Abstract
We studied intracellular pH (pHi) regulation in the absence of HCO3- in single mesangial cells (MCs) with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(and -6)carboxyfluorescein. Our approach was to acid load the cells by an NH+4 prepulse and to monitor the subsequent pHi recovery. Previous work on MCs and other cells has shown that the recovery is prevented by adding ethylisopropyl amiloride (EIPA) or removing Na+ before the recovery begins, suggesting that at low pHi only Na-H exchange contributes to the recovery. This conclusion is often extrapolated to the entire pHi range. To test this, we interrupted the recovery with EIPA at various pHi values, finding that EIPA unmasked a background acidification that was negligible at pHi less than approximately 6.7 but increased steeply at higher pHi values. Correcting the total recovery rate for this EIPA-insensitive component, we found that the EIPA-sensitive (Na-H exchange) rate fell steeply with increasing pHi between 6.3 and 6.7 but was relatively pHi insensitive between 6.7 and 7.2. Thus, the recovery halts as pHi approaches approximately 7.2 not so much because Na-H exchange slows, but because acid loading accelerates. Applying the mitogen arginine vasopressin (AVP; 100 nM) caused a rapid pHi decrease of approximately 0.4, followed by a slower increase to a level approximately 0.15 higher than the initial pHi. Coincident with this biphasic change in pHi was a biphasic change in Na-H exchange kinetics. In the early phase (i.e., pHi recovery commencing approximately 8 min after AVP addition), AVP linearized the pHi dependence of the exchanger; its rate was unaffected by AVP at pHi less than approximately 6.7 but was progressively inhibited at higher pHi values. In the later phase (i.e., pHi recovery commencing approximately 14 min after AVP addition), AVP shifted this linear pHi dependence in the alkaline direction; the exchanger was stimulated at pHi less than 6.9 but was modestly inhibited at higher pHi values (i.e., in the physiological range). At all times, AVP greatly inhibited background acid loading. Thus, AVP raises steady-state pHi not because Na-H exchange is stimulated but because, although the exchanger is inhibited, acid loading is inhibited even more.
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