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An amino acid channel activated by hypotonically induced swelling of Leishmania major promastigotes.

ABSTRACT: Leishmania promastigotes accumulate amino acids (AAs) by an uphill transport mechanism that is dependent on membrane potential. The accumulated AAs provide the cell with an osmotic reservoir that can be utilized for osmoregulation. Exposure of Leishmania promastigotes to hypotonic media induced a rapid release of AAs that was proportional to the imposed osmotic gradients and independent of the ionic strength or the presence of Cl-, K+, Na+ or Ca2+ in the medium. The hypotonically activated AA release pathway was of relatively low chemical specificity. The solutes released included most of the zwitterionic and anionic AAs, predominantly alanine, hydroxyproline, glycine and glutamic acid, whereas cationic AAs were virtually excluded. AA release was markedly blocked by classical anion transport inhibitors such as the disulphonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) and its dihydro derivative H2DIDS and others, by restoration of isotonicity or by lowering the temperature (4 degrees C). The temperature profile of AA release showed a low energy of activation (Ea 46 +/- 1.3 (S.E.M.) kJ/mol) in the range 15-30 degrees C and a very high Ea (147 +/- 8 kJ/mol) in the range 4-15 degrees C. Parasites exposed to hypotonic media containing AAs also showed a hypotonically stimulated AA uptake under favourable solute concentration gradients. This uptake was analogous for L- and D-isomers of threonine. After hypotonic exposure, cells underwent a depolarization that was largely prevented by anion transport blockers. On the basis of all these results we propose that after hypotonic stress Leishmania promastigotes restore their internal volume by a regulated release of AAs, which involves activation of channels that allow the passage of both neutral and anionic AAs and possibly other anionic substances.

SUBMITTER: Vieira LL

PROVIDER: S-EPMC1217844 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Project description:All cellular processes, including those involved in normal cell metabolism to those responsible for cell proliferation or death, are finely controlled by cell signaling pathways, whose core proteins constitute the family of phosphatidylinositol 3-kinase-related kinases (PIKKs). Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3 related (ATR) are two important PIKK proteins that act in response to DNA damage, phosphorylating a large number of proteins to exert control over genomic integrity. The genus Leishmania belongs to a group of early divergent eukaryotes in evolution and has a highly plastic genome, probably owing to the existence of signaling pathways designed to maintain genomic integrity. The objective of this study was to evaluate the use of specific human inhibitors of ATR and ATM in Leishmania major. Bioinformatic analyses revealed the existence of the putative PIKK genes ATR and ATM, in addition to mTOR and DNA-PKcs in Leishmania spp. Moreover, it was possible to suggest that the inhibitors VE-821 and KU-55933 have binding affinity for the catalytic sites of putative L. major ATR and ATM, respectively. Promastigotes of L. major exposed to these inhibitors show slight growth impairment and minor changes in cell cycle and morphology. It is noteworthy that treatment of promastigotes with inhibitors VE-821 and KU-55933 enhanced the oxidative damage caused by hydrogen peroxide. These inhibitors could significantly reduce the number of surviving L. major cells following H2O2 exposure whilst also decreasing their evaluated IC50 to H2O2 to less than half of that observed for non-treated cells. These results suggest that the use of specific inhibitors of ATR and ATM in Leishmania interferes in the signaling pathways of this parasite, which can impair its tolerance to DNA damage and affect its genome integrity. ATR and ATM could constitute novel targets for drug development and/or repositioning for treatment of leishmaniases.

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An amino acid channel activated by hypotonically induced swelling of Leishmania major promastigotes.

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