DIDS-SENSITIVE PH(I) REGULATION IN SINGLE-RAT CARDIAC MYOCYTES IN NOMINALLY HCO3-FREE CONDITIONS

The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BC ECF) was used to measure pH(i) 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 bl ocked, the intrinsic buffering power (beta(i)) in SHR myocytes was sig nificantly higher than when both the Na-H exchanger and 4,4'-diisothio cyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pH(i) regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. S imilar 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 pH(i) slowly alkalin ized (by 0.16+/-0.02 and 0.11+/-0.02 pH units, respectively); this alk alinization was also DIDS sensitive. The reacidification during NH4+ p erfusion was inhibited 30.2+/-7.4% by DIDS. In addition, in the nomina l absence of HCO3-, 100 mu mol/L ATP acidified the pH(i) in both norma l and SHR myocytes (by 0.21+/-0.03 and 0.33+/-0.03 pH units, respectiv ely); this acidification was totally inhibited by 0.1 mmol/L DIDS. It has been shown, in rat cardiac myocytes, that ATP acidifies the pH(i) by 0.35 pH unit via stimulation of a DIDS-sensitive Cl-HCO3 exchanger in HCO3-containing solutions. Finally, we have shown, in normal cardia c myocytes, that two potent Na-H exchanger blockers, N-5-ethylisopropy l amiloride (EIPA) and N-5-methyl-N-isobutyl amiloride (MIA), only par tially inhibited the pH(i) recovery from internal acidosis under nomin ally bicarbonate-free conditions. When DIDS was added at the same time as EIPA, pH(i) recovery from an internal acid loading was completely inhibited. Our results clearly demonstrate that in both normal and SHR cardiac myocytes, bicarbonate-dependent pH(i) regulators can be signi ficantly activated under resting or acidified pH(i) in HEPES-buffered medium, probably because of the cellular production of CO2. The contri bution of these bicarbonate-dependent pH(i) regulators, ie, the Na-HCO 3 cotransporter and the Cl-HCO3, exchanger, cannot therefore be ignore d even under nominally HCO3-free conditions.