BACKGROUND AND PURPOSET16Ainh-A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16Ainh-A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone. EXPERIMENTAL APPROACHSingle channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery. KEY RESULTSIn rabbit pulmonary artery myocytes T16Ainh-A01 (1-30 mM) inhibited single calcium (Ca -activated Cl -channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, Fact (10 mM), increased single channel and whole cell Ca 2+ -activated Cl -currents in rabbit pulmonary arteries. In isometric tension studies, T16Ainh-A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC50 of 1.6 mM and suppressed the methoxamine concentration-effect curve. T16Ainh-A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 mM T16Ainh-A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K + ) channel blockers. T16Ainh-A01 (10 mM) also relaxed human visceral adipose arteries by 88 Ϯ 3%. CONCLUSIONS AND IMPLICATIONST16Ainh-A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels. Abbreviations
Cerebral blood flow is dynamically regulated by neurovascular coupling to meet the dynamic metabolic demands of the brain. We hypothesized that TRPA1 channels in capillary endothelial cells are stimulated by neuronal activity and instigate a propagating retrograde signal that dilates upstream parenchymal arterioles to initiate functional hyperemia. We find that activation of TRPA1 in capillary beds and post-arteriole transitional segments with mural cell coverage initiates retrograde signals that dilate upstream arterioles. These signals exhibit a unique mode of biphasic propagation. Slow, short-range intercellular Ca2+ signals in the capillary network are converted to rapid electrical signals in transitional segments that propagate to and dilate upstream arterioles. We further demonstrate that TRPA1 is necessary for functional hyperemia and neurovascular coupling within the somatosensory cortex of mice in vivo. These data establish endothelial cell TRPA1 channels as neuronal activity sensors that initiate microvascular vasodilatory responses to redirect blood to regions of metabolic demand.
Cerebral parenchymal arterioles (PA) regulate blood flow between pial arteries on the surface of the brain and the deeper microcirculation. Regulation of PA contractility differs from that of pial arteries and is not completely understood. Here, we investigated the hypothesis that the Ca(2+) permeable vanilloid transient receptor potential (TRPV) channel TRPV3 can mediate endothelium-dependent dilation of cerebral PA. Using total internal reflection fluorescence microscopy (TIRFM), we found that carvacrol, a monoterpenoid compound derived from oregano, increased the frequency of unitary Ca(2+) influx events through TRPV3 channels (TRPV3 sparklets) in endothelial cells from pial arteries and PAs. Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20) than previously characterized TRPV4 (ΔF/F0 = 0.06) or TRPA1 (ΔF/F0 = 0.13) sparklets, suggesting that TRPV3-mediated Ca(2+) influx could have a robust influence on cerebrovascular tone. In pressure myography experiments, carvacrol caused dilation of cerebral PA that was blocked by IPP. Carvacrol-induced dilation was nearly abolished by removal of the endothelium and block of intermediate (IK) and small-conductance Ca(2+)-activated K(+) (SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating IK and SK channels in the endothelium.
The angiotensin II receptor type 1b (AT R ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT R activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT R or AT R with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT R mRNA is ∼30-fold higher than AT R in whole cerebral arteries and ∼45-fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT R for pressure-induced vasoconstriction ABSTRACT: Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT R - AT R and AT R - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT R increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT R knockout animals compared with controls. In patch-clamp experiments using native cerebral arterial myocytes, membrane stretch-induced cation currents were blocked by the TRPM4 inhibitor 9-phenanthrol in both groups. Further, the AT R blocker losartan (1 μm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT R mRNA was ∼30-fold greater than AT R in cerebral arteries, and knockdown of AT R selectively diminished myogenic constriction. We conclude that AT R , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.
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