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Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and 3H-deoxyglucose-6-phosphate release from
M S Jiang1, J E Fletcher, L A Smith
1Department of Anesthesiology, Hahnemann University, Philadelphia, PA 19102-1192.
Insights
Naja naja kaouthia cardiotoxin induces cell damage, with calcium enhancing effects at high toxin doses. Manganese ions effectively block cardiotoxin activity, suggesting distinct lysis mechanisms.
Area of Science:
- Toxicology
- Biochemistry
- Cell Biology
Background:
- Cardiotoxins from Naja naja kaouthia venom are known to affect cell membranes.
- The precise mechanisms and ionic dependencies of cardiotoxin-induced cell damage require further elucidation.
Purpose of the Study:
- To investigate the effects of various divalent cations (Ca2+, Sr2+, Ba2+, Mn2+) on cardiotoxin-induced release of 3H-deoxyglucose-6-phosphate (3H-dGlu-6-p) and hemoglobin from red blood cells.
- To explore the influence of cardiotoxin concentration, pH, and cation concentration on these release processes.
Main Methods:
- Red blood cells were treated with Naja naja kaouthia cardiotoxin at different concentrations (3 microM and >=10 microM).
- The effects of divalent cations (Ca2+, Sr2+, Ba2+, Mn2+ at 2 mM and 0.5 mM for Ca2+) on 3H-dGlu-6-p and hemoglobin release were measured.
- Experiments were conducted across a pH range (7.0 to 8.5) to assess pH dependency.
Main Results:
- At high cardiotoxin concentrations (>=10 microM), Ca2+ significantly enhanced 3H-dGlu-6-p and hemoglobin release, while Sr2+ and Ba2+ had no significant effect.
- Mn2+ (2 mM) almost completely inhibited both 3H-dGlu-6-p release and hemolysis at both low and high cardiotoxin concentrations.
- Low concentrations of Ca2+ (0.5 mM) enhanced cardiotoxin-induced release, but higher concentrations caused dose-dependent inhibition.
- pH increase from 7.0 to 8.5 significantly enhanced cardiotoxin-induced 3H-dGlu-6-p release.
- The similar release kinetics of 3H-dGlu-6-p and hemoglobin suggest lysis of a subpopulation of cells rather than pore formation in all cells.
Conclusions:
- Cardiotoxin-induced cell lysis has at least two components, one of which is Ca2+-dependent and more apparent at higher cardiotoxin concentrations.
- The Ca2+-dependent lysis may be attributed to trace phospholipase A2 contamination in the cardiotoxin fraction.
- Mn2+ acts as an effective antagonist against cardiotoxin-induced red blood cell damage.
Abstract:
At a low concentration of Naja naja kaouthia cardiotoxin (3 microM) Ca2+, Sr2+ and Ba2+ (2 mM), had little to no effect on 3H-deoxyglucose-6-phosphate (3H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (greater than or equal to 10 microM), Ca2+ (2 mM), but not Sr2+ or Ba2+, significantly enhanced 3H-dGlu-6-p and hemoglobin release. Mn2+ (2 mM) almost completely inhibited 3H-dGlu-6-p release and hemolysis at both the 3 microM and 10 microM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 microM). Ca2+ at low concentrations (0.5 mM) enhanced 3H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 microM) induced 3H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. 3H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between 3H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca2+-dependent. The Ca2+-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A2 contamination in the toxin fraction. Mn2+ is an effective antagonist of cardiotoxin action.