RESUMEN
Premenopausal women are relatively protected from developing hypertension compared to men. Perivascular adipose tissue (PVAT) has been shown to mediate vasoactive effects; however, a sex-dependent difference in PVAT function in the setting of hypertension has not yet been explored. We investigated the effect of PVAT on resistance vessel biology in male and female 16 week old stroke prone spontaneously hypertensive rats (SHRSP). This preclinical model of hypertension exhibits a sex-dependent difference in the development of hypertension similar to humans. Wire myography was used to assess vascular function in third-order mesenteric arteries. KATP channel-mediated vasorelaxation by cromakalim was significantly impaired in vessels from SHRSP males + PVAT relative to females (maximum relaxation: male + PVAT 46.9 ± 3.9% vs. female + PVAT 97.3 ± 2.7%). A cross-over study assessing the function of male PVAT on female vessels confirmed the reduced vasorelaxation response to cromakalim associated with male PVAT (maximum relaxation: female + PVATfemale 90.6 ± 1.4% vs. female + PVATmale 65.8 ± 3.5%). In order to explore the sex-dependent differences in PVAT at a molecular level, an adipokine array and subsequent western blot validation identified resistin expression to be increased approximately 2-fold in PVAT from male SHRSP vessels. Further wire myography experiments showed that pre-incubation with resistin (40 ng/ml) significantly impaired the ability of female + PVAT vessels to relax in response to cromakalim (maximum relaxation: female + PVAT 97.3 ± 0.9% vs. female + PVAT + resistin[40ng/ml] 36.8 ± 2.3%). These findings indicate a novel role for resistin in mediating sex-dependent vascular function in hypertension through a KATP channel-mediated mechanism.
Asunto(s)
Tejido Adiposo/patología , Hipertensión/patología , Hipertensión/fisiopatología , Arterias Mesentéricas/fisiopatología , Resistina/metabolismo , Caracteres Sexuales , Tejido Adiposo/metabolismo , Animales , Cromakalim/metabolismo , Femenino , Regulación de la Expresión Génica , Hipertensión/metabolismo , Masculino , Ratas , Ratas Endogámicas SHRRESUMEN
ATP-sensitive K(+) channels (K(ATP)) are an octameric complex of inwardly rectifying K(+) channels (Kir6.1 and Kir6.2) and sulfonylurea receptors (SUR1 and SUR2A/B), which are involved in several diseases. The tissue-selective expression of the subunits leads to different channels; however, the composition and role of the functional channel in native muscle fibers is not known. In this article, the properties of K(ATP) channels of fast-twitch and slow-twitch muscles were compared by combining patch-clamp experiments with measurements of gene expression. We found that the density of K(ATP) currents/area was muscle-type specific, being higher in fast-twitch muscles compared with the slow-twitch muscle. The density of K(ATP) currents/area was correlated with the level of Kir6.2 expression. SUR2A was the most abundant subunit expressed in all muscles, whereas the vascular SUR2B subunit was expressed but at lower levels. A significant expression of the pancreatic SUR1 was also found in fast-twitch muscles. Pharmacological experiments showed that the channel response to the SUR1 agonist diazoxide, SUR2A/B agonist cromakalim, SUR1 antagonist tolbutamide, and the SUR1/SUR2A/B-antagonist glibenclamide matched the SURs expression pattern. Muscle-specific K(ATP) subunit compositions contribute to the physiological performance of different muscle fiber types and determine the pharmacological actions of drugs modulating K(ATP) activity in muscle diseases.
Asunto(s)
Adenosina Trifosfato/química , Biofisica/métodos , Canales de Potasio/química , Proteínas Recombinantes de Fusión/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiarrítmicos/farmacología , Antihipertensivos/farmacología , Cromakalim/metabolismo , Cromakalim/farmacología , Cartilla de ADN/química , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Gliburida/farmacología , Hipoglucemiantes/farmacología , Canales KATP , Cinética , Masculino , Modelos Estadísticos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculos/metabolismo , Técnicas de Placa-Clamp , Fenotipo , Potasio/química , Potasio/metabolismo , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Ratas , Ratas Wistar , Receptores de Droga/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores de Sulfonilureas , Tolbutamida/farmacologíaRESUMEN
Responses to human calcitonin gene-related peptide (hCGRP) and human adrenomedullin (hADM) hAmylin were investigated in isolated mesenteric resistance arteries from the rat. The results of the present investigation show that hCGRP, hAmylin, and hADM induce dose-related vasodilator responses in isolated resistance arteries from the rat mesenteric vascular bed. Vasodilator responses to hCGRP and hAmylin were not altered after denuding the vascular endothelium, after administration of the nitric oxide synthase inhibitor L-NA, or after administration of the soluble guanylate cyclase inhibitor ODQ, suggesting that vasodilator responses to hCGRP and hAmylin are not mediated by the release of nitric oxide from the vascular endothelium and the subsequent increase in cGMP. Vasodilator responses to hCGRP, hAmylin, and hADM were not altered by the vascular selective K+(ATP) channel antagonist U-37883A. The role of the CGRP1 receptor was investigated and responses to hCGRP and hAmylin, but not hADM, were significantly reduced following administration of hCGRP-(8-37). Moreover, vasodilator responses to hCGRP and hAmylin, but not hADM, were significantly reduced by hAmylin-(8-37), suggesting that an hAmylin-(8-37)-sensitive receptor mediates responses to hCGRP and hAmylin in the rat mesenteric artery. These data suggest that hCGRP and hAmylin have direct vasodilator effects in the isolated mesenteric resistance artery that are mediated by hAmylin-(8-37)- and hCGRP-(8-37)-sensitive receptors.
Asunto(s)
Adamantano/análogos & derivados , Amiloide/farmacología , Arginina/análogos & derivados , Péptido Relacionado con Gen de Calcitonina/farmacología , Arterias Mesentéricas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Receptores de Péptido Relacionado con el Gen de Calcitonina/fisiología , Receptores de Péptidos/fisiología , Acetilcolina/farmacología , Adamantano/farmacología , Adrenomedulina , Animales , Arginina/farmacología , Cromakalim/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Polipéptido Amiloide de los Islotes Pancreáticos , Arterias Mesentéricas/fisiología , Morfolinas/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxadiazoles/farmacología , Perfusión , Bloqueadores de los Canales de Potasio , Quinoxalinas/farmacología , Ratas , Receptores de Péptido Relacionado con el Gen de Calcitonina/efectos de los fármacos , Receptores de Polipéptido Amiloide de Islotes Pancreáticos , Receptores de Péptidos/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
K(ATP) channel openers are a diverse group of drugs with a wide range of potential therapeutic uses. Their molecular targets, the K(ATP) channels, exhibit tissue-specific responses because they possess different types of regulatory sulfonylurea receptor subunits. It is well recognized that complex interactions occur between K(ATP) channel openers and nucleotides, but the cloning of the K(ATP) channel has introduced a new dimension to the study of these events and has furthered our understanding of the molecular basis of the action of K(ATP) channel openers.
Asunto(s)
Minoxidil/análogos & derivados , Canales de Potasio/efectos de los fármacos , Adenosina Trifosfato/fisiología , Animales , Sitios de Unión , Cromakalim/metabolismo , Cromakalim/farmacología , Diazóxido/metabolismo , Diazóxido/farmacología , Humanos , Minoxidil/metabolismo , Minoxidil/farmacología , Nicorandil/metabolismo , Nicorandil/farmacología , Pinacidilo/metabolismo , Pinacidilo/farmacología , Canales de Potasio/metabolismo , Canales de Potasio/fisiologíaRESUMEN
1. The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a KD value of 9.2 +/- 5.2 nM and a binding capacity (BMax) of 140 +/- 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5-10 per microns2 plasmalemma was estimated. 3. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 +/- 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 +/- 40 nM and 870 +/- 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 microM) was completely reversed by glibenclamide with an IC50 value of 86 +/- 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 microM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 microM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 microM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 micron-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.