1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10μM), isoproternol (1 or 10 μM), phenylephrine (1 μM), carbamoylcholine (0.1 or 1 μM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (> 3h) of probably a similar dimension. Cholinergic and α-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas β-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (β-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).

Rat parotid gland homogenates were fractionated into mitochondrial, heavy microsomal and light microsomal fractions by differential centrifugation. ATP-dependent 45Ca2+ uptake by the subcellular fractions paralleled the distribution of NADPH-cytochrome c reductase, an enzyme associated with the endoplasmic reticulum. The highest rate of Ca2+ uptake was found in the heavy microsomal fraction. Ca2+ uptake by this fraction was dependent on the presence of ATP and was sustained at a linear rate by 5 mM-oxalate. Inhibitors of mitochondrial Ca2+ transport had no effect on the rate of Ca2+ uptake. Na+ and K+ stimulated Ca2+ uptake. At optimal concentrations. Na+ stimulated Ca2+ uptake by 120% and K+ stimulated Ca2+ uptake by 260%. Decreasing the pH from 7.4 to 6.8 had little effect on Ca2+ uptake. The Km for Ca2+ uptake was 3.7 microM free Ca2+ and 0.19 mM-ATP. Vanadate inhibited Ca2+ uptake; 60 microM-vanadate inhibited the rate of Ca2+ accumulation by 50%. It is concluded that the ATP-dependent Ca2+ transport system is located on the endoplasmic reticulum and may play a role in maintaining intracellular levels of free Ca2+ within a narrow range of concentration.

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.