Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells

We have previously shown that the stimulatory effect of TRH on alpha-MSH se cretion from the frog pars intermedia is associated with Ca2+ influx throug h voltage-dependent Ca2+ channels, activation of a phospholipase C and mobi lization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (A C), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A( 2), and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Inc ubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13 -acetate (24 h), which causes desensitization of PKC, or with the PKC inhib itor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha- MSH release. In most melanotrope cells, TRH induces a sustained and biphasi c increase in cytosolic Ca2+ concentration ([Ca2+](i)). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase o f the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is in volved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and t he CAM KII inhibitor KN-93 did not significantly reduce the response to TRH . Similarly, the phospholipase A(2) inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST6 38 and Tyr-A23 had no effect on TRH-induced [Ca2+](i) increase but inhibite d in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22 x 10 (-5) M and ED50 = 1.47 x 10(-5) M, respectively). Taken together, these dat a indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH -induced alpha-MSH secretion. Activation of PKC is responsible for the sust ained phase of the increase in [Ca2+](i), whereas activation of PTK does no t affect Ca2+ mobilization.