1. Intracellular pH (pHi) was measured by spectrofluorometry in perfused mandibular salivary glands isolated from the rat and loaded with the pH‐sensitive fluoroprobe 2',7'‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). Cell volume changes were estimated from changes in intracellular water content measured by proton NMR spectroscopy. 2. Stimulation with 1 microM acetylcholine (ACh) led to a 15 +/‐ 2% decrease in cell volume. A transient decrease in pHi was followed by a sustained increase (0.17 +/‐ 0.03 pH units) that has previously been attributed to the upregulation of the Na(+)‐H+ exchanger. 3. Increasing perfusate osmolarity by addition of 60 mM sucrose caused a 19 +/‐ 2% decrease in cell volume and a sustained increase in pHi (0.12 +/‐ 0.01 pH units) that was abolished by 1 mM amiloride. Acid loading experiments indicated that the increase in pHi was due to an alkaline shift in the pH dependence of the Na(+)‐H+ exchanger. 4. A 20% reduction in perfusate osmolarity prevented the cell shrinkage normally associated with ACh stimulation and largely abolished the ACh‐induced increase in pHi. 5. Steady‐state Na(+)‐H+ exchanger activity, estimated from the initial rate of change in pHi following addition of amiloride, increased 9‐fold during stimulation with ACh. When cell shrinkage was prevented by simultaneous exposure to the hypotonic solution, the activity of the exchanger still increased 7‐fold in response to ACh. 6. We conclude that, although cell shrinkage leads to upregulation of the Na(+)‐H+ exchanger, this factor alone is insufficient to account for the marked increase in exchanger activity that follows muscarinic stimulation.
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