γ3-MSH has recently been shown to be a biologically active peptide in the rat anterior pituitary. It induces a sustained rise in intracellular free calcium levels ([Ca²⁺]_i) in a relatively small population of immature pituitary cells. The present study was intended to identify the target cells of this peptide and to discern the signal-transducing melanocortin (MC) receptor. In dispersed pituitary cells from 14-day-old rats, increasing doses ofγ 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 responsive cells, 53% showed GH immunoreactivity (-ir), 12% showed PRL-ir, 2% showed TSHβ-ir, 5% showed LHβ-ir, and 10% showed ACTH-ir, whereas 18% did not express any hormone-ir to a detectable level. As assessed by single cell RT-PCR for the presence of pituitary hormone messenger RNA (mRNA), 26% of theγ 3-MSH-responsive cells contained only GH mRNA, 5% contained only PRL mRNA, and 4% contained only TSHβ mRNA. Twenty-two percent contained mRNA of GH, PRL, and TSHβ in various dual or triple combinations. About 24% of the γ3-MSH-responsive cells expressed POMC mRNA, mostly together with other mRNAs, i.e. with GH mRNA and/or PRL mRNA or with mRNA of GH, PRL, and TSHβ. Eighteen percent of the responsive cells expressed LHβ, all of them together with mRNA of GH, PRL, and TSHβ 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 pituitary cell population), the proportion of cells expressing two or multiple hormone mRNAs was twice as low as that in the γ3-MSH-responsive population, whereas the proportion of cells expressing a single hormone mRNA was twice as high (about two thirds of all cells). Moreover, unlike in theγ 3-MSH-responsive cell population, randomly chosen cells were found that coexpressed POMC mRNA with LHβ mRNA.

The effect of γ3-MSH on [Ca²⁺]_i was blocked by the MC-3 receptor antagonist SHU9119 (used up to a 1000-fold excess) in 46% or less of the responsive cells. SHU9119 failed to block the[ Ca²⁺]_i response to γ3-MSH in PRL-, GH-, and TSHβ-ir cells, but it did block the response in most ACTH-ir cells and in cells expressing no hormone to a detectable level. Single cell RT-PCR revealed that expression of MC-3 receptor mRNA was detected in only 16% of γ3-MSH-responsive cells.

The present data suggest that the target cells of γ3-MSH in terms of[ Ca²⁺]_i responses in the immature rat pituitary constitute subpopulations of all main pituitary cell types, including nonhormonal (or low expression hormonal) cells. However, in contrast to the total pituitary cell population, most of these cells display multilineage gene activation at the mRNA level, i.e. express mRNA of GH, PRL, TSHβ, POMC, and LHβ in dual, triple, or quadruple combinations. Although γ3-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 receptor or through an MC-3 receptor with unconventional binding characteristics.