Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin.

The pars tuberalis (PT) of the anterior pituitary is notable for the expression of levels of melatonin receptors that consistently exceed those in all other tissues in mammals. For this reason and because of its anatomical position, it has been suggested that the PT may play a role in seasonal reproductive responsiveness. However, no data have been forthcoming on the nature of the melatonin-responsive cells in this tissue or on the interaction of melatonin with other hormonal signals in the control of PT cells. A number of recent studies have reported that the tubero-infundibular region of the pituitary in several species contains binding sites for insulin-like growth factor-1 (IGF-1). The present study, therefore, sought to address the question of whether functional receptors for IGF-1 exist in the ovine PT (oPT). Primary cultures of cells from the oPT contained a widespread distribution of cells staining positively with a monoclonal antibody to the human IGF-1 receptor, with the strongest staining occurring over the small phase-bright cells that predominate in this culture system and are thought to constitute the melatonin-responsive cell type. As a functional assay for responsiveness to IGF-1, primary cultures of oPT cells were assayed for activation of mitogen-activated protein kinase (MAPK) using a previously validated phosphotransferase assay. Cytosolic extracts from PT cells treated with IGF-1 (100 pM-10 nM) caused a dose-dependent increase in the rate of phosphorylation of myelin basic protein; in contrast, treatment with melatonin had no significant effect on myelin basic protein phosphorylation. Immunostaining of Western blots of PT cell extracts with a pan-extracellular regulated kinase antibody demonstrated that both p42 and p44 MAPK are strongly expressed in this tissue. To confirm that the effects observed in the cytosol assay were indeed attributable to increased activation of p42/p44, gel renaturation assays of protein kinase activity were performed. These experiments revealed that IGF-1 (10 nM) and forskolin (1 microM) were both potent activators of 42- and 44-kDa moeities; however, neither of these agents had any significant effect on the phosphotransferase activity associated with several other higher molecular weight kinases also detected by the gel-renaturation assay procedure. Melatonin (10 nm) was consistently found to be a highly potent inhibitor of the activation of MAPK induced by forskolin; in contrast, melatonin did not inhibit the activation of MAPK induced by IGF-1.(ABSTRACT TRUNCATED AT 400 WORDS)