Nitric Oxide Inhibits Functional Coupling Among Endothelial TRPV4 Channels and Limits Vasodilation in Small Pulmonary Arteries

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Vasodilatory function of endothelial cells (ECs) is regulated by localized increases in intracellular Ca2+. TRPV4 (transient receptor potential vanilloid 4) channels on the EC membrane are a major Ca2+ influx pathway in the systemic and pulmonary endothelium, and a key determinant of endothelial vasodilations to physiological stimuli. We recently showed that localized, unitary Ca2+ influx events through TRPV4 channels (TRPV4 Ca2+ sparklets) on the EC membranes dilate resistance‐sized pulmonary arteries (PAs) through selective activation of endothelial nitric oxide synthase (eNOS) (Marziano et al., FASEB J, 2016) and NO release. Studies in the expression system have shown that NO can modify the activity of TRPV4 channels. Based on the predominant role for NO in TRPV4‐dependent vasodilations in the PAs, we hypothesized that NO released by TRPV4‐eNOS activation alters TRPV4 channel function in the native PA endothelium, exerting a finer control over TRPV4‐mediated vasodilations. In en face 4th order PAs loaded with Fluo‐4, NOS inhibitor (L‐NG‐Nitroarginine or L‐NNA; 100 μM) produced a 2‐fold increase in TRPV4 sparklet activity, whereas a NO donor (Spermine NONOate or NONOate; 30 μM) inhibited TRPV4 sparklet activity by ~70%, confirming NO‐inhibition of TRPV4 sparklet activity. Inhibitors of S‐nitrosylation did not alter the effect of NO on TRPV4 channel activity ruling out a role for S‐nitrosylation in NO‐inhibition of the channel. However, guanylyl cyclase (GC) and protein kinase G (PKG) inhibitors (ODQ; 3 μM and Rp‐8‐Br‐PET‐cGMPS or PET; 30 μM, respectively) caused a 2‐fold increase in TRPV4 sparklet activity, and abolished the effect of NONOate on TRPV4 sparklets. Concurrently, cannulated and pressurized 4th order PAs showed a significantly higher dilation to the specific TRPV4 channel activator GSK1016790A (3–30 nM) in the presence of ODQ when compared to control arteries. These results revealed the negative feedback inhibition of TRPV4 activity and vasodilations by NO‐GC‐PKG signaling in PAs. Recent studies demonstrate that cooperative openings of TRPV4 channels at an individual sparklet site determine Ca2+ influx through the channels, and are dependent on Ca2+‐potentiation of the channel (Sonkusare et al., Sci Signal., 2014). To determine whether PKG altered TRPV4 channel cooperativity we used a coupled Markov chain model to examine the coupling strength/cooperativity among TRPV4 channels at individual sparklet sites. Whereas L‐NNA stimulated the coupling among TRPV4 channels, NONOate reduced the coupling. Moreover, both PET and ODQ increased the coupling strength and inhibited the effect of NONOate. Chelating local Ca2+ with membrane permeable EGTA‐AM (5 μM) also inhibited the effect of NONOate on sparklet activity, indicating that NO‐GC‐PKG activation disrupted Ca2+‐dependent cooperativity among TRPV4 channels. Together these data reveal a novel NO‐dependent regulatory mechanism that mediates and limits TRPV4‐vasodilations in PAs. Perturbations in the endothelial GC‐PKG dependent negative‐feedback mechanism can be particularly important in pulmonary disorders including pulmonary hypertension, pulmonary edema and lung injury that are associated with endothelial dysfunction.Support or Funding InformationThis study was supported by a grant from NIH (HL121484‐01) to S.K.S and (4T32HL007284‐40) to C.M.