BackgroundRecent studies demonstrate that spatially restricted, local Ca2+ signals are key regulators of endothelium‐dependent vasodilation in systemic circulation. There are drastic functional differences between pulmonary arteries (PAs) and systemic arteries, but the local Ca2+ signals that control endothelium‐dependent vasodilation of PAs are not known. Localized, unitary Ca2+ influx events through transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, regulate endothelium‐dependent vasodilation in resistance‐sized mesenteric arteries via activation of Ca2+‐dependent K+ channels. The objective of this study was to determine the unique functional roles, signaling targets, and endogenous regulators of TRPV4 sparklets in resistance‐sized PAs.Methods and ResultsUsing confocal imaging, custom image analysis, and pressure myography in fourth‐order PAs in conjunction with knockout mouse models, we report a novel Ca2+ signaling mechanism that regulates endothelium‐dependent vasodilation in resistance‐sized PAs. TRPV4 sparklets exhibit distinct spatial localization in PAs when compared with mesenteric arteries, and preferentially activate endothelial nitric oxide synthase (eNOS). Nitric oxide released by TRPV4‐endothelial nitric oxide synthase signaling not only promotes vasodilation, but also initiates a guanylyl cyclase‐protein kinase G‐dependent negative feedback loop that inhibits cooperative openings of TRPV4 channels, thus limiting sparklet activity. Moreover, we discovered that adenosine triphosphate dilates PAs through a P2 purinergic receptor‐dependent activation of TRPV4 sparklets.ConclusionsOur results reveal a spatially distinct TRPV4‐endothelial nitric oxide synthase signaling mechanism and its novel endogenous regulators in resistance‐sized PAs.

Background: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca 2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca 2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca 2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. Methods: We used endothelium-specific knockout mice and high-fat diet–fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. Results: We show that Ca 2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. Conclusions: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

These results demonstrate that SMC GPCR stimulation triggers inositol 1,4,5-trisphosphate-dependent activation of endothelial TRPV4 channels to limit vasoconstriction.

Key points Endothelial cell TRPV4 (TRPV4EC) channels exert a dilatory effect on the resting diameter of resistance mesenteric and pulmonary arteries. Functional intermediate‐ and small‐conductance K+ (IK and SK) channels and endothelial nitric oxide synthase (eNOS) are present in the endothelium of mesenteric and pulmonary arteries. TRPV4EC sparklets preferentially couple with IK/SK channels in mesenteric arteries and with eNOS in pulmonary arteries. TRPV4EC channels co‐localize with IK/SK channels in mesenteric arteries but not in pulmonary arteries, which may explain TRPV4EC‐IK/SK channel coupling in mesenteric arteries and its absence in pulmonary arteries. The presence of the nitric oxide‐scavenging protein, haemoglobin α, limits TRPV4EC‐eNOS signalling in mesenteric arteries. Spatial proximity of TRPV4EC channels with eNOS and the absence of haemoglobin α favour TRPV4EC‐eNOS signalling in pulmonary arteries. Abstract Spatially localized Ca2+ signals activate Ca2+‐sensitive intermediate‐ and small‐conductance K+ (IK and SK) channels in some vascular beds and endothelial nitric oxide synthase (eNOS) in others. The present study aimed to uncover the signalling organization that determines selective Ca2+ signal to vasodilatory target coupling in the endothelium. Resistance‐sized mesenteric arteries (MAs) and pulmonary arteries (PAs) were used as prototypes for arteries with predominantly IK/SK channel‐ and eNOS‐dependent vasodilatation, respectively. Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4EC) channels played an important role in controlling the baseline diameter of both MAs and PAs. TRPV4EC channel activity was similar in MAs and PAs. However, the TRPV4 channel agonist GSK1016790A (10 nm) selectively activated IK/SK channels in MAs and eNOS in PAs, revealing preferential TRPV4EC‐IK/SK channel coupling in MAs and TRPV4EC‐eNOS coupling in PAs. IK/SK channels co‐localized with TRPV4EC channels at myoendothelial projections (MEPs) in MAs, although they lacked the spatial proximity necessary for their activation by TRPV4EC channels in PAs. Additionally, the presence of the NO scavenging protein haemoglobin α (Hbα) within nanometer proximity to eNOS limits TRPV4EC‐eNOS signalling in MAs. By contrast, co‐localization of TRPV4EC channels and eNOS at MEPs, and the absence of Hbα, favour TRPV4EC‐eNOS coupling in PAs. Thus, our results reveal that differential spatial organization of signalling elements determines TRPV4EC‐IK/SK vs. TRPV4EC‐eNOS coupling in resistance arteries.

Impaired endothelium‐dependent vasodilation is a hallmark finding of obesity‐induced hypertension. Recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. Here, we show that Ca2+ influx through TRPV4 channels at myoendothelial projections (MEPs) to smooth muscle cells lowers resting blood pressure in non‐obese mice, a response that is diminished in obese mice. Counter‐intuitively, release of nitric oxide (NO) from endothelial cells attenuates TRPV4 channel activity and TRPV4‐mediated vasodilation in the obese animals. Increased activities of iNOS and NOX1 correlated with elevated levels of NO and superoxide radicals at the MEPs in obese mice, ultimately reacting to form peroxynitrite. Subsequent findings revealed that peroxynitrite causes cysteine oxidation of the regulatory protein AKAP150 to inhibit TRPV4 channel activity at the MEPs. TRPV4 channel activity and vasodilation are also impaired in the arteries from obese patients, and are restored by strategies that lower peroxynitrite levels. These data suggest that defective TRPV4 channel–mediated vasodilation may contribute to obesity‐induced hypertension, and imply that inhibiting peroxynitrite may represent a strategy to normalize endothelial TRPV4 channel activity, vasodilation and blood pressure during obesity. Support or Funding Information HL121484‐01, HL138496