We investigated the effect of ischemia and reperfusion on the cardiac ryanodine receptor, which corresponds to the sarcoplasmic reticulum Ca2+ channel. Isolated working rat hearts were subjected to 10 to 30 minutes of global ischemia, followed or not by reperfusion. Ischemia produced significant reduction in the density of high-affinity 3H-ryanodine binding sites, determined either in whole-heart homogenate (Bmax, 220 +/- 22, 203 +/- 12, and 228 +/- 14 fmol/mg protein after 10, 20, and 30 minutes of ischemia versus 298 +/- 18 fmol/mg protein in the control condition; P < .01) or in a fraction enriched in sarcoplasmic reticulum (Bmax, 1.08 +/- 0.15 pmol/mg protein after 20 minutes of ischemia versus 1.69 +/- 0.08 pmol/mg protein in the control condition; P < .01). The Kd (1.5 +/- 0.1 nmol/L) and the Ca2+ dependence of high-affinity 3H-ryanodine binding were not affected by ischemia. The density of low-affinity 3H-ryanodine binding sites was also reduced after 20 minutes of ischemia (14.0 +/- 2.3 versus 34.0 +/- 8.2 pmol/mg protein in the sarcoplasmic reticulum fraction, P < .05), without significant changes in Kd (4.7 +/- 1.2 versus 2.4 +/- 1.0 mumol/L). All these changes persisted after 20 minutes of reperfusion. Analysis of tissue fractions showed that 55% of the ryanodine binding sites were retained in the pellet of a low-speed centrifugation ("nuclear pellet") and that the effects of ischemia concerned only the receptors released in the supernatant ("postnuclear supernatant"). In parallel experiments, we evaluated the effect of ryanodine on oxalate-supported Ca2+ uptake, which represents sarcoplasmic reticulum Ca2+ uptake. As expected, we found that high concentrations of ryanodine stimulated Ca2+ uptake, owing to channel blockade. The response to 900 mumol/L ryanodine was slightly reduced in crude homogenate and significantly reduced in postnuclear supernatant obtained from ischemic hearts. In conclusion, the number of ryanodine receptors is reduced after ischemia; this effect concerns a subpopulation of the receptors, persists after reperfusion, and might contribute to modify sarcoplasmic reticulum function.
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.
3 Gallopamil decreased the dissociation rate constant of 1 pM [3HJ-ryanodine. While gallopamil alone did not affect the dissociation of 4 nM [3H]-ryanodine, gallopamil and micromolar ryanodine slowed it to a greater extent than micromolar ryanodine alone. 4 Our results are consistent with the hypothesis that the ryanodine receptor is a negatively cooperative oligomer, which undergoes a sequential alteration after ryanodine binding. Gallopamil has complex actions: it inhibits ryanodine binding to its low affinity site(s), and probably modulates the cooperativity of ryanodine binding and/or the transition to a receptor state characterized by slow ryanodine dissociation. These molecular actions could account for the previously reported effect of gallopamil on the sarcoplasmic reticulum calcium release channel.
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