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We have previously shown that the melanotrope population of the pituitary intermediate lobe of Rana ridibunda is composed of two subpopulations, of low (LD) and high density (HD), that show distinct ultrastructural features and display different synthetic and secretory rates. To investigate whether LD and HD melanotrope cells also differ in proopiomelanocortin (POMC) processing, we have analyzed the POMC-end products in single cells from both subpopulations by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mass spectra revealed the presence of 8 POMC-derived peptides in HD and LD melanotrope cells, indicating a similar processing of the precursor in both subpopulations. However, the relative abundance of three POMC-end products (i.e. lys-gamma1-MSH, acetyl-alpha-MSH, and CLIP fragment) was higher in the HD subset. Moreover, two peptides with molecular weights of 1030 and 1818 Da, respectively, were detected that could not be assigned to any product deduced from the frog POMC sequence. The relative amount of the 1030 Da peptide was higher in LD melanotrope cells. Taken together, our results suggest that POMC processing is differentially regulated in the two melanotrope cell subsets.

Ca2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca2+ sources and of cAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca2+]i rises that could be followed by a refractory plateau (type 1 response), or by a decrease in [Ca2+]i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca2+ profiles in response to both peptides were similar, PACAP38-induced [Ca2+]i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca2+, blocking Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC), or inhibiting PKA. Conversely, Ca2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca2+]i induced by PACAP38. We conclude that both peptides stimulate extracellular Ca2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.