u-50488 and Brain-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

It remains unclear whether the kappa-opioid receptor (kappa-OR) is altered during ischemia and reperfusion. Therefore, the present study was designed to investigate changes in the kappa-OR. Additionally, the anti-arrhythmic effect induced by kappa-OR stimulation was also determined during ischemia and reperfusion (I/R).. Rats were randomly divided into different groups according to two experimental protocols. The anti-arrhythmic effects of U50,488H, a selective kappa-OR agonist, in an I/R model of 15-min ischemia were studied followed by 15 min of reperfusion. The content of kappa-OR mRNA and protein were measured by RT-PCR and Western blotting techniques in an I/R model of 30-min ischemia followed by 360 min of reperfusion.. Limited numbers of premature ventricular contractions (PVCs) were revealed in the control group. Administration of U50,488H in the control group had no effect on occurrence of PVCs. Incidence of arrhythmia in the I/R group was significantly increased. Treated with U50,488H in the I/R group, the incidence of arrhythmia was significantly reduced. With prior use of nor-BNI, a selective kappa-OR antagonist, the anti-arrhythmic effect of U50,488H was completely blocked. Compared with the control group, the content of kappa-OR mRNA and the density of kappa-OR protein increased significantly at 0 min, 60 min, and 180 min during reperfusion.. The present study provides evidence for the first time that the expressions of kappa-OR mRNA and protein are upregulated in the heart of I/R rats. This alteration may produce a strengthened anti-arrhythmic effect upon kappa-OR stimulation during I/R.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Anti-Arrhythmia Agents; Brain Ischemia; Gene Expression Regulation; Male; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Reperfusion; RNA, Messenger

Substantiating evidence has raised the possibility that sigma ligands may have therapeutic potential as neuroprotective agents in brain ischemia. It has been suggested that the neuroprotective capacity of sigma ligands is related primarily to their affinity for the NMDA receptor complex and not to any selective action at the sigma binding site. However, sigma specific ligands, devoid of significant affinity for the NMDA receptor, are also neuroprotective via an inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. In the present study, we have investigated the potential neuroprotective effect of a comprehensive series of sigma ligands, with either significant (sigma/PCP) or negligible (sigma) affinity for the PCP site of the NMDA receptor, in order to delineate a selective sigma site-dependent neuroprotective effect. For this aim, we have employed two different neuronal culture toxicity paradigms implicating either postsynaptic-mediated neurotoxicity, (brief exposure of cultures to a low concentration of NMDA or Kainate) or pre- and postsynaptic mechanisms (exposure to hypoxic/hypoglycemic conditions). Only sigma ligands with affinity for the NMDA receptor [(+) and (-) cyclazocine, (+) pentazocine, (+) SKF-10047, ifenprodil and haloperidol] were capable of attenuating NMDA-induced toxicity whereas the sigma [(+)BMY-14802, DTG, JO1784, JO1783, and (+)3-PPP] and kappa-opioid [CI-977, U-50488H] ligands, with very low affinity for the NMDA receptor, were inactive. The rank order of potency, based on the 50% protective concentration (PC50) value, of sigma/PCP ligands against NMDA-mediated neurotoxicity correlates with their affinity for the PCP site of the NMDA receptor, and not with their affinity for the sigma site. In addition sigma/PCP, sigma or kappa-opioid ligands failed to attenuate kainate-mediated neurotoxicity. On the other hand, sigma/PCP, sigma and kappa-opioid ligands were potent inhibitors of hypoxia/hypoglycemia-induced neurotoxicity, although their neuroprotective potency did not correlate with their affinity for either the sigma or PCP binding sites. In conclusion, the ability of sigma and kappa-opioid ligands to attenuate hypoxia/hypoglycemia, but not NMDA or kainate-induced toxicity, suggests that these drugs exert their neuroprotective role by a predominantly presynaptic mechanism possibly by inhibiting ischemic-mediated glutamate release.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Anti-Arrhythmia Agents; Antihypertensive Agents; Benzofurans; Brain Ischemia; Cell Death; Cells, Cultured; Dizocilpine Maleate; Hypoxia; Kainic Acid; Ligands; N-Methylaspartate; Neurons; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Phencyclidine; Receptors, sigma

The effects of a novel opioid kappa-receptor agonist U-50488H on Na(+)-K(+)-adenosine triphosphatase (ATPase) activity and regional cerebral blood flow (rCBF) were studied in the acute ischemic brain of rats after middle cerebral artery (MCA) occlusion. Administration of U-50488H 15 minutes prior to MCA occlusion attenuated ischemic reduction in Na(+)-K(+)-ATPase activity 15 minutes after MCA occlusion. The effect was statistically significant at a dosage of 30 mg/kg, but not at lower doses (0.3 and 3 mg/kg). There was no effect on rCBF before MCA occlusion, and the decreased flow after occlusion was enhanced with a significant fall in systemic blood pressure at a dosage of 30 mg/kg. These results indicate that U-50488H has therapeutic potential in cerebral ischemia by mechanisms other than improvement in CBF.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Antihypertensive Agents; Blood Pressure; Brain; Brain Damage, Chronic; Brain Ischemia; Male; Pyrrolidines; Rats; Rats, Wistar; Receptors, Opioid, kappa; Regional Blood Flow; Sodium-Potassium-Exchanging ATPase; Synaptic Membranes

The effect of opioids on delayed neuronal death was evaluated in the gerbil hippocampus. Male Mongolian gerbils were subjected to transient forebrain ischemia and neuronal density was evaluated in the hippocampus 7 days following ischemia. When hypothermia during and after ischemia was prevented, treatment with morphine, U-50488H, or naloxone provided no significant protection. In contrast, a spontaneous drop in rectal temperature to 32 degrees C at the end of ischemia produced near-complete protection of CA1 pyramidal neurons. No opioids modulate the protective effect of hypothermia.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Body Temperature; Brain Ischemia; Cell Count; Cell Death; Gerbillinae; Hippocampus; Male; Morphine; Naloxone; Neurons; Pyrrolidines; Receptors, Opioid

The effect of U-50,488H, a selective kappa-receptor agonist, on memory functions in an animal model of cerebral ischemia was investigated by use of a three-panel runway task. A 5-min period of ischemia caused a significant increase in the number of errors (pushes made on the two incorrect panels of the three panel-gates at four choice points) in a working memory task but it did not impair a reference memory task. U-50,488H at 10 and 32 mg/kg, administered i.p. immediately after blood flow restoration significantly reduced the increase in errors expected to occur in a working memory task assessed 24 h after 5 min of ischemia. This protective effect of U-50,488H on amnesia in the ischemic rat was antagonized by the kappa-receptor antagonist, MR-2266. We conclude that U-50,488H prevents the impairment of working memory following transient forebrain ischemia, an event mediated by the activation of the kappa-opioid receptor.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Behavior, Animal; Brain; Brain Ischemia; Male; Memory Disorders; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa

U-50,488H, a kappa (kappa) opioid ligand with moderate potency at sigma (sigma) receptors, protects against mechanical and ischemia-induced injury. The purpose of this study was to evaluate the possibility that sigma-receptors may be involved in mediating the neuroprotective actions of U-50,488H. This possibility was examined by testing the potential of a series of U-50,488H analogs, which are potent sigma-ligands with minimal activity at kappa-opioid receptors, to protect against ischemia-induced neuronal damage in the gerbil. Like U-50,488H, BD-449 (20 mg/kg), the cis-diastereomer of U-50,4888H, protected against ischemia-induced neuronal damage as did BD-737 (50 and 30 mg/kg) and BD-738 (50 mg/kg). All 3 compounds interacted selectively with sigma-receptors. In contrast, BD-601 (50 mg/kg), did not protect against ischemia-induced neuronal damage, although it also interacted potently with sigma-receptors. One difference between the compounds that were neuroprotective and BD-601 is that only BD-601 produced sigma-like behavioral effects in the rat. Thus, it is possible that BD-601 may interact differently or at a different sigma-subtype than BD-449, BD-737 and BD-738 with sigma-receptors. However, these results clearly indicate that an interaction with kappa-opioid receptors is not required for anti-ischemic activity, and that sigma-receptors may play a role in neuroprotection.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Behavior, Animal; Brain Ischemia; Gerbillinae; Male; Nervous System Diseases; Neurons; Pyrrolidines; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, sigma

The ability of the kappa-opioid receptor agonists U50488H and U62066E (spiradoline mesylate) compared with the non-kappa close structural analogue U54494A to affect postischemic necrosis of the selectively vulnerable hippocampal CA1 neurons was examined in male Mongolian gerbils. The gerbils were treated with either saline vehicle or 10 mg/kg i.p. of one of the test drugs 30 minutes before and again 2 hours after a 10-minute period of bilateral carotid artery occlusion or sham occlusion under light methoxyflurane anesthesia. Seven days after ischemia and reperfusion the brains were perfusion-fixed, and hippocampal CA1 cells were counted in a blind fashion. In ischemic gerbils that received only vehicle, there was a 78.9% loss of CA1 neurons compared with sham-occluded gerbils. In contrast, in U50488H-treated gerbils, mean cell loss was reduced to 33.9% (p less than 0.01 vs. vehicle-treated group). U62066E was even more effective in reducing postischemic CA1 degeneration to only 20.7% (p less than 0.0001 vs. vehicle-treated group). However, treatment with the non-kappa analogue U54494A did not cause any apparent protection; the gerbils in this group showed an 80.7% loss of CA1 neurons. Our results are consistent with the hypothesis that kappa-receptor stimulation is associated with improved postischemic neuronal preservation.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain Ischemia; Gerbillinae; Hippocampus; Male; Necrosis; Neurons; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa

U-50488 is a specific kappa opioid agonist which produces in rats water diuresis resulting in an elevation of plasma osmolarity. Pretreatment with U-50488H (the methanesulfonate salt) in Fisher rats prior to 4 h of bilateral carotid occlusion prevented the development of edema in the forebrain, and the effect was greater than that from pentobarbital anesthesia. An additional injection of an antidiuretic hormone which prevented the plasma hyperosmolarity also significantly reduced the anticerebral edemic effects of U-50488H. The plasma osmotic effect, however, may not completely account for the ischemic protection produced by U-50488H.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Body Water; Brain Chemistry; Brain Edema; Brain Ischemia; Diuresis; Male; Osmolar Concentration; Potassium; Pyrrolidines; Rats; Rats, Inbred F344; Receptors, Opioid; Sodium; Vasopressins