| Research Abstract |
We previously demonstrated that the prostaglandin E (PGE) receptor in bovine adrenal chromaffin cells is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to stimulation of phosphoinositide (PI) metabolism via a pertussis toxin- insensitive manner, the latter of which may mediate the stimulatory effect of PGE_2 on catecholamine release from the cells. We further extended this approach this year and obtained the following results; 1) a GTP-binding protein (G-protein) is involved in the coupling of PGE receptor to PI metabolism by NaF, an activator of G-proteins. 2) PI hydrolysis produces two second messengers, diacylglycerol and IP3, which activates protein kinase C and increases intracellular free Ca^<2+> concentration ([Ca^<2+>]i), respectively. We demonstrated that PGE_2 may increase [Ca^<2+>]i in chromaffin cells by release of Ca^<2+> from intracellular stores and by Ca^<2+> entry through Ca^<2+> channels. Activation of Ca^<2+> channels
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by PGE_2 is confirmed by electrophysiological techniques. 3) PGE_2-enhanced catecholamine release is mediated by activation of protein kinase C, rather than by Ca^<2+> mobilization, resulting from phosphoinositide metabolism. 4) Activation of protein kinase C in turn activates the Na^+, H^+ antiport on the membrane, and secondly induces a sustained elevation of [Ca^<2+>]i resulting in a gradual secretion of catecholamines. Thus we have revealed a sequence of biological events from PGE receptor activation to catecholamine release from chromaffin cells. We have accomplished the characterization of the PGE receptor and its signal transduction systems more than we made the plans for the first year. On the other hand, according to the second-year plan, we synthesized a biotinyl derivative of PGE_2 and tried to purify a complex of PGE_2 receptor-G-protein (HRG complex) by two affinity column chromatographies of WGA and avidin. We analyzed a partially purified HRG complex by SDS gel electrophoresis and detected two protein bands, a broad band with a Mr of around 100 K and a sharp one with a Mr of 40 K on the gel. However, it will be quite difficult to identify and purify the PGE receptor from bovine adrenal medulla due to the limitation of its amount and lack of a specific and potent antagonist. At present we plan to isolate the PGE receptor by several approaches including monoclonal antibodies to the receptor and cDNA cloning. Less
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