Stimulation of intracellular free calcium in GH3 cells by gamma 3-melanocyte-stimulating hormone. Involvement of a novel melanocortin receptor?

The melanocortin (MC) gamma 3MSH is a peptide that can be generated from th e N-terminal domain of POMC and is believed to signal through the MC3 recep tor. We recently showed that it induces a sustained rise in intracellular f ree calcium levels ([Ca2+](i)) in a subpopulation of pituitary cells, parti cularly in the lactosomatotroph lineage. In the present study we report tha t gamma 3MSH and some analogs increase [Ca2+](i) in the GH- and PRL-secreti ng GH3 cell. line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved. A dose as low as 1 pM gamma 3MSH induced an oscillating [Ca2+](i) increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nM. gam ma SMSH, alpha MSH, and NDP-alpha MSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not af fect the number of cells showing a [Ca2+](i) rise in response to gamma 3MSH . SHU9119 had also no effect when added alone. MTII, a potent synthetic ago nist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of gamma 3MSH showed low potency in increasing [Ca2+](i) in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide cor responding to the gamma 2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca2+](i) in GH3 cells, but was about 50 times less potent than g amma2- or gamma 3MSH in stimulating cAMP accumulation in the MC3 receptor e xpressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetec table, but messenger RNA of the MC5 receptor was clearly present. These data suggest that the GH3 cell line does not mediate the effect of ga mma 3MSH through the MC3 receptor. The involvement of the MC5 receptor is u nlikely, but cannot definitely be excluded. The findings animate the hypoth esis that there exists a second, hitherto unidentified, MC receptor that di splays high affinity for gamma 3MSH.