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

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.