Stimulation of Ca2+ Entry in Lactotrophs and Somatotrophs from Immature Rat Pituitary by N-Terminal Fragments of Proopiomelanocortin

We have previously shown that 10–12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC_1–76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC_1–26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration (Ca²⁺]_i) in response to POMC_1–76 and POMC_1–26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca²⁺) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC_1–76 or POMC_1–26 induced a maintained oscillating Ca²⁺]_i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of Ca²⁺]_i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC_1–76 or 5 nM POMC_1–26, and 11–13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a Ca²⁺]_i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, Ca²⁺]_i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca²⁺ response to POMC_1–76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC_1–26. 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 medium or addition of cadmium completely abolished both the POMC_1–76- and POMC_1–26-induced Ca²⁺]_i increase. Nifedipine inhibited the Ca²⁺ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca²⁺ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC_1–76- or POMC_1–26-induced Ca²⁺]_i increase. The present data show that, in immature pituitary cells, POMC_1–76 induces an increase in Ca²⁺]_i through extracellular Ca²⁺ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC_1–76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC_1–76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.