Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced nitric oxide production and vasorelaxation...

Stimulation of calcium-sensing receptors (CaSR) by increasing the external calcium concentration (Ca²⁺]_o) induces endothelium-dependent vasorelaxation through nitric oxide (NO) production and activation of intermediate Ca²⁺-activated K⁺ currents (IK_Ca) channels in rabbit mesenteric arteries. The present study investigates the potential role of heteromeric TRPV4-TRPC1 channels in mediating these CaSR-induced vascular responses. Immunocytochemical and proximity ligation assays showed that TRPV4 and TRPC1 proteins were expressed and co-localised at the plasma membrane of freshly isolated endothelial cells (ECs). In wire myography studies, increasing [Ca²⁺]_o between 1 and 6mM induced concentration-dependent relaxations of methoxamine (MO)-induced pre-contracted tone, which were inhibited by the TRPV4 antagonists RN1734 and HC067047, and the externally-acting TRPC1 blocking antibody T1E3. In addition, CaSR-evoked NO production in ECs measured using the fluorescent NO indicator DAF-FM was reduced by RN1734 and T1E3. In contrast, [Ca²⁺]_o-evoked perforated-patch IK_Ca currents in ECs were unaffected by RN1734 and T1E3. The TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent relaxation of MO-evoked pre-contracted tone and increased NO production, which were inhibited by the NO synthase inhibitor L-NAME, RN1734 and T1E3. GSK activated 6pS cation channel activity in cell-attached patches from ECs which was blocked by RN1734 and T1E3. These findings indicate that heteromeric TRPV4-TRPC1 channels mediate CaSR-induced vasorelaxation through NO production but not IK_Ca channel activation in rabbit mesenteric arteries. This further implicates CaSR-induced pathways and heteromeric TRPV4-TRPC1 channels in regulating vascular tone.