RESUMEN

Transient receptor potential canonical type 3 channels (TRPC3) are expressed in neural, cardiac, respiratory and vascular tissues, with both similarities and differences between human and animal models for the same cell types. In common with all members of the six subfamilies of TRP channels, TRPC3 are non-voltage gated, non-selective cation channels that are mainly permeated by Ca2+, and have distinct molecular, biophysical, anatomical and functional properties. TRP channels are present in excitable and non-excitable cells where they sense and respond to a wide variety of physical and chemical stimuli. TRPC3 are expressed in the endothelium and/or smooth muscle of specific intact arteries, such as mesenteric, cerebral and myometrial, where they are critical for the control of vascular tone, and show altered activity in development and disease. In artery endothelium, TRPC3 contributes to endothelium-derived hyperpolarization and nitric oxide-mediated vasodilation. In artery smooth muscle, TRPC3 contributes to constrictor mechanisms. In both endothelium and smooth muscle, TRPC3 contributes to function via caveolae-caveolin dependent and independent mechanisms. In different cell types and states, like other TRP channels, TRPC3 can form complexes with other TRP proteins and associated channels and accessory proteins, including those associated with endo(sarco)plasmic reticulum (ER/SR), thereby facilitating Ca2+ channel activation and/or refilling ER/SR Ca2+ stores. The diversity of TRPC3 interactions with other vascular signaling components is a potential target for artery specific control mechanisms. This brief perspective highlights recent advances in understanding the functional diversity of TRPC3, with an emphasis on vascular health and disease.

Asunto(s)

Calcio/metabolismo , Endotelio Vascular/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Humanos , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Especificidad de la Especie , Enfermedades Vasculares/metabolismo , Vasodilatación/fisiología

RESUMEN

This study investigated the expression and function of transient receptor potential vanilloid type-3 ion channels (TRPV3) in uterine radial arteries isolated from non-pregnant and twenty-day pregnant rats. Immunohistochemistry (IHC) suggested TRPV3 is primarily localized to the smooth muscle in arteries from both non-pregnant and pregnant rats. IHC using C' targeted antibody, and qPCR of TRPV3 mRNA, suggested pregnancy increased arterial TRPV3 expression. The TRPV3 activator carvacrol caused endothelium-independent dilation of phenylephrine-constricted radial arteries, with no difference between vessels from non-pregnant and pregnant animals. Carvacrol-induced dilation was reduced by the TRPV3-blockers isopentenyl pyrophosphate and ruthenium red, but not by the TRPA1 or TRPV4 inhibitors HC-030031 or HC-067047, respectively. In radial arteries from non-pregnant rats only, inhibition of NOS and sGC, or PKG, enhanced carvacrol-mediated vasodilation. Carvacrol-induced dilation of arteries from both non-pregnant and pregnant rats was prevented by the IKCa blocker TRAM-34. TRPV3 caused an endothelium-independent, IKCa-mediated dilation of the uterine radial artery. NO-PKG-mediated modulation of TRPV3 activity is lost in pregnancy, but this did not alter the response to carvacrol.

Asunto(s)

Canales Catiónicos TRPV/metabolismo , Arteria Uterina/metabolismo , Vasodilatación , Animales , Presión Sanguínea , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Cimenos , Relación Dosis-Respuesta a Droga , Femenino , Técnicas In Vitro , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Monoterpenos/farmacología , Óxido Nítrico/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Regulación hacia Arriba , Arteria Uterina/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología

RESUMEN

In pregnancy, the vasculature of the uterus undergoes rapid remodelling to increase blood flow and maintain perfusion to the fetus. The present study determines the distribution and density of caveolae, transient receptor potential vanilloid type 4 channels (TRPV4) and myoendothelial gap junctions, and the relative contribution of related endothelium-dependent vasodilator components in uterine radial arteries of control virgin non-pregnant and 20-day late-pregnant rats. The hypothesis examined is that specific components of endothelium-dependent vasodilator mechanisms are altered in pregnancy-related uterine radial artery remodelling. Conventional and serial section electron microscopy were used to determine the morphological characteristics of uterine radial arteries from control and pregnant rats. TRPV4 distribution and expression was examined using conventional confocal immunohistochemistry, and the contribution of endothelial TRPV4, nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type activity determined using pressure myography with pharmacological intervention. Data show outward hypertrophic remodelling occurs in uterine radial arteries in pregnancy. Further, caveolae density in radial artery endothelium and smooth muscle from pregnant rats was significantly increased by ~94% and ~31%, respectively, compared with control, whereas caveolae density did not differ in endothelium compared with smooth muscle from control. Caveolae density was significantly higher by ~59% on the abluminal compared with the luminal surface of the endothelium in uterine radial artery of pregnant rats but did not differ at those surfaces in control. TRPV4 was present in endothelium and smooth muscle, but not associated with internal elastic lamina hole sites in radial arteries. TRPV4 fluorescence intensity was significantly increased in the endothelium and smooth muscle of radial artery of pregnant compared with control rats by ~2.6- and 5.5-fold, respectively. The TRPV4 signal was significantly higher in the endothelium compared with the smooth muscle in radial artery of both control and pregnant rats, by ~5.7- and 2.7-fold, respectively. Myoendothelial gap junction density was significantly decreased by ~37% in radial artery from pregnant compared with control rats. Pressure myography with pharmacological intervention showed that NO contributes ~80% and ~30%, and the EDH-type component ~20% and ~70% of the total endothelium-dependent vasodilator response in radial arteries of control and pregnant rats, respectively. TRPV4 plays a functional role in radial arteries, with a greater contribution in those from pregnant rats. The correlative association of increased TRPV4 and caveolae density and role of EDH-type activity in uterine radial artery of pregnant rats is suggestive of their causal relationship. The decreased myoendothelial gap junction density and lack of TRPV4 density at such sites is consistent with their having an integral, albeit complex, interactive role in uterine vascular signalling and remodelling in pregnancy.

Asunto(s)

Caveolas/ultraestructura , Uniones Comunicantes/ultraestructura , Arteria Radial/ultraestructura , Canales Catiónicos TRPV/fisiología , Arteria Uterina/ultraestructura , Útero/anatomía & histología , Animales , Endotelio Vascular/ultraestructura , Femenino , Inmunohistoquímica , Microscopía Electrónica , Embarazo , Ratas , Ratas Sprague-Dawley , Vasodilatación/fisiología

RESUMEN

In pregnancy, α-adrenoceptor-mediated vasoconstriction is augmented in uterine radial arteries and is accompanied by underlying changes in smooth muscle (SM) Ca(2+) activity. This study aims to determine the Ca(2+) entry channels associated with altered vasoconstriction in pregnancy, with the hypothesis that augmented vasoconstriction involves transient receptor potential canonical type-3 (TRPC3) and L- and T-type voltage-dependent Ca(2+) channels. Immunohistochemistry showed TRPC3, L-type Cav1.2 (as the α1C subunit), T-type Cav3.1 (α1G), and Cav3.2 (α1H) localization to the uterine radial artery SM. Fluorescence intensity of TRPC3, Cav1.2, and Cav3.2 was increased, and Cav3.1 decreased in radial artery SM from pregnant rats. Western blot analysis confirmed increased TRPC3 protein expression in the radial artery from pregnant rats. Pressure myography incorporating pharmacological intervention to examine the role of these channels in uterine radial arteries showed an attenuation of phenylephrine (PE)-induced constriction with Pyr3 {1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid}-mediated TRPC3 inhibition or with nifedipine-mediated L-type channel block alone in vessels from pregnant rats; both effects of which were diminished in radial arteries from nonpregnant rats. Combined TRPC3 and L-type inhibition attenuated PE-induced constriction in radial arteries, and the residual vasoconstriction was reduced and abolished with T-type channel block with NNC 55-0396 in arteries from nonpregnant and pregnant rats, respectively. With SM Ca(2+) stores depleted and in the presence of PE, nifedipine, and NNC 55-0396, blockade of TRPC3 reversed PE-induced constriction. These data suggest that TRPC3 channels act synergistically with L- and T-type channels to modulate radial artery vasoconstriction, with the mechanism being augmented in pregnancy.

Asunto(s)

Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Contracción Muscular/fisiología , Canales Catiónicos TRPC/metabolismo , Arteria Uterina/metabolismo , Vasoconstricción/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miografía , Nifedipino/farmacología , Fenilefrina/farmacología , Embarazo , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Arteria Uterina/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología

RESUMEN

OBJECTIVE: To determine whether impairment of endothelial connexin40 (Cx40), an effect that can occur in hypertension and aging, contributes to the arterial dysfunction and stiffening in these conditions. APPROACH AND RESULTS: A new transgenic mouse strain, expressing a mutant Cx40, (Cx40T202S), specifically in the vascular endothelium, has been developed and characterized. This mutation produces nonfunctional hemichannels, whereas gap junctions containing the mutant are electrically, but not chemically, patent. Mesenteric resistance arteries from Cx40T202S mice showed increased sensitivity of the myogenic response to intraluminal pressure in vitro, compared with wild-type mice, whereas transgenic mice overexpressing native Cx40 (Cx40Tg) showed reduced sensitivity. In control and Cx40Tg mice, the sensitivity to pressure of myogenic constriction was modulated by both NO and endothelium-derived hyperpolarization; however, the endothelium-derived hyperpolarization component was absent in Cx40T202S arteries. Analysis of passive mechanical properties revealed that arterial stiffness was enhanced in vessels from Cx40T202S mice, but not in wild-type or Cx40Tg mice. CONCLUSIONS: Introduction of a mutant form of Cx40 in the endogenous endothelial Cx40 population prevents endothelium-derived hyperpolarization activation during myogenic constriction, enhancing sensitivity to intraluminal pressure and increasing arterial stiffness. We conclude that genetic polymorphisms in endothelial Cx40 can contribute to the pathogenesis of arterial disease.

Asunto(s)

Conexinas/fisiología , Endotelio Vascular/metabolismo , Polimorfismo Genético , Rigidez Vascular , Animales , Presión Sanguínea , Peso Corporal , Conexinas/análisis , Conexinas/genética , Conductividad Eléctrica , Uniones Comunicantes/fisiología , Frecuencia Cardíaca , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Transgénicos , Proteína alfa-5 de Unión Comunicante , Proteína alfa-4 de Unión Comunicante

RESUMEN

Diet-induced obesity induces changes in mechanisms that are essential for the regulation of normal artery function, and in particular the function of the vascular endothelium. Using a rodent model that reflects the characteristics of human dietary obesity, in the rat saphenous artery we have previously demonstrated that endothelium-dependent vasodilation shifts from an entirely nitric oxide (NO)-mediated mechanism to one involving upregulation of myoendothelial gap junctions and intermediate conductance calcium-activated potassium channel activity and expression. This study investigates the changes in NO-mediated mechanisms that accompany this shift. In saphenous arteries from controls fed a normal chow diet, acetylcholine-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylyl cyclase inhibitors, but in equivalent arteries from obese animals sensitivity to these agents was reduced. The expression of endothelial NO synthase (eNOS) and caveolin-3 in rat saphenous arteries was unaffected by obesity, whilst that of caveolin-1 monomer and large oligomeric complexes of caveolins-1 and -2 were increased in membrane-enriched samples. The density of caveolae was increased at the membrane and cytoplasm of endothelial and smooth muscle cells of saphenous arteries from obese rats. Dissociation of eNOS from caveolin-1, as a prerequisite for activation of the enzyme, may be compromised and thereby impair NO-mediated vasodilation in the saphenous artery from diet-induced obese rats. Such altered signaling mechanisms in obesity-related vascular disease represent significant potential targets for therapeutic intervention.

Asunto(s)

Caveolas/metabolismo , Caveolina 1/biosíntesis , Dieta Alta en Grasa/efectos adversos , Óxido Nítrico/metabolismo , Obesidad/metabolismo , Vasodilatación , Animales , Caveolina 1/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

AIMS: Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. METHODS AND RESULTS: TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. CONCLUSION: The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Asunto(s)

Factores Biológicos/metabolismo , Endotelio Vascular/metabolismo , Arterias Mesentéricas/metabolismo , Canales Catiónicos TRPC/metabolismo , Vasodilatación , Animales , Presión Arterial , Western Blotting , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/ultraestructura , Células HEK293 , Humanos , Inmunohistoquímica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Masculino , Potenciales de la Membrana , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/ultraestructura , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Inmunoelectrónica , Miografía , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Canales Catiónicos TRPC/efectos de los fármacos , Canales Catiónicos TRPC/genética , Transfección , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología

RESUMEN

Obesity is a risk factor for hypertension and other vascular disease. The aim of this study was to examine the effect of diet-induced obesity on endothelium-dependent dilation of rat cremaster muscle arterioles. Male Sprague-Dawley rats (213 ± 1 g) were fed a cafeteria-style high-fat or control diet for 16-20 wk. Control rats weighed 558 ± 7 g compared with obese rats 762 ± 12 g (n = 52-56; P < 0.05). Diet-induced obesity had no effect on acetylcholine (ACh)-induced dilation of isolated, pressurized (70 mmHg) arterioles, but sodium nitroprusside (SNP)-induced vasodilation was enhanced. ACh-induced dilation of arterioles from control rats was abolished by a combination of the K(Ca) blockers apamin, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), and iberiotoxin (IBTX; all 0.1 µmol/l), with no apparent role for nitric oxide (NO). In arterioles from obese rats, however, IBTX had no effect on responses to ACh while the NO synthase (NOS)/guanylate cyclase inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 µmol/l)/1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µmol/l) partially inhibited ACh-induced dilation. Furthermore, NOS activity (but not endothelial NOS expression) was increased in arteries from obese rats. L-NAME/ODQ alone or removal of the endothelium constricted arterioles from obese but not control rats. Expression of caveolin-1 and -2 oligomers (but not monomers or caveolin-3) was increased in arterioles from obese rats. The number of caveolae was reduced in the endothelium of arteries, and caveolae density was increased at the ends of smooth muscle cells from obese rats. Diet-induced obesity abolished the contribution of large-conductance Ca(2+)-activated K(+) channel to ACh-mediated endothelium-dependent dilation of rat cremaster muscle arterioles, while increasing NOS activity and inducing an NO-dependent component.

Asunto(s)

Arteriolas/metabolismo , Caveolas/metabolismo , Endotelio Vascular/metabolismo , Músculo Liso/irrigación sanguínea , Óxido Nítrico/metabolismo , Obesidad/metabolismo , Canales de Potasio/metabolismo , Acetilcolina/farmacología , Animales , Apamina/farmacología , Arteriolas/efectos de los fármacos , Arteriolas/fisiopatología , Caveolas/efectos de los fármacos , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Dieta Alta en Grasa , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Masculino , Músculo Liso/metabolismo , Músculo Liso/fisiopatología , Nitroprusiato/farmacología , Obesidad/fisiopatología , Péptidos/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacología

RESUMEN

Vascular tone refers to the balance between arterial constrictor and dilator activity. The mechanisms that underlie tone are critical for the control of haemodynamics and matching circulatory needs with metabolism, and thus alterations in tone are a primary factor for vascular disease etiology. The dynamic spatiotemporal control of intracellular Ca(2+) levels in arterial endothelial and smooth muscle cells facilitates the modulation of multiple vascular signaling pathways. Thus, control of Ca(2+) levels in these cells is integral for the maintenance of tone and blood flow, and intimately associated with both physiological and pathophysiological states. Hence, understanding the mechanisms that underlie the modulation of vascular Ca(2+) activity is critical for both fundamental knowledge of artery function, and for the development of targeted therapies. This brief review highlights the role of Ca(2+) signaling in vascular endothelial function, with a focus on contact-mediated vasodilator mechanisms associated with endothelium-derived hyperpolarization and the longitudinal conduction of responses over distance.

Asunto(s)

Señalización del Calcio , Calcio/fisiología , Endotelio Vascular/fisiología , Transducción de Señal/fisiología , Acetilcolina/farmacología , Adenosina Trifosfato/fisiología , Animales , Humanos , Canales Catiónicos TRPC/fisiología , Vasodilatación/efectos de los fármacos

Asunto(s)

Endotelio Vascular/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Vasodilatación/fisiología , Animales , Western Blotting , Regulación de la Expresión Génica/fisiología , Ratas , Transducción de Señal

RESUMEN

Mechanisms underlying obesity-related vascular dysfunction are unclear. This study examined the effect of diet-induced obesity on expression and function of large conductance Ca(2+)-activated potassium channel (BK(Ca)) in rat pressurized small resistance vessels with myogenic tone. Male Sprague-Dawley rats fed a cafeteria-style high fat diet (HFD; ∼30% energy from fat) for 16-20 wk were ∼30% heavier than controls fed standard chow (∼13% fat). Obesity did not alter BK(Ca) α-subunit function or α-subunit protein or mRNA expression in vessels isolated from the cremaster muscle or middle-cerebral circulations. In contrast, BK(Ca) ß(1)-subunit protein expression and function were significantly reduced in cremaster muscle arterioles but increased in middle-cerebral arteries from obese animals. Immunohistochemistry showed α- and ß(1)-subunits were present exclusively in the smooth muscle of both vessels. Cremaster muscle arterioles from obese animals showed significantly increased medial thickness, and media-to-lumen ratio and pressurized arterioles showed increased myogenic tone at 30 mmHg, but not at 50-120 mmHg. Myogenic tone was not affected by obesity in middle-cerebral arteries. The BK(Ca) antagonist iberiotoxin constricted both cremaster muscle and middle-cerebral arterioles from control rats; this effect of iberiotoxin was abolished in cremaster muscle arteries only from obese rats. Diet-induced obesity has contrasting effects on BK(Ca) function in different vascular beds, through differential effects on ß(1)-subunit expression. However, these alterations in BK(Ca) function had little effect on overall myogenic tone, suggesting that the mechanisms controlling myogenic tone can be altered and compensate for altered BK(Ca) expression and function.

Asunto(s)

Arteriolas/metabolismo , Arterias Cerebrales/metabolismo , Dieta , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Canales de Potasio/biosíntesis , Animales , Western Blotting , Grasas de la Dieta/farmacología , Ingestión de Energía/fisiología , Frecuencia Cardíaca/fisiología , Hiperinsulinismo/etiología , Hiperinsulinismo/fisiopatología , Hipertensión/etiología , Hipertensión/fisiopatología , Inmunohistoquímica , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Leptina/sangre , Masculino , Microscopía Electrónica , Tono Muscular/fisiología , Músculo Esquelético/irrigación sanguínea , Canales de Potasio/agonistas , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Aumento de Peso/fisiología

RESUMEN

BACKGROUND: The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study. METHODOLOGY/PRINCIPAL FINDINGS: Membrane potential, vessel diameter and luminal pressure were recorded in 4(th) order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16-20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK(Ca)/IK(Ca)) inhibition; with such activity being impaired in obesity. SK(Ca)-IK(Ca) activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK(Ca)-mediated EDH contribution was increased in obesity, and associated with altered IK(Ca) distribution and elevated expression. In contrast, the SK(Ca)-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K(ir)) and Na(+)/K(+)-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K(ir) expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density. CONCLUSION/SIGNIFICANCE: In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK(Ca) and Na(+)/K(+)-ATPase, and decreased K(ir) underlie changes in the EDH mechanism.

Asunto(s)

Dieta/efectos adversos , Endotelio Vascular/patología , Potenciales de la Membrana , Obesidad/fisiopatología , Canales de Potasio/análisis , Transducción de Señal/fisiología , Animales , Uniones Comunicantes , Obesidad/etiología , Ratas , Distribución Tisular , Vasodilatación

RESUMEN

Myoendothelial microdomain signaling via localized calcium-activated potassium channel (K(Ca)) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K(Ca) mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K(Ca) and Cx activity. In mesenteric arteries from intestinal surgery patients, endothelium-dependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K(Ca) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 µM) inhibited bradykinin (0.1 nM-3 µM)-induced vasodilation, whereas the small (S) K(Ca) blocker apamin (50 and 100 nM) had no effect. Direct IK(Ca) activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10-300 µM) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (1-30 µM), the SK(Ca) activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10-100 µM) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 µM), with remaining dilation blocked by N-nitro l-arginine methyl ester (100 µM) and [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (10 µM), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK(Ca) activity are consistent with Cx37 and IK(Ca) microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK(Ca), and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.

Asunto(s)

Factores Relajantes Endotelio-Dependientes/fisiología , Uniones Comunicantes/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Arterias Mesentéricas/fisiología , Óxido Nítrico/fisiología , Conexinas/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Vasodilatación/fisiología , Proteína alfa-4 de Unión Comunicante

RESUMEN

The mechanisms involved in altered endothelial function in obesity-related cardiovascular disease are poorly understood. This study investigates the effect of chronic obesity on endothelium-dependent vasodilation and the relative contribution of nitric oxide (NO), calcium-activated potassium channels (K(Ca)), and myoendothelial gap junctions (MEGJs) in the rat saphenous artery. Obesity was induced by feeding rats a cafeteria-style diet (∼30 kJ as fat) for 16 to 20 weeks, with this model reflecting human dietary obesity etiology. Age- and sex-matched controls received standard chow (∼12 kJ as fat). Endothelium-dependent vasodilation was characterized in saphenous arteries by using pressure myography with pharmacological intervention, Western blotting, immunohistochemistry, and ultrastructural techniques. In saphenous artery from control, acetylcholine (ACh)-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylate cyclase inhibition, whereas in obese rats, the ACh response was less sensitive to such inhibition. Conversely, the intermediate conductance K(Ca) (IK(Ca)) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H pyrazole attenuates ACh-mediated dilation in obese, but not control, vessels. In a similar manner, putative gap junction block with carbenoxolone increased the pEC(50) for ACh in arteries from obese, but not control, rats. IK1 protein and MEGJ expression was up-regulated in the arteries of obese rats, an observation absent in control. Addition of the small conductance K(Ca) blocker apamin had no effect on ACh-mediated dilation in either control or obese rat vessels, consistent with unaltered SK3 expression. Up-regulation of distinct IK(Ca)- and gap junction-mediated pathways at myoendothelial microdomain sites, key mechanisms for endothelial-derived hyperpolarization-type activity, maintains endothelium-dependent vasodilation in diet-induced obese rat saphenous artery. Plasticity of myoendothelial coupling mechanisms represents a significant potential target for therapeutic intervention.

Asunto(s)

Endotelio Vascular/fisiología , Uniones Comunicantes/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Músculo Liso Vascular/metabolismo , Obesidad/fisiopatología , Vasodilatación/fisiología , Animales , Western Blotting , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Uniones Comunicantes/fisiología , Uniones Comunicantes/ultraestructura , Inmunohistoquímica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/agonistas , Masculino , Microscopía Electrónica , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/ultraestructura , Miografía , Obesidad/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos

RESUMEN

1. Coordinated oscillations in diameter occur spontaneously in cerebral vessels and depend on the opening of voltage dependent calcium channels. However, the mechanism that induces the initial depolarisation has remained elusive. We investigated the involvement of canonical transient receptor potential (TRPC) channels, which encode nonselective cation channels passing Na(+) and Ca(2+) currents, by measuring changes in diameter, intracellular Ca(2+) and membrane potential in branches of juvenile rat basilar arteries. 2. Removal of extracellular Ca(2+) abolished vasomotion and relaxed arteries, but paradoxically produced depolarisation. 3. Decrease in temperature to 24 degrees C or inhibition of phospholipase C (PLC) abolished vasomotion, hyperpolarised and relaxed arteries and decreased intracellular Ca(2+). 4. Reduction in the driving force for Na(+) through decrease in extracellular Na(+) produced similar effects and prevented the depolarisation elicited by removal of extracellular Ca(2+). 5. Nonselective TRP channel blockers, SKF96365 and gadolinium, mimicked the effects of inhibition of the PLC pathway. 6. Depolarisation of vessels in which TRP channels were blocked with SKF96365 reinstated vascular tone and vasomotion. 7. Quantitative polymerase chain reaction revealed TRPC1 as the predominantly expressed TRPC subtype. 8. Incubation with a function blocking TRPC1 antibody delayed the onset of vasomotion. 9. We conclude that nonselective cation channels contribute to vasoconstriction and vasomotion of cerebral vessels by providing an Na(+)-induced depolarisation that activates voltage dependent calcium channels. Our antibody data suggest the involvement of TRPC1 channels that might provide a target for treatment of therapy-refractory vasospasm.

Asunto(s)

Arteria Basilar/metabolismo , Canales Catiónicos TRPC/fisiología , Vasoconstricción , Vasodilatación , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Gadolinio/farmacología , Imidazoles/farmacología , Inmunohistoquímica , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/metabolismo , Canales Catiónicos TRPC/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos

RESUMEN

The potential physiological role of plasmalemmal large-conductance calcium-activated potassium channels (BK(Ca)) in vascular endothelial cells is controversial. Studies of freshly isolated and cultured vascular endothelial cells provide disparate results, both supporting and refuting a role for BK(Ca) in endothelial function. Most studies using freshly isolated, intact, healthy arteries provide little support for a physiological role for BK(Ca) in the endothelium, although recent work suggests that this may not be the case in diseased vessels. In isolated and cultured vascular endothelial cells, the autocrine action of growth factors, hormones, and vasoactive substances results in phenotypic drift. Such an induced heterogeneity is likely a primary factor accounting for the apparent differences, and often enhanced BK(Ca) expression and function, in isolated and cultured vascular endothelial cells. In a similar manner, heterogeneity in endothelial BK(Ca) expression and function in intact arteries may be representative of normal and disease states, BK(Ca) being absent in normal intact artery endothelium and upregulated in disease where dysfunction induces signals that alter channel expression and function. Indeed, in some intact vessels, there is evidence for the presence of BK(Ca), such as mRNA and/or specific BK subunits, an observation that is consistent with the potential for rapid upregulation, as may occur in disease. This perspective proposes that the disparity in the results obtained for BK(Ca) expression and function from freshly isolated and cultured vascular endothelial cells is largely due to variability in experimental conditions and, furthermore, that the expression of BK(Ca) in intact artery endothelium is primarily associated with disease. Although answers to physiologically relevant questions may only be available in atypical physiological conditions, such as those of isolation and culture, the limitations of these methods require open and objective recognition.

Asunto(s)

Endotelio Vascular/fisiología , Canales de Potasio Calcio-Activados/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Humanos , Técnicas In Vitro , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Canales de Potasio Calcio-Activados/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética

RESUMEN

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+-activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nM or 5 microM in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 microM). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 microM Ca2+, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 +/- 1.9 vs 45.5 +/- 1.7 pA pF(-1) at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 microM intracellular [Ca2+] were largely due to activity of BKCa. 17beta-Oestradiol (1 microM), reported to potentiate BKCa current via the channel's beta-subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the alpha-subunit-selective BKCa opener, NS-1619 (20 microM), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the beta1 subunit and a decrease in the beta1:alpha ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the beta1:alpha-subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both beta-subunit regulation and interaction with SR-mediated Ca2+ signalling.

Asunto(s)

Arterias/fisiología , Músculo Liso Vascular/fisiología , Canales de Potasio Calcio-Activados/fisiología , Animales , Arteriolas/fisiología , Western Blotting , Arterias Cerebrales/citología , Arterias Cerebrales/fisiología , Electrofisiología , Indicadores y Reactivos , Masculino , Músculo Esquelético/fisiología , Músculo Esquelético/ultraestructura , Técnicas de Placa-Clamp , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resistencia Vascular/fisiología

RESUMEN

1. Constriction of cerebral arteries is considered to depend on L-type voltage-dependent calcium channels (VDCCs); however, many previous studies have used antagonists with potential non-selective actions. Our aim was to determine the expression and function of VDCCs in the rat basilar artery. 2. The relative expression of VDCC subtypes was assessed using quantitative polymerase chain reaction and immunohistochemistry. Data were correlated with physiological studies of vascular function. Domains I-II of the T channel subtypes expressed in the rat basilar artery were cloned and sequenced. 3. Blockade of L-type channels with nifedipine had no effect on vascular tone. In contrast, in the presence of nifedipine, hyperpolarization of short arterial segments produced relaxation, whereas depolarization of quiescent segments evoked constriction. 4. The mRNA and protein for L- and T-type VDCCs were strongly expressed in the main basilar artery and side branches, with Ca(V)3.1 and Ca(V)1.2 the predominant subtypes. 5. T-Type VDCC blockers (i.e. 1 micromol/L mibefradil, 10 micromol/L pimozide and 100 micromol/L flunarizine) decreased intracellular calcium in smooth muscle cells, relaxed and hyperpolarized arteries, whereas nickel chloride (100 micromol/L) had no effect. In contrast with nifedipine, 10 micromol/L nimodipine produced hyperpolarization and relaxation. 6. When arteries were relaxed with 10 micromol/L U73122 (a phospholipase C inhibitor) in the presence of nifedipine, 40 mmol/L KCl evoked depolarization and constriction, which was significantly reduced by 1 micromol/L mibefradil. 7. Sequencing of domains I-II revealed splice variants of Ca(V)3.1, which may impact on channel activity. 8. We conclude that vascular tone of the rat basilar artery results from calcium influx through nifedipine-insensitive VDCCs with pharmacology consistent with Ca(V)3.1 T-type channels.

Asunto(s)

Arteria Basilar/fisiología , Canales de Calcio/metabolismo , Animales , Arteria Basilar/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/clasificación , Canales de Calcio/genética , Clonación Molecular , Regulación de la Expresión Génica/fisiología , Masculino , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología

RESUMEN

Molecular diversity of T-type/Ca(v)3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I-II linker in gating and surface expression of Ca(v)3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Delta8b variant revealed enhanced current density compared to Ca(v)3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Delta8b channels at the plasma membrane. We conclude that alternative splicing of Ca(v)3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased.

Asunto(s)

Empalme Alternativo , Canales de Calcio Tipo T/biosíntesis , Membrana Celular/metabolismo , Animales , Canales de Calcio Tipo T/genética , Variación Genética , Masculino , Potenciales de la Membrana , Ratas , Ratas Wistar , Eliminación de Secuencia

RESUMEN

BACKGROUND: Hypertension is a complex disease with many contributory genetic and environmental factors. We aimed to identify common targets for therapy by gene expression profiling of a resistance artery taken from animals representing two different models of hypertension. We studied gene expression and morphology of a saphenous artery branch in normotensive WKY rats, spontaneously hypertensive rats (SHR) and adrenocorticotropic hormone (ACTH)-induced hypertensive rats. RESULTS: Differential remodeling of arteries occurred in SHR and ACTH-treated rats, involving changes in both smooth muscle and endothelium. Increased expression of smooth muscle cell growth promoters and decreased expression of growth suppressors confirmed smooth muscle cell proliferation in SHR but not in ACTH. Differential gene expression between arteries from the two hypertensive models extended to the renin-angiotensin system, MAP kinase pathways, mitochondrial activity, lipid metabolism, extracellular matrix and calcium handling. In contrast, arteries from both hypertensive models exhibited significant increases in caveolin-1 expression and decreases in the regulators of G-protein signalling, Rgs2 and Rgs5. Increased protein expression of caveolin-1 and increased incidence of caveolae was found in both smooth muscle and endothelial cells of arteries from both hypertensive models. CONCLUSION: We conclude that the majority of differences in gene expression found in the saphenous artery taken from rats with two different forms of hypertension reflect distinctive morphological and physiological alterations. However, changes in common to caveolin-1 expression and G protein signalling, through attenuation of Rgs2 and Rgs5, may contribute to hypertension through augmentation of vasoconstrictor pathways and provide potential targets for common drug development.

Asunto(s)

Vasos Sanguíneos/metabolismo , Caveolina 1/genética , Perfilación de la Expresión Génica , Hipertensión/genética , Modelos Genéticos , Proteínas RGS/genética , Animales , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Especificidad de la Especie