Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Gßγ subunit signalling underlies neuropeptide Y-stimulated vasoconstriction in rat mesenteric and coronary arteries.

BACKGROUND AND PURPOSE: Raised serum concentrations of the sympathetic co-transmitter neuropeptide Y (NPY) are linked to cardiovascular diseases. However, the signalling mechanism for vascular smooth muscle (VSM) constriction to NPY is poorly understood. Therefore, the present study investigated the mechanisms of NPY-induced vasoconstriction in rat small mesenteric (RMA) and coronary (RCA) arteries. EXPERIMENTAL APPROACH: Third-order mesenteric or intra-septal arteries from male Wistar rats were assessed in wire myographs for isometric tension, VSM membrane potential and VSM intracellular Ca2+ events. KEY RESULTS: NPY stimulated concentration-dependent vasoconstriction in both RMA and RCA, which was augmented by blocking NO synthase or endothelial denudation in RMA. NPY-mediated vasoconstriction was blocked by the selective Y1 receptor antagonist BIBO 3304 and Y1 receptor protein expression was detected in both the VSM and endothelial cells in RMA and RCA. The selective Gßγ subunit inhibitor gallein and the PLC inhibitor U-73122 attenuated NPY-induced vasoconstriction. Signalling via the Gßγ-PLC pathway stimulated VSM Ca2+ waves and whole-field synchronised Ca2+ flashes in RMA and increased the frequency of Ca2+ flashes in myogenically active RCA. Furthermore, in RMA, the Gßγ pathway linked NPY to VSM depolarization and generation of action potential-like spikes associated with intense vasoconstriction. This depolarization activated L-type voltage-gated Ca2+ channels, as nifedipine abolished NPY-mediated vasoconstriction. CONCLUSIONS AND IMPLICATIONS: These data suggest that the Gßγ subunit, which dissociates upon Y1 receptor activation, initiates VSM membrane depolarization and Ca2+ mobilisation to cause vasoconstriction. This model may help explain the development of microvascular vasospasm during raised sympathetic nerve activity.

Tracking endothelium-dependent NO release in pressurized arteries.

Background: Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries. Methods: Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), Cu2FL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries. Intact rat mesenteric arteries were studied using pressure myography or en face imaging to visualize vascular smooth muscle cells (SMCs) and endothelial cells (ECs) under physiological conditions. Both NO-dyes irreversibly bind NO, so the time course of accumulated fluorescence during basal, EC-agonist (ACh, 1 µM), and NO donor (SNAP, 10 µM) responses were assessed and compared in all experimental conditions. To avoid motion artefact, we introduced the additional step of labelling the arterial elastin with AF-633 hydrazide (AF) and calculated the fluorescence ratio (FR) of NO-dye/elastin over time to provide data as FR/FR0. Results: In cell-free chambers using either Cu2FL2E or DAR-4M AM, the addition of SNAP caused a time-dependent and significant increase in fluorescence compared to baseline. Next, using pressure myography we demonstrate that both Cu2FL2E and DAR-4M AM could be loaded into arterial cells, but found each also labelled the elastin. However, despite the use of different approaches and the clear observation of NO-dye in SMCs or ECs, we were unable to measure increases in fluorescence in response to either ACh or SNAP when cells were loaded with Cu2FL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR0 following stimulation with either ACh or SNAP. The addition of each agent evoked an accumulating, time-dependent, and statistically significant increase in fluorescence within 30 min compared to time controls. These experiments were repeated in the presence of L-NAME, an NO synthase inhibitor, which blocked the increase in fluorescence on addition of ACh but not to SNAP. Conclusion: These data advance our understanding of vascular function and in the future will potentially allow us to establish whether ECs continuously release NO, even under basal conditions.

Endothelium-Dependent Hyperpolarization: The Evolution of Myoendothelial Microdomains.

ABSTRACT: Endothelium-derived hyperpolarizing factor (EDHF) was envisaged as a chemical entity causing vasodilation by hyperpolarizing vascular smooth muscle (VSM) cells and distinct from nitric oxide (NO) ([aka endothelium-derived relaxing factor (EDRF)]) and prostacyclin. The search for an identity for EDHF unraveled the complexity of signaling within small arteries. Hyperpolarization originates within endothelial cells (ECs), spreading to the VSM by 2 branches, 1 chemical and 1 electrical, with the relative contribution varying with artery location, branch order, and prevailing profile of VSM activation. Chemical signals vary likewise and can involve potassium ion, lipid mediators, and hydrogen peroxide, whereas electrical signaling depends on physical contacts formed by homocellular and heterocellular (myoendothelial; MEJ) gap junctions, both able to conduct hyperpolarizing current. The discovery that chemical and electrical signals each arise within ECs resulted in an evolution of the single EDHF concept into the more inclusive, EDH signaling. Recognition of the importance of MEJs and particularly the fact they can support bidirectional signaling also informed the discovery that Ca2+ signals can pass from VSM to ECs during vasoconstriction. This signaling activates negative feedback mediated by NO and EDH forming a myoendothelial feedback circuit, which may also be responsible for basal or constitutive release of NO and EDH activity. The MEJs are housed in endothelial projections, and another spin-off from investigating EDH signaling was the discovery these fine structures contain clusters of signaling proteins to regulate both hyperpolarization and NO release. So, these tiny membrane bridges serve as a signaling superhighway or infobahn, which controls vasoreactivity by responding to signals flowing back and forth between the endothelium and VSM. By allowing bidirectional signaling, MEJs enable sinusoidal vasomotion, co-ordinated cycles of widespread vasoconstriction/vasodilation that optimize time-averaged blood flow. Cardiovascular disease disrupts EC signaling and as a result vasomotion changes to vasospasm.

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

BACKGROUND: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. METHODS: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. RESULTS: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. CONCLUSIONS: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.

Endothelial Nitric Oxide Suppresses Action-Potential-Like Transient Spikes and Vasospasm in Small Resistance Arteries.

Endothelial dysfunction in small arteries is a ubiquitous, early feature of cardiovascular disease, including hypertension. Dysfunction reflects reduced bioavailability of endothelium-derived nitric oxide (NO) and depressed endothelium-dependent hyperpolarization that enhances vasoreactivity. We measured smooth muscle membrane potential and tension, smooth muscle calcium, and used real-time quantitative polymerase chain reaction in small arteries and isolated tubes of endothelium to investigate how dysfunction enhances vasoreactivity. Rat nonmyogenic mesenteric resistance arteries developed vasomotion to micromolar phenylephrine (α1-adrenoceptor agonist); symmetrical vasoconstrictor oscillations mediated by L-type voltage-gated Ca2+ channels (VGCCs). Inhibiting NO synthesis abolished vasomotion so nanomolar phenylephrine now stimulated rapid, transient depolarizing spikes in the smooth muscle associated with chaotic vasomotion/vasospasm. Endothelium-dependent hyperpolarization block also enabled phenylephrine-vasospasm but without spikes or chaotic vasomotion. Depolarizing spikes were Ca2+-based and abolished by either T-type or L-type VGCCs blockers with depressed vasoconstriction. Removing NO also enabled transient spikes/vasoconstriction to Bay K-8644 (L-type VGCC activator). However, these were abolished by the L-type VGCC blocker nifedipine but not T-type VGCC block. Phenylephrine also initiated T-type VGCC-transient spikes and enhanced vasoconstriction after NO loss in nonmyogenic arteries from spontaneously hypertensive rats. In contrast to mesenteric arteries, myogenic coronary arteries displayed transient spikes and further vasoconstriction spontaneously on loss of NO. T-type VGCC block abolished these spikes and additional vasoconstriction but not myogenic tone. Therefore, in myogenic and nonmyogenic small arteries, reduced NO bioavailability engages T-type VGCCs, triggering transient depolarizing spikes in normally quiescent vascular smooth muscle to cause vasospasm. T-type block may offer a means to suppress vasospasm without inhibiting myogenic tone mediated by L-type VGCCs.

Intrinsic regulation of microvascular tone by myoendothelial feedback circuits.

The endothelium is an important regulator of arterial vascular tone, acting to release nitric oxide (NO) and open Ca2+-activated K+ (KCa) channels to relax vascular smooth muscle cells (VSMCs). While agonists acting at endothelial cell (EC) receptors are widely used to assess the ability of the endothelium to reduce vascular tone, the intrinsic EC-dependent mechanisms are less well characterized. In small resistance arteries and arterioles, the presence of heterocellular gap junctions termed myoendothelial gap junctions (MEGJs) allows the passage of not only current, but small molecules including Ca2+ and inositol trisphosphate (IP3). When stimulated to contract, the increase in VSM Ca2+ and IP3 can therefore potentially pass through MEGJs to activate adjacent ECs. This activation releases NO and opens KCa channels, which act to limit contraction. This myoendothelial feedback (MEF) is amplified by EC Ca2+ influx and release pathways, and is dynamically modulated by processes regulating gap junction conductance. There is a remarkable localization of key signaling and regulatory proteins within the EC projection toward VSM, and the intrinsic EC-dependent signaling pathways occurring with this highly specialized microdomain are reviewed.

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries.

The role of vascular gap junctions in the conduction of intercellular Ca2+ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 µm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s-1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18ß-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.

VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells.

KEY POINTS: Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca2+ release and subsequent activation of intermediate conductance Ca2+ -activated K+ (IKCa ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca2+ responses and the EDH-vasodilatation mediated by IKCa activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IKCa channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases. ABSTRACT: Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca2+ -activated K+ (IKCa ) channels. Importantly, VEGF-A had no direct effect against IKCa channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca2+ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.

Hyperglycaemia disrupts conducted vasodilation in the resistance vasculature of db/db mice.

Vascular dysfunction in small resistance arteries is observed during chronic elevations in blood glucose. Hyperglycaemia-associated effects on endothelium-dependent vasodilation have been well characterized, but effects on conducted vasodilation in the resistance vasculature are not known. Small mesenteric arteries were isolated from healthy and diabetic db/db mice, which were used as a model of chronic hyperglycaemia. Endothelium-dependent vasodilation via the Gq/11-coupled proteinase activated receptor 2 (PAR2) was stimulated with the selective agonist SLIGRL. The Ca2+-sensitive fluorescent indicator fluo-8 reported changes in endothelial cell (EC) [Ca2+]i, and triple cannulated bifurcating mesenteric arteries were used to study conducted vasodilation. Chronic hyperglycaemia did not affect either EC Ca2+ or local vasodilation to SLIGRL. However, both acute and chronic exposure to high glucose or the mannitol osmotic control attenuated conducted vasodilation to 10µM SLIGRL. This investigation demonstrates for the first time that a hypertonic solution containing glucose or mannitol can interfere with the spread of a hyperpolarizing current along the endothelium in a physiological setting. Our findings reiterate the importance of studying the effects of hyperglycaemia in the vasculature, and provide the basis for further studies regarding the modulation of junctional proteins involved in cell to cell communication by diseases such as diabetes.

Vasorelaxation to the Nitroxyl Donor Isopropylamine NONOate in Resistance Arteries Does Not Require Perivascular Calcitonin Gene-Related Peptide.

Nitroxyl (HNO) donors offer considerable therapeutic potential for the treatment of hypertension-related cardiovascular disorders, particularly heart failure, as they combine an inotropic action with peripheral vasodilation. Angeli's salt is the only HNO donor whose mechanism has been studied in depth, and recently, Angeli's salt vasodilation was suggested to be indirect and caused by calcitonin gene-related peptide (CGRP) released from perivascular nerves after HNO activates TRPA1 (transient receptor potential cation channel subfamily A member 1) channels. We investigated resistance artery vasorelaxation to the HNO donor, isopropylamine NONOate (IPA/NO), one of the structures providing a template for therapeutic development. Wire myography in combination with measurements of smooth muscle membrane potential was used to characterize the effect of IPA/NO in mesenteric resistance arteries. Immunohistochemistry was assessed in pressurized arteries. IPA/NO concentration dependently hyperpolarized and relaxed arteries precontracted with the α1-adrenoreceptor agonist, phenylephrine. These effects were blocked by the soluble guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by the KATP channel inhibitor, glibenclamide. Vasorelaxation persisted in the presence of raised [K+]o, used to block hyperpolarization, capsaicin to deplete perivascular CGRP, or HC030031 (2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4 isopropylphenyl) acetamide) to block TRPA1 receptors. Without preconstriction, hyperpolarization to IPA/NO was suppressed by glibenclamide, capsaicin, or HC030031. Hyperpolarization but not vasorelaxation to exogenous CGRP was inhibited with glibenclamide. Thus, vascular hyperpolarization is not necessary for vasorelaxation to the HNO donor IPA/NO, even though both effects are cGMP dependent. The reduced hyperpolarization after depletion of perivascular CGRP or block of TRPA1 receptors indicates some release of CGRP, but this does not contribute to HNO vasorelaxation. Therefore, HNO-TRPA1-CGRP signaling does not seem important for vasodilation to IPA/NO in resistance arteries.

Voltage-dependent Ca2+ entry into smooth muscle during contraction promotes endothelium-mediated feedback vasodilation in arterioles.

Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca2+ signals passing from smooth muscle through gap junctions to endothelial cells, which protrude through holes in the internal elastic lamina to make contact with vascular smooth muscle cells. Although hypothetically either Ca2+ or inositol trisphosphate (IP3) may provide the intercellular signal, it is generally thought that IP3 diffusion is responsible. We provide evidence that Ca2+ entry through L-type voltage-dependent Ca2+ channels (VDCCs) in vascular smooth muscle can pass to the endothelium through positions aligned with holes in the internal elastic lamina in amounts sufficient to activate endothelial cell Ca2+ signaling. In endothelial cells in which IP3 receptors (IP3Rs) were blocked, VDCC-driven Ca2+ events were transient and localized to the endothelium that protrudes through the internal elastic lamina to contact vascular smooth muscle cells. In endothelial cells in which IP3Rs were not blocked, VDCC-driven Ca2+ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca2+-activated K+ (IKCa) channels, thereby providing feedback that effectively suppressed vasoconstriction and enabled cycles of constriction and dilation called vasomotion. Thus, agonists that stimulate vascular smooth muscle depolarization provide Ca2+ to endothelial cells to activate a feedback circuit that protects tissue blood flow.

Scaffolding builds to reduce blood pressure.

Endothelial cells provide vasodilator signals to reduce blood pressure. In the small resistance arteries and arterioles, which determine the distribution and pressure of blood, the major signal is hyperpolarization reflecting the endothelial activity of calcium-activated potassium channels (KCa). In this issue of Science Signaling, Sonkusare et al. report that the scaffold protein AKAP150 is required for the kinase PKC and the calcium channel TRPV4 to enable receptor-mediated relaxation signaling. This scaffold enhances TRPV4 gating cooperativity and markedly amplifies the Ca(2+) signal, which ultimately activates (mainly) IKCa channels. Normally restricted to tiny endothelial projections, AKAP150 localization and associated signaling is disrupted in a model of hypertension, thereby diminishing hyperpolarization and vasodilation.

Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone.

Endothelial cell (EC) Ca(2+)-activated K channels (SK(Ca) and IK(Ca) channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK(Ca) channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca(2+) events (Ca(2+) puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (~2 min(-1)) inositol 1,4,5-trisphosphate receptor-based Ca(2+) events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca(2+) events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca(2+) increases, which on occasion led to intracellular Ca(2+) waves. The concurrent vasodilation associated with increases in Ca(2+) event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IK(Ca) channel blocker), but not by apamin (SK(Ca) channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca(2+) event frequency; at low pressures the consequence is activation of EC IK(Ca) channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.

Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (K(Ca)2.3) in cerebral arteries.

BACKGROUND: In rat middle cerebral and mesenteric arteries the K(Ca)2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K(Ca)2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K(Ca)2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). METHODS: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. RESULTS: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K(Ca)2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K(Ca)2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. CONCLUSIONS: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K(Ca)2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K(Ca)2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.

EDHF: spreading the influence of the endothelium.

Our view of the endothelium was transformed around 30 years ago, from one of an inert barrier to that of a key endocrine organ central to cardiovascular function. This dramatic change followed the discoveries that endothelial cells (ECs) elaborate the vasodilators prostacyclin and nitric oxide. The key to these discoveries was the use of the quintessentially pharmacological technique of bioassay. Bioassay also revealed endothelium-derived hyperpolarizing factor (EDHF), particularly important in small arteries and influencing blood pressure and flow distribution. The basic idea of EDHF as a diffusible factor causing smooth muscle hyperpolarization (and thus vasodilatation) has evolved into one of a complex pathway activated by endothelial Ca(2+) opening two Ca(2+) -sensitive K(+) -channels, K(Ca)2.3 and K(Ca)3.1. Combined application of apamin and charybdotoxin blocked EDHF responses, revealing the critical role of these channels as iberiotoxin was unable to substitute for charybdotoxin. We showed these channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, and close to interendothelial gap junctions. Activation of K(Ca) channels hyperpolarizes ECs, and K(+) efflux through them can act as a diffusible 'EDHF' stimulating Na(+) /K(+) -ATPase and inwardly rectifying K-channels. In parallel, hyperpolarizing current can spread from the endothelium to the smooth muscle through myoendothelial gap junctions upon endothelial projections. The resulting radial hyperpolarization mobilized by EDHF is complemented by spread of hyperpolarization along arteries and arterioles, effecting distant dilatation dependent on the endothelium. So the complexity of the endothelium still continues to amaze and, as knowledge evolves, provides considerable potential for novel approaches to modulate blood pressure.

A novel role for HNO in local and spreading vasodilatation in rat mesenteric resistance arteries.

Nitric oxide-mediated vasodilatation has previously been attributed to the uncharged form of the molecule (NO(•)), but increasing evidence suggests that nitroxyl (HNO) may play a significant role in endothelium-dependent relaxation. The aim of this study was to investigate the mechanisms underlying HNO-mediated vasodilatation in phenylephrine pre-constricted pressurized (70 mmHg) mesenteric arteries, and on membrane currents in isolated smooth muscle cells using whole cell and perforated patch clamp recordings. Angeli's salt (AS: nitroxyl donor), evoked concentration-dependent vasodilatation that was insensitive to the NO(•) scavengers carboxy-PTIO and hydroxocobalamin (HXC), but sensitive to either the HNO scavenger L-cysteine, K-channel blockers (4-AP and iberiotoxin), raised [K(+)](o), or inhibition of soluble guanylyl cyclase (ODQ). AS-evoked smooth muscle hyperpolarization significantly augmented K(V) current in an ODQ sensitive manner, and also increased the BK(Ca) current. Importantly, 30 µM AS initiated conducted or spreading vasodilatation, and following blockade of endothelial K-channels (TRAM-34 and apamin), ACh was able to evoke similar L-cysteine-sensitive conducted dilatation. These data show that vasodilatation induced by HNO is mediated by both K(V) and BK(Ca) channels, and suggest a physiological role in vasodilatation through the vasculature.

Compromised vascular endothelial cell SK(Ca) activity: a fundamental aspect of hypertension?

Smooth muscle hyperpolarization originating in the endothelium and commonly referred to as the EDHF (endothelium-derived hyperpolarizing factor) response provides a very significant drive to vasodilatation particularly in small resistance arteries. Together with other endothelium-dependent dilator pathways 'EDHF' hyperpolarization is compromised by cardiovascular disease, including hypertension. However, although attenuated vascular hyperpolarization has been described in animal models of hypertension, the underlying mechanisms are not fully understood. In the current issue of the British Journal of Pharmacology, Weston et al. combine classic pharmacological approaches with electrophysiological and molecular techniques to suggest that attenuated endothelium-dependent hyperpolarization (and as a consequence vasodilatation) reflects major disruption of pathways associated with the activation of endothelial small conductance Ca(2+)-activated K-channels (SK(Ca)) in mesenteric arteries from spontaneously hypertensive rats. In addition to reductions in SK(Ca) and K(IR) proteins, changes in caveolin-1 isomers were also detected, possibly indicating channel realignment within plasmalemmal structures.

Nitric oxide suppresses cerebral vasomotion by sGC-independent effects on ryanodine receptors and voltage-gated calcium channels.

BACKGROUND/AIMS: In cerebral arteries, nitric oxide (NO) release plays a key role in suppressing vasomotion. Our aim was to establish the pathways affected by NO in rat middle cerebral arteries. METHODS: In isolated segments of artery, isometric tension and simultaneous measurements of either smooth muscle membrane potential or intracellular [Ca(2+)] ([Ca(2+)](SMC)) changes were recorded. RESULTS: In the absence of L-NAME, asynchronous propagating Ca(2+) waves were recorded that were sensitive to block with ryanodine, but not nifedipine. L-NAME stimulated pronounced vasomotion and synchronous Ca(2+) oscillations with close temporal coupling between membrane potential, tone and [Ca(2+)](SMC). If nifedipine was applied together with L-NAME, [Ca(2+)](SMC) decreased and synchronous Ca(2+) oscillations were lost, but asynchronous propagating Ca(2+) waves persisted. Vasomotion was similarly evoked by either iberiotoxin, or by ryanodine, and to a lesser extent by ODQ. Exogenous application of NONOate stimulated endothelium-independent hyperpolarization and relaxation of either L-NAME-induced or spontaneous arterial tone. NO-evoked hyperpolarization involved activation of BK(Ca) channels via ryanodine receptors (RYRs), with little involvement of sGC. Further, in whole cell mode, NO inhibited current through L-type voltage-gated Ca(2+) channels (VGCC), which was independent of both voltage and sGC. CONCLUSION: NO exerts sGC-independent actions at RYRs and at VGCC, both of which normally suppress cerebral artery myogenic tone.

Modulation of endothelial cell KCa3.1 channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries.

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of K(Ca)3.1 (IK(Ca)) channels and K(Ca)2.3 (SK(Ca)) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate K(Ca)3.1 and K(Ca)2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. K(Ca)3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca(2+)](o) but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca(2+)](i) increases stimulated by phenylephrine depolarization. Imaging [Ca(2+)](i) within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca(2+)](i) during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca(2+)](i) evoked by phenylephrine. If gap junctions were uncoupled, K(Ca)3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na(+)/K(+)-ATPase. There was no evidence for an equivalent link through K(Ca)2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed K(Ca)2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas K(Ca)3.1 channels and Na(+)/K(+)-ATPase alpha(2)/alpha(3) subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca(2+)](o) appears to modify K(Ca)3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca(2+)](i). The resulting hyperpolarization links to arterial relaxation largely through Na(+)/K(+)-ATPase, possibly reflecting K(+) acting as an EDHF. In contrast, K(Ca)2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K(+) and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.