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.

The adverse effects of angiotensin II (Ang II) are primarily mediated through the Ang II type 1 receptor (AT 1 R). A silent polymorphism (؉1166 A/C) in the human AT 1 R gene has been associated with cardiovascular disease, possibly as a result of enhanced AT 1 R activity. Because this polymorphism occurs in the 3-untranslated region of the human AT 1 R gene, the biological importance of this mutation has always been questionable. Computer alignment demonstrated that the ؉1166 A/C polymorphism occurred in a cis-regulatory site, which is recognized by a specific microRNA (miRNA), miR-155. miRNAs are noncoding RNAs that silence gene expression by base-pairing with complementary sequences in the 3-untranslated region of target RNAs. When the ؉1166 C-allele is present, base-pairing complementarity is interrupted, and the ability of miR-155 to interact with the cis-regulatory site is decreased. As a result, miR-155 no longer attenuates translation as efficiently as demonstrated by luciferase reporter and Ang II radioreceptor binding assays. In situ hybridization experiments demonstrated that mature miR-155 is abundantly expressed in the same cell types as the AT 1 R (e.g. endothelial and vascular smooth muscle). Finally, when human primary vascular smooth muscle cells were transfected with an antisense miR-155 inhibitor, endogenous human AT 1 R expression and Ang II-induced ERK1/2 activation were significantly increased. Taken together, our study demonstrates that the AT 1 R and miR-155 are co-expressed and that miR-155 translationally represses the expression of AT 1 R in vivo. Therefore, our study provides the first feasible biochemical mechanism by which the ؉1166 A/C polymorphism can lead to increased AT 1 R densities and possibly cardiovascular disease.

Cold-induced vasoconstriction in cutaneous blood vessels is mediated by increased constrictor activity of vascular alpha2-adrenoceptors (alpha2-ARs). In mouse cutaneous arteries, alpha2-AR constriction at 37 degrees C is mediated by alpha2A-ARs, whereas after cold exposure (28 degrees C), alpha2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of alpha2-AR responsiveness. The goals of the present study were to develop a cell model of cutaneous thermoregulation and to determine the mechanisms underlying the thermosensitivity of alpha2C-ARs. Human embryonic kidney 293 cells were transiently transfected with the mouse alpha2A- or alpha2C-AR. In cells expressing alpha2A-ARs, UK-14,304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine), an alpha2-AR agonist, inhibited (10 pM) and stimulated (1-10 nM) the accumulation of cAMP evoked by forskolin. Similar responses were obtained at 37 degrees C and 28 degrees C. In contrast, in cells expressing alpha2C-ARs, UK-14,304 did not affect forskolin-stimulated cAMP accumulation at 37 degrees C but did cause a concentration-dependent inhibitory effect at 28 degrees C. Subcellular fractionation revealed that at 37 degrees C alpha2C-ARs were localized predominantly to Golgi compartments, whereas alpha2A-ARs localized predominantly to the plasma membrane. After cooling (28 degrees C), alpha2C-ARs relocated from Golgi compartments to the plasma membrane, whereas the alpha2A-AR remained at the plasma membrane. Immunofluorescence microscopy confirmed that, at 37 degrees C, alpha2A-ARs were localized to the cell surface, whereas alpha2C-ARs colocalized with a trans-Golgi marker. Cooling did not affect localization of alpha2A-ARs, but shifted alpha2C-ARs to the cell surface. Moderate cooling, therefore, caused a selective redistribution of alpha2C-ARs from the Golgi compartments to the cell surface, allowing the rescue of the alpha2C-adrenergic functional response. This mechanism may explain the role of alpha2-ARs in thermoregulation of the cutaneous circulation.

Abstract-Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (␣-actin, calponin, and SM1 and SM2 myosin), but not ␤-actin. ROS activity, determined using the H 2 O 2 -sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (