Cooling Evokes Redistribution of α2C-Adrenoceptors from Golgi to Plasma Membrane in Transfected Human Embryonic Kidney 293 Cells

Abstract

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.

Cold-induced cutaneous vasoconstriction is mediated by Rho kinase in vivo in human skin

“…Alternatively, it is possible that cold-induced Rho kinase activity augments norepinephrine-mediated VC through its actions on ␣ 2C -adrenoceptors. Taking into consideration 1) the results of the present study suggesting a cold-specific Ca 2ϩ -dependent element in Rho kinase-mediated VC, 2) the established role of Rho kinase in mediating cold-induced ␣ 2C -adrenoceptor translocation in vitro (2,3,10,22), and 3) the preponderance of in vivo human literature suggesting that early-phase cold-induced VC is mediated almost exclusively by ␣ 2 -, but not ␣ 1 -, adrenoceptors (despite the presence of functional ␣ 1 -adrenoceptors in skin), it is plausible that Rho kinase may mediate VC, at least in part, through stimulation of ␣ 2C -adrenoceptor translocation. However, this speculation requires further testing to verify its validity in an in vivo model.…”

“…In vitro, cutaneous vessel cooling leads to increased mitochondrial production of reactive oxygen species, which stimulate Rho kinase activity (2,3). Cold-induced Rho kinase activity in cutaneous vascular smooth muscle cells can drive VC via two distinct pathways: 1) stimulation of ␣ 2C -adrenoceptor translocation from intracellular storage in the Golgi to the vascular smooth muscle cell surface (2,3,10,22) and 2) enhanced intracellular Ca 2ϩ sensitization (2,4,15,17,36,37). These findings suggest that Rho kinase may participate in early and/or later phases of cutaneous cold-induced VC in vivo.…”

Limb-specific differences in the skin vascular responsiveness to adrenergic agonists

“…The ␣ 2 -adrenoceptor function in the skin vasculature plays a pivotal role in thermoregulation under cool conditions, because cutaneous vasoconstriction in response to direct skin cooling is mediated by the ␣ 2c -adrenoceptor subtype, which translocates from the Golgi compartment to the extracellular membrane in the adrenergic pathways (2,17). It is thought, however, that the cold-induced translocation in ␣ 2c -adrenoceptors did not occur while adrenergic agents were administered, because the local skin temperature was maintained at 34°C throughout the experiments in this study.…”

Distinct cAMP signaling pathways differentially regulate α2C-adrenoceptor expression: role in serum induction in human arteriolar smooth muscle cells

“…logical constriction in response to cooling (7,19). Furthermore, in VSMs from human cutaneous arterioles, serum stimulation selectively increases ␣ 2C -AR expression and function (8).…”

“…␣ 2 -ARs mediate constriction of cutaneous arterioles and veins, and vascular smooth muscle cells (VSMs) from these vessels demonstrate high expression of ␣ 2C -ARs (8). These cutaneous ␣ 2C -ARs have a unique function and role, mediating physiological constriction in response to cooling (7,19). Furthermore, in VSMs from human cutaneous arterioles, serum stimulation selectively increases ␣ 2C -AR expression and function (8).…”