THE EFFECTS OF NA+ REPLACEMENT ON INTRACELLULAR PH AND (CA-2+) IN RABBIT SALIVARY-GLAND ACINAR-CELLS

1. The role of Na+-dependent mechanisms in regulating the intracellular pH (pH(i)) and free calcium concentration ([Ca2+]i) in acinar cells of the rabbit mandibular salivary gland was examined. The fluorescent dyes BCECF and Fura-2 were used to measure pH(i) and [Ca2+]i respectively in suspensions of isolated acini. 2. Replacement of all of the extracellular Na+ with N-methyl-D-glucamine (NMDG) decreased resting pH(i) from a control value of 7.1-7.2 to 6.8-6.9. Re-addition of Na+ or Li+ caused a recovery of pH(i) towards control values. This recovery was blocked by 10-50-mu-M-ethylisopropylamiloride (EIPA), suggesting that it was mediated by Na+-H+ exchange. The rate of recovery of pH(i) when Na+ was reintroduced increased with Na+ concentration with an apparent K(m) for Na+ of around 30 mM. 3. Replacement of all of the extracellular Na+ with Li+ caused only a small decrease in resting pH(i). 4. Stimulation of acini with 1-mu-M-acetylcholine (ACh) evoked an intracellular acidosis both under control conditions and when acini were bathed in Na+-free media. Following the acidosis pH(i) recovered in acini bathed in either control medium or Na+-free (Li+) medium, but not in acini bathed in Na+-free (NMDG) medium or in control medium containing EIPA. 5. Stimulation of acini bathed in Na+-fre, HCO3--free medium with ACh did not cause any change in pH(i). 6. Re-addition of Na+ to acini bathed in Na+-free, HCO3--free medium evoked the same rate of alkalinization whether or not the acini had been stimulated with ACh, suggesting that receptor stimulation per se did not lead to an activation of acid extrusion. 7. Resting [Ca2+]i was elevated in acini bathed in Na+-free (NMDG) medium, but not in acini bathed in Na+-free (Li+) medium. 8. ACh evoked a maintained rise in [Ca2+]i in acini bathed in control medium and in Na+-free media with either NMDG or Li+ as the Na+ substitute. 9. Experiments in which external Ca2+ was reduced to low levels (by the addition of EGTA) just prior to addition of ACh showed that ACh released intracellular Ca2+ stores under both control and Na+-free conditions. 10. In acini bathed in Na+-free (NMDG) solution and stimulated with ACh, re-addition of either Na+ or Li+ reduced [Ca2+]i. The reduction of [Ca2+]i on Na+ re-addition was blocked by EIPA. [Ca2+]i could also be reduced under these conditions by alkalinizing the cytosol using the weak base trimethylamine. 11. Our results suggest that Na+-H+ exchange is the major acid extrusion mechanism in isolated rabbit mandibular acinar cells. The exchanger is responsible for maintaining pH(i) above equilibrium, and also underlies the recovery of pH(i) when the cytosol is acidified by stimulation with ACh. 12. Removal of external Na+ does not appear to interfere with receptor-operated Ca mobilization in rabbit mandibular acinar cells. The effects of Na+ removal and re-addition on [Ca2+]i appear to be secondary to the changes in pH(i), and are most easily explained by a direct interaction between Ca2+ and H+.