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.
The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 mM-chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant (KD) is 8 nM for [3H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.
Cytosolic Ca2+ overload plays a major role in the development of irreversible injury during myocardial ischemia. Such overload is due at least in part to the release of Ca2+ from the sarcoplasmic reticulum. Therefore, we investigated whether dantrolene, a blocker of the sarcoplasmic reticulum Ca2+ release channel, may protect from ischemic injury. In binding experiments, we determined the effect of dantrolene on [3H]-ryanodine binding in rat cardiac tissue. In perfusion experiments, isolated rat hearts were perfused for 20 min in the working mode, in the presence of 0-45 microM dantrolene. The hearts were then subjected to 30 min of global ischemia and 120 min of retrograde reperfusion. Tissue injury was evaluated on the basis of triphenyltetrazolium chloride (TTC) staining and LDH release. The binding experiments showed that dantrolene displaced 4 nM [3H]-ryanodine with IC50 of 34 microM. In the perfusion experiments, tissue necrosis (i.e., TTC-negative tissue) averaged 28.3 +/- 1.6% of the ventricular mass under control conditions. Dantrolene was protective at micromolar concentrations: tissue necrosis decreased to 21.4 +/- 1.0% and 8.4 +/- 1.4% with 1 microM and 45 microM dantrolene, respectively (P < 0.05 and P < 0.01). Similar results were obtained with regard to LDH release. At low concentrations (up to 4 microM), dantrolene did not produce any significant hemodynamic effect, except for a slight increase in coronary flow, whereas at higher concentration a negative inotropic effect was apparent. In conclusion, dantrolene reduced ischemic injury even at concentrations that did not affect contractile performance. Modulation of sarcoplasmic reticulum Ca2+ release might represent a new cardioprotective strategy.
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