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.

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.

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.