RESUMEN

Patch clamp electrophysiology is a powerful tool that has been important in isolating and characterizing the ion channels that govern cellular excitability under physiological and pathophysiological conditions. The ability to enzymatically dissociate blood vessels and acutely isolate vascular smooth muscle cells has enabled the application of patch clamp electrophysiology to the identification of diverse voltage dependent ion channels that ultimately control vasoconstriction and vasodilation. Since intraluminal pressure results in depolarization of vascular smooth muscle, the channels that control the voltage dependent influx of extracellular calcium are of particular interest. This chapter describes methods for isolating smooth muscle cells from resistance vessels, and for recording, isolating, and characterizing voltage dependent calcium channel currents, using patch clamp electrophysiological and pharmacological protocols.

Asunto(s)

Canales de Calcio Tipo T/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Electrofisiología , Técnicas In Vitro , Ratones , Técnicas de Placa-Clamp , Ratas

RESUMEN

Mutant forms of connexin40 (Cx40) exist in the human population and predispose carriers to atrial fibrillation. Since endothelial expression of Cx40 is important for electrical and chemical communication within the arterial wall, carriers of mutant Cx40 proteins may be predisposed to peripheral arterial dysfunction and dysregulation of blood pressure. We have therefore studied mice expressing either a chemically dysfunctional mutant, Cx40T202S, or wild-type Cx40, with native Cx40, specifically in the endothelium. Blood pressure was measured by telemetry under normal conditions and during cardiovascular stress induced by locomotor activity, phenylephrine or nitric oxide blockade (N(É·)-nitro-L-arginine methyl ester hydroxide, L-NAME). Blood pressure of Cx40T202STg mice was significantly elevated at night when compared with wild-type or Cx40Tg mice, without change in mean heart rate, pulse pressure or locomotor activity. Analysis over 24 h showed that blood pressure of Cx40T202STg mice was significantly elevated at rest and additionally during locomotor activity. In contrast, neither plasma renin concentration nor pressor responses to phenylephrine or L-NAME were altered, the latter indicating that nitric oxide bioavailability was normal. In isolated, pressurised mesenteric arteries, hyperpolarisation and vasodilation evoked by SKA-31, the selective modulator of SKCa and IKCa channels, was significantly reduced in Cx40T202STg mice, due to attenuation of the SKCa component. Acetylcholine-induced ascending vasodilation in vivo was also significantly attenuated in cremaster muscle arterioles of Cx40T202STg mice, compared to wild-type and Cx40Tg mice. We conclude that endothelial expression of the chemically dysfunctional Cx40T202S reduces peripheral vasodilator capacity mediated by SKCa-dependent hyperpolarisation and also increases blood pressure.

Asunto(s)

Conexinas/metabolismo , Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Canales de Potasio Calcio-Activados/metabolismo , Vasodilatación/fisiología , Animales , Presión Sanguínea , Conexinas/genética , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Proteína alfa-5 de Unión Comunicante

RESUMEN

Cardiovascular disease is characterised by reduced nitric oxide bioavailability resulting from oxidative stress. Our previous studies have shown that nitric oxide deficit per se increases the contribution of T-type calcium channels to vascular tone through increased superoxide from NADPH oxidase (Nox). The aim of the present study was therefore to identify the Nox isoform responsible for modulating T-type channel function, as T-type channels are implicated in several pathophysiological conditions involving oxidative stress. We evaluated T-channel function in skeletal muscle arterioles in vivo, using a novel T-channel blocker, TTA-A2 (3 µmol/L), which demonstrated no cross reactivity with L-type channels. Wild-type and Nox2 knockout (Nox2ko) mice were treated with the nitric oxide synthase inhibitor L-NAME (40 mg/kg/day) for 2 weeks. L-NAME treatment significantly increased systolic blood pressure and the contribution of T-type calcium channels to arteriolar tone in wild-type mice, and this was not prevented by Nox2 deletion. In Nox2ko mice, pharmacological inhibition of Nox1 (10 µmol/L ML171), Nox4 (10 µmol/L VAS2870) and Nox4-derived hydrogen peroxide (500 U/mL catalase) significantly reduced the effect of chronic nitric oxide inhibition on T-type channel function. In contrast, in wild-type mice, ML171 and VAS2870, but not catalase, reduced the contribution of T-type channels to vascular tone, suggesting a role for Nox1 and non-selective actions of VAS2870. We conclude that Nox1, but not Nox2 or Nox4, is responsible for the upregulation of T-type calcium channels elicited by chronic nitric oxide deficit. These data point to an important role for this isoform in increasing T-type channel function during oxidative stress.

Asunto(s)

Arteriolas/metabolismo , Canales de Calcio Tipo T/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/deficiencia , Animales , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/irrigación sanguínea , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Regulación hacia Arriba , Vasoconstricción

RESUMEN

During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.

Asunto(s)

Conexinas/deficiencia , Endotelio Vascular/metabolismo , Hipertensión/etiología , Hipertensión/fisiopatología , Condicionamiento Físico Animal/efectos adversos , Animales , Presión Sanguínea/fisiología , Conexinas/genética , Conexinas/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Uniones Comunicantes/fisiología , Frecuencia Cardíaca/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Vasodilatación/fisiología , Proteína alfa-5 de Unión Comunicante

RESUMEN

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R), and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R) protein.

Asunto(s)

Células Endoteliales/metabolismo , Expresión Génica , Represoras Lac/fisiología , Animales , Conexinas/biosíntesis , Conexinas/genética , Endotelio Vascular/citología , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Homocigoto , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas , Receptor TIE-2/genética , Activación Transcripcional , Transgenes , Proteína alfa-5 de Unión Comunicante

RESUMEN

Low-voltage-activated T-type calcium channels play an important role in regulating cellular excitability and are implicated in conditions, such as epilepsy and neuropathic pain. T-type channels, especially Cav3.1 and Cav3.2, are also expressed in the vasculature, although patch clamp studies of isolated vascular smooth muscle cells have in general failed to demonstrate these low-voltage-activated calcium currents. By contrast, the channels which are blocked by T-type channel antagonists are high-voltage activated but distinguishable from their L-type counterparts by their T-type biophysical properties and small negative shifts in activation and inactivation voltages. These changes in T-channel properties may result from vascular-specific expression of splice variants of Cav3 genes, particularly in exon 25/26 of the III-IV linker region. Recent physiological studies suggest that T-type channels make a small contribution to vascular tone at low intraluminal pressures, although the relevance of this contribution is unclear. By contrast, these channels play a larger role in vascular tone of small arterioles, which would be expected to function at lower intra-vascular pressures. Upregulation of T-type channel function following decrease in nitric oxide bioavailability and increase in oxidative stress, which occurs during cardiovascular disease, suggests that a more important role could be played by these channels in pathophysiological situations. The ability of T-type channels to be rapidly recruited to the plasma membrane, coupled with their subtype-specific localisation in signalling microdomains where they could modulate the function of calcium-dependent ion channels and pathways, provides a mechanism for rapid up- and downregulation of vasoconstriction. Future investigation into the molecules which govern these changes may illuminate novel targets for the treatment of conditions such as therapy-resistant hypertension and vasospasm.

Asunto(s)

Canales de Calcio Tipo T/fisiología , Músculo Liso Vascular/fisiología , Vasoconstricción/fisiología , Vasodilatación/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Isoformas de Proteínas/fisiología

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

AIMS: As cardiovascular disease is characterized by reduced nitric oxide bioavailability, our aim was to determine the impact of this change on the mechanism underlying vascular tone of pressurized arteries in vitro and in vivo. METHODS AND RESULTS: We used pressurized cerebral and mesenteric arteries in vitro and skeletal muscle arterioles in vivo to study the contribution of L-type (1 µmol/L nifedipine) and T-type (1 µmol/L mibefradil, 3 µmol/L NNC 55-0396) calcium channels to vascular tone, following acute or chronic inhibition of nitric oxide. Acute inhibition with l-NAME (10 µmol/L) significantly increased the T-type, but not the L-type, channel contribution to vascular tone in vitro and in vivo, and altered the smooth muscle expression of the Cav3.1 and Cav3.2 T-type channels. In pressurized mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with l-NAME, the contribution of T-type channels relative to L-type channels was significantly reduced, compared with arteries from wild-type mice.Chronic l-NAME treatment (40 mg/kg/day; 14-18 days) increased blood pressure, vascular superoxide, and the contribution of T-type channels to vascular tone in vivo. The latter was reversed by acute scavenging of superoxide with tempol (1 mmol/L), or inhibition of NADPH oxidase with apocynin (500 µmol/L) or DPI (5 µmol/L). CONCLUSION: We conclude that nitric oxide deficit produces a significant increase in the contribution of Cav3.1 and Cav3.2 T-type calcium channels to vascular tone, by regulating the bioavailability of reactive oxygen species produced by NADPH oxidase. Our data provide evidence for a novel causal link between nitric oxide deficit, oxidative stress, and T-type calcium channel function.

Asunto(s)

Canales de Calcio Tipo T/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo , Vasoconstricción , Animales , Arteriolas/metabolismo , Presión Sanguínea , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/efectos de los fármacos , Canales de Calcio Tipo T/genética , Arterias Cerebrales/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Depuradores de Radicales Libres/farmacología , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Superóxidos/metabolismo , Factores de Tiempo , Vasoconstricción/efectos de los fármacos

RESUMEN

Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin-angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 µmol l(-1) NNC 55-0396), relative to L-type (1 µmol l(-1) nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin-angiotensin system (candesartan, 1.0 mg kg(-1) day(-1) for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l(-1)). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.

Asunto(s)

Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Estrés Oxidativo , Angiotensina II/fisiología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Arteriolas/fisiología , Bencimidazoles/farmacología , Compuestos de Bifenilo , Canales de Calcio Tipo L/fisiología , Canales de Calcio Tipo T/fisiología , Conexinas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación , Modelos Cardiovasculares , Renina/sangre , Tetrazoles/farmacología , Vasodilatación , Proteína alfa-5 de Unión Comunicante

RESUMEN

Tudor Griffith's untimely death cut short a research career focused on the mechanisms regulating vascular tone and blood flow. This brief review highlights the contribution that Tudor's work made to 3 main areas: the early days of study toward elucidating the identity of the endothelium-derived relaxing factor (or nitric oxide), the use of computational modeling to unravel the mechanisms underlying the rhythmical arterial contractions known as vasomotion, and the role played by gap junctions in the vasodilatation attributed to endothelium-derived hyperpolarization. Tudor's pioneering application of the connexin mimetic peptides as selective gap junction antagonists has contributed substantially to the current state of knowledge on the role of cell coupling in arterial function. Together, these studies have reemphasized the importance of electromechanical coupling by which changes in membrane potential can rapidly control vessel diameter and blood flow.

Asunto(s)

Factores Biológicos/metabolismo , Uniones Comunicantes/metabolismo , Vasodilatación/fisiología , Animales , Factores Biológicos/historia , Conexinas/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Potenciales de la Membrana/fisiología , Modelos Biológicos , Óxido Nítrico/metabolismo , Flujo Sanguíneo Regional/fisiología

RESUMEN

The mechanism enabling coordination of the resistance of feed arteries with microcirculatory arterioles to rapidly regulate tissue blood flow in line with changes in metabolic demand has preoccupied scientists for a quarter of a century. As experiments uncovered the underlying electrical events, it was frequently questioned how vasodilation could conduct over long distances without appreciable attenuation. This perspective reviews the data pertinent to this phenomenon and provides evidence that this remarkable response could be made possible by a simple mechanism based on the steep relationship between membrane potential and calcium entry demonstrated by the voltage-dependent calcium channels which mediate the control of vascular tone in vivo.

Asunto(s)

Canales de Calcio/metabolismo , Calcio/metabolismo , Fenómenos Electrofisiológicos , Microcirculación , Regeneración , Vasodilatación , Animales , Impedancia Eléctrica , Humanos

RESUMEN

While a close correlation exists in obese humans between sympathetic, adrenergic hyperactivity and structural and functional organ damage, a role for the co-transmitter, ATP, in vascular function remains unexplored. We therefore studied sympathetic nerve-mediated responses of pressurised small mesenteric arteries from control and obese rats. Diet-induced obesity significantly increased the amplitude of vasoconstriction to transmural nerve stimulation (1-10 Hz; P <0.05). At 1 and 5 Hz, both adrenergic and purinergic responses were significantly augmented, while only the purinergic component was increased at 10 Hz (P <0.05). Nerve stimulation at 1 Hz evoked contractions and underlying excitatory junction potentials (EJPs), which were both significantly increased in amplitude during obesity (P <0.05) and abolished by αß-methylene ATP (1 µM; desensitises purinergic receptors). The rise time and rate of decay of these EJPs were significant decreased (P <0.05), without change in resting membrane potential. Amplitude and frequency of spontaneous EJPs and the density of perivascular sympathetic nerves were also significantly increased (P <0.05). Inhibition of sensory neurotransmitter release (capsaicin; 10 µM) significantly increased the amplitude of nerve-mediated contraction (P <0.05), with a greater effect in control than obese animals, although the density of sensory nerves was unaffected by obesity. We demonstrate that sympathetic nerve-mediated vasoconstriction is enhanced by diet-induced obesity due to upregulation of purinergic, in addition to adrenergic, neurotransmission. Changes result from increased perivascular sympathetic innervation and release of ATP. We conclude that augmented sympathetic control of vasoconstriction induced by obesity could contribute directly to hypertension and global organ damage. A decrease in sensitivity to sensory vasodilatory neurotransmitters may also affect these processes.

Asunto(s)

Fibras Adrenérgicas/fisiología , Arterias Mesentéricas/fisiología , Obesidad/fisiopatología , Receptores Purinérgicos/fisiología , Vasoconstricción/fisiología , Animales , Masculino , Potenciales de la Membrana/fisiología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley

RESUMEN

Blood flow is adjusted to tissue demand through rapidly ascending vasodilatations resulting from conduction of hyperpolarisation through vascular gap junctions. We investigated how these dilatations can spread without attenuation if mediated by an electrical signal. Cremaster muscle arterioles were studied in vivo by simultaneously measuring membrane potential and vessel diameter. Focal application of acetylcholine elicited hyperpolarisations which decayed passively with distance from the local site,while dilatation spread upstream without attenuation. Analysis of simultaneous recordings at the local site revealed that hyperpolarisation and dilatation were only linearly related over a restricted voltage range to a threshold potential, beyond which dilatation was maximal. Experimental data could be simulated in a computational model with electrotonic decay of hyperpolarisation but imposition of this threshold. The model was tested by reducing the amplitude of the local hyperpolarisation which led to entry into the linear range closer to the local site and decay of dilatation. Serial section electron microscopy and light dye treatment confirmed that the spread of dilatation occurred through the endothelium and that the two cell layers were tightly coupled. Generality of the mechanism was demonstrated by applying the model to the attenuated propagation of dilatation found in larger arteries.We conclude that long distance spread of locally initiated dilatations is not due to a regenerative electrical phenomenon, but rather a restricted linear relationship between voltage and vessel tone, which minimises the impact of electrotonic decay of voltage. Disease-related alterations in endothelial coupling or ion channel expression could therefore decrease the ability to adjust blood flow to meet metabolic demand.

Asunto(s)

Modelos Biológicos , Dinámicas no Lineales , Vasodilatación/fisiología , Acetilcolina/farmacología , Animales , Arteriolas/citología , Arteriolas/efectos de los fármacos , Arteriolas/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Uniones Comunicantes/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología

Asunto(s)

Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Desarrollo Embrionario/genética , Mutación/genética , Virus SSPE/genética , Panencefalitis Esclerosante Subaguda/genética , Replicación Viral/genética , Animales , Antígenos CD/metabolismo , Línea Celular , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Proteínas de Unión al ARN , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria

RESUMEN

While L-type voltage-dependent calcium channels have long been considered the predominant source of calcium for myogenic constriction, recent studies of both cerebral and systemic circulations have provided evidence for the prominent expression of other members of the voltage-dependent calcium channel family, in particular the low voltage activated T-type channels. Although physiological studies have not supported the involvement of a classical low voltage activated, T-type channel in vascular function, evidence is accumulating that points to the involvement of a non-L-type, high voltage activated channel with sensitivity to T-type channel antagonists. We propose that this may arise due to expression of a T-type channel splice variant with unique biophysical characteristics resulting in a more depolarised profile. Expression of these channels in smooth muscle cells would broaden the voltage range over which sustained calcium influx occurs, while expression of T-type channels in endothelial cells could provide a feedback mechanism to prevent excessive vasoconstriction. Perturbation of this balance during pathophysiological conditions by upregulation of channel expression and endothelial dysfunction could contribute to vasospastic conditions and therapy-refractory hypertension.

Asunto(s)

Vasos Sanguíneos/fisiología , Canales de Calcio Tipo T/fisiología , Vasoconstricción/fisiología , Animales , Presión Sanguínea/fisiología , Calcio/metabolismo , Canales de Calcio Tipo L/fisiología , Fenómenos Fisiológicos Cardiovasculares , Humanos

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

Although dihydropyridines are widely used for the treatment of vasospasm, their effectiveness is questionable, suggesting that other voltage-dependent calcium channels (VDCCs) contribute to control of cerebrovascular tone. This study therefore investigated the role of dihydropyridine-insensitive VDCCs in cerebrovascular function. Using quantitative PCR and immunohistochemistry, we found mRNA and protein for L-type (Ca(V)1.2) and T-type (Ca(V)3.1 and Ca(V)3.2) channels in adult rat basilar and middle cerebral arteries and their branches. Immunoelectron microscopy revealed both L- and T-type channels in smooth muscle cell (SMC) membranes. Using patch clamp electrophysiology, we found that a high-voltage-activated calcium current, showing T-type channel kinetics and insensitivity to nifedipine and nimodipine, comprised approximately 20% of current in SMCs of the main arteries and approximately 45% of current in SMCs from branches. Both components were abolished by the T-type antagonists mibefradil, NNC 55-0396, and efonidipine. Although nifedipine completely blocked vasoconstriction in pressurized basilar arteries, a nifedipine-insensitive constriction was found in branches and this increased in magnitude as vessel size decreased. We conclude that a heterogeneous population of VDCCs contributes to cerebrovascular function, with dihydropyridine-insensitive channels having a larger role in smaller vessels. Sensitivity of these currents to nonselective T-type channel antagonists suggests that these drugs may provide a more effective treatment for therapy-refractory cerebrovascular constriction.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Arterias Cerebrales/metabolismo , Circulación Cerebrovascular/efectos de los fármacos , Dihidropiridinas/farmacología , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Arterias Cerebrales/ultraestructura , Circulación Cerebrovascular/fisiología , Masculino , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Ratas , Ratas Wistar , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología

RESUMEN

Reduction in endothelium-derived hyperpolarizing factor (EDHF)-mediated dilatory function in large, elastic arteries during hypertension is reversed after blood pressure normalization. We investigated whether similar mechanisms occurred in smaller mesenteric resistance arteries from aged Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs), and SHRs treated with the angiotensin-converting enzyme inhibitor, enalapril, using immunohistochemistry, serial-section electron microscopy, electrophysiology and wire myography. Unlike the superior mesenteric artery, EDHF relaxations in muscular mesenteric arteries were not reduced in SHRs, although morphological differences were found in the endothelium and smooth muscle. In WKY rats, SHRs and enalapril-treated SHRs, relaxations were mediated by small-, large-, and intermediate-conductance calcium-activated potassium channels, which were distributed in the endothelium, smooth muscle, and both layers, respectively. However, only WKY hyperpolarizations and relaxations were sensitive to gap junction blockers, and these arteries expressed more endothelial and myoendothelial gap junctions than arteries from SHRs. Responses in WKY rats, but not SHRs, were also reduced by inhibitors of epoxyeicosatrienoic acids (EETs), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and miconazole, although sensitivity to EET regioisomers was endothelium-independent in all rats. Enalapril treatment of SHRs reduced blood pressure and restored sensitivity to 14,15-EEZE, but not to gap junction blockers, and failed to reverse the morphological changes. In conclusion, the mechanisms underlying EDHF in muscular mesenteric arteries differ between WKY rats and SHRs, with gap junctions and EETs involved only in WKY rats. However, reduction of blood pressure in SHRs with enalapril restored a role for EETs, but not gap junctions, without reversing morphological changes, suggesting a differential control of chemical and structural alterations.

Asunto(s)

Ácidos Araquidónicos/metabolismo , Factores Biológicos/metabolismo , Enalapril/uso terapéutico , Uniones Comunicantes/metabolismo , Hipertensión/metabolismo , Arterias Mesentéricas/metabolismo , Animales , Enalapril/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/ultraestructura , Hipertensión/tratamiento farmacológico , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/ultraestructura , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Vasodilatación/efectos de los fármacos , Vasodilatación/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

1. Modulation of vascular cell calcium is critical for the control of vascular tone, blood flow and pressure. 2. Specialized microdomain signalling sites associated with calcium modulation are present in vascular smooth muscle cells, where spatially localized channels and calcium store receptors interact functionally. Anatomical studies suggest that such sites are also present in endothelial cells. 3. The characteristics of these sites near heterocellular myoendothelial gap junctions (MEGJs) are described, focusing on rat mesenteric artery. The MEGJs enable current and small molecule transfer to coordinate arterial function and are thus critical for endothelium-derived hyperpolarization, regulation of smooth muscle cell diameter in response to contractile stimuli and vasomotor conduction over distance. 4. Although MEGJs occur on endothelial cell projections within internal elastic lamina (IEL) holes, not all IEL holes have MEGJ-related projections (approximately 0-50% of such holes have MEGJ-related projections, with variations occurring within and between vessels, species, strains and disease). 5. In rat mesenteric, saphenous and caudal cerebellar artery and hamster cheek pouch arteriole, but not rat middle cerebral artery or cremaster arteriole, intermediate conductance calcium-activated potassium channels (IK(Ca)) localize to endothelial cell projections. 6. Rat mesenteric artery MEGJ connexins and IK(Ca) are in close spatial association with endothelial cell inositol 1,4,5-trisphosphate receptors and endoplasmic reticulum. 7. Data suggest a relationship between spatially associated endothelial cell ion channels and calcium stores in modulation of calcium release and action. Differences in spatial relationships between ion channels and calcium stores in different vessels reflect heterogeneity in vasomotor function, representing a selective target for the control of endothelial and vascular function.

Asunto(s)

Células Endoteliales/citología , Células Endoteliales/fisiología , Microdominios de Membrana/fisiología , Transducción de Señal/fisiología , Animales , Cricetinae , Uniones Comunicantes/fisiología , Ratones , Ratas