α1-Adrenergic regulation of peptidylglycine α-amidating monooxygenase gene expression in cultured rat cardiac myocytes:: transcriptional studies and messenger ribonucleic acid stability

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1. 14. 17. 3) is a bifunctional protein containing two enzymes that act sequentially to catalyse the alpha-amidation of neuroendocrine peptides. Previous studies have demonstrated that alpha-adrenergic stimulation results in an increase in intracellular volume and protein content of cultured neonatal rat myocardial cells. The present study examined the regulated expression of PAM during alpha-adrenergic stimulation, alpha 1-adrenergic stimulation activates the expression and release of PAM from myocytes. Following phenylephrine treatment, myocardial cells displayed a several fold increase in PAM activity, and a 2-4-fold increase in the steady state levels of PAM mRNA. This effect of alpha-adrenergic stimulation was dependent on the concentration and duration of exposure to the agonist, and displayed alpha 1-adrenergic receptor specificity. The transcription rate experiments indicated that these alpha-adrenergic effects were not due to increased PAM gene activity, suggesting that a post-transcriptional mechanism was involved. The most common mechanism of post-transcriptional regulation affects cytoplasmic mRNA stability. Cardiomyocytes cultures from atria and ventricles in the presence of 5,6 dichloro-1-beta ribofuranosyl benzamidazole (DRB) showed that phenylephrine treatment increased the half-life of PAM mRNA from 13 +/- 1 to 21 +/- 1 h in atrial cells and from 8 +/- 1 to 12 +/- 1 h in ventricle cells. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that increased PAM expression after phenylephrine treatment was not due to intranuclear stabilisation of the primary transcript. Protein kinase C inhibitors H7 and GF109203x, completely blocked the phenylephrine stimulated PAM expression. These results suggest that alpha-adrenergic agonist induces PAM mRNA levels by increasing its stability in the cytoplasm. They indicate that PAM gene expression augments through a H7 and GF109203x sensitive pathway, involving the activation of protein kinase C. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.