The ovine pars tuberalis (PT) still offers the best model for the study of signal transduction pathways regulated by the melatonin receptor. From the evidence accumulated so far, it seems likely that the cAMP signal transduction pathway will be a major effector of a stimulatory signal to the PT which can be regulated by melatonin. Thus a principal action of melatonin in the PT may be the repression of biochemical processes driven by cAMP. However, through the phenomenon of sensitization, melatonin may also act to amplify a stimulatory input to the cAMP signal transduction pathway in the PT. These events are mediated via the melatonin receptor, which is itself a target for regulation by the melatonin signal. Studies using the PT have identified several signalling pathways that may serve to positively or negatively regulate the expression of the melatonin receptor. These and other studies in the PT have alluded to cAMP-independent pathways regulated by the melatonin receptor.

The cocaine-amphetamine regulated transcript (CART) encodes for a protein which has an important role in the regulation of appetite and body weight. To date, no details of the molecular events and signal transduction pathways which regulate this gene are available. We report the identification of CART gene expression in the GH3 pituitary cell line. We have used activators of the cAMP or protein kinase C (PKC) signal transduction pathways to show that, in GH3 cells, CART is transcriptionally up-regulated by activators of the cAMP signal transduction pathway. We also identify CART gene expression in ovine pars tuberalis (PT) tissue and primary cell cultures. In PT cells in contrast to GH3 cells, CART gene expression is upregulated by activators of the PKC signal transduction pathway. Cultured cells have provided a valuable resource for the detailed analysis of specific regulatory mechanisms underlying transcriptional or translational regulation of genes, signal transduction events and many other cellular processes. GH3 and PT cells may therefore provide a resource for the further detailed molecular analysis of the events regulating CART gene expression and processing.

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Mimicking short photoperiod melatonin signals (16 h exposure) on primary cell cultures of melatonin target cells of the ovine pars tuberalis (PT) results in an enhanced cAMP response to forskolin stimulation relative to untreated cells, a phenomenon termed sensitization. The sensitized response of PT cells may be an important aspect of the interpretation of the melatonin signal to initiate appropriate seasonal physiological responses. The aim of this study is to add to our understanding of the molecular mechanisms involved in the sensitization of PT cells by melatonin. We demonstrate that sensitization of PT cells by melatonin is mediated via a G(i)-coupled melatonin receptor. The sensitized cAMP response is not only obtained with the pharmacological tool forskolin, but also with cholera toxin, an activator of G(salpha). Changes in the level of G(salpha) or G(ialpha) G-protein subunits are ruled out as part of the sensitization mechanism. However, changes in tyrosine phosphorylation may be involved as tyrosine kinase inhibitors sensitize ovine PT cells and tyrosine phosphatase inhibitors significantly blunt adenylate cyclase activity, including the sensitized response to melatonin. The adenylate cyclase isoforms mediating the sensitized response may be broad as 7 of the 9 isoforms of adenylate cyclase are expressed in the PT.

The expression of the melatonin receptor is positively regulated by cAMP and negatively regulated by melatonin in the ovine pars tuberalis (PT). Furthermore, when PT cells are dispersed in primary culture, both messenger RNA (mRNA) and protein levels spontaneously increase through a process that can be blocked by melatonin, but does not involve cAMP. This suggests that other second messengers may be regulated by melatonin, which, in turn, regulates melatonin receptor mRNA and protein levels. In this study using ribonuclease protection assays, ligand binding, protein kinase C (PKC), and cAMP analysis, we demonstrate that the levels of Mel 1a mRNA and protein expression in ovine PT are reduced by phorbol 12-myristate 13-acetate in a cAMP-independent process. This is indicative of an inhibitory role for PKC in receptor regulation. Melatonin, however, does not act through PKC activation to reduce Mel 1a mRNA or protein levels. Basal PKC activity in PT cells can be inhibited by the PKC inhibitor Ro 31-8220, and this suggests that basal PKC activity may suppress Mel 1a receptor expression. Paradoxically, however, Ro 31-8220 also inhibits melatonin receptor mRNA and protein levels in PT cells by a cAMP-independent mechanism. This suggests that other undefined pathways must play an important role in the physiological self-regulation of Mel 1a receptor expression by melatonin. (Endocrinology 139: [163][164][165][166][167][168][169][170][171] 1998) D AILY release of melatonin from the pineal gland occurs during the hours of darkness to provide a sustained hormonal signal of between 4 -16 h in a 24-h period. The action of melatonin is mediated by a G protein-coupled receptor (1, 2) at central sites within the brain or peripheral tissues and, in particular, the pars tuberalis (PT) of the anterior pituitary gland (3, 4). The effects of melatonin are to entrain the circadian clock on a daily basis to a 24-h light-dark cycle (5, 6) and, in particular through a change in the length of the melatonin signal during successively lengthening nights of the fall-winter seasons, initiate a number of circannual physiological, metabolic, and behavioral changes required for optimal survival (7-10).The melatonin receptor expressed in the plasma membrane of the cells of the ovine PT appears to mediate one of several key events in response to a lengthening melatonin signal, i.e. a reduction in PRL output from the pars distalis (11). Recent evidence suggests that the physiological function of melatonin receptors within the PT may be to modulate the output of a factor called tuberalin (12,13). This factor can stimulate the release of PRL from pars distalis cells and probably contributes to the seasonal changes in plasma PRL levels, which can occur in the absence of neuroendocrine factors (11). In the ovine PT, the melatonin receptor is present as a part of a large protein complex (14) that mediates the inhibition of the cAMP second messenger pathway via pertussis toxin-sensitive and insensitive mechanisms (15). This inhibition of cA...