DEPENDENCE OF INTRACELLULAR SIGNALING AND NEUROSECRETION ON PHOSPHOLIPASE-D ACTIVATION IN IMMORTALIZED GONADOTROPIN-RELEASING-HORMONE NEURONS

The excitability of gonadotropin-releasing hormone (GnRH) neurons is e ssential for episodic neuropeptide release, but the mechanism by which electrical activity controls GnRH secretion is not well characterized , The role of phospholipase D (PLD) in mediating the activity-dependen t secretory pathway was investigated in immortalized GT1 neurons, whic h both secrete GnRH and express GnRH receptors, Activation of these Ca 2+-mobilizing receptors was associated with transient hyperpolarizatio n of GT1 cells, followed by sustained firing of action potentials, Thi s was accompanied by an increase in PLD activity, as indicated by elev ated phosphatidylethanol (PEt) production. GnRH-induced PEt production was reduced by inhibition of phospholipase C-dependent phosphoinositi de hydrolysis by U73122 and neomycin, suggesting that signaling from p hospholipase C led to activation of PLD. The intermediate role of prot ein kinase C (PKC) in this process was indicated by the ability of pho rbol 12-myristate 13-acetate to induce time- and dose-dependent increa ses in PEt and diacylglycerol, but not inositol trisphosphate, and by reduction of GnRH-induced PEt accumulation in PKC-depleted cells, Cons istent with the role of action potential-driven Ca2+ entry in this pro cess, agonist-induced PLD activity was also reduced by nifedipine and low extracellular Ca2+. Inhibition of the PLD pathway by ethanol and p ropranolol reduced diacylglycerol production and caused a concomitant fall in GnRH release. These data indicate that voltage-gated Ca2+ entr y and PKC act in an independent but cooperative manner to regulate PLD activity, which contributes to the secretory response in GT1 cells, T hus, the electrical activity of the GnRH-secreting neuron participates in the functional coupling between GnRH receptors and PLD pathway.