Abstract
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 (IKCa) 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 IKCa 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 IKCa channel activation in rabbit mesenteric arteries. This further implicates CaSR-induced pathways and heteromeric TRPV4-TRPC1 channels in regulating vascular tone.
Highlights
Stimulation of plasmalemmal calcium-sensing receptors (CaSR) by an increase in the extracellular Ca2 + concentration ([Ca2 +]o) is involved in maintaining plasma Ca2+ homeostasis through the regulation of parathyroid hormone synthesis and secretion from the parathyroid gland, intestinal Ca2+ absorption, and renal Ca2+ excretion [6,7,27]
We examined the expression of transient receptor potential vanilloid-4 (TRPV4), canonical transient receptor potential channel 1 (TRPC1), and potential co-localisation between these two channel proteins in freshly isolated rabbit mesenteric artery endothelial cell (EC)
In this series of experiments, we investigated the effect of the TRPV4 channel blockers RN1734 and HC067047 [3,51,56], and the externallyacting TRPC1 antibody T1E3, which is known to act as a TRPC1 channel blocking agent [46,61] on CaSR-induced vasorelaxation and nitric oxide (NO) production
Summary
Stimulation of plasmalemmal calcium-sensing receptors (CaSR) by an increase in the extracellular Ca2 + concentration ([Ca2 +]o) is involved in maintaining plasma Ca2+ homeostasis through the regulation of parathyroid hormone synthesis and secretion from the parathyroid gland, intestinal Ca2+ absorption, and renal Ca2+ excretion [6,7,27]. It is increasingly apparent that CaSR are expressed in tissues not involved in plasma Ca2+ homeostasis, including the cardiovascular system [42,49,60]. CaSR in the vasculature is considered physiologically possible as localised [Ca2+]o is likely to rise sufficiently at the surface of cells due to active Ca2 + transport mechanisms such as the Ca2 +-ATPase and the Na+-Ca2+ exchanger, as well as opening and closing of voltage-dependent Ca2 + channels [16,27,28,40,44]. There is currently little consensus on the function of CaSR in the vasculature, with findings suggesting that stimulation of CaSR induce both vasoconstriction and vasorelaxation through diverse cellular mechanisms [9,16,24,28,30,57,58,60].
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