Connolly E, Nedergaard J
Wenner-Gren Institute, University of Stockholm, Sweden.
J Biol Chem. 1988 Aug 5;263(22):10574-82.
Rapid, unidirectional Ca2+ influx was examined in isolated brown adipocytes by short incubations (30 s) with 45Ca2+. Ca2+ uptake was found to be large in the resting brown adipocyte, but was markedly inhibited when the cells were presented with norepinephrine. Specific alpha 1-adrenergic stimulation was without effect on Ca2+ uptake. The effect of norepinephrine (which had an EC50 of 140 nM) could be inhibited by beta-adrenergic blockade and could be mimicked by forskolin (an adenylate cyclase activator) and theophylline (a phosphodiesterase inhibitor). Exogenous free fatty acids such as octanoate and palmitate (classical stimulators of respiration in brown adipocytes) were also able to dramatically inhibit Ca2+ uptake by the cells. The artificial mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induced a large reduction in cellular Ca2+ uptake (even in the presence of the ATPase inhibitor oligomycin), and in the presence of FCCP the inhibitory effect of norepinephrine on Ca2+ uptake was significantly reduced. The effect of beta-adrenergic stimulation on Ca2+ uptake was not directly caused by the large increase in respiration that occurs in response to norepinephrine because the respiratory inhibitor rotenone did not affect the Ca2+ response of the cells to the hormone. The evidence suggests that beta-adrenergic stimulation of brown adipocyte metabolism leads to a partial inhibition of Ca2+ uptake into the mitochondrial Ca2+ pool and we discuss the possibility that this represents the effect of a reduced membrane potential (and thus reduced Ca2+ uniport activity) in the partially uncoupled mitochondria of the thermogenically active brown adipocyte.
通过将分离的褐色脂肪细胞与45Ca2+短时间孵育（30秒）来检测快速、单向的Ca2+内流。发现静息褐色脂肪细胞中的Ca2+摄取量很大，但当细胞暴露于去甲肾上腺素时，Ca2+摄取受到显著抑制。特异性α1-肾上腺素能刺激对Ca2+摄取没有影响。去甲肾上腺素（其EC50为140 nM）的作用可被β-肾上腺素能阻断所抑制，并且可被福斯可林（一种腺苷酸环化酶激活剂）和茶碱（一种磷酸二酯酶抑制剂）模拟。外源性游离脂肪酸如辛酸和棕榈酸（褐色脂肪细胞中呼吸的经典刺激物）也能够显著抑制细胞的Ca2+摄取。人工线粒体解偶联剂羰基氰化物对三氟甲氧基苯腙（FCCP）导致细胞Ca2+摄取大幅减少（即使在存在ATP酶抑制剂寡霉素的情况下），并且在FCCP存在下，去甲肾上腺素对Ca2+摄取的抑制作用显著降低。β-肾上腺素能刺激对Ca2+摄取的作用不是由去甲肾上腺素引起的呼吸大幅增加直接导致的，因为呼吸抑制剂鱼藤酮不影响细胞对该激素的Ca2+反应。证据表明，褐色脂肪细胞代谢的β-肾上腺素能刺激导致进入线粒体Ca2+池的Ca2+摄取部分受到抑制，并且我们讨论了这种情况可能代表产热活跃的褐色脂肪细胞部分解偶联线粒体中膜电位降低（从而Ca2+单向转运活性降低）的效应的可能性。
