Beta-adrenergic modulation of Ca2+ uptake by isolated brown adipocytes. Possible involvement of mitochondria

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.