pH Regulatory Transport Systems in a Smooth Muscle-Like Cell Line

The membrane transport systems responsible for pH regulation in BC3H-1 cells were studied using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In nominally CO ₂ -free Na N - 2 -hydroxyethylpiperazine- N' -2-ethanesulfonic acid buffer (NHB) recovery from acidification after an NH ₄ Cl pulse was reversibly inhibited by 1 mM amiloride or by Na-free solutions. On exposure to 5% CO ₂ -HCO ₃ (external pH constant at 7.4), BC3H-1 cells alkalinized by approximately 0.3-0.4 pH unit. This CO ₂ -induced alkalinization was unaffected by 1 mM amiloride, markedly reduced by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and inhibited by Na-free solutions. On readdition of Na, cells rapidly alkalinized, even in the presence of 1 mM amiloride. Exposure to Cl-free CO ₂ -HCO ₃ solutions caused a rapid alkalinization of nearly 1 pH unit that was abolished by SITS, largely independent of Na, unaffected by amiloride, and unchanged by membrane depolarization in high external K solutions. CO ₂ -induced alkalinization was slowed by approximately 75% after prolonged exposure of cells to Cl-free NHB, but a distinct recovery from acidification remained in these Cl-depleted cells. This recovery was Na-dependent, SITS-inhibitable, and unaffected by depolarization in high-K solutions. In the presence of CO ₂ , the acidification seen in response to NH ₄ Cl-induced alkalinization was reduced 50% by 0.5 mM SITS. These data suggest that the regulation of pH in BC3H-1 cells is mediated by at least three transport systems: 1 ) Na-H exchange; 2 ) Cl-HCO ₃ exchange; and 3 ) electroneutral (Na + HCO ₃ )-Cl exchange. A fourth transport system, which appears to be electroneutral, SITS-inhibitable, dependent on Na and HCO ₃ and independent of Cl, may also exist.