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We have previously shown that gamma3-MSH stimulates DNA replication in lactotrophs, somatotrophs and thyrotrophs of early postnatal rat pituitary in culture. Since the melanocortin-3 (MC-3) receptor is the only known receptor displaying high affinity for gamma3-MSH, the present study explored whether mRNA of the latter receptor is present in the pituitary and whether the receptor is functional. RT-PCR of RNA extracts from 14-day-old rat pituitary revealed the presence of MC-3 receptor mRNA in both the anterior and the neurointermediate lobe. The identity of the amplified products was confirmed by sequence analysis. Dispersed cells of 14-day-old female rats (24 h in culture) were exposed to gamma3-MSH, and changes in intracellular free calcium levels ([Ca2+]i) were assessed by means of fluo-3 video imaging. Gamma3-MSH evoked a rapid and maintained oscillating [Ca2+]i increase in 5%, 10% and 15% of the cells at a dose of 0.1, 1 and 10 nM, respectively. The MC-3/MC-4 receptor antagonist Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (SHU 9119) blocked the effect of gamma3-MSH in about 50% of the responsive cells. The present data suggest that the MC-3 receptor is expressed in the rat pituitary but that this receptor mediates only half of the effects of its putative ligand, gamma3-MSH, on [Ca2+]i. Part of the effect of gamma3-MSH may be mediated by a MC-3 receptor in a functional state different from the one studied previously in transfected cell lines or by a hitherto unidentified MC receptor.

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A pituitary cell population of 14-day-old female rats, containing lactotropes and somatotropes but deprived of gonadotropes, was prepared by unit gravity sedimentation through a serum albumin gradient and allowed to reaggregate either as such or after mixing this population with cells of the gonadotropic αT3-1 cell line. In a perifusion system gonadotropin-releasing hormone (GnRH) had no effect on prolactin (PRL) and growth hormone (GH) release in the former aggregates but stimulated PRL release in the latter. In the αT3-l cell-containing aggregates GnRH showed a biphasic effect on GH release: inhibition during exposure to GnRH followed by a rebound secretion upon removal of the peptide. The aggregation capacity of αT3-l cells with the normal pituitary cells was demonstrated by using an αT3-l cell clone stably transfected with the reporter gene β-galactosidase. Perifusion of the gonado-trope-deprived aggregates with medium conditioned by αT3-1 cell provoked a rapid stimulation of PRL release and a biphasic effect on GH release. Medium conditioned by the cortico-tropic cell line AtT20 also stimulated PRL release but had no concomitant effect on GH release. Medium conditioned by αT3-l cells, when added for 40 h to aggregates of 14-day-old rat pituitary, provoked an increase in the number of ³H-thymidine (³H-T)-labelled lactotropes and a decreased in the number of ³H-T-labelled somatotropes. The conditioned medium was concentrated on Sep-Pak C18 and ultrafiltrated through an Amicon membrane with 3-kD molecular weight cut-off and the retained molecules separated by reversed-phase HPLC. The material stimulating ³H-T labelling of lactotropes eluted from the column with a different retention time than material inhibiting ³H-T labelling of somatotropes, suggesting that the effect on lactotropes is mediated by (a) molecule(s) different from that affecting somatotropes. The effects of αT3-l cells and their secretion products on lactotropes and somatotropes were comparable to those we previously observed using enriched populations of normal gonadotropes. The HPLC elution profiles of the substances affecting ³H-T incorporation as well as the specificity of these effects were also similar to that of the substances isolated previously from gonadotrope-conditioned medium. The present data, therefore, support previous conclusions on the paracrine control of the lactotrope/somatotrope lineage by the gonadotropes. Further purification and chemical characterization of the growth factors with selective action on lactotropes and somatotropes may lead to a better understanding of the development of the latter lineage.

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