Direct measurement of Na influx by23Na NMR during secretion with acetylcholine in perfused rat mandibular gland

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Abstract
Intracellular Na content (Nain) in the perfused rat mandibular gland was measured by using a23Na NMR spectroscopy at 24°C. An aqueous chemical shift reagent, dysprosium triethylenetetramine-N,N,N′,N″,N‴N‴-hexaacetic acid [Dy(TTHA)] was used in order to discriminate between the intracellular and the extracellular Na signal. The mandibular gland of rat was perfused arterially with a modified Krebs solution containing 10 mM Dy(TTHA). At rest, Nain was not changed by blocking the Na+/K+ ATPase with ouabain (1 mM) and atropine (3 μM), implying that, in the absence of stimulation, the spontaneous Na influx across the plasma membrane must have been negligibly small. Following onset of stimulation with acetylcholine (1 μM), Nain increased by 9.1±1.5 mmol/l intracellular fluid (mean±SEM,n=13), and remained at this level during stimulation. In the initial phase of secretion (0–5 min), about 50 mmol/min/l intracellular fluid of Na was secreted into the luminal space (estimated from the secretory rate by assuming an isotonic primary secretion) but, in spite of the higher secretion rate, Nain increased only at an initial rate of 4.1 mmol/min/l intracellular fluid. During the steady phase of secretion (15–30 min) evoked by acetylcholine (1 μM), ouabain (1 mM) caused an increment of Nain of 44±8 mmol/l intracellular fluid (mean±SEM,n=4). From the rate of Nain increment, the Na influx rate at the steady phase was estimated as 4.5 mmol/min/l intracellular fluid. These results suggest that the influx of Na is caused by stimulation with acetylcholine. The observed Na influx rate was about 50% of the Na secretory rate at the steady phase of secretion, estimated from the secretory rate by assuming an isotonic primary secretion. This is fully compatible with the operation of Na−K-2Cl contransport system for which one would expect a Na influx rate exactly half of the rate of Na and Cl secretion.

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