The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Ca2+ handling and sensitivity in airway smooth muscle: emerging concepts for mechanistic understanding and therapeutic targeting.

Free calcium ions within the cytosol serve as a key secondary messenger system for a diverse range of cellular processes. Dysregulation of cytosolic Ca(2+) handling in airway smooth muscle (ASM) has been implicated in asthma, and it has been hypothesised that this leads, at least in part, to associated changes in both the architecture and function of the lung. Significant research is therefore directed towards furthering our understanding of the mechanisms which control ASM cytosolic calcium, in addition to those regulating the sensitivity of its downstream effector targets to calcium. Key aspects of the recent developments in this field were discussed at the 8th Young Investigators' Symposium on Smooth Muscle (2013, Groningen, The Netherlands), and are outlined in this review.

ß1-Adrenoceptor stimulation suppresses endothelial IK(Ca)-channel hyperpolarization and associated dilatation in resistance arteries.

BACKGROUND AND PURPOSE: In small arteries, small conductance Ca²âº-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²âº-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI2-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether ß-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. EXPERIMENTAL APPROACH: Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²âº]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. KEY RESULTS: Intraluminal perfusion of ß-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 µM) without modifying the associated changes in endothelial cell [Ca²âº]i . The inhibitory effect of ß-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the ß-adrenoceptor antagonists propranolol (non-selective), atenolol (ß1) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (ß2) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by ß-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 µM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented ß-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. CONCLUSIONS AND IMPLICATIONS: In resistance arteries, endothelial cell ß1-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

Vascular hyperpolarization to ß-adrenoceptor agonists evokes spreading dilatation in rat isolated mesenteric arteries.

BACKGROUND AND PURPOSE: ß-Adrenoceptor stimulation causes pronounced vasodilatation associated with smooth muscle hyperpolarization. Although the hyperpolarization is known to reflect K(ATP) channel activation, it is not known to what extent it contributes to vasodilatation. EXPERIMENTAL APPROACH: Smooth muscle membrane potential and tension were measured simultaneously in small mesenteric arteries in a wire myograph. The spread of vasodilatation over distance was assessed in pressurized arteries following localized intraluminal perfusion of either isoprenaline, adrenaline or noradrenaline. KEY RESULTS: Isoprenaline stimulated rapid smooth muscle relaxation associated at higher concentrations with robust hyperpolarization. Noradrenaline or adrenaline evoked a similar hyperpolarization to isoprenaline if the α(1)-adrenoceptor antagonist prazosin was present. With each agonist, glibenclamide blocked hyperpolarization without reducing relaxation. Focal, intraluminal application of isoprenaline, noradrenaline or adrenaline during block of α(1)-adrenoceptors evoked a dilatation that spread along the entire length of the isolated artery. This response was endothelium-dependent and inhibited by glibenclamide. CONCLUSIONS AND IMPLICATIONS: Hyperpolarization is not essential for ß-adrenoceptor-mediated vasodilatation. However, following focal ß-adrenoceptor stimulation, this hyperpolarization underlies the ability of vasodilatation to spread along the artery wall. The consequent spread of vasodilatation is dependent upon the endothelium and likely to be of physiological relevance in the coordination of tissue blood flow.

An atomic force microscope nanoscalpel for nanolithography and biological applications.

We present the fabrication of specialized nanotools, termed nanoscalpels, and their application for nanolithography and nanomechanical manipulation of biological objects. Fabricated nanoscalpels have the shape of a thin blade with the controlled thickness of 20-30 nm and width of 100-200 nm. They were fabricated using electron beam induced deposition at the apex of atomic force microscope probes and are hard enough for a single cut to penetrate a approximately 45 nm thick gold layer; and thus can be used for making narrow electrode gaps required for fabrication of nanoelectronic devices. As an atomic force microscope-based technique the nanoscalpel provides simultaneous control of the applied cutting force and the depth of the cut. Using mammalian cells as an example, we demonstrated their ability to make narrow incisions and measurements of local elastic and inelastic characteristics of a cell, making nanoscalpels also useful as a nanosurgical tool in cell biology. Therefore, we believe that the nanoscalpel could serve as an important tool for nanofabrication and nanosurgery on biological objects.

Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries.

BACKGROUND AND PURPOSE: Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries. EXPERIMENTAL APPROACH: The non-hydrolyzable selective P2Y1 agonist ADPbetaS (3 microM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca(2+)](i) were obtained in the presence and absence of inhibitors of protein kinase C (PKC). KEY RESULTS: ADPbetaS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 microM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 microM) or BIS-VIII (1 microM) tended to augment concentration-dependent dilatation to ADPbetaS (0.1-3 microM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 microM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca(2+)](i) in pressurized arteries confirmed the P2Y1 receptor but not M(3) muscarinic receptor desensitization. CONCLUSIONS AND IMPLICATIONS: These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.