STIMULATION OF CA2-TERMINAL FRAGMENTS OF PROOPIOMELANOCORTIN( ENTRY IN LACTOTROPHS AND SOMATOTROPHS FROM IMMATURE RAT PITUITARY BY N)

We have previously shown that 10-12 kDa N-terminal fragments of rat pr oopiomelanocortin (POMC) and human POMC1-76 stimulate mitosis and/or d ifferentiation in lactotrophs of early postnatal rat pituitary. A trun cated form, POMC1-26 mimics the differentiation-inducing but not the m itogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the in tracellular free calcium concentration ([Ca2+](i)) in response to POMC 1-76 and POMC1-26 in isolated pituitary cells from 14-day-old female r ats. Calcium (Ca2+) responses were also used as a guide to determine w hether the responsive cells belong to the lactosomatotroph lineage. Ap plication of POMC1-76 or POMC1-26 induced a maintained oscillating [Ca 2+](i) increase in a small population of cells. Increasing doses of th e peptides did not affect the magnitude and the frequency of [Ca2+](i) oscillations but clearly augmented the number of responding cells. Ap proximately 2% of the cells responded at 0.1 nM POMC1-76 or 5 nM POMC1 -26, and 11-13% of the cells responded at 10 nM and 500 nM of the resp ective peptides. About one-third of the cells responsive to these pept ides also showed a [Ca2+](i) increase in response to growth hormone-re leasing peptide-6 (GHRP-6) while, in a small number of responsive cell s, [Ca2+](i) was depressed by dopamine, suggesting that the former cel ls are somatotrophs and the latter lactotrophs. This interpretation wa s confirmed by immunocytochemical identification of prolactin and grow th hormone (GH) in the cells. Of the cells showing Ca2+ response to PO MC1-76, approximately one-third contained GH and another third prolact in. The remainder contained neither GH nor prolactin. Comparable resul ts were obtained with POMC1-26. The rise of [Ca2+](i) induced by the N -terminal POMC peptides persisted after depletion of intracellular Ca2 + stores by thapsigargin. Removal of Ca2+ from the extracellular mediu m or addition of cadmium completely abolished both the POMC1-76- and P OMC1-26-induced [Ca2+](i) increase. Nifedipine inhibited the Ca2+ resp onse to both peptides, although only in 55% of the responsive cells. D epletion of some isoforms of protein kinase C by preincubation with th e phorbol ester PMA for 24 h did not modify the Ca2+ responses. in con trast, blockade of the protein kinase A pathway with Rp-cAMPs partiall y inhibited the POMC1-76- or POMC1-26-induced [Ca2+](i) increase. The present data show that, in immature pituitary cells, POMC1-76 induces an increase in [Ca2+](i) through extracellular Ca2+ influx, possibly m ediated in part by protein kinase A activation. The active domain of P OMC1-76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC1-76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the pep tide mobilizes different subsets of cells within this lineage, by a me chanism determined by its concentration.