Cholinergic regulation of amylase gene expression in the rat parotid gland. Inhibition by two distinct post-transcriptional mechanisms

Abstract

Stimulation of the beta-adrenergic or cholinergic muscarinic receptors are the principal mechanisms by which parotid salivary secretion is regulated in vivo. In this study we have examined the effects of cholinergic stimulation on amylase gene expression in dispersed rat parotid cells. [3H]Leucine incorporation into amylase and total protein was inhibited by carbamylcholine. Within 5 min of its addition, 10 microM carbamylcholine induced a 50-60% reduction in the rate of amylase synthesis which was sustained for more than 2 h. Blockade of the muscarinic receptor with atropine 8 min after addition of 10 microM 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. Carbamylcholine 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 microM carbamylcholine was 46% of control levels. Actinomycin D (5 micrograms/ml) lowered amylase mRNA by 8%; cycloheximide and phorbol 12-myristate 13-acetate had no effect. Isoprenaline (isoproterenol; at a concentration of 10 microM), which is an inducer of amylase gene transcription, elevated the amylase mRNA content by 30% after 2h. The calcium ionophore A23187 mimicked the effect of carbamylcholine by inhibiting [3H]leucine incorporation into amylase and lowering amylase mRNA content. The results suggest that acute stimulation of the muscarinic cholinergic receptor inhibits amylase biosynthesis in parotid cells not only by rapid attenuation of translation but also by causing a gradual loss of amylase mRNA, apparently by a Ca(2+)-dependent destabilization of the mRNA.

Microarray analysis of the rat lacrimal gland following the loss of parasympathetic control of secretion

“…Since the messages for secretory proteins have long half-lives (>6 h) and are often found associated with ribonucleoprotein complexes, the transcriptional regulation may not change rapidly after the loss of stimulation. On the other hand, loss of muscarinic stimulation may have a nondestabilizing effect on secretory protein mRNAs, as was found for amylase mRNA in the parotid gland after cholinergic stimulation (37).…”

Parasympathetic Non‐Adrenergic, Non‐Cholinergic‐Induced Protein Synthesis and Mitogenic Activity in Rat Parotid Glands

“…infusions of bethanechol neither affect the polyamine synthesis ) nor prevent the fall in weight of the parotid gland subjected to atrophying influences (M ansson et al 1990). In vitro studies on rat parotid gland cells show that muscarinic agonists decrease the secretory protein synthesis, while VIP seems to be without effect (Scott & Baum, 1985;Liu et al 1995). In vitro protein synthesis in rat submandibular acinar cells has, however, been reported to increase in response to a low concentration of carbacholine, but to decrease in response to a high concentration of this drug (Anderson, 1988).…”

Recombinant Antitoxic and Antiinflammatory Factor from the Nonvenomous Snake Python reticulatus: Phospholipase A2 Inhibition and Venom Neutralizing Potential,

“…Cloning and Nucleotide Sequencing. Total RNA was isolated from the frozen liver tissue of P. reticulatus by a modified guanidinium isothiocynate extraction method (19) and analyzed by denaturing formaldehyde agarose gel electrophoresis (20). A Marathon cDNA amplification kit (Clontech) was used to obtain an uncloned library of adapterligated double-stranded cDNA, which was amplified by the polymerase chain reaction in a Perkin-Elmer Cetus thermal cycler (model 480) for 30 cycles (94 °C for 1 min; 50 °C for 1 min; 72 °C for 2 min; 72 °C for 10 min/cycle).…”