Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle alpha(2)-adrenergic receptors (alpha(2)-ARs). Experiments were performed to determine the role of alpha(2)-AR subtypes (alpha(2A)-, alpha(2B)-, alpha(2C)-ARs) in this response. Stimulation of alpha(1)-ARs by phenylephrine or alpha(2)-ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to alpha(2)-AR activation but less responsive to activation of alpha(1)-ARs. Cold augmented constriction to alpha(2)-AR activation in distal arteries but did not affect the response to alpha(1)-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of alpha(2A)- and alpha(2C)-ARs in tail arteries: expression of alpha(2C)-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the alpha(2A)-AR increased in distal arteries. At 37 degrees C, alpha(2)-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of alpha(2A)-ARs (BRL-44408) but not by selective inhibition of alpha(2B)-ARs (ARC-239) or alpha(2C)-ARs (MK-912). In contrast, during cold exposure (28 degrees C), the augmented response to UK-14,304 was inhibited by the alpha(2C)-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of alpha(2)-ARs is mediated by alpha(2C)-ARs that are normally silent in these cutaneous arteries. Blockade of alpha(2C)-ARs may prove an effective treatment for Raynaud's Phenomenon.

Short chain fatty acid (SCFA) metabolites are byproducts of gut microbial metabolism that are known to affect host physiology via host G protein-coupled receptor (GPCRs). We previously showed that an acute SCFA bolus decreases blood pressure (BP) in anesthetized mice, an effect mediated primarily via Gpr41. In this study, our aims were to identify the cellular localization of Gpr41 and to determine its role in BP regulation. We localized Gpr41 to the vascular endothelium using RT-PCR: Gpr41 is detected in intact vessels (with endothelium) but is absent from denuded vessels (without endothelium). Furthermore, using pressure myography we confirmed that SCFAs dilate resistance vessels in an endothelium-dependent manner. Since we previously found that Gpr41 mediates a hypotensive response to acute SCFA administration, we hypothesized that Gpr41 knockout (KO) mice would be hypertensive. Here, we report that Gpr41 KO mice have isolated systolic hypertension compared with wild-type (WT) mice; diastolic BP was not different between WT and KO. Older Gpr41 KO mice also exhibited elevated pulse wave velocity, consistent with a phenotype of systolic hypertension; however, there was no increase in ex vivo aorta stiffness (measured by mechanical tensile testing). Plasma renin concentrations were also similar in KO and WT mice. The systolic hypertension in Gpr41 KO is not salt sensitive, as it is not significantly altered on either a high- or low-salt diet. In sum, these studies suggest that endothelial Gpr41 lowers baseline BP, likely by decreasing active vascular tone without altering passive characteristics of the blood vessels, and that Gpr41 KO mice have hypertension of a vascular origin.

Abstract-Cold-induced vasoconstriction in cutaneous blood vessels is mediated in part by increased activity of vascular smooth muscle ␣2-adrenoceptors (VSM ␣2-ARs). In mouse cutaneous arteries, ␣2C-ARs are normally silent at 37°C but mediate cold-induced augmentation of ␣2-AR responsiveness. In transfected HEK293 cells, this functional rescue is mediated by cold-induced translocation of ␣2C-ARs from the Golgi to the plasma membrane. Experiments were performed to determine the role of Rho/Rho kinase signaling in this process. Inhibition of Rho kinase (fasudil, Y27632 or H-1152) did not affect constriction of isolated mouse tail arteries to the ␣2-AR agonist UK 14 304 at 37°C but dramatically reduced the augmented responses to the agonist at 28°C. After Rho kinase inhibition, cooling no longer increased constriction evoked by ␣2-AR stimulation. Cooling (to 28°C) activated Rho in VSM cells and increased the calcium sensitivity of constriction in ␣ toxin-permeabilized arteries. Stimulation of ␣2-ARs in VSM cells had no effect on Rho activity or calcium sensitivity at 37°C or 28°C. In HEK293 cells transfected with ␣2C-ARs, cooling (to 28°C) stimulated the translocation of ␣2C-ARs to the plasma membrane and this effect was prevented by inhibition of Rho kinase, using fasudil or RNA interference. Consistent with inhibition of the spatial rescue of ␣2C-ARs, fasudil inhibited ␣2-AR-mediated mobilization of calcium in tail arteries at 28°C but not 37°C. Therefore, cold-induced activation of Rho/Rho kinase can mediate cold-induced constriction in cutaneous arteries by enabling translocation of ␣2C-ARs to the plasma membrane and by increasing the calcium sensitivity of the contractile process. Key Words: raynaud phenomenon Ⅲ siRNA Ⅲ thermoregulation Ⅲ HEK293 cells C old-induced vasoconstriction in the cutaneous circulation is a protective physiological response that acts to reduce heat loss. The constriction results from a reflex increase in sympathetic output and a direct local effect of cold to increase the activity of nerve-released norepinephrine. 1 This local effect is mediated by a cold-induced, selective augmentation of ␣2-adrenoceptor (␣2-AR) reactivity on vascular smooth muscle cells (VSMs). [2][3][4] Local cold-sensitivity is increased in patients with Raynaud phenomenon and Scleroderma who exhibit cold-induced peripheral vasospasm, which can be prevented by ␣2-AR blockade. 5 ␣2-ARs have been classified by pharmacological and molecular techniques into ␣2A-AR, ␣2B-AR, and ␣2C-AR subtypes. 6 Thermosensitivity of cutaneous blood vessels is mediated by ␣2C-ARs. 7 In cutaneous arteries of the mouse tail, ␣2-AR constriction at 37°C is mediated by ␣2A-ARs, with no apparent contribution from ␣2C-ARs. However, after moderate cooling, ␣2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of ␣2-AR reactivity. After transfection in HEK 293 cells, ␣2A-ARs are expressed on the cell surface and respond to activation by regulating adenylyl cyclase activity. Moderate cooling does not influence ␣2A-A...