AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension

Abstract

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 vaso-dilation, 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.

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…Endothelial denudation was performed by passing an air bubble through the artery for ≈60 s. Complete removal of the endothelial layer was verified by the absence of dilation to NS309. Changes in arterial diameter were recorded at a 60‐ms frame rate using a charge‐coupled device camera and edge‐detection software (IonOptix LLC, Westwood, MA) 4, 5. All drug treatments were incubated for 10 minutes.…”

“…Measurement of Ca 2+ events in the native ECs from PAs was performed as previously described 4, 5. Briefly, Andor Revolution WD (with Borealis) spinning‐disk confocal imaging system described above was used to record Ca 2+ influx events in en face PAs.…”

“…Representative F/F 0 traces were filtered using a Gaussian filter and a cutoff corner frequency of 4 Hz. Sparklet activity, all‐points histograms, and coupling coefficients were determined as described previously using the custom‐designed SparkAn software, Clampfit, and OriginPro7.5 4, 5…”

“…Regions of interest (1.7 μm 2 ) corresponding to the peak sparklet fluorescence were overlaid onto the IEL staining image. The regions of interest within 5 μm from the center of the holes in the IEL were counted as MEP sparklet sites and the remaining regions of interest were counted as non‐MEP sparklet sites, as we have done previously 5. A sparklet site displays spread of calcium away from the site of initiation.…”

“…Recent studies in systemic arteries—including mesenteric, cremaster, and cerebral arteries—have demonstrated that endothelium‐dependent vasodilation is controlled by local, spatially restricted increases in endothelial Ca 2+ while the whole‐cell Ca 2+ level remains unchanged 1, 2, 3, 4, 5. In contrast to systemic arteries, pulmonary arteries (PAs) comprise a low‐pressure, high‐flow circulation.…”