DIDS-sensitive pHi regulation in single rat cardiac myocytes in nominally HCO3-free conditions.

Abstract

The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi in the spontaneously hypertensive rat (SHR) and in normal rat cardiac myocytes under nominally HCO3-free (20 mmol/L HEPES-buffered) conditions. When only the Na-H exchanger was blocked, the intrinsic buffering power (beta i) in SHR myocytes was significantly higher than when both the Na-H exchanger and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pHi regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. Similar low values for beta i were also found for normal rat myocytes in Na(+)-free conditions. In Cl(-)-free solution under nominally HCO3-free conditions, in both normal and SHR myocytes, the pHi slowly alkalinized (by 0.16 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively); this alkalinization was also DIDS sensitive. The reacidification during NH4+ perfusion was inhibited 30.2 +/- 7.4% by DIDS. In addition, in the nominal absence of HCO3-, 100 mumol/L ATP acidified the pHi in both normal and SHR myocytes (by 0.21 +/- 0.03 and 0.33 +/- 0.03 pH units, respectively); this acidification was totally inhibited by 0.1 mmol/L DIDS. It has been shown, in rat cardiac myocytes, that ATP acidifies the pHi by 0.35 pH unit via stimulation of a DIDS-sensitive Cl-HCO3 exchanger in HCO3-containing solutions. Finally, we have shown, in normal cardiac myocytes, that two potent Na-H exchanger blockers, N-5-ethylisopropyl amiloride (EIPA) and N-5-methyl-N-isobutyl amiloride (MIA), only partially inhibited the pHi recovery from internal acidosis under nominally bicarbonate-free conditions. When DIDS was added at the same time as EIPA, pHi recovery from an internal acid loading was completely inhibited. Our results clearly demonstrate that in both normal and SHR cardiac myocytes, bicarbonate-dependent pHi regulators can be significantly activated under resting or acidified pHi in HEPES-buffered medium, probably because of the cellular production of CO2. The contribution of these bicarbonate-dependent pHi regulators, ie, the Na-HCO3 cotransporter and the Cl-HCO3 exchanger, cannot therefore be ignored even under nominally HCO3-free conditions.