alpha 1-Adrenergic regulation of peptidylglycine alpha-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 b ifunctional protein containing two enzymes that act sequentially to catalys e the alpha-amidation of neuroendocrine peptides. Previous studies have dem onstrated that alpha-adrenergic stimulation results in an increase in intra cellular volume and protein content of cultured neonatal rat myocardial cel ls. The present study examined the regulated expression of PAM during alpha -adrenergic stimulation, alpha 1-adrenergic stimulation activates the expre ssion 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 alp ha-adrenergic stimulation was dependent on the concentration and duration o f exposure to the agonist, and displayed alpha 1-adrenergic receptor specif icity. The transcription rate experiments indicated that these alpha-adrene rgic 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. Cardiom yocytes cultures from atria and ventricles in the presence of 5,6 dichloro- 1-beta ribofuranosyl benzamidazole (DRB) showed that phenylephrine treatmen t 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 nucle ar RNA with probes specific for PAM intron sequences shows that increased P AM expression after phenylephrine treatment was not due to intranuclear sta bilisation of the primary transcript. Protein kinase C inhibitors H7 and GF 109203x, completely blocked the phenylephrine stimulated PAM expression. Th ese results suggest that alpha-adrenergic agonist induces PAM mRNA levels b y increasing its stability in the cytoplasm. They indicate that PAM gene ex pression augments through a H7 and GF109203x sensitive pathway, involving t he activation of protein kinase C. (C) 1999 Elsevier Science Ireland Ltd. A ll rights reserved.