Stachowiak_1991_Mol.Cell.Neurosci_2_213

The purpose of this study was to examine the effects of angiotensin on the release of enkephalin peptides and the expression of the proenkephalin (pEK) gene. Incubation of cultured bovine adrenal medullary (AM) cells in serum-free medium resulted in calcium- and time-dependent accumulation of [Met(5)]-enkephalin (MEK) in the medium. Fifteen minutes to three hours of incubation with 2 nM [Sar(1)]-angiotensin II (s(1)-AII) did not affect basal secretion of MEK; however, longer incubations (24 h) resulted in four- to fivefold increases. Northern and dot blot analyses with bovine pEK cDNA demonstrated increases in the relative abundance of pEK mRNA in angiotensin-treated cells, suggesting that the long-term increases in MEK release may reflect increased expression of pEK gene and MEK synthesis. Stimulation of MEK release and induction of pEK mRNA were concentration dependent (ED(50) approximately 1 nM. Changes in pEK mRNA levels were not observed until 12 h of incubation with s(1)-AII and continued to increase during an additional 12 h of incubation. Addition of an angiotensin antagonist, saralasin, at 0-16 h, but not at 18-20 h, to cells incubated continuously for 24 h with s(1)-AII inhibited changes in pEK mRNA and MEK release. These observations demonstrate the absence of apparent desensitization of angiotensin receptor function and indicate that long-term receptor-ligand interactions are required to induce changes in gene expression and MEK release. Induction of pEK mRNA and stimulation of MEK release were additive to the effects of veratridine and forskolin, respectively, indicating that the effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Angiotensin-induced increases in pEK mRNA were partially inhibited by nifedipine and also by dantrolene and TMB-8, drugs that inhibit voltage-dependent calcium channels and mobilization of calcium from intracellular stores, respectively. s(1)-AII-induced changes in pEK mRNA were inhibited with calmidazolium, suggesting involvement of calmodulin. The participation of protein kinase C in the induction of pEK gene and long-term stimulation of MEK release was indicated by inhibition of the s(1)-AII effects by pretreatment of cells with protein kinase C inhibitor sphingosine. Effects of s(1)-AII on induction of pEK mRNA by angiotensin and by nicotine were prevented by the translational inhibitor cycloheximide. In conclusion, angiotensin receptors were found to control expression of the pEK gene and secretion of MEK. Unlike nicotinic receptors, which may control secretion of enkephalin peptides directly by stimulating exocytosis and indirectly by controlling peptide synthesis, the effects of angiotensin appear to be mediated indirectly at the level of pEK gene expression.