Expression of the proenkephalin a gene and [Met⁵]enkephalin secretion induced by prostaglandin E₂ in bovine adrenal medullary chromaffin cells: Involvement of second messengers

We have reported that prostaglandin E₂ (PGE₂) increases the long-term secretion of [Met⁵]enkephalin (ME) and the expression of proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin (BAMC) cells. In order to characterize the underlying mechanisms for the PGE₂-induced responses, we have now studied the effects of PGE₂ on intracellular free calcium [Ca²⁺]_i levels. The interactions of PGE₂ with several second messenger systems were also studied. Treatment with PGE₂ (10 μM) produced a transient increase followed by a prolonged plateau in the levels of [Ca²⁺]_i. Ionomycin (3 × 10^-6 M), which depletes intracellular calcium pools, did not inhibit the PGE₂-induced responses. Nimodipine (1 × 10^-6 M), an L-type calcium channel blocker, did not block the initial transient but blocked the plateau phase of the PGE₂-induced [Ca²⁺]_i response. Long-term (24 h) treatment with PGE₂ (10 μM) increased both the secretion of ME and the expression of proENK mRNA. Pretreatment of BAMC cells with nimodipine but not with ω-conotoxin GVIA inhibited the secretion of ME and the expression of proENK mRNA induced by PGE₂. Calmidazolium, a calmodulin antagonist, also significantly inhibited PGE₂-induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 μM), was ineffective in blocking PGE₂-induced responses. In addition, the down-regulation of PKC by phorbol myristate acetate (PMA) (0.1 μM) for 48 h did not inhibit the PGE₂-induced responses. Furthermore, PGE₂ did not affect AP-1 DNA binding activity, while PMA (1 μM) increased AP-1 DNA binding activity. Forskolin (an adenyl cyclase activator) alone increased ME secretion as well as proENK mRNA levels and when coincubated with PGE₂ showed less than an additive effect on the secretion of ME and the levels of the proENK mRNA. The results suggest that the Ca²⁺/ calmodulin pathway, but not the protein kinase A or PKC pathways, appear to be involved in mediating the PGE₂-induced increases of the long-term secretion of ME and the levels of proENK mRNA.