The present study has characterized the gonadotrophin-releasing hormone (GnRH) receptor in immortalized alpha T3-1 pituitary gonadotroph cells. GnRH and GnRH analogues produced both a dose- and time-dependent increase in total inositol phosphate (IP) accumulation. The rank order of potency of these analogues was the same as that obtained in parallel receptor-binding studies in alpha T3-1 cells. These responses were abolished following pretreatment with a GnRH antagonist. The use of a specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) assay demonstrated a rapid but short-lived rise in Ins(1,4,5)P3 production. Intracellular calcium ([Ca2+]i) was subsequently measured in alpha T3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. GnRH produced a biphasic rise in [Ca2+]i. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. G-protein involvement in the IP response to GnRH in alpha T3-1 cells was investigated using sodium fluoride (NaF) and pertussis toxin (PTx) which activate and inactivate G-proteins respectively. Like GnRH, NaF produced a dose- and time-dependent increase in IP accumulation. Activation of phospholipase C in these cells by either GnRH or NaF was PTx-insensitive, suggesting that the G-protein involved was neither Gi nor Go but more probably Gq. Immunoblot analysis of alpha T3-1 cell membranes using antisera raised against the predicted C-terminal decapeptide of the alpha subunit of Gq demonstrated the presence of Gq in alpha T3-1 cells. Collectively these results show that the GnRH receptors expressed in alpha T3-1 cells are coupled to the phosphatidylinositol second messenger pathway via a specific G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)