THE ROLE OF ANIONS IN PH REGULATION OF LEISHMANIA-MAJOR PROMASTIGOTES

The pH regulation of Leishmania major promastigotes was studied as a f unction of the ionic composition of the medium and in response to acid and alkali load. Intracellular pH (pH(i)) was monitored by on-line ra tio fluorescence using the fluorescence dependent pH indicator 2',7'-b is-(carboxyethyl)-5,6-carboxyfluorescein (BCECF). In Cl--based medium (pH 7.4, 30 degrees C), the steady state pH(i) was maintained at 6.75 +/- 0.01. Only a minor (less than or equal to 0.07 +/- 0.02 unit) decr ease in steady state pH(i) was observed when parasites were treated wi th H+-ATPase inhibitors such as vanadate, N-ethylmaleimide, or bafilom ycin. After treatment with the impermeantanion transport blocker DIDS, or in the presence of the reduced analog H2DIDS, pH(i) decreased by g reater than or equal to 0.2 unit. In gluconate-based medium, however, pH(i) gradually decreased to 6.53 +/- 0.05 and showed a swift but time dependent recovery (alkalinization) when Cl- or other halides or nitr ate were restored to the medium. That recovery was also inhibited by p retreating cells with DIDS or exposing them to H2DIDS. The findings pr ovide evidence for Cl- transport mechanisms that support a pH, regulat ory process which is operative in acidic-neutral cytoplasmic milieu. U nder alkali load induced by weak base treatment, parasites undergo a r apid alkalinization which was followed first by a fast but limited aci dification and subsequently by a slower but more robust acidification (recovery) to reach a pH(i) of 6.85 +/- 0.05. The recovery of pH(i) wa s markedly reduced in the presence of H2DIDS and/or in the absence of Cl- in the medium. Based on these results and on the fact that the nat ural parasite environment is both alkaline and rich in HCO3-CO32- ions , we propose (Cl-)(o) (HCO3-)(i) or (Cl-)(o)-(OH-)(i) exchange as the major mechanism of regulatory cell acidification which is operative up on cell alkalinization. The possibility that similar pH regulatory mec hanisms are operative in Leishmania promastigotes in both acidic and a lkaline conditions is considered. The putative pH regulatory mechanism s might serve as potential targets for therapeutic intervention.