Stimulation of the calcium-sensing receptor induces relaxations through CGRP and NK1 receptor-mediated pathways in male rat mesenteric arteries.

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.

Stimulation of the calcium-sensing receptor induces relaxations of rat mesenteric arteries by endothelium-dependent and -independent pathways via BKCa and KATP channels.

Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca2+ concentration ([Ca2+ ]o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca2+ ]o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca2+ ]o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca2+ -activated K+ channels (BKCa , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca2+ ]o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K+ channel (KATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K+ channels (Kir ), Kv7 channels, and small conductance Ca2+ -activated K+ channels (SKCa ) on [Ca2+ ]o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BKCa channels and an endothelium-independent pathway involving stimulation of KATP channels.

Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced relaxations and nitric oxide production in mesenteric arteries: comparative study using wild-type and TRPC1-/- mice.

We have previously provided pharmacological evidence that stimulation of calcium-sensing receptors (CaSR) induces endothelium-dependent relaxations of rabbit mesenteric arteries through activation of heteromeric TRPV4/TRPC1 channels and nitric oxide (NO) production. The present study further investigates the role of heteromeric TRPV4/TRPC1 channels in these CaSR-induced vascular responses by comparing responses in mesenteric arteries from wild-type (WT) and TRPC1-/- mice. In WT mice, stimulation of CaSR induced endothelium-dependent relaxations of pre-contracted tone and NO generation in endothelial cells (ECs), which were inhibited by the TRPV4 channel blocker RN1734 and the TRPC1 blocking antibody T1E3. In addition, TRPV4 and TRPC1 proteins were colocalised at, or close to, the plasma membrane of endothelial cells (ECs) from WT mice. In contrast, in TRPC1-/- mice, CaSR-mediated vasorelaxations and NO generation were greatly reduced, unaffected by T1E3, but blocked by RN1734. In addition, the TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent vasorelaxations which were blocked by RN1734 and T1E3 in WT mice, but only by RN1734 in TRPC1-/- mice. Moreover, GSK activated cation channel activity with a 6pS conductance in WT ECs but with a 52 pS conductance in TRPC1-/- ECs. These results indicate that stimulation of CaSR activates heteromeric TRPV4/TRPC1 channels and NO production in ECs, which are responsible for endothelium-dependent vasorelaxations. This study also suggests that heteromeric TRPV4-TRPC1 channels may form the predominant TRPV4-containing channels in mouse mesenteric artery ECs. Together, our data further implicates CaSR-induced pathways and heteromeric TRPV4/TRPC1 channels in the regulation of vascular tone.

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

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.