Roles of KATP channels in delayed cardioprotection and intracellular Ca(2+) in the rat heart as revealed by kappa-opioid receptor stimulation with U50488H.

The effect of preconditioning with U50488 H (UP), a selective kappa-opioid receptor (kappa-OR) agonist, on infarct size and intracellular Ca2+ ([Ca2+]i) in the heart subjected to ischaemic insults were studied and evaluated. U50488 H administered intravenously reduced the infarct size 18-48 h after administration in isolated hearts subjected to regional ischaemia/reperfusion (I/R). The effect was dose dependent. A peak effect was reached at 10 mg x kg-1 U50488 H and at 24 h after administration. The effect of 10 mg x kg-1 U50488 H at 24 h after administration was abolished by nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, indicating the effect was kappa-OR mediated. The infarct reducing effect of U50488 H was attenuated when a selective blocker of mitochondrial (5-hydroxydecanoic acid, 5-HD) or sarcolemmal (HRM-1098) ATP-sensitive potassium channel (KATP) was coadministered with U50488 H 24 h before ischaemia or when 5-HD was administered just before ischaemia. U50488 H also attenuated the elevation in [Ca2+]i and reduction in electrically induced [Ca2+]i transient in cardiomyocytes subjected to ischaemic insults. The effects were reversed by blockade of KATP channel, which abolished the protective effect of preconditioning with U50488 H. The results indicated that mitochondrial KATP channel serves as both a trigger and a mediator, while sarcolemmal KATP channel as a trigger only, of delayed cardioprotection of kappa-OR stimulation. The effects of these channels may result from prevention/attenuation of [Ca2+]i overload induced by ischaemic insults.

Effects of heat shock protein 70 activation by metabolic inhibition preconditioning or kappa-opioid receptor stimulation on Ca2+ homeostasis in rat ventricular myocytes subjected to ischemic insults.

Heat shock protein 70 (HSP70) mediates delayed cardioprotection of preconditioning. Cytosolic calcium ([Ca(2+)])(i) overload precipitates injury, whereas attenuation of [Ca(2+)](i) overload is believed to be responsible for cardioprotection. There is evidence suggesting a link between HSP70 and [Ca(2+)](i) homeostasis. We hypothesize that activation of HSP70 by preconditioning may restore [Ca(2+)](i) homeostasis altered by ischemic insults. To test the hypothesis, we determined the effects of preconditioning with metabolic inhibition or pretreating with U50,488H [trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (a kappa-opioid receptor agonist)] on viability and injury, HSP70 expression, and [Ca(2+)](i) in ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), with blockade of HSP70 synthesis. In myocytes with vehicle pretreatment, the percentage of dead cells determined by trypan blue exclusion, the injury reflected by release of lactate dehydrogenase, and the resting [Ca(2+)](i) measured by spectrofluorometry significantly increased, whereas the amplitude of electrically induced [Ca(2+)](i) transient decreased, after 10 min with 10 mM 2-deoxy-d-glucose and 10 mM sodium dithionite, known to cause MI/A. However, when myocytes were subjected for 30 min to either 20 mM lactate and 10 mM 2-deoxy-d-glucose (MIP) or 30 microM U50,488H (UP) 20 h before MI/A, the changes in viability and injury, and [Ca(2+)](i) responses were significantly attenuated. These were accompanied by a significantly increased HSP70 expression. Furthermore, blockade of HSP70 synthesis with selective antisense oligonucleotides abolished the beneficial effects of MIP or UP. This study provides first evidence that activation of HSP70 induced by preconditioning, which conferred delayed cardioprotection, restored partially the [Ca(2+)](i) homeostasis altered by ischemic insults.