We investigated the effect of sulfhydryl and
disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca2+ release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global
ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global
ischemia and 120 minutes of retrograde reperfusion.
Necrosis was evaluated on the basis of
lactate dehydrogenase release and
triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca2+ release and [3H]-
ryanodine binding were determined before the sustained
ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca2+ release and reduced [3H]-
ryanodine binding. The
disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion
buffer either before or after the preconditioning procedure. In preconditioned hearts,
dithiothreitol increased sarcoplasmic reticulum Ca2+ release and
ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents
4,4'-dithiodipyridine (25 micromol/L) and
N-ethylmaleimide (16 micromol/L) increased the resistance to
ischemia and reduced sarcoplasmic reticulum Ca2+ release and [3H]-
ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and
disulfide reagents produced similar effects on Ca2+ release and [3H]-
ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca2+ release.