Background and Purpose Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary blood flow. Ca2+-activated chloride channels contribute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca2+-activated chloride channels, TMEM16A, in rat coronary arteries. Experimental Approach We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel inhibitors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs). Key Results TMEM16A was expressed in rat coronary arteries. The TMEM16A-specific inhibitor, MONNA, hyperpolarised the membrane potential in U46619. MONNA, T16A(inh)-A01, and Ani9 attenuated 5-HT/U46619-induced contractions. MONNA and T16A(inh)-A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5-HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5-HT. Conclusions and Implications In conclusion, TMEM16A is a key regulator of coronary blood flow and is implicated in the altered contractility of coronary arteries from SHRs.

Key points KV7 channels are a family of voltage‐dependent K+ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability. Drugs that target KV7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how. In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow. Urinary bladder smooth muscle had no measurable KV7 channel currents. However, the KV7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that KV7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness. AbstractKV7 channels are voltage‐dependent K+ channels that open in response to membrane depolarization to regulate cell excitability. KV7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of KV7 channels as regulators of murine urinary bladder function. The KV7 activator retigabine (10 μM) had no effect on voltage‐dependent K+ currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine‐sensitive KV7 channels. The KV7 inhibitor XE‐991 (10 μM) inhibited UBSM K+ currents; the properties of these currents, however, were typical of KV2 channels and not KV7 channels. Retigabine inhibited voltage‐dependent Ca2+ channel (VDCC) currents and reduced steady‐state contractions to 60 mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co‐incubation with XE‐991, suggesting specific activation of KV7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non‐specific effects on VDCCs and KV2 channels.

The muscarinic receptor antagonist, Oxybutynin (Ox), and the β3‐receptor agonist, mirabegron (Mb), are used in the treatment of overactive bladder syndrome (OAB). However, the exact downstream mechanisms by which Ox and Mb opposes the enhanced contractility in OAB is unknown. The KCNQ genes encode five Kv7 K+ channel subunits (KV7.1–Kv7.5). Of these, KV7.2–KV7.5 channels have been proposed to be involved in regulation of smooth muscle tone, i.e. the contractile state of the bladder. In this study, we investigated the involvement of KV7.2–KV7.5 channels in Ox‐ and Mb‐induced relaxation of the bladder. Furthermore, the direct effect of KV7.2–KV7.5 channel‐activation on bladder relaxation was determined. Patch clamp recordings from HEK cells expressing KV7.1–KV7.5 channels were used to determine the relative potency of five different activators; ICA‐27243, ML213, NS15370, Retigabine, and SciFluor, on the KV7.2–KV7.5 channels. Bladder strips from rats mounted in organ baths and contracted with electrical field stimulation were used to investigate the functional effects of inhibiting and activating KV7.2–KV7.5 channels. This study found that inhibiting KV7 channels with XE991 markedly reduced Ox‐ and Mb‐induced relaxation of bladder strips. Furthermore, bladder strip EC50 values corresponded well with HEK cell EC50 values for both KV7.2–3 and Kv7.4 channels, and arranged the five activators with the most potent first accordingly: NS15370>ML213=SciFluor>Retigabine>ICA‐27243. In conclusion, this study showed that KV7.2–KV7.5 channels are probably involved in the therapeutic relaxation of the bladder induced by Ox and Mb in OAB patients. In addition, direct pharmacological activation of KV7.2–KV7.5 channels induced a potent relaxation of the bladder, suggesting that KV7.2–KV7.5 channel activators may be a new target for treating OAB ‐ either as standalone treatment or as enhancers of the effect of currently available treatment options.Support or Funding InformationThis study was funded by the Innovation Fund Denmark.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BACKGROUND AND PURPOSE PDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone. EXPERIMENTAL APPROACH In rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings. KEY RESULTS In rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation inmesenteric arteries. Inhibition of NOS with L-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect. CONCLUSIONS AND IMPLICATIONS Pde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms.

We investigated the acute effects of glucagon-like peptide-1 (GLP-1), GLP-1(1-36), and GLP-1(7-36) on vascular endothelial growth factor-A (VEGFA)-induced endothelium-dependent signaling and vasodilation. Our hypothesis was that GLP-1 released from intestinal l-cells modulates processes related to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries. In isolated rat mesenteric resistance arteries precontracted with the thromboxane analog U46619 to 80-90% of maximum contraction, VEGFA (25 ng/ml) caused a small and gradual relaxation (28.9 ± 3.9%). Pretreatment of arteries with either GLP-1(1-36) (500 nM) or GLP-1(7-36) (1 nM) abolished the VEGFA-induced relaxation. VEGFA-induced relaxations were also inhibited in endothelial-denuded arteries and in arteries pretreated with the nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester (100 μM). In vivo studies on male Wistar rats also revealed that GLP-1(7-36) inhibited VEGFA-induced vasodilation of the same arteries. In isolated endothelial cells, GLP-1(1-36) and GLP-1(7-36) caused a reduction in VEGFA-induced phosphorylation of PLCγ. Ca2+ imaging of endothelial cells and rat mesenteric resistance arteries using fura-2, revealed that both GLP-1 analogs caused a reduction in VEGFA-induced Ca2+ signaling. GLP-1(1-36) also reduced VEGFA-induced eNOS phosphorylation in HDMEC. In conclusion, GLP-1 reduced relaxation induced by VEGFA in resistance arteries by inhibiting VEGFR2-mediated Ca2+ signaling and endothelial NO synthesis. GLP-1, on its own, also induced phosphorylation of Src and ERK1/2 that can lead to proliferation and is implicated in vessel permeability.

The incretine GLP‐1 enhances insulin release and thereby causes a diminished blood glucose peak in response to food intake. We explored the potential roles of GLP‐1 in vascular function by studying signaling in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells (HRMEC) and rat mesenteric resistance arteries. We found that HDMEC, and HRMEC and resistance vessels express the GLP‐1R by use of RT‐PCR. GLP‐1, on its own, induced phosphorylation of Src and ERK 1/2. By use of Ca 2+ imaging we found that pre‐incubation for 15 min with GLP‐1 caused a reduction in VEGF‐A (25 ng/ml) induced Ca 2+ signaling due to a reduction in phosphorylation of PLCγ. In addition, GLP‐1 also caused a reduction in the [Ca 2+ ] i rise evoked by ATP 4− which is partially due to PLC activation and IP 3 synthesis through the P 2 X 7 receptor. We also found a reduction in VEGF‐A induced Ca 2+ increase in rat mesenteric resistance arteries preincubated with GLP‐1. A VEGFA induced increase in [Ca 2+ ] i is mediated through PLCγ and it has been shown that PLCγ activation is uniquely dependent on a G αi protein (Yang et al., J. Biol. Chem. (266:33, 1991). In accordance with this observation we found a strong inhibition of VEGFA‐induced Ca 2+ signaling after preincubation with pertussin toxin (100 ng/ml), which is an inhibitor of G αi . Our data are thus consistent with the GLP‐1 signaling being evoked through an enhanced Gs/βγ signaling over that of G αi since the G αi inhibition with pertussis toxin elicits a similar response thus favoring the Gs/βγ signaling pathway. In rat mesenteric resistance arteries mounted in a wire myograph and precontracted with the thromboxane analogue U46619 to 90% of their maximum contraction, VEGF‐A caused partial relaxation (−35±4%), an effect that was blocked by GLP‐1(7–36) (1000 pM). VEGF‐A induced relaxation was also inhibited in endothelial denuded arteries and in arteries pretreated with the NOS inhibitor N ω ‐nitro‐l‐arginine methyl ester (100 μM). In addition, preincubation with GLP‐1 caused a reduction in VEGF‐A induced eNOS phosphorylation consistent with endothelial NO synthesis being responsible for the relaxation. In resistance vessels VEGF is released from smooth muscle cells. Our data are consistent with GLP‐1 inhibiting VEGFR signaling by modulating G i induced processes such as PLCγ phosphorylation by enhancing signaling of Gs/βγ signaling over that of Gαi. The blockade of VEGF‐A vessel relaxation by GLP‐1 is in accordance with the diminished eNOS phosphorylation seen when endothelial cells are preincubated with GLP‐1. Thus, GLP‐1 can reduce relaxation induced by VEGF‐A in resistance vessels by inhibiting the VEGFR induced endothelial NO synthesis. We hypothesize that GLP‐1 released from intestinal L‐cells after food intake modulates physiological processes related to PLCγ activation such as endothelial control of vessel tone, endothelial cell migration and proliferation.

Key points Increase in endothelial cell (EC) calcium activates calcium‐sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium‐dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium‐dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium‐dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. AbstractEndothelium‐dependent vasodilators, such as acetylcholine, increase intracellular Ca2+ through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca2+‐sensitive intermediate and small conductance K+ (IK and SK, respectively) channels. Although strong inward rectifier K+ (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC‐dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC‐dependent vasodilatation of resistance‐sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC‐specific Kir2.1 knockdown (EC‐Kir2.1−/−) mice. Elevation of extracellular K+ to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba2+, ML‐133) or in the arteries from EC‐Kir2.1−/− mice. Potassium‐induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba2+, did not affect currents through TRPV4, IK or SK channels. Endothelial cell‐dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC‐Kir2.1−/−. In angiotensin II‐induced hypertension, the Kir channel function was not altered, although the endothelium‐dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca2+‐dependent activation of IK and SK channels.

Sepsis is characterized by systemic inflammation, edema formation and hypo-perfusion leading to organ dysfunction and ultimately death. Activation of the transient receptor potential vanilloid type 4 (TRPV4) channel is associated with edema formation and circulatory collapse. Here, we show that TRPV4 channels are involved in the hyper-inflammatory response and mortality associated with sepsis. Pharmacological inhibition of TRPV4 channels in mice reduced mortality in lipopolysaccharide and cecal-ligation-and-puncture models of sepsis, but not in a tumor necrosis factor-α (TNFα)-induced sepsis model. These protective effects of TRPV4 channel inhibition were attributable to prevention of the sepsis-induced surge of a broad spectrum of pro-inflammatory cytokines, including TNFα, interleukin (IL)-1 and IL-6 and subsequent preservation of endothelial cell function, including Ca2+ signaling, integrity and endothelium-dependent vasodilation. These results suggest that TRPV4 antagonists may be of therapeutic utility in the management of sepsis.

Significance Small vessel disease (SVD) of the brain refers to a group of pathological processes leading to cerebral lesions, cognitive decline, and stroke. Despite the importance of SVD, there is no specific treatment, mainly due to a limited understanding of the disease pathogenesis. Using a recently developed mouse model of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, a hereditary form of SVD, we determined the basis of altered brain artery function at an early stage of disease progression. We found that cerebrospecific up-regulation of the voltage-gated potassium channel, K V 1, prevents intracerebral arterioles from constricting in response to physiological levels of intraluminal pressure. This impairment of a fundamental vascular function is expected to impact cerebral blood flow autoregulation and local dilation in response to neuronal activity (functional hyperemia).

Background and PurposeThe intermediate conductance calcium/calmodulin-regulated K+ channel K(Ca)3.1 produces hyperpolarizing K+ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl- and fluid movements. We investigated whether a deficiency in K(Ca)3.1 (K(Ca)3.1(-/-)) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels.Experimental ApproachAn opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and K(Ca)3.1(-/-) mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC).Key ResultsIn wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, K(Ca)3.1(-/-) mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of K(Ca)3.1(-/-) mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and K(Ca)3.1(-/-) mice, which co-activated K(Ca)3.1 and disrupted membrane resistance in wt PAEC, but not in K(Ca)3.1(-/-) PAEC.Conclusions and ImplicationsOur findings show that a genetic deficiency of K(Ca)3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of K(Ca)3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.

Smooth muscle cells (SMCs) in systemic resistance (mesenteric) arteries lack strong inward rectifier K+ (Kir) channels, which are responsible for external K+‐induced dilations of cerebral arteries. Here we report that pressurized, resistance‐sized (3rd‐order branches) mesenteric arteries from mice dilate to an elevation of external K+ from 6 to 14 mM, which is prevented by Kir blockers Ba2+ (100 μM) and ML 133 (20 μM) or removal of the endothelium. Freshly isolated endothelial cells (ECs) exhibited robust Kir currents (‐7 pA/pF at ‐140 mV, 60 mM external K+). Kir channel currents and K+‐induced dilations were absent in myocytes or arteries from EC‐specific Kir2.1 knockout mice. We have recently shown that muscarinic receptor activation causes vasodilation through membrane hyperpolarization via stimulation of EC TRPV4 (transient receptor potential vanilloid 4) and IK (intermediate conductance calcium activated potassium) channels (Sonkusare et al, Science, 2012; Science Signaling, 2014). We therefore tested the hypothesis that EC Kir channels act as amplifiers to this signaling pathway to enhance EC‐dependent vasodilation. Indeed, vasodilation to stimulation of each element of this pathway (muscarinic receptors, TRPV4, IK) was attenuated by EC Kir channel blockers or in the absence of EC‐Kir2.1. These results suggest that EC Kir2.1 channels have a central role in vasodilation to external K+ and endothelial‐dependent vasodilators.

Perivascular retina has been shown to regulate retinal vascular tone. In the present study, we evaluated an ex vivo retina preparation, and investigated whether hydrogen sulfide (H2S) mediates an inhibitory effect of retina and/or hypoxia on arteriolar tone. In retina, immunolabeling showed an increase of glial fibrillary acidic protein, but not vimentin over time in Müller cells, and the presence of necrotic cells after 2 h and apoptotic cells after 8 h. Isometric tension recordings showed endothelin-1(ET-1) to induce concentration-dependent contractions, which were reduced in the presence of retina. In arterioles with retina no change was observed in ET-1 contractions after 5 h compared to 8 h. Hypoxia (1% O2) reduced ET-1 contraction in arterioles with and without retina. The H2S donor, GYY4137 and the salt, sodium hydrogen sulfide, induced concentration-dependent relaxations in ET-1 contracted retinal arterioles. Inhibition of the H2S producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), with carboxymethoxylamine (AOA) and L-propargylglycine (PPG) enhanced ET-1 contractions. This effect was more pronounced in hypoxic conditions. However, even in the presence of AOA and PPG ET-1 induced less contraction in the presence of perivascular retina compared to isolated vessels. These findings suggest that both the presence of perivascular retina and hypoxia reduce arteriolar vasoconstriction and that both H2S and another factor mediate this effect. Finally, H2S donors, as well as endogenous H2S, can reduce retinal arteriolar tone, suggesting a potential therapeutic role for enhanced H2S bioavailability in the treatment of retinal disease.

The carbonic anhydrase (CA) inhibitor acetazolamide (AZ) is used routinely to estimate cerebrovascular reserve capacity in patients, as it reliably increases cerebral blood flow (CBF). However, the mechanism by which AZ accomplishes this CBF increase is not entirely understood. We recently discovered that CA can produce nitric oxide (NO) from nitrite, and that AZ enhances this NO production in vitro. In fact, this interaction between AZ and CA accounted for a large part of AZ's vasodilatory action, which fits well with the known vasodilatory potency of NO. The present study aimed to assess whether AZ acts similarly in vivo in the human cerebrovascular system. Hence, we increased or minimized the dietary intake of nitrate in 20 healthy male participants, showed them a full-field flickering dartboard, and measured their CBF response to this visual stimulus with arterial spin labeling. Doing so, we found a significant positive interaction between the dietary intake of nitrate and the CBF modulation afforded by AZ during visual stimulation. In addition, but contrary to studies conducted in elderly participants, we report no effect of nitrate intake on resting CBF in healthy human participants. The present study provides in vivo support for an enhancing effect of AZ on the NO production from nitrite catalyzed by CA in the cerebrovascular system. Furthermore, our results, in combination with the results of other groups, indicate that nitrate may have significant importance to vascular function when the cerebrovascular system is challenged by age or disease.

Endothelial cell (EC) dysfunction is a hallmark of hypertension. We recently discovered that local Ca2+ influx through clusters of functionally coupled TRPV4 channels (detected optically as “TRPV4 sparklets”) drives physiological vasodilation. Here, we show that stimulation of EC muscarinic receptors activated TRPV4 sparklets exclusively at discrete sites at myoendothelial projections (MEPs)—specialized regions of ECs that contact adjacent smooth muscle cells. This activation was dependent on protein kinase C (PKC) and was absent in mice lacking the PKC‐anchoring protein AKAP150 (A‐kinase anchoring protein 150), which was localized predominantly to MEPs. Cooperative gating of TRPV4 channels within a cluster amplified Ca2+ influx at MEPs by more than 2‐fold. This cooperativity was largely absent at non‐MEP sites and was virtually eliminated by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting AKAP150‐dependent potentiation of TRPV4 activity by Ca2+ influx via adjacent channels. Notably, MEP‐localization of AKAP150 was disrupted in angiotensin II‐induced hypertension, leading to a complete loss of muscarinic activation of TRPV4 channels, much weaker coupling among TRPV4 channels at MEPs, and approximately an 80% reduction in carbachol‐induced vasodilation. Our results support the concept that endothelial‐dependent vasodilation of resistance arteries is enabled by MEP‐localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and coupled channel gating necessary for efficient communication of the endothelium to the smooth muscle cells in arteries—a molecular configuration that is disrupted in hypertension.Grant Funding Source: Supported by HL044455, 1P01HL095488, R37DK053832, R01HL098243 to MTN

Endothelial cell dysfunction, characterized by a diminished response to endothelial cell-dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vasodilation, a function mediated by local Ca2+ influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca2+. We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor-mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca2+ influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting that Ca2+ entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II-induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension.