Further characterisation of vascular responses to ligands of the calcium-sensing receptor family

K. Sabarwal, P. Tate, R. Bhamra, V. Ho. St George's University of London, London, UNITED KINGDOM.

Introduction: We previously showed that the two positive allosteric modulators of calcium-sensing receptor (CaSR), calindol and cinacalcet are potent vasorelaxants, but CaSR may play a minor role in comparison with voltage-gated calcium channels1. Since then, a diverse range of endogenous and exogenous compounds, for example modulators structurally distinct from calindol and cinacalcet, and aromatic and basic L-amino acids, have been reported to activate CaSR and its related receptor, GPRC6. Thus far, the vascular actions of these ligands are poorly characterised.

Method: Male Wistar rats (10-12 weeks) were killed by cervical dislocation and small mesenteric arteries from the gut were isolated for isometric tension recording. Relaxant responses in methoxamine-precontracted vessels are shown as mean±sem (n=4-6) and analysed by two-way analysis of variance or Student t-test, where appropriate.

Results: AC-265347 (AC), a more recently developed positive CaSR modulator, induced relaxation in mesenteric arteries (pEC50=6.3±0.1, Emax=102±4%), which was more potent than calindol (pEC50=6.0±0.1, Emax=100±5%, P<0.05) or cinacalcet (pEC50=5.7±0.1, Emax=101±7%, P<0.01). However, only modest relaxation was seen with other novel positive CaSR modulators, S-methylglutathione (Emax=33±3%), Lglutathione (Emax=27±6%), L-tryptophan (1mM: 19.3±7.6%), or Lphenylalanine (1mM: -1.6±10%). Relaxation to AC was insensitive to endothelial removal (pEC50=6.2±0.1, Emax=99±9%) or two structurally distinct negative CaSR modulators (+calhex231: pEC50=6.3±0.1, Emax=105±9%; +NPS-2143: pEC50=6.4±0.2, Emax=101±7%). However, it was reduced by a lower extracellular [Ca2+] (0.5mM; pEC50=6.1±0.1, Emax=95±11%, P<0.01), high extracellular [K+] (60mM; pEC50=5.5±0.3, Emax=96±14%, P<0.01), or desensitisation of Transient Receptor Potential Vanilloid type 1 receptor system by 10µM capsaicin (pEC50=5.9±0.3, Emax=107±23%, P<0.01), similar to our previous findings with calindol and cinacalcet. Contractions to 3µM BayK8644, a voltage-gated Ca2+ channel activator, were also inhibited by AC (control: 3.1±0.9mN; +3µM AC: 0.9±0.3mN; P<0.05). On the other hand, L-arginine, a GPRC6 agonist and a precursor of nitric oxide, induced endothelium-dependent relaxation (at 1mM; with endothelium: 46±5%, without endothelium: 16±5%, P<0.01), whereas the selective GPRC6agonist, L-ornithine had little effect (1mM: 4±4%). Pretreatment with L-ornithine alone or in combination with other amino acids (L-tryptophan, Lphenylalanine, L-arginine and L-serine) also had no effect on CaSR-mediated relaxation to extracellular Ca2+ (data not shown).

Conclusion: The vascular profile of CaSR and GPRC6 ligands is complex. Selected positive CaSR modulators induce mesenteric relaxation mainly through CaSR-independent mechanisms, whereas other CaSR/GPRC6 ligands including L-amino acids have limited effect on vascular tone.

References:

(1) Thakore P and Ho WSV (2011). Br J Pharmacol 162: 749-762