The calcium-sensing receptor induces nitric oxide mediated vasorelaxations involving TRPV4 and TRPC1 on the endothelium

Calcium-sensing receptors (CaRs) in the parathyroid gland, kidney, bone and intestine play an important role in regulating calcium homeostasis(1). CaRs are also expressed in the vasculature, including in the endothelium(2), where they have been reported to play a role in regulating vascular tone. However the mechanisms by which endothelial CaRs do this remain unclear. This work examines endothelial CaR mechanisms involved in vascular tone regulation. Male New Zealand White Rabbits (2-3kg) were killed by I.V. injection of sodium pentobarbitone (120mg kg−1) in accordance with Schedule I of the UK Animals Scientific Procedures Act, 1986. 2nd-order branches of the superior mesenteric artery (SMA) were dissected and mounted in a wire myograph. CaR stimulation by increasing [Ca2+]o from 1 - 6mM, evoked vasorelaxations of methoxamine precontracted arteries with ~98% relaxation seen at 6mM [Ca2+]o (n=7). This was mediated by the production of nitric oxide, with only ~8% relaxation observed at 6mM [Ca2+]o in the presence of the eNOS inhibitor L-NAME (p<0.05 vs. controls, 2-Way ANOVA followed by Bonferroni Test, n=7). CaR evoked-vasorelaxations were also significantly inhibited by the TRPV4 antagonists RN1734 and HC067047 (~16% and ~20% relaxation respectively at 6mM [Ca2+]o, p<0.05 vs controls, n=6) and a significant attenuation of the response was also seen in the presence of the anti-TRPC1 antibody T1E3, with only ~35% relaxation observed at 6mM [Ca2+]o (p<0.05, n=5). Furthermore, direct TRPV4 activation with GSK1016790A (1 – 30nM), induced nitric-oxide mediated vasorelaxations (~97% relaxation at 30nM) that were significantly inhibited by T1E3 (~60% relaxation in the presence of T1E3, p<0.05, n=5). In immunocytochemistry staining on freshly isolated endothelial cells (ECs), co-localisation of TRPC1 and TRPV4 proteins was observed at the plasma membrane, and proximal ligation assays also demonstrated the close proximity of TRPV4 and TRPC1 proteins (<40nm apart). Taken together this data suggests that TRPV4 and TRPC1 may be functionally linked in rabbit SMA endothelial cells, similar to previous reports of a TRPV4/C1 heteromeric ion channel in mouse aortic ECs(3), and that activation of this channel by the CaR results in nitric oxide-mediated vasorelaxations of precontracted arteries. Further work is needed to confirm the direct activation of this channel following CaR stimulation.

(1) Brown EM and MacLeod RJ (2001) Physiol Rev 81: 239-297

(2) Weston AH et al. (2005) Circ Res 97: 391-8.

(3) Ma X et al. (2011) Cell Calcium 50: 502-509