No abstract

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was deperndent on the phenylalanine concentration in the medium and proceeded linearly for up to 3 h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187 required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10pM extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200pM. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both a-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.

SUMMARY1. The secretion of amylase, deoxyribonuclease, ribonuclease, protein and Ca2+ by the rat parotid gland in vitro was studied.2. Isoproterenol and carbamoyleholine elicited a parallel discharge of amylase, deoxyribonuclease, ribonuclease and protein over a 40 min time period.3. The composition of the secretion was independent of the secretogogue used for stimulation. When gland slices from the same animal were stimulated with isoproterenol, adrenaline, phenylephrine or carbamoyleholine, secretary enzymes and protein were secreted in constant proportions.4. 45Ca injected intraperitoneally 16 h before stimulation with either isoproterenol or carbamoyleholine was released in parallel with amylase and protein.5. The relative proportions of amylase, ribonuclease, deoxyribonuclease, protein and Ca present in isolated parotid gland secretary granules was identical to that of isoproterenol stimulated gland secretion.6. It is concluded that the secretary proteins and Ca2+ are discharged in constant proportions by the rat parotid gland regardless of the mode of stimulation or the rate of secretion. The similarity in the composition of gland secretion and granule contents also suggests that enzymes and Ca2+ are released by exocytosis and not by diffusion across the apical plasma membrane.

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.

A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [( Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane.