Expression of the Proenkephalin A Gene and [Met 5]enkephalin Secretion Induced by Prostaglandin E 2 in Bovine Adrenal Medullary Chromaffin Cells: Involvement of Second Messengers

Abstract

We have reported that prostaglandin E 2 (PGE 2) increases the long-term secretion of [Met 5]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 PGE 2-induced responses, we have now studied the effects of PGE 2 on intracellular free calcium [Ca 2+] i levels. The interactions of PGE 2 with several second messenger systems were also studied. Treatment with PGE 2 (10 μ M) produced a transient increase followed by a prolonged plateau in the levels of [Ca 2+] i. Ionomycin (3 × 10 -6 M), which depletes intracellular calcium pools, did not inhibit the PGE 2-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 PGE 2-induced [Ca 2+] i response. Long-term (24 h) treatment with PGE 2 (10 μ M) 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 PGE 2. Calmidazolium, a calmodulin antagonist, also significantly inhibited PGE 2-induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 μ M), was ineffective in blocking PGE 2-induced responses. In addition, the down-regulation of PKC by phorbol myristate acetate (PMA) (0.1 μ M) for 48 h did not inhibit the PGE 2-induced responses. Furthermore, PGE 2 did not affect AP-1 DNA binding activity, while PMA (1 μ M) 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 PGE 2 showed less than an additive effect on the secretion of ME and the levels of the proENK mRNA. The results suggest that the Ca 2+/ calmodulin pathway, but not the protein kinase A or PKC pathways, appear to be involved in mediating the PGE 2-induced increases of the long-term secretion of ME and the levels of proENK mRNA.