Measuring T-Type Calcium Channel Currents in Isolated Vascular Smooth Muscle Cells.

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.

Loss of functional endothelial connexin40 results in exercise-induced hypertension in mice.

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.

Perturbation of chemical coupling by an endothelial Cx40 mutant attenuates endothelium-dependent vasodilation by KCa channels and elevates blood pressure in mice.

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.

Nox1 upregulates the function of vascular T-type calcium channels following chronic nitric oxide deficit.

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.

Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo.

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.

Role of T-type channels in vasomotor function: team player or chameleon?

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.

Polymorphism in endothelial connexin40 enhances sensitivity to intraluminal pressure and increases arterial stiffness.

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.

Spreading vasodilatation in the murine microcirculation: attenuation by oxidative stress-induced change in electromechanical coupling.

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.

Chronic deficit in nitric oxide elicits oxidative stress and augments T-type calcium-channel contribution to vascular tone of rodent arteries and arterioles.

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.

Tudor Griffith, gap junctions and conducted vasodilatation: electromechanical coupling back in the limelight.

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.

Long distance conduction of vasodilation: a passive or regenerative process?

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.

Sympathetic overdrive in obesity involves purinergic hyperactivity in the resistance vasculature.

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.

Non-linear relationship between hyperpolarisation and relaxation enables long distance propagation of vasodilatation.

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.

T-type calcium channels and vascular function: the new kid on the block?

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.

Involvement of nonselective cation channels in the depolarisation initiating vasomotion.

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.

Dihydropyridine-insensitive calcium currents contribute to function of small cerebral arteries.

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.

Enalapril treatment alters the contribution of epoxyeicosatrienoic acids but not gap junctions to endothelium-derived hyperpolarizing factor activity in mesenteric arteries of spontaneously hypertensive rats.

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.

What's where and why at a vascular myoendothelial microdomain signalling complex.

1. Modulation of vascular cell calcium is critical for the control of vascular tone, blood flow and pressure. 2.

Alternative splicing within the I-II loop controls surface expression of T-type Ca(v)3.1 calcium channels.

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.

Limited intravascular coupling in the rodent brainstem and retina supports a role for glia in regional blood flow.

Regional synaptic activity induces local increases in perfusion that are coupled to upstream vasodilation and improved blood flow. In the cerebral circulation, it has been proposed that astrocytes mediate the link between the initiating stimulus and local vasodilation through propagated intracellular calcium waves. In the systemic circulation the mechanism by which local vasodilation triggers upstream alterations in blood flow involves electrotonic propagation of hyperpolarization via endothelial gap junctions, although less is known concerning the cerebral circulation.

Non-L-type voltage-dependent calcium channels control vascular tone of the rat basilar artery.

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.

Vascular microarray profiling in two models of hypertension identifies caveolin-1, Rgs2 and Rgs5 as antihypertensive targets.

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.

Connexins 37 and 40 transduce purinergic signals mediating renal autoregulation.

Our previous data indicated that various subtypes of connexin (Cx) were expressed in the juxtaglomerular apparatus. Experiments were performed to characterize the effects on renal autoregulation of specific mimetic peptides that inhibit these Cx subtypes in Wistar-Kyoto rats. Intrarenal infusion of (Cx37,43)GAP27 increased autoregulatory index of renal plasma flow (0.