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

Abstract

Cold constricts cutaneous blood vessels by selectively increasing the activity of smooth muscle α₂-adrenoceptors (α₂-ARs). In mouse tail arteries, α₂-AR constriction is mediated by α_2A-ARs at 37°C, whereas the cold-induced augmentation in α₂-AR activity is mediated entirely by α_2C-ARs. Cold causes translocation of α_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°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 μmol/l), the antioxidant N-acetylcysteine (NAC; 20 mmol/l), or the cell-permeable mimic of superoxide dismutase MnTMPyP (50 μmol/l) did not affect vasoconstriction to α₂-AR stimulation (UK-14304) at 37°C but dramatically inhibited the response at 28°C. Indeed, these ROS inhibitors abolished the cold-induced increase in α₂-AR constrictor activity. NAC (20 mmol/l) or MnTMPyP (50 μmol/l) also abolished the cold-induced activation of RhoA in human cultured vascular smooth muscle cells and the cold-induced mobilization of α_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 α_2C-ARs to the cell surface. Altered activity of ROS may contribute to cold-induced vasospasm occurring in Raynaud's phenomenon.