Sodium-potassium pump in guinea-pig parotid gland: secretagogue stimulation of ouabain binding to dispersed acini

Abstract

Dispersed acini were prepared from guinea-pig parotid glands and incubated at 37 degrees C in HEPES-buffered Ringer (HR) containing [3H]ouabain. Acini bound the Na+-K+-pump inhibitor with an estimated equilibrium dissociation constant (Kd) of 2.05 microM and a capacity of 2.9 X 10(6) ouabain-binding sites/cell. Carbachol and adrenaline each increased the equilibrium level of binding attained at a medium [3H]ouabain concentration of 0.1 microM by up to 250%, but had no effect on binding at a medium ouabain concentration of 10 microM, a near-saturating level of the glycoside. These results indicate that the two secretagogues elicit increases in Na+-K+-pump activity without increasing the number of pump sites available. The observed stimulation by carbachol was readily reversed by atropine and that evoked by adrenaline was reversed by phentolamine. Incubation of acini in Ca2+-free HR with 0.2 mM-EGTA did not alter [3H]ouabain binding in the absence of agonists, but decreased by over 80% the response to both carbachol and adrenaline. The full response to either could be restored by adding 1.5 mM-Ca2+ to the Ca2+-free medium after challenge with agonists. The response to a maximally effective dose of carbachol could not be augmented by adrenaline or vice versa in either the presence or absence of extracellular Ca2+. Carbachol and adrenaline also stimulated the ouabain-sensitive component of acinar oxygen uptake by 230-260%, but had no significant effect on ouabain-insensitive respiration. In the absence of extracellular Ca2+, stimulation of oxygen uptake by either agonist was reduced by over 80% and the stimulatory effects disappeared within 5 min. We conclude that alterations in equilibrium level of ouabain bound at 0.1 microM-[3H]ouabain and in ouabain-sensitive oxygen uptake by guinea-pig parotid acini both reflect changes in acinar Na+-K+-pump activity. Muscarinic cholinergic and alpha-adrenergic receptor occupancy strongly stimulates pump activity in a Ca2+-dependent manner. Na+-K+ pumps are the primary energy-requiring ion-transport component that is activated by these secretagogues.