Intracellular pH in mammalian stages of Trypanosoma cruzi is K +-dependent and regulated by H +-ATPases

Abstract

Regulation of intracellular pH (pH i) was investigated in Trypanosoma cruzi amastigotes and trypomastigotes using 2′,7′-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). pH i was determined to be 7.33±0.08 and 7.35±0.07 in amastigotes and trypomastigotes, respectively, and there were no significant differences in the regulation of pH i between the two stages. Steady-state pH i, recovery of pH i from acidification, and H +-efflux were all decreased markedly by the H +-ATPase inhibitors N, N′-dicyclohexylcarbodi-imide (DCCD), diethylstilbestrol (DES) and N-ethylmaleimide (NEM) supporting a significant role for a plasma membrane H +-ATPase in the regulation of pH i. pH i was maintained at neutrality over a range of external pH (pH e) from 5–8 in parasites suspended in a buffer containing Na + and K + (standard buffer) but was acidified at low pH e in the absence of these cations (choline buffer). The pH i of trypomastigotes decreased significantly when they transformed into amastigotes. The rate of recovery of pH i by acidified parasites was similar in Na +-free buffer and standard buffer but was slower in the absence of K + (K +-free or choline buffer) and parasites suspended in choline buffer were acidic by 0.25 pH units as compared with controls. Ba 2+ and Cs + decreased the pH i of parasites suspended in standard but not choline buffer suggesting the presence of an inward directed K + channel. The pH i of amastigotes and trypomastigotes suspended in Cl −-free buffer was decreased by 0.13 and 0.2 pH units, respectively, supporting the presence of a chloride conductive channel. No evidence of pH regulation via a Na +/H + or Cl −/HCO 3 − exchanger was found. These results are consistent with the presence of a plasma membrane H +-ATPase that regulates pH i and is supported by K + and Cl − channels.