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

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Abstract
Dispersed acini were prepared from guinea-pig parotid glands and incubated at 37.degree. C in HEPE[N-2-hydroxyethyl-piperizine-N''-2-ethanesulfonic acid]-buffered Ringer (HR) containing [3H]ouabain. Acini bound the Na+-K+-pump inhibitor with an estimated equilibrium dissociation constant (Kd) of 205 .mu.M and a capacity of 29 .times. 106 ouabain-binding sites/cell. Carbachol and adrenaline [epinephrine] each increased the equilibroum level of binding attained at a medium [3H]ouabain concentration of 0.1 .mu.M by up to 250%, but had no effect on binding at a medium ouabain concentration of 10 .mu.M, a near-saturating level of the glycoside. The 2 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 [ethylene glycol bis (.beta.-aminoethylether) N, N, N'',N, -tetraacetic acid] did not alter [3H]ouabain binding in the absence of agonist, but decreased by > 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 > 80% and the stimulatory effects disappeared within 5 min. Alterations in equilibrium level of ouabain bound at 0.1 .mu.M-[3H]ouabain and in ouabain-sensitive oxygen uptake by guinea-pig parotid acini both reflect changes in acinar Na+-K+-pump activity. Muscarine 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.