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Background and Purpose In small arteries, small conductance Ca2+-activated K+ channels ( SKCa) and intermediate conductance Ca2+-activated K+ channels ( IKCa) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI2-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IKCa channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/ PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. Experimental Approach Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [ Ca2+]i, mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. Key Results Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 μM) without modifying the associated changes in endothelial cell [ Ca2+]i. The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol ( β1) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 ( β2) or glibenclamide ( ATP-sensitive K+ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IKCa {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole ( TRAM-34)}, but not SKCa (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. Conclusions and Implications In resistance arteries, endothelial cell β1-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IKCa channels and may be one consequence of raised circulating catecholamines. [ABSTRACT FROM AUTHOR]

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 Ca 2+ -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, Ca 2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, N G -(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism., Results: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α 1 -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 N G -(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca 2+ 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., Competing Interests: None.

Discovered almost 40 years ago, the potent vasoconstrictor peptide endothelin-1 (ET-1) has a wide range of roles both physiologically and pathologically. In recent years, there has been a focus on the contribution of ET-1 to disease. This has led to the development of various ET receptor antagonists, some of which are approved for the treatment of pulmonary arterial hypertension, while clinical trials for other diseases have been numerous yet, for the most part, unsuccessful. However, given the vast physiological impact of ET-1, it is both surprising and disappointing that therapeutics targeting the ET-1 pathway remain limited. Strategies aimed at the pathways influencing the synthesis and release of ET-1 could provide new therapeutic avenues, yet research using cultured cells in vitro has had little follow up in intact ex vivo and in vivo preparations. This article summarises what is currently known about the synthesis, storage and release of ET-1 as well as the role of ET-1 in several diseases including cardiovascular diseases, COVID-19 and chronic pain. Unravelling the ET-1 pathway and identifying therapeutic targets has the potential to treat many diseases whether through disease prevention, slowing disease progression or reversing pathology.

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), Cu 2 FL2E 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/FR 0 . Results: In cell-free chambers using either Cu 2 FL2E 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 Cu 2 FL2E 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 Cu 2 FL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR 0 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wallis, Donovan, Johnston, Phillips, Lin, Garland and Dora.)

Aims: Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study., Methods and Results: Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial (RA) appendage and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy. All RA- and LV-IMCAs from organ donors and pigs developed circa 25% MT. In contrast, 44% of human RA-IMCAs from 88 patients with heart valve disease had poor (<10%) MT yet retained cell viability and an ability to raise cytoplasmic Ca2+ in response to vasoconstrictor agents. Comparing across human heart chambers and species, we found that based on patient medical history and six tests, the strongest predictor of poor MT in IMCAs was increased expression of the synthetic marker caldesmon relative to the contractile marker SM-myosin heavy chain. In addition, high resolution imaging revealed a distinct layer of longitudinally aligned SMCs between ECs and radial SMCs, and we show poor MT was associated with disruptions in these cellular alignments., Conclusion: These data demonstrate the first use of atrial and ventricular biopsies from patients and pigs to reveal that impaired coronary MT reflects a switch of viable SMCs towards a synthetic phenotype, rather than a loss of SMC viability. These arteries represent a model for further studies of coronary microvascular contractile dysfunction., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

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., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Ca 2+ handling within cardiac myocytes underpins coordinated contractile function within the beating heart. This protocol enables high spatial and temporal Ca 2+ imaging of ex vivo multicellular myocardial strips. The endocardial surface is retained, and strips of 150-300-µm thickness are dissected, loaded with Ca 2+ indicators and mounted within 1.5 h. A list of the equipment and reagents used and the key methodological aspects allowing the use of this technique on strips from any chamber of the mammalian heart are described. We have successfully used this protocol on human, pig and rat biopsy samples. On use of this protocol with intact endocardial endothelium, we demonstrated that the myocytes develop asynchronous spontaneous Ca 2+ events, which can be ablated by electrically evoked Ca 2+ transients, and subsequently redevelop spontaneously after cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca 2+ signaling underpinning cardiomyocyte contraction, in both healthy and diseased tissue., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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 (PLM 3SA ; 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 PLM 3SA 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 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 (α 1 -adrenoceptor agonist); symmetrical vasoconstrictor oscillations mediated by L-type voltage-gated Ca 2+ 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 Ca 2+ -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.

The endothelium is an important regulator of arterial vascular tone, acting to release nitric oxide (NO) and open Ca 2+ -activated K + (K Ca ) 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 Ca 2+ and inositol trisphosphate (IP 3 ). When stimulated to contract, the increase in VSM Ca 2+ and IP 3 can therefore potentially pass through MEGJs to activate adjacent ECs. This activation releases NO and opens K Ca channels, which act to limit contraction. This myoendothelial feedback (MEF) is amplified by EC Ca 2+ 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., (© 2020 Elsevier Inc. All rights reserved.)

The role of vascular gap junctions in the conduction of intercellular Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca 2+ waves and vasoconstriction that spread along an entire artery segment 3000 μm long. Ca 2+ waves spread as nifedipine-sensitive Ca 2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca 2+ and vasoconstriction spread at circa 3 mm s -1 and were independent of the endothelium. The spread but not the generation of Ca 2+ spikes was reversibly blocked by the gap junction inhibitor 18β-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca 2+ -based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Key Points: Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca 2+ release and subsequent activation of intermediate conductance Ca 2+ -activated K + (IK Ca ) 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 Ca 2+ responses and the EDH-vasodilatation mediated by IK Ca activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IK Ca 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) Ca 2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca 2+ -activated K + (IK Ca ) channels. Importantly, VEGF-A had no direct effect against IK Ca 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 (IP 3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP 3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca 2+ 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., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

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 G q/11 -coupled proteinase activated receptor 2 (PAR2) was stimulated with the selective agonist SLIGRL. The Ca 2+ -sensitive fluorescent indicator fluo-8 reported changes in endothelial cell (EC) [Ca 2+ ] i , and triple cannulated bifurcating mesenteric arteries were used to study conducted vasodilation. Chronic hyperglycaemia did not affect either EC Ca 2+ 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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 α 1 -adrenoreceptor agonist, phenylephrine. These effects were blocked by the soluble guanylyl cyclase inhibitor, ODQ (1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by the K ATP 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-7 H -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., (© 2017 American Heart Association, Inc.)

Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca 2+ 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 Ca 2+ or inositol trisphosphate (IP 3 ) may provide the intercellular signal, it is generally thought that IP 3 diffusion is responsible. We provide evidence that Ca 2+ entry through L-type voltage-dependent Ca 2+ 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 Ca 2+ signaling. In endothelial cells in which IP 3 receptors (IP 3 Rs) were blocked, VDCC-driven Ca 2+ 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 IP 3 Rs were not blocked, VDCC-driven Ca 2+ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca 2+ -activated K + (IK Ca ) 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 Ca 2+ to endothelial cells to activate a feedback circuit that protects tissue blood flow., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies., Competing Interests: The authors declare no conflict of interest.

Endothelium-dependent hyperpolarizing factor (EDHF) is a powerful vasodilator influence in small resistance arteries and thus an important modulator of blood pressure and flow. As the name suggests, EDHF was thought to describe a diffusible factor stimulating smooth muscle hyperpolarization (and thus vasodilatation). However, this idea has evolved with the recognition that a factor can operate alongside the spread of hyperpolarizing current from the endothelium to the vascular smooth muscle (VSM). As such, the pathway is now termed endothelium-dependent hyperpolarization (EDH). EDH is activated by an increase in endothelial [Ca 2+ ] i , which stimulates two Ca 2+ -sensitive K channels, SK C a and IK C a . This was discovered because apamin and charybdotoxin applied in combination blocked EDHF responses, but iberiotoxin - a blocker of BK C a - was not able to substitute for charybdotoxin. SK C a and IK C a channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, which are rich in IK C a channels and close to interendothelial gap junctions where SK C a channels, are prevalent. K C a activation hyperpolarizes endothelial cells, and K + efflux through them can act as a diffusible 'EDHF' by stimulating VSM Na + ,K + -ATPase and inwardly rectifying K channels (K IR ). In parallel, hyperpolarizing current spreads from the endothelium to the smooth muscle through myoendothelial gap junctions located on endothelial projections. The resulting radial EDH is complemented by the spread of 'conducted' hyperpolarization along the endothelium of arteries and arterioles to affect conducted vasodilatation (CVD). Retrograde CVD effectively integrates blood flow within the microcirculation, but how the underlying hyperpolarization is sustained is unclear., (© 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Aim: During exercise in humans, circulating levels of ATP and K + increase at a time when blood flow increases to satisfy metabolic demand. Both molecules can activate arteriolar K + channels to stimulate vasodilatation; here, it is established whether conducted dilatation is observed in a skeletal muscle bed., Methods: Isolated and cannulated rat cremaster arterioles were used to assess both local and conducted responses. Agents were either added to the bath, focally pulse-ejected to the downstream end of arterioles, or in triple-cannulated arterioles, luminally perfused into the downstream branches to assess both local and conducted responses., Results: The endothelium-dependent agonist ACh and the K ATP channel opener levcromakalim each stimulated both local and conducted vasodilatation. Focal, bolus delivery of ATP (10 μm) or KCl (33 mm) to the outside of arterioles stimulated a biphasic vasomotor response: rapid vasoconstriction followed by dilatation as each washed away. At lower concentrations of KCl (19 mm), constriction was avoided, and instead, Ba 2+ -sensitive local dilatation and conducted dilatation were both observed. Luminal perfusion of ATP avoided constriction and activated P2Y 1 receptors stimulating vasodilatation secondary to opening of K C a channels. In triple-cannulated arterioles, either ATP (10 μm) or K + (15 mm) luminally perfused into daughter branches of a bifurcation stimulated local dilatation which conducted into the parent arteriole., Conclusion: The recognized physiological autocrine and paracrine mediators ATP and K + each act to evoke both local and conducted vasodilatation in rat cremaster arterioles. Therefore, in situations when circulating levels are raised, such as during exercise, these agents can act as important regulators of blood flow., (© 2016 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

The SMCs of skeletal muscle arterioles are intricately sensitive to changes in membrane potential. Upon increasing luminal pressure, the SMCs depolarize, thereby opening VDCCs, which leads to contraction. Mechanisms that oppose this myogenic tone can involve voltage-dependent and independent dilator pathways, and can be endothelium-dependent or independent. Of particular interest are the pathways leading to hyperpolarization of SMCs, as these can potentially evoke both local and conducted dilation. This review focuses on three agonists that cause local and conducted dilation in skeletal muscle: ACh, ATP, and KCl. The mechanisms for the release of these agonists during motor nerve stimulation and/or hypoxia, and their actions to open either Ca 2+ -activated K + channels (K C a ) or inwardly rectifying K + channels (K IR ) are described. By causing local and conducted dilation, each agonist has the ability to improve skeletal muscle blood flow during exercise and ischemia., (© 2016 John Wiley & Sons Ltd.)

Background: Pulmonary dysfunction is a known complication after cardiac surgery using cardiopulmonary bypass, ranging from subclinical functional changes to prolonged postoperative ventilation, acute lung injury, and acute respiratory distress syndrome. Whether human pulmonary arterial function is compromised is unknown. The aim of the present study was to compare the structure and function of isolated and cannulated human pulmonary arteries obtained from lung biopsies after the chest was opened (pre-cardiopulmonary bypass) to those obtained at the end of cardiopulmonary bypass (post-cardiopulmonary bypass) from patients undergoing coronary artery bypass graft surgery., Methods and Results: Pre- and post-cardiopulmonary bypass lung biopsies were received from 12 patients undergoing elective surgery. Intralobular small arteries were dissected, cannulated, pressurized, and imaged using confocal microscopy. Functionally, the thromboxane mimetic U46619 produced concentration-dependent vasoconstriction in 100% and 75% of pre- and post-cardiopulmonary bypass arteries, respectively. The endothelium-dependent agonist bradykinin stimulated vasodilation in 45% and 33% of arteries pre- and post-cardiopulmonary bypass, respectively. Structurally, in most arteries smooth muscle cells aligned circumferentially; live cell viability revealed that although 100% of smooth muscle and 90% of endothelial cells from pre-cardiopulmonary bypass biopsies had intact membranes and were considered viable, only 60% and 58%, respectively, were viable from post-cardiopulmonary bypass biopsies., Conclusions: We successfully investigated isolated pulmonary artery structure and function in fresh lung biopsies from patients undergoing heart surgery. Pulmonary artery contractile tone and endothelium-dependent dilation were significantly reduced in post-cardiopulmonary bypass biopsies. The decreased functional responses were associated with reduced cell viability., Clinical Trial Registration: URL: http://www.isrctn.com/ISRCTN34428459. Unique identifier: ISRCTN 34428459., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background and Purpose: Smooth muscle transient receptor potential melastatin 4 (TRPM4) channels play a fundamental role in the development of the myogenic arterial constriction that is necessary for blood flow autoregulation. As TRPM4 channels are present throughout the vasculature, we investigated their potential role in non-myogenic resistance arteries using the TRPM4 inhibitor 9-phenanthrol., Experimental Approach: Pressure and wire myography were used to assess the reactivity of rat arteries, the latter in combination with measurements of smooth muscle membrane potential. Immunohistochemistry (IHC) and endothelial cell (EC) calcium changes were assessed in pressurized vessels and patch clamp measurements made in isolated ECs., Key Results: The TRPM4 inhibitor 9-phenanthrol reversibly hyperpolarized mesenteric arteries to circa EK and blocked α1 -adrenoceptor-mediated vasoconstriction. Hyperpolarization was abolished and vasoconstriction re-established by damaging the endothelium. In mesenteric and cerebral artery smooth muscle, 9-phenanthrol hyperpolarization was effectively blocked by the KCa 3.1 inhibitor TRAM-34. 9-Phenanthrol did not increase mesenteric EC [Ca(2+)]i , and Na(+) substitution with N-methyl-D-glucamine only increased the muscle resting potential by 10 mV. Immunolabelling for TRPM4 was restricted to the endothelium and perivascular tissue., Conclusions and Implications: These data reveal a previously unrecognized action of the TRPM4 inhibitor 9-phenanthrol - the ability to act as an activator of EC KCa 3.1 channels. They do not indicate a functionally important role for TRPM4 channels in the reactivity of non-myogenic mesenteric arteries., (© 2014 The British Pharmacological Society.)

Our understanding of the relationship between EC membrane potential and Ca(2+) entry has been shaped historically by data from cells in culture. Membrane hyperpolarization was associated with raised cytoplasmic [Ca(2+) ] ascribed to the increase in the inward electrochemical gradient for Ca(2+) , as ECs are generally thought to lack VGCC. Ca(2+) influx was assumed to reflect the presence of an undefined Ca(2+) "leak" channel, although the original research articles with isolated ECs did not elucidate which Ca(2+) influx channel was involved or indeed if a transporter might contribute. Overall, these early studies left many unanswered questions, not least whether a similar mechanism operates in native ECs that are coupled to each other and, in many smaller arteries and arterioles, to the adjacent vascular SMCs via gap junctions. This review discusses whether Ca(2+) leak through constitutively active EC Ca(2+) channels or a more defined, gated pathway might underlie the reported link between enhanced Ca(2+) entry and hyperpolarization. Electrophysiological evidence from ECs in isolation is compared with those in intact arteries and arterioles and the possible physiological relevance of EC Ca(2+) entry driven by hyperpolarization discussed., (© 2013 John Wiley & Sons Ltd.)

The use of single- and dual-wavelength Ca(2+)-sensitive fluorescent dyes to monitor changes in endothelial and/or smooth muscle intracellular Ca(2+) levels has provided information linking Ca(2+) events to changes in arterial function. Here we describe the in vitro techniques used to selectively load Ca(2+) indicators into either the endothelium or the smooth muscle of cannulated rat cremaster arteries. These vessels normally develop spontaneous myogenic tone that is largely unaffected by the loading of Ca2+ indicators or the subsequent imaging procedures. This suggests that there is minimal Ca2+ buffering or damage, and that the fluorescent indicator-loaded vessels behave similarly to unloaded preparations. Importantly, these approaches are applicable to both isobaric and isometric preparations and have been also used for the study of a number of vascular beds including cerebral, mesenteric, coronary, and skeletal muscle vasculatures.

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.

Objective: Despite the role that extracellular matrix (ECM) plays in vascular signaling, little is known of the complex structural arrangement between specific ECM proteins and vascular smooth muscle cells. Our objective was to examine the hypothesis that adventitial elastin fibers are dominant in vessels subject to longitudinal stretch., Methods and Results: Cremaster muscle arterioles were isolated, allowed to develop spontaneous tone, and compared with small cerebral arteries. 3D confocal microscopy was used to visualize ECM within the vessel wall. Pressurized arterioles were fixed and stained with Alexa 633 hydrazide (as a nonselective ECM marker), anti-elastin, or anti-type 1 collagen antibody and a fluorescent nuclear stain. Exposure of cremaster muscle arterioles to elastase for 5 minutes caused an irreversible lengthening of the vessel segment that was not observed in cerebral arteries. Longitudinal elastin fibers were demonstrated on cremaster muscle arterioles using 3D imaging but were confirmed to be absent in cerebral vessels. The fibers were also distinct from type I collagen fibers and were degraded by elastase treatment., Conclusions: These results indicate the importance of elastin in bearing longitudinal stress in the arteriolar wall and that these fibers constrain vascular smooth muscle cells. Differences between skeletal muscle and cerebral small arteries may reflect differences in the local mechanical environment, such as exposure to longitudinal stretch.

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., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)