Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries

Cold constricts cutaneous blood vessels by selectively increasing the activity of smooth muscle alpha(2)-adrenoceptors (alpha(2)-ARs). In mouse tail arteries, alpha(2)-AR constriction is mediated by alpha(2A)-ARs at 37 degrees C, whereas the cold-induced augmentation in alpha(2)-AR activity is mediated entirely by alpha(2C)-ARs. Cold causes translocation of alpha(2C)-ARs from the trans-Golgi to the plasma membrane, mediated by cold-induced activation of RhoA and Rho kinase. The present experiments analyzed the mechanisms underlying these responses. Mouse tail arteries were studied in a pressure myograph. Cooling the arteries ( 28 degrees C) caused a rapid increase in reactive oxygen species (ROS) in smooth muscle cells, determined by confocal microscopy of arteries loaded with the ROS-sensitive probes, dichlorodihydrofluorescein or reduced Mitotracker Red. The inhibitor of mitochondrial complex I rotenone (10 mu mol/l), the antioxidant N-acetylcysteine (NAC; 20 mmol/l), or the cell-permeable mimic of superoxide dismutase MnTMPyP (50 mu mol/l) did not affect vasoconstriction to alpha(2)-AR stimulation (UK-14304) at 37 degrees C but dramatically inhibited the response at 28 degrees C. Indeed, these ROS inhibitors abolished the cold-induced increase in alpha(2)-AR constrictor activity. NAC ( 20 mmol/l) or MnTMPyP ( 50 mu mol/l) also abolished the cold-induced activation of RhoA in human cultured vascular smooth muscle cells and the cold-induced mobilization of alpha(2C)-ARs to the cell surface in human embryonic kidney 293 cells transfected with the receptor. The combined results suggest that cold-induced constriction is mediated by redox signaling in smooth muscle cells, initiated by mitochondrial generation of ROS, which stimulate RhoA/Rho kinase signaling and the subsequent mobilization of alpha(2C)-ARs to the cell surface. Altered activity of ROS may contribute to cold-induced vasospasm occurring in Raynaud's phenomenon.