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In this study, a sequential analysis of pathways involved in the regulation of GH secretagogue receptor subtype 1a (GHSR-1a) signaling has been undertaken to characterize the process of rapid desensitization that is observed after ghrelin binding. This process was evaluated by studying the binding of [(125)I]ghrelin, measurement of intracellular calcium mobilization, and confocal microscopy. The results indicate that GHSR-1a is mainly localized at the plasma membrane under unstimulated conditions and rapidly desensitizes after stimulation. The agonist-dependent desensitization is not mediated by protein kinase C because phorbol ester, phorbol-12-myristate-13-acetate, failed to block the ghrelin-induced calcium response. The ghrelin/GHSR-1a complex progressively disappears from the plasma membrane after 20 min exposure to ghrelin and accumulates in the perinuclear region after 60 min. Colocalization of the internalized GHSR-1a with the early endosome marker (EEA1) after 20 min exposure to ghrelin suggests that endocytosis occurs via clathrin-coated pits, which is consistent with the lack of internalization of this receptor observed after potassium depletion. Different from other G protein-coupled receptors, GHSR-1a showed slow recycling. Surface binding slowly recovered after agonist treatment and returned to control levels within 360 min. Furthermore, inhibition of vacuolar H(+)-ATPases prevented recycling of the receptor, suggesting that the nondissociation of the ligand/receptor complex is responsible for this effect. The GHSR-1a internalization may explain the characteristic physiological responses mediated by this receptor.

In vivo FFA block basal and stimulated GH secretion and have been implicated in the pathogenesis of the altered GH secretion present in obesity and Cushing's syndrome. Although a direct action on the somatotroph cell has been postulated, the FFA mechanism of action is unknown. The main biological target for FFA action is the cellular membrane, and it has been shown that these metabolites can block the activity of a number of plasma membrane pumps, channels, and receptor systems. In the present work, it was observed using different types of pituitary cells (GH3, GH4C1, and rat pituitary primary cultures) that cis-unsaturated fatty acids, such as oleic, 1) do not perturb TRH binding or the homologous desensitization of the TRH receptor; 2) inhibit TRH-induced inositol 1,4,5-trisphosphate/diacylglycerol generation, probably by a direct perturbation of phospholipase C; 3) reduce the TRH-induced intracellular Ca2+ redistribution and the ensuing changes in membrane potential; 4) completely inhibit the [Ca2+]i rise due to the TRH-induced opening of voltage-gated Ca2+ channels; and 5) abolish the TRH-induced Ca2+ efflux through plasma membrane Ca2+ pumps. These results suggest that cis-unsaturated FFA such as oleic acid selectively perturb the function of integral membrane proteins such as enzymes, channels, and pumps without perturbing the binding of ligands to receptors.