Effects of 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis.
Cell viability requires the perfect functioning of the processes controlling ATP and Ca(2+) homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with a disruption of ATP and Ca(2+) homeostasis. This study shows that 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits Trypanosoma cruzi epimastigote cell growth. This thiol-reagent thiocyanate derivative was able to inhibit two ecto-enzymes present in this parasite. The ecto-ATPase and ecto-phosphatase activities were inhibited in a dose-dependent manner (K(i)=47.7 and 472.5 microM, respectively), but the 5'nucleotidase and 3'nucleotidase activities were not. DIDS uptake was approached by fluorescence microscopy. Pulse-chase experiments revealed the DIDS accumulation in compartments, presumably endocytic, in the posterior region of epimastigotes. In addition, we show that the T. cruzi mitochondria studied in permeabilized cells are able to accumulate and retain medium Ca(2+) in the absence of DIDS. However, in the presence of increasing concentrations of DIDS (50-200 microM), Ca(2+) transport was inhibited in a dose-dependent manner. DIDS also caused a disruption of the mitochondrial membrane potential, in the same concentration range, thus explaining its effect on Ca(2+) uptake. The presence of EGTA prevented the elimination of the mitochondrial membrane potential (DeltaPsi), supporting previous data suggesting that the binding of Ca(2+) to the mitochondrial membrane exposes buried thiols to react with DIDS. This thiocyanate derivative was also able to inhibit Ca(2+) uptake by the endoplasmic reticulum in a dose-dependent manner. Taken together, the data presented here provide further insights into the mechanisms underlying the antiproliferative actions of DIDS in T. cruzi.
Bernardes
C F
CF
Instituto de Ciências Biológicas e Química, Departamento de Química e Bioquímica, PUC-Campinas, 13020-904, Campinas, Brazil.
Meyer-Fernandes
J R
JR
Saad-Nehme
J
J
Vannier-Santos
M A
MA
Peres-Sampaio
C E
CE
Vercesi
A E
AE
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000493:pharmacokinetics / Q000494:pharmacology*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D007425:Intracellular Membranes / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism
D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; D004072:Digitonin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium