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Increased calcium permeability is not responsible for the rapid lethal effects of amphotericin B on Leishmania sp
B E Cohen1, G Benaim, M C Ruiz
1Centro de Biología Celular, Facultad de Ciencias, Universidad Central de Venezuela, Caracas.
Abstract:
The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration [( Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell permeabilization to EB.
Insights
Amphotericin B (AmB) can increase intracellular calcium but does not rely on this for its cell-permeabilizing effects. Its leishmanicidal activity is actually enhanced without external calcium, suggesting a different primary mechanism of action.
Area of Science:
- Microbiology
- Parasitology
- Pharmacology
Background:
- Amphotericin B (AmB) is a crucial antifungal and antileishmanial drug, yet its precise mechanism of action remains elusive.
- A potential lethal mechanism involves increased intracellular calcium concentration ([Ca2+]i), which can be toxic to cells.
- Understanding AmB's action is vital for optimizing treatment of systemic fungal infections and visceral leishmaniasis.
Purpose of the Study:
- To investigate the role of intracellular calcium ([Ca2+]i) in the cell permeabilization and leishmanicidal effects of amphotericin B (AmB).
- To determine if AmB's action is mediated by an increase in intracellular calcium or by direct cell membrane effects.
- To elucidate the primary mechanism of action of AmB in *Leishmania* parasites.
Main Methods:
- Assessing cell viability using ethidium bromide (EB) permeabilization in *Leishmania* promastigotes.
- Quantifying intracellular calcium concentration ([Ca2+]i) using quin-2 fluorescence.
- Evaluating the effect of AmB and the calcium ionophore A23187 on cell permeabilization and viability.
Main Results:
- Amphotericin B (AmB) demonstrated efficacy as a calcium ionophore, increasing intracellular calcium levels.
- However, AmB-induced cell permeabilization to ethidium bromide (EB) was not dependent on elevated intracellular calcium ([Ca2+]i).
- The leishmanicidal effect of AmB was potentiated in the absence of external calcium, and the calcium ionophore A23187 did not cause EB permeabilization.
Conclusions:
- Amphotericin B's (AmB) rapid cell permeabilization mechanism is independent of intracellular calcium ([Ca2+]i) increase.
- The drug's leishmanicidal activity is not primarily driven by calcium influx, and may be enhanced by its absence.
- These findings suggest that AmB's primary mode of action involves direct membrane disruption rather than calcium-mediated toxicity.