were not inhibited by U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or GF109203X, a protein kinase C inhibitor, but were abolished by D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). However, D609 had no effect on those induced by 1 µM phenylephrine. The U46619-induced responses were also partially inhibited by cation channel blockers, 2-APB and LOE908. The inhibition by LOE908 was abolished in the presence of verapamil, suggesting that LOE908-sensitive cation channels lead to the activation of VDCC by depolarizing plasma membrane. In contrast, 2-APB further diminished the U46619-induced [Ca(2+)](i) elevation in the presence of verapamil. In conclusion, TP receptor stimulation is suggested to be coupled with PC-PLC. Diacylglycerol produced by PC-PLC seems to activate two types of cation channels independently of PKC, which in turn leads to VDCC-dependent and independent Ca(2+) influx, thereby eliciting contraction.
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Aorta/citología , Calcio/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Fosfatidilcolinas/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Fosfolipasas de Tipo C/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Canales Iónicos/metabolismo , Masculino , Ratas , Ratas Wistar , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Vasoconstricción/efectos de los fármacosRESUMEN
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.
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Actinas/fisiología , Toxinas Marinas/toxicidad , Microtúbulos/efectos de los fármacos , Actinas/química , Actinas/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Estabilidad de Medicamentos , Humanos , Toxinas Marinas/química , Toxinas Marinas/uso terapéutico , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/fisiologíaRESUMEN
Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca(2+)-mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase Cgamma in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice.
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Proteínas de la Membrana/fisiología , Mutación , Células de Purkinje/fisiología , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Cerebelo/citología , Cerebelo/metabolismo , Estimulación Eléctrica , Perfilación de la Expresión Génica , Immunoblotting , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Proteína Quinasa C/metabolismo , Células de Purkinje/metabolismoRESUMEN
Notch signaling plays an important role in the process of cell-fate assignation during nervous system development. DNER is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats and is expressed in somatodendritic regions. In vitro studies demonstrated that DNER mediates Notch signaling by cell-cell interaction. In the cerebellum, DNER is abundantly expressed in Purkinje cells and moderately in granule cells. DNER-knockout mice showed motor discoordination. The mutant cerebellum showed morphological impairments of Bergmann glia and multiple innervation between climbing fibers and Purkinje cells. Moreover, glutamate clearance at the synapses between parallel fibers and Purkinje cells was significantly weakened, and the expression of GLAST, a glutamate transporter in Bergmann glia, was reduced in the mutant cerebellum. Therefore, DNER contributes to the morphological and functional maturation of Bergmann glia via the Notch signaling pathway, and is essential for precise cerebellar development.