CHOLINERGIC REGULATION OF AMYLASE GENE-EXPRESSION IN THE RAT PAROTID-GLAND - INHIBITION BY 2 DISTINCT POSTTRANSCRIPTIONAL MECHANISMS

Stimulation of the beta-adrenergic or cholinergic muscarinic receptors are the principal mechanisms by which parotid salivary secretion is r egulated in vivo. In this study we have examined the effects of cholin ergic stimulation on amylase gene expression in dispersed rat parotid cells. [H-3]Leucine incorporation into amylase and total protein was i nhibited by carbamylcholine. Within 5 min of its addition, 10 mu M car bamylcholine induced a 50-60 % reduction in the rate of amylase synthe sis which was sustained for more than 2 h. Blockade of the muscarinic receptor with atropine 8 min after addition of 10 mu M carbamylcholine reversed the carbamylcholine-induced inhibition of amylase synthesis. When cells were exposed to carbamylcholine for 2 h before addition of atropine, there was only a slight reversal of inhibition. Carbamylcho line had no significant effect on the rate of total RNA synthesis but caused a progressive loss of amylase mRNA. After 2 h, amylase mRNA in cells treated with 10 mu M carbamylcholine was 46% of control levels. Actinomycin D (5 mu g/ml) lowered amylase mRNA by 8 %; cycloheximide a nd phorbol 12-myristate 13-acetate had no effect. Isoprenaline (isopro terenol; at a concentration of 10 mu M), which is an inducer of amylas e gene transcription, elevated the amylase mRNA content by 30% after 2 h. The calcium ionophore A23187 mimicked the effect of carbamylcholin e by inhibiting [H-3]leucine incorporation into amylase and lowering a mylase mRNA content. The results suggest that acute stimulation of the muscarinic cholinergic receptor inhibits amylase biosynthesis in paro tid cells not only by rapid attenuation of translation but also by cau sing a gradual loss of amylase mRNA, apparently by a Ca2(+) dependent destabilization of the mRNA.