Contracture induction by snake venom cardiotoxin in skeletal muscle from humans and rats

Mode of Action of Membrane Perturbing Agents: Snake Venom Cardiotoxins and Phospholipases A

“…Contractures of skeletal muscle are believed to reflect the "true" cardiotoxic mechanism of CTXs. A special advantage of this preparation is that we have access to human skeletal muscle, which differs from the commonly used rat diaphragm preparation in Ca 2 + dependence of CTX action (Fletcher and Lizzo, 1987). Disadvantages of skeletal muscle are the great degree of biological variability involved in examining contracture induction, the small number of studies that can be run in a single day and the inability to conveniently use fluorescence probes to directly monitor myoplasmic Ca 2 ÷ concentrations or patch-clamp electrophysiology to directly examine ion currents.…”

“…PLA2 is a Ca 2 *-dependent enzyme (Shipolýni et al, 1971;Tsai et al, 1985) that catalyzes the hydrolysis of phospholipids at the #2 position releasing lysophospholipids and, primarily, unsaturated free fatty acids (Hanahan et al, 1960). The bee and cobra (Naja naJa) venom PLA2 enzymes readily hydrolyze biological phospholipid substrates, but are unable to penetrate membrane bilayers (Zwaal et al, 1975;Sundler et al, 1978;Fletcher et al, 1987). The inability to hydrolyze the inner phospholipids of the bilayer does not relate to interactions of these primarily negativelycharged phospholipids with spectrin (Raval and Allan, 1984), and can even be observed in pure phospholipid vesicles (Sundler et al, 1978;Wilschut et al, 1979).…”

“…Therefore, PLA2 activity has been considered to be essential for the hemolytic action of CTXs on erythrocytes, suggesting the nmechanism of action of CTX on the red blood cell does not reflect the same mechanism in skeletal muscle (Harvey, 1985). However, more recent studies have demonstrated that higher concentrations of PLA2 do act in synergy with CTX in skeletal muscle and that the apparent difference in action of the CTXs may ba related to differences in the concentration and type of free fatty acids in the two tissues in the absence of CTX (Fletcher and Lizzo, 1987), not differences in mode of CTX action.…”

“…The inability to hydrolyze the inner phospholipids of the bilayer does not relate to interactions of these primarily negativelycharged phospholipids with spectrin (Raval and Allan, 1984), and can even be observed in pure phospholipid vesicles (Sundler et al, 1978;Wilschut et al, 1979). Hemolysis of fresh human red blood cells is not induced to any significant extent by bee venom PLA2, even though almost all the phospholipids in the outer leaflet of the membrane bilayer have been hydrolyzed (Zwaal et al, 1975;Fletcher et al, 1987). Louw and Visser (1978) reported that some CTX fractions were contaminated with trace amounts of venom PLAz, which greatly potentiated the hemolytic activity of the CTX protein.…”

“…The rapid time to onset of effect (seconds; Fletcher and Lizzo, 1987) suggests signal transduction is a more likely mechanism for CTX action. Regardless the actual location of the toxin molecule (inside or outside of the cell), it is important in developing prophylactic and therapeutic measures to determine acce3sibility of antibodies to the toxin site for neutralization.…”