Role of cell calcium in alpha-1 adrenergic receptor control of arachidonic acid release from brown adipocytes

Abstract

Exposure of brown fat cells to phenylephrine, an agonist alpha-1 adrenergic receptors, activates a phospholipase A 2 which releases arachidonic acid. Since receptor activation of phospholipase A 2 requires calcium, experiments were undertaken to define more precisely the role played by calcium in the regulation of enzyme activity. In this study, adipocytes were loaded with the fluorescent calcium chelator quin2 in order to buffer intracellular calcium and block receptor stimulated changes in its concentration. When quin2 loaded adipocytes were incubated in buffer containing 0.10 mM clacium, the ability of phenylephrine to stimulate release of arachidonic acid was severely reduced. At an intracellular quin2 concentration of 6.6 nM stimulated arachidonic acid release was inhibited by more than 50% and at 13 mM it was completely blocked. In contrast, phenylephrine stimulation of inositol phosphate accumulation was unaffected by quin2. Quin2 also did not affect the liberation of arachidonic acid in response to exogenous phospholipase C, A23187 or forskolin. The intracellular calcium antagonist TMB-8 also ihibited phenylephrine-stimulation of arachidonic acid release and this effect was reversed by ionomycin. Basal phospholipase A 2 activity was increased by introduction of high calcium concentrations into cells rendered permeable with digitonin, but phenylephrine still caused a further increase in enzyme activity. These findings show a selective inhibition of phenylephrine activation of phospholipase A 2 by either the chelation of intracellular calcium with quin2 or by the calcium antagonist TMB-8 and suggest an essential role for intracellular calcium in alpha adrenergic stimulation of enzyme activity. However, because phenylephrine still stimulates enzymes activity in cells rendered permeable with digitonin, we suggest that the action of phenylephrine cannot be attributed solely to changes in intracellular calcium.