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.

1 Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). 2 Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of N0-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M,), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3).3 The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 ± 0.07 (EC5,,). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (-logM) 9.48 ± 0.07, 6.74 ± 0.22, 6.30 ± 0.20 and 9.39 ± 0.22 respectively. The potency of acetylcholine in inducing endotheliumdependent relaxation was 7.82 ± 0.09 (ICW). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (-log M) 9.15 ± 0.23 and 9.63 ± 0.28, respectively. These results suggest that the muscarinic M3 receptor subtype mediates both endothelium-dependent relaxation and contraction to acetylcholine in SHR aorta.

Contraction of canine pulmonary artery to hypoxia in vitro is both endothelium dependent and independent. The mechanisms which underlie this phenomenon were studied. Rings of canine pulmonary artery were suspended for isometric force recording in tissue baths containing modified Krebs-Ringer bicarbonate solution. Tissues were first contracted with norepinephrine [effective dose at 35% (ED35) concentration]. Subsequent exposure to hypoxia induced a triphasic response: an initial phasic transient contraction (phase 1), a transient reduction in force (phase 2), followed by a sustained tonic contraction (phase 3). In the absence of endothelium, all phases of the hypoxic response were reduced, and phase 2 was reversed from a contraction to a relaxation (with endothelium: 0.68 +/- 0.2 g; without endothelium: -0.34 +/- 0.1 g). Similar data were obtained in the presence of nitro-L-arginine (3 x 10(-5) M). In the absence of endothelium, indomethacin (10(-5) M) abolished the phase 2 relaxation and converted phase 3 from a contraction to a relaxation (control: 0.99 +/- 0.2 g; indomethacin: -0.44 +/- 0.1 g); and ONO-3708 (thromboxane A2/prostaglandin H2 receptor antagonist) diminished phase 3 (control: 0.99 +/- 0.2 g; ONO-3708: 0.3 +/- 0.04 g). In the absence of endothelium, but in the presence of indomethacin (10(-5) M), K(+)-free solution diminished phase 1 (contraction) and converted phase 2 (relaxation) to a contraction (control: -0.74 +/- 0.1 g; K(+)-free solution: 0.1 +/- 0.06 g). Similar results were obtained with ouabain (4 x 10(-7) M), and cooling of the bathing medium (20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

The responsiveness of airway smooth muscle is influenced by the functional integrity of the respiratory epithelium. The nature of this regulatory action by the epithelium remains largely unresolved. Several explanations may account for the epithelium-dependent responses induced by numerous stimuli. This review will present and discuss the evidence suggesting that the epithelium generates an inhibitory signal or signals that function to modulate the responsiveness of the underlying smooth muscle. In addition, the possible candidates for the identity of this epithelium-derived relaxing factor or factors will be assessed. Finally, the mechanisms by which the epithelium-derived relaxing factors may act to modulate bronchomotor tone will be discussed.

Coronary endothelial cells are the main site of induction of 70 kd heat shock protein in the heart and appear to contribute to the protective effects of heat stress on the recovery of mechanical and endothelial function.