ADRENERGIC REGULATION OF AMYLASE GENE EXPRESSION IN THE RAT PAROTID GLAND

Abstract

Protein secretion by rat parotid glands is regulated primarily by stimulation of the adrenergic and cholinergic receptors. The role of these receptors in regulating the biosynthesis of secretory proteins such as a -amylase has not been well characterized. The aim of this study was therefore to gain a better understanding of the regulation of amylase gene expression by ?- and ?- adrenergic receptors. Acute stimulation of parotid glands with an injection of isoproterenol induced almost complete discharge of amylase within 1 h. Amylase mRNA was elevated after 1 hand was 2-3 fold higher than controls after 4 h. In vitro, ?-adrenergic stimulation of parotid cells leads to a more than 2-fold increase in [3H]uridine incorporation into total RNA, by a cyclic AMP and protein kinase A (PKA)-dependent pathway. Concentrations of isoproterenol and epinephrine which stimulated RNA synthesis were similar to those required for elevating amylase secretion suggesting that protein secretion and induction of gene transcription are likely to occur concurrently. Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C transiently stimulated the incorporation of [ 3H]uridine into RNA. This induction of gene transcription appeared to be independent of the B-adrenergic receptor-activated pathway and probably involves a different set of genes. Both isoproterenol (10 µM) or epinephrine (10 µM) elevated amylase mRNA levels by about 100% after 2.5 h. The epinephrine-induced effect was reversed by the B-adrenergic antagonist propranolol. Actinomycin D abolished the stimulatory effect of epinephrine suggesting that epinephrine stimulates transcription of the a-amylase gene. Alpha-adrenergic and cholinergic agonists had no significant effect on [ 3H]uridine incorporation. Activation of these receptors however decreased amylase mRNA levels. The calcium ionophore A23187, but not PMA, induced a similar effect. The results indicate that these agents may destabilize amylase mRNA via a novel, Ca2+-dependent mechanism. Thus cyclic AMP and Ca2+ appear to act antagonistically in controlling amylase mRNA levels in these cells. Isoproterenol rapidly increased [3H]leucine incorporation into amylase and total protein. The stimulation of amylase and total protein synthesis was apparently mediated through an increase in intracellular cyclic AMP and activation of PKA. Different isoforms of PKA may mediate the B-adrenergic receptor-induced stimulation of transcription and translation, since they show differential sensitivity to inhibition by H-8. Isoproterenol had no effect on the ribosomal transit time and indirect evidence suggested that its stimulation of protein synthesis is most likely due to an enhanced rate of translational initial ion. Stimulation of the a-adrenergic receptor with 10 µM epinephrine rapidly inhibited [ 3H]leucine incorporation into amylase and total protein synthesis. Thus, activation of the ?-adrenergic receptor inhibits amylase gene transcription not only by destabilizing its mRNA but also by attenuating translation. In contrast, activation of the ?-adrenergic receptor stimulates amylase biosynthesis by accelerating the rate of gene transcription as well as translation of its mRNA.