Target cells of gamma 3-melanocyte-stimulating hormone detected through intracellular Ca2+ responses in immature rat pituitary constitute a fraction of all main pituitary cell types, but mostly express multiple hormone phenotypes at the messenger ribonucleic acid level. Refractoriness to melanocortin-3 receptor blockade in the lacto-somatotroph lineage

gamma 3-MSH has recently been shown to be a biologically active peptide in the rat anterior pituitary. It induces a sustained rise in intracellular fr ee calcium levels ([Ca2+](i)) in a relatively small population of immature pituitary cells. The present study was intended to identify the target cell s of this peptide and to discern the signal-transducing melanocortin (MC) r eceptor. In dispersed pituitary cells from 14-day-old rats, increasing dose s of gamma 3-MSH (0.1, 1, and 10 nM) evoked a sustained oscillating [Ca2+]( i) rise in an increasing number of cells (up to 14.5%). Within the responsi ve cells, 53% showed GH immunoreactivity (-ir), 12% showed PRL-ir, 2% showe d TSH beta-ir, 5% showed LH beta-ir, and 10% showed ACTH-ir, whereas 18% di d not express any hormone-ir to a detectable level. As assessed by single c ell RT-PCR for the presence of pituitary hormone messenger RNA (mRNA), 26% of the gamma 3-MSH-responsive cells contained only GH mRNA, 5% contained on ly PRL mRNA, and 4% contained only TSH beta mRNA. Twenty-two percent contai ned mRNA of GK, PRL, and TSH beta in various dual or triple combinations. A bout 24% of the gamma 3-MSH-responsive cells expressed POMC mRNA, mostly to gether with other mRNAs, i.e. with GH mRNA and/or PRL mRNA or with mRNA of GH, PRL, and TSH beta. Eighteen percent of the responsive cells expressed L H beta, all of them together with mRNA of GK, PRL, and TSH beta in various combinations. The absence of hormone mRNA was found in less than 1% of the responsive cells. In cells chosen at random (representative of the total pi tuitary cell population), the proportion of cells expressing two or multipl e hormone mRNAs was twice as low as that in the gamma 3-MSH-responsive popu lation, whereas the proportion of cells expressing a single hormone mRNA wa s twice as high (about two thirds of all cells). Moreover, unlike in the ga mma 3-MSH-responsive cell population, randomly chosen cells were found that coexpressed POMC mRNA with LH beta mRNA. The effect of gamma 3-MSH on [Ca2+](i) was blocked by the MC-3 receptor ant agonist SHU9119 (used up to a 1000-fold excess) in 46% or less of the respo nsive cells. SHU9119 failed to block the [Ca2+](i) response to gamma 3-MSH in PRL-, GH-, and TSH beta-ir cells, but it did block the response in most ACTH-ir cells and in cells expressing no hormone to a detectable level. Sin gle cell RT-PCR revealed that expression of MC-3 receptor mRNA was detected in only 16% of gamma 3-MSH-responsive cells. The present data suggest that the target cells of gamma 3-MSH in terms of [ Ca2+](i) responses in the immature rat pituitary constitute sub-populations of all main pituitary cell types, including nonhormonal (or low expression hormonal) cells. However, in contrast to the total pituitary cell populati on, most of these cells display multilineage gene activation at the mRNA le vel, i.e. express mRNA of GH, PRL, TSH beta, POMC, and LH beta in dual, tri ple, or quadruple combinations. Although gamma 3-MSH may act through the MC -3 receptor in a portion of these cells, most of these cells (mainly in the lacto-somatotroph lineage) may transduce the signal through another recept or or through an MC-3 receptor with unconventional binding characteristics.