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.

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels.

The present study investigates the effect of commonly used negative and positive allosteric modulators of the calcium-sensing receptor (CaSR) on vascular reactivity. In wire myography studies, increasing [Ca2+]o from 1mM to 6mM induced concentration-dependent relaxations of methoxamine-induced pre-contracted rabbit mesenteric arteries, with 6mM [Ca2+]o producing almost complete relaxation. [Ca2+]o-induced relaxations were attenuated in the presence of the calcilytics Calhex-231 and NPS 2143, and abolished by the removal of the endothelium. In addition to their calcilytic effects, Calhex-231 and NPS 2143 also produced concentration-dependent inhibitions of methoxamine- or KCl-induced precontracted tone, which were unaffected by removal of the endothelium and unopposed in the presence of the calcimimetic Calindol. In vessels with depleted Ca2+ stores, contractions mediated by Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) were inhibited by Calhex231. In freshly isolated single rabbit mesenteric artery smooth muscle cells, Calhex-231 and NPS 2143 inhibited whole-cell VGCC currents. Application of Calindol also inhibited methoxamine- and KCl-induced pre-contracted tone, and inhibited whole-cell VGCC currents. In conclusion, in addition to their CaSR-mediated actions in the vasculature, Calhex-231, NPS 2143 and Calindol reduce vascular contractility via direct inhibition of VGCCs.

Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels.

Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K(+) channels in these responses. In wire myography studies, increasing [Ca(2+)]o from 1mM to 6mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca(2+)]o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca(2+)]o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca(2+)-activated K(+) channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca(2+)]o-induced vasorelaxations. Increasing [Ca(2+)]o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations.