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. 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.

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. 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.

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 Cav3.2, 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.

T-type Ca(2+) channels facilitate NO-formation, vasodilatation and NO-mediated modulation of blood pressure.

Voltage-gated calcium channels are involved in the vascular excitation-contraction mechanism and regulation of arterial blood pressure. It was hypothesized that T-type channels promote formation of nitric oxide from the endothelium. The present experiments determine the involvement of T-type channels in depolarization-dependent dilatation of mesenteric arteries and blood pressure regulation in Cav3.1 knock-out mice. Nitric oxide-dependent vasodilatation following depolarization-mediated vasoconstriction was reduced significantly in mesenteric arteries from Cav3.1(-/-) compared to wild type mice. Four days of systemic infusion of a nitric oxide (NO)-synthase-inhibitor to conscious wild type elicited a significant increase in mean arterial blood pressure that was absent in Cav3.1(-/-) mice. Immunoprecipitation and immunofluorescence labeling showed co-localization of Cav3.1 and endothelial nitric oxide synthase (eNOS) in arteries from wild type mice. Nitric oxide release measured as DAF fluorescence and cGMP levels were significantly lower in depolarized Cav3.1(-/-) compared to wild type arteries. In summary, the absence of T-type Cav3.1 channels attenuates NO-dependent dilatation in mesenteric arteries in vitro, as well as the hypertension after L-NAME infusion in vivo. Furthermore, Cav3.1 channels cluster with eNOS and promote formation of nitric oxide by the endothelium. The present findings suggest that this mechanism is important for the systemic impact of NO on peripheral resistance.

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. 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.

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. 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.

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. Together, these studies have reemphasized the importance of electromechanical coupling by which changes in membrane potential can rapidly control vessel diameter and blood flow.

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 <0.05). 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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.

Depolarization evoked by acetylcholine in mesenteric arteries of hypertensive rats attenuates endothelium-dependent hyperpolarizing factor.

OBJECTIVE: During blockade of endothelium-dependent hyperpolarizing factor (EDHF), acetylcholine evoked larger and faster depolarization in mesenteric arteries of spontaneously hypertensive rats (SHR) than normotensive Wistar-Kyoto (WKY) rats. We studied the mechanism underlying this response and its role in the attenuation of EDHF. METHODS: Electrophysiology, computational modelling and myography were used to study changes in membrane potential and effects on contractility. RESULTS: The large acetylcholine-evoked depolarization in SHR was accompanied by contraction, but this was not seen in WKY rats. The depolarization depended on release of intracellular Ca2+ but was unaffected by nonselective cation channel inhibitors, gadolinium, lanthanum or amiloride. The depolarization was significantly reduced by the Ca2+-dependent Cl- channel inhibitors, niflumic acid or flufenamic acid, or alterations in Cl- gradients using bumetanide (Na/K/Cl transporter inhibitor) or external Cl- replacement with isethionate. These drugs altered the time course of EDHF-evoked hyperpolarizations in SHR, making them indistinguishable from those in WKY rats. EDHF-induced relaxation was less sensitive to acetylcholine in SHR than in WKY rats, but this difference was eliminated following artery pretreatment with bumetanide. Computational modelling in which the SHR fast depolarizing response was selectively modulated mimicked physiologically acquired results obtained in SHR and WKY rats during Cl- -channel blockade. CONCLUSIONS: Acetylcholine evokes a fast depolarization in SHR but not in WKY rats, mediated by the opening of Ca2+-dependent Cl- channels. The depolarization is responsible for a constriction that reduces EDHF-mediated relaxation. Data suggest that Ca2+-dependent Cl- channels may provide a novel therapeutic target for improvement of endothelial dysfunction during hypertension.

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. 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.