Melatonin-sensitive, serum-stimulated signalling in ovine pars tuberalis

In primary cultures of ovine pars tuberalis (oPT), serum acts through melatonin-sensitive mechanisms independent of cyclic AMP to increase the phosphorylation of the Ca2+/cyclic AMP response element binding protein (CREB). Immunocytochemical and biochemical assays were used to characterize the active components of serum and the signalling pathways through which they and melatonin function in oPT. The stimulatory effect of serum was heat-labile, sensitive to precipitation by methanol, and required components with a mass greater than 10 KDa implicating peptide or protein factors as the active agent. Serum increased the cytosolic free Ca2+ concentration ([Ca2+]i) of oPT cells. Serum also enhanced the release of [3H]-choline and [3H]-arachidonic acid from prelabeled cells, demonstrating that factors present in serum increase the breakdown of cellular phospholipids. This effect, however, was not blocked by melatonin (1 microM). Serum also caused a dose-dependent increase in levels of immediate early gene immunoreactivity, confirming that factors in serum have the ability to control transcription in the oPT. Down-regulation of protein kinase C (PKC) by treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA, 100 nM) or treatment with a specific PKC inhibitor (RO-31-8220, 1 microM), did not affect protein kinase A-mediated stimulation of CREB phosphorylation. However, down-regulation of PKC blocked the acute stimulatory effects of TPA (100 nM) and of serum (1%). Moreover, RO-31-8220 abolished the stimulatory effect of TPA (100 nM) and strongly attenuated that of serum (1%). These results demonstrate that serum increases the phosphorylation of CREB by stimulating cyclic AMP-independent, PKC-dependent, signalling pathways within the oPT. PKC may be activated through increased phospholipid catabolism and/or raised [Ca2+]i.