“Spatial localization determines preferential calcium signal‐target coupling in microvascular endothelium”
Authors: Ottolini Matteo , Daneva Zdravka , Cope Eric Lee , Chen Yen-Lin , Sonkusare Swapnil K. 
Published: April 2020

Endothelial cell layer is an important regulator of vascular resistance. Localized, spatially restricted Ca2+ signals in endothelial cells promote vasodilation in systemic and pulmonary arteries, thereby lowering vascular resistance. Ca2+ signals are known to activate two main vasodilatory targets in endothelial cells: intermediate and small conductance Ca2+‐activated K+ (IK and SK, respectively) channels and endothelial nitric oxide synthase (eNOS). The goal of this study was to determine the specific mechanisms underlying preferential Ca2+ signal to vasodilatory target coupling. Based on the literature, we used resistance mesenteric arteries (MA) as a prototype for Ca2+ signal‐IK/SK channel coupling, and resistance pulmonary arteries (PA) as a prototype for Ca2+ signal‐eNOS coupling. Unitary Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, dilate both mesenteric and pulmonary arteries, thereby lowering resting systemic and pulmonary arterial pressures. Therefore, the study focused on the coupling of endothelial TRPV4 channels with their vasodilator targets. Whole‐cell patch clamp studies in freshly isolated endothelial cells showed that TRPV4 channel activator GSK1016790A (GSK101, 10 nM) induced IK/SK currents in MA endothelial cells, but not in PA endothelial cells. However, direct IK/SK channel activator NS309 (1 mM) or 3 mM intracellular Ca2+ was able to elicit similar IK/SK currents in both MA and PA endothelial cells, confirming the presence of IK/SK channels in endothelial cells from PA and supporting a lack of TRPV4‐IK/SK channel coupling in PAs. NO measurements with fluorescent indicator DAF‐FM showed increased endothelial NO levels in response to GSK101 in PAs but not in MAs, supporting a lack of TRPV4 sparklet‐eNOS coupling in MAs. Because of the localized nature of TRPV4 sparklets, we hypothesized that spatial co‐localization determines Ca2+ signal‐vasodilator target coupling. While TRPV4 channels were localized at endothelial projections to smooth muscle or myoendothelial projections (MEPs) in both MAs and PAs, IK/SK channels were seen at MEPs only in MAs, supporting the importance of spatial co‐localization for TRPV4‐IK/SK channel coupling. Interestingly, eNOS was localized at MEPs in both MAs and PAs, raising the question why TRPV4 sparklets cannot activate eNOS in MAs despite the spatial proximity. Therefore, we postulated that hemoglobin α (Hbα), which is known to bind eNOS and scavenge NO, silences TRPV4 channel‐eNOS coupling in MAs. Hbα immunostaining was seen mostly at MEPs in MAs, but was absent in PA endothelium. Moreover, Hbα existed in nanometer proximity with eNOS in MAs. Finally, a synthetic peptide that disrupts eNOS‐Hbα interaction uncovered the TRPV4‐eNOS coupling in MAs. Our results suggest that spatial co‐localization and the presence of Hbα determine Ca2+ signal‐vasodilator target coupling in endothelial cells. Understanding the differential coupling mechanisms may lead to novel therapeutic approaches that can selectively dilate pulmonary versus systemic vasculature.

Support or Funding Information

HL121484‐01, HL138496