PHORBOL ESTER-INDUCED LH-RELEASE IN PITUITARY GONADOTROPHS - EFFECTS OF ANTAGONISTS OF CALMODULIN AND GNRH

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of Ca2+-and phospholipid-dependent protein kinase (C kinase), stimulates luteinizing hormone (LH) release from rat pituitary cells. The actions of TPA upon LH release were compared with those of the GnRH superagonist [D-Ala6] des-Glyl0-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. LH release was stimulated by 0.1 nM TPA and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. The ED50 values for TPA and GnRHa were 1.2 and 0.037 nM, respectively, and the maximum stimulatory effects of TPA and GnRHa on LH release were not additive. GnRHa-stimulated LH release was decreased by calmodulin (CaM) antagonists including pimozide, trifluoperazine, W5 and W7, being most effectively reduced (by 70%) by 10-mu-M pimozide. In contrast to their inhibition of GnRH action, these antagonists enhanced TPA-stimulated LH release, so that 10-mu-M pimozide and W7 doubled the maximum LH response. The potent GnRH antagonist [Ac-D-p-Cl-Phe1,2, D-Trp3, D-Lys6, D-Ala10]GnRH, which completely inhibited GnRHa-stimulated LH release with ID50 of 6.8 nM, also reduced maximum TPA-stimulated LH release by about 50%. These results suggest that both Ca2+/CaM and C kinase pathways are involved in the LH release mechanism, and indicate that C kinase plays a major role in the action of GnRH upon gonadotropin secretion. The synergism between CaM antagonists and TPA suggests that blockade of CaM-mediated processes leads to enhanced activation of the C kinase pathway, possibly by removal of an inhibitory influence. Furthermore, the partial inhibition of TPA-stimulated LH release by a GnRH antagonist suggests that the pathway(s), specifically connected with LH release in the diverse effects of C kinase, might be locked by the continuous receptor inactivation by antagonist and indicates the complicated pathways which diverge from the receptor and converge into specific cellular response.