The role of TRPV4 (transient receptor potential vanilloid 4) in regulating vascular contraction in hypertensive mice is poorly established. We tested the hypothesis that TRPV4 regulates endothelium-dependent contractions in aortas from hypertensive mice through the activation of cytosolic cPLA (phospholipase A) and COX2 (cyclooxygenase 2) and identified the possible endothelium-derived contracting factor generated by COX2. Using myography, we demonstrated that GSK1016790A (a TRPV4 agonist) and acetylcholine (ACh) trigger endothelium-dependent contractions in aortas from hypertensive mice, and the contractions were abolished with TRPV4 deletion. PLA assay and Western blotting showed that cPLA activity was higher in salt-induced hypertension and HC067047 or a Ca chelator inhibited cPLA activity. Contractions induced by TRPV4 and ACh were inhibited by the cPLA inhibitor or removal of extracellular Ca COX2 expression was enhanced in the endothelium from hypertensive mice and contractions induced by TRPV4 or ACh were inhibited by the COX2 inhibitor. Enzyme immunoassay showed that the release of prostaglandin F (PGF) was increased in hypertensive mice. GSK1016790A or ACh triggered the release of PGF and this was inhibited by HC067047, the cPLA inhibitor, and COX2 inhibitor. GSK1016790A, ACh, and PGF induced contractions were significantly reduced by S18886 in salt-induced hypertensive mice. The present study demonstrates that PGF generated by COX2 in the endothelium is the most likely endothelium-derived contracting factor underlying endothelium-dependent, TRPV4-mediated contraction in hypertensive mice. This contraction involved increased intracellular Ca concentrations and cPLA activity. These results suggested an important role of TRPV4 in endothelium-dependent contraction in mice during hypertension.

The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta‐blockers, constipation by Ca2+ channel blockers, and dry cough by ACEI, lead to non‐compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca2+‐activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high‐salt diet, N(G)‐nitro‐l‐arginine intake, or angiotensin II delivery, showed decreased TRPV4‐KCa2.3 interaction in ECs. Perturbation of the TRPV4‐KCa2.3 interaction in mouse ECs by overexpressing full‐length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small‐molecule drug, JNc‐440, which showed affinity for both TRPV4 and KCa2.3. JNc‐440 significantly strengthened the TRPV4‐KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc‐440 specifically targeted the impaired TRPV4‐KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4‐KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.

Aim: TRPV4-C1 heteromeric channels contribute to store-operated Ca 2+ entry in vascular endothelial cells. However, the negative regulation of these channels is not fully understood. This study was conducted to investigate the inhibitory effect of PKG1α on TRPV4-C1 heteromeric channels. Methods: Immuno-fluorescence resonance energy transfer (FRET) was used to explore the spatial proximity of PKG1α and TRPC1.Phosphorylation of endogenous TRPC1 was tested by phosphorylation assay. [Ca 2+ ] i transients and cation current in MAECs were assessed with Fura-2 fluorescence and whole-cell recording, respectively. In addition, rat mesenteric arteries segments were prepared, and vascular relaxation was examined with wire myography. Results: In immuno-FRET experiments, after exposure of these cells to 8-Br-cGMP, more PKG1α was observed in the plasma membrane, and PKG1α and TRPC1 were observed to be in closer proximity. TAT-TRPC1 S172 and TAT-TRPC1 T313 peptide fragments, which contain the PKG targeted residues Ser172 and Thr313, respectively, were introduced into isolated endothelial cells to abrogate the translocation of PKG1α. Furthermore, a phosphorylation assay demonstrated that PKG directly phosphorylates TRPC1 at Ser172 and Thr313 in endothelial cells. In addition, PKG activator 8-Br-cGMP markedly reduced the magnitude of the 4αPDD-induced and 11,12-EET-induced [Ca 2+ ] i transients, the cation current and vascular relaxation. Conclusion: This study uncovers a novel mechanism by which PKG negatively regulates endothelial heteromeric TRPV4-C1 channels through increasing the spatial proximity of TRPV4-C1 to PKG1α via translocation and through phosphorylating Ser172 and Thr313 of TRPC1.