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The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

ABSTRACT: Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.

SUBMITTER: Schimmel RJ

PROVIDER: S-EPMC1149262 | biostudies-other | 1988 Jul

REPOSITORIES: biostudies-other

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Project description:The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.

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The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

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