COUPLING OF I-1-IMIDAZOLINE RECEPTORS TO DIACYLGLYCERIDE ACCUMULATIONIN PC12 RAT PHEOCHROMOCYTOMA CELLS

The I-1-subtype of imidazoline binding sites has been characterized co ncerning binding specificity and tissue localization, and several phys iological functions have been ascribed to it, However, the signaling p athways coupled to this putative receptor are not known. Pheochromocyt oma PC12 cells express I-1-imidazoline binding sites in plasma membran e and lack cr,adrenergic receptors, which recognize many I-1-imidazoli ne ligands. In this cellular model, diacylglycerol (DAG), a second mes senger, is generated in response to the putative I-1-imida zoline agon ist moxonidine. Using radioflux with [H-3]myristate and direct measure ments of DAG mass, we showed a rapid and transient peak of DAG in undi fferentiated PC12 cells within the first 1 min of agonist exposure. In PC12 cells treated with nerve growth factor to initiate differentiati on, DAG accumulation at 15 sec was facilitated, and the increase in DA G mass persisted throughout 10 min of agonist treatment. Efaroxan, a p utative I-1-antagonist, attenuated the effect of moxonidine on DAG acc umulation in nerve growth factor-treated cells, as did D609, an inhibi tor of phosphatidylcholine-selective phospholipase C. Phospholipase D did not seem to be involved in generation of DAG in response to I-1-re ceptor activation, nor was there accumulation of phosphatidic acid, Th ese findings suggest coupling of I-1-imidazoline receptors to a phosph olipase C to generate DAG as a second messenger, a process regulated b y neuronal differentiation and possibly participating in the physiolog ical responses to I-1-imidazoline receptor activation.