u-50488 and Ischemia

To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Brain Edema; Brain Infarction; Brain Ischemia; Cells, Cultured; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glutamic Acid; Ischemia; Motor Activity; Neurons; Neuroprotective Agents; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reperfusion Injury; Severity of Illness Index; Stroke

Previous research has demonstrated that pretreatment with kappa opioid receptor (KOR) agonist protects against ischemia reperfusion (I/R) injury in cardiomyocytes and neuron cells through activation of protein kinase C. The purpose of this study is to investigate the KOR agonist's effect on I/R-injured cremaster muscle and its underlying mechanism.. Male Sprague Dawley rats were randomized into 3 groups (n = 6 each group). Group I was the ischemia reperfusion (I/R) injury group (4 hours of ischemia followed by 90 minutes of reperfusion). Group II was the U-50488H (selective KOR agonist)-pretreated group (KOR agonist + I/R injury). Group III was pretreated with U-50488H + nor-binaltorphimine (NBI, a selective KOR antagonist) (KOR agonist + antagonist + I/R injury). The numbers of leukocyte rolling, adhering, and transmigrating, functional capillary, and swelling index of the vessel wall of the postcapillary venule were observed under intravital videomicroscopy. Biochemically, the lactate dehydrogenase, creatine phosphokinase isoenzyme, expression of E-selectin, and intercellular adhesion molecule-1 (ICAM-1) were analyzed.. The U-50488H-pretreated group had significantly decreased the number of leukocyte sticking (P < 0.001) and transmigrating (P < 0.001) as compared with the I/R-injury group and the U-50488H + NBI-pretreated group. The numbers of functional capillary in the U-50488H-pretreated group were significantly less attenuated compared with the I/R-injury group and U-50488H + NBI-pretreated group (P < 0.001). The expression of the ICAM-1 in the cremaster muscle was evidently reduced in the U-50488H-pretreated group than in the I/R-injury group or the U-50488H + NBI-pretreated group.. Administration of KOR agonist protects the muscle flap microcirculation from I/R injury, which can be abolished by concomitant KOR antagonist administration. The KOR agonist-induced protection from ischemia reperfusion injury may be related to decreased expression of adhesion molecule ICAM-1.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Cell Adhesion; Endothelium, Vascular; Intercellular Adhesion Molecule-1; Ischemia; Leukocytes; Male; Microcirculation; Muscle, Skeletal; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Reperfusion Injury; Surgical Flaps

Studies that have evaluated the beneficial effect of pre-ischemic treatment of kappa-opioid receptor agonists have used short-term reperfusion intervals. We examined the long-term impact of the pre-ischemic peripheral injection of U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide), a selective kappa-opioid receptor agonist, on neuronal damage and behavioral deficits following global ischemia in rats. Four groups of ischemic rats were pretreated with various doses of U50,488H (i.p. 0, 5, 15, 30 mg/kg) 15 min prior to vessel occlusion. Two groups of sham-operated animals that received either saline or U50,488H (30 mg/kg) acted as controls. The injection of 30 mg/kg U50,488H led to a 65% increase in CA1 neuron survival 35 days post-ischemia. CA1 neuronal protection translated into significant improvement of ischemia-induced spatial memory deficits assessed in the 8-arm radial maze. However, there was no difference in activity in the open field. We also found that the pre-ischemic intracerebroventricular injection of 5 mug of the delta1-opioid receptor agonist DPDPE ([d-Pen(2,5)]-enkephalin) produced a 59% increase in CA1 neuron survival 7 days post-ischemia. Similar to U50,488H, DPDPE had no significant impact on locomotor activity. These findings support a role for kappa- and delta-opioid receptors in attenuation of ischemia-induced hippocampal damage and cognitive impairments.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Analysis of Variance; Animals; Behavior, Animal; Cell Death; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Enkephalin, D-Penicillamine (2,5)-; Exploratory Behavior; Hippocampus; Ischemia; Male; Maze Learning; Memory Disorders; Neurons; Rats; Rats, Wistar; Recovery of Function; Time Factors

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca(2+) at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and Na(+)/Ca(2+) exchanger (NCX) handled Ca(2+) in rat ventricular myocytes preconditioned with a kappa-opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca(2+) transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca(2+) homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca(2+) fluxes via NCX by the use of (45)Ca(2+) in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca(2+) from the sarcoplasmic reticulum via RyR and in the removal of Ca(2+) from the cytoplasm by reuptake of Ca(2+) to the SR via SERCA and by extrusion of Ca(2+) out of the cell via NCX. UP also reduced mitochondrial Ca(2+) accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca(2+) homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca(2+) overload, improved recovery in contractile function, and cardioprotection.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Antihypertensive Agents; Caffeine; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Heart Ventricles; Homeostasis; HSP70 Heat-Shock Proteins; Ischemia; Male; Mitochondria; Myocytes, Cardiac; Oligonucleotides, Antisense; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger

In the present study, we found that complement C3a exerted central effects after intracerebroventricular administration in mice. At doses of 3 and 10 pmol/mouse, the peptide showed an antagonistic effect on analgesia induced by morphine and U-50488H, known to be mu- and kappa-opioid receptor agonists, respectively. Moreover, complement C3a improved scopolamine- and ischemia-induced amnesia at a dose of 10 pmol/mouse. Anti-analgesia was not observed by C3a des-Arg at 10 pmol/mouse. The present findings suggest that complement C3a may act as a peptide with anti-opioid activity in the central nervous system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Avoidance Learning; Cerebral Ventricles; Complement C3a; Dose-Response Relationship, Drug; Electroshock; Humans; Injections, Intraventricular; Ischemia; Male; Mice; Mice, Inbred Strains; Morphine; Oligopeptides; Pain; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu