Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

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Abstract
The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50± 10-8M). Accumulation of α-melanocyte-stimulating hormone (α-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10-8M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10-8M, whereas the inactive phorbol ester 4α-phorbol at 5 X 10-8M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on α-MSH secretion. After 8 h of PMA treatment, peptide accumulation in the medium was significantly increased, whereas a 24-h treatment with PMA inhibited α-MSH secretion as compared with untreated controls. Treatments with neither CAM antagonists nor phorbol esters decreased [3H]uridine incorporation into trichloroacetic acid-precipitable material, results suggesting that the observed effects on gene expression are limited to POMC or to a restricted number of genes. Taken together, these results suggest that in the IL of the pituitary, Ca2+ modifies hormone secretion and POMC gene expression via the CAM and PKC pathways.