The effectiveness of thyrotropin-releasing hormone (TRH) and vasoactive intestinal peptide (VIP) to release prolactin (PRL) after a brief interruption of the tonic inhibitory action of dopamine (DA) was investigated in enzymatically dispersed anterior pituitary cells in superfusion. We also studied the involvement of cAMP and Ca2 + /protein kinase C second messenger systems in the mediation of the stimulated PRL release. Anterior pituitary cells from lactating or E2-treated rats were superfused for 10 min with secretagogues either during continual dopamine administration or 10–20 min after a 10-min transient interruption of DA (500 nM). Removal of DA for 10 min resulted in a significant increase in PRL release which had returned to basal levels 10 min after the return of DA to the superfusion. During continuous DA exposure, TRH administration (10 nM) did not alter the rate of PRL release from cells from lactating rats; however, TRH caused a 2-fold increase after the transient interruption of DA. The transient escape from DA inhibition also increased the effectiveness of TRH (100 nM) to release PRL from cells from E2-treated rats (from a 4- to a 15-fold stimulation). In contrast, VIP (0.5 or 5 µM) caused a 2-fold stimulation of PRL release in both cells treated with continuous or transiently interrupted DA. The transient removal of DA also potentiated PRL release induced by treatment with the Ca2 + ionophore A23187 (5 and 20 µM) and/or the protein kinase C stimulator 12–0-tetradecanoyl-phorbol-3-acetate (TPA) (50 nM), but not following treatment with the adenylate cyclase activator forskolin (10 µM) or the cAMP analog 8-Br-cAMP (2.5 mM). These data suggest that a transient escape from DA inhibition increases the responsiveness of lactotrophs to the PRL-re-leasing action of TRH but not to VIP. It appears that the potentiated action of TRH involves a Ca2 + /protein kinase C-mediated p