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.

Role of oxidative stress in calcific aortic valve disease and its therapeutic implications.

Calcific aortic valve disease (CAVD) is the end result of active cellular processes that lead to the progressive fibrosis and calcification of aortic valve leaflets. In western populations, CAVD is a significant cause of cardiovascular morbidity and mortality, and in the absence of effective drugs, it will likely represent an increasing disease burden as populations age. As there are currently no pharmacological therapies available for preventing, treating, or slowing the development of CAVD, understanding the mechanisms underlying the initiation and progression of the disease is important for identifying novel therapeutic targets. Recent evidence has emerged of an important causative role for reactive oxygen species (ROS)-mediated oxidative stress in the pathophysiology of CAVD, inducing the differentiation of valve interstitial cells into myofibroblasts and then osteoblasts. In this review, we focus on the roles and sources of ROS driving CAVD and consider their potential as novel therapeutic targets for this debilitating condition.

MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2.

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity.

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.

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.