u-50488 and chelerythrine

To determine the effect of activation of lambda-opioid receptor with U50, 488H, a selective kappa-opioid receptor agonist, on the changes in electrical coupling during prolonged ischemia and to explore the possible mechanism.. The isolated rat heart was perfused in a Langendorff apparatus. The effect of U50, 488H on electrical coupling parameters including onset of uncoupling, plateau time, slope and fold increase in r(t) was observed in isolated perfused rat heart subjected to global no-flow ischemia. The effect of U50, 488H on connexin 43 (Cx43) expression of ventricular muscle during ischemia was determined by immunohistochemistry.. In the prolonged ischemia model, U50, 488H concentration dependently delayed the onset of uncoupling, increased time to plateau, and decreased the maximal rate of uncoupling during ischemia. The effect of U50, 488H on electrical uncoupling parameters during ischemia was abolished by a selective kappa-opioid receptor antagonist nor-BNI or a PKC inhibitor chelerythrine. The amount of Cx43 immunoreactive signal in ventricular muscle was greatly reduced after ischemia. U50, 488H markedly increased Cx43 expression during ischemia and its effect was also attenuated by nor-BNI or chelerythrine.. These results demonstrated that U50, 488H delayed the onset of uncoupling and plateau time, decreased the maximal rate of uncoupling and increased Cx43 expression of ventricular muscle during ischemia, and these effects of U50, 488H were mediated by kappa-opioid receptor, in which activation of PKC was involved. The effect of U50, 488H on electrical coupling during ischemia was probably correlated with preservation of Cx43 in cardiac muscle.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzophenanthridines; Connexin 43; Female; Heart; In Vitro Techniques; Myocardial Ischemia; Myocardium; Naltrexone; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Signal Transduction

Intravenous pretreatment with kappa-opioid receptor antagonist (-)-U-50,488 (1 mg/kg) improved heart resistance to the arrhythmogenic effect of coronary occlusion and reperfusion. Selective kappa1-opioid receptor antagonist norbinaltorphimine and nonselective blocker of peripheral opioid receptors methylnaloxone abolished this antiarrhythmic effect. Preliminary blockade of protein kinase C with chelerythrine or inhibition of ATP-dependent K+ channels (K(ATP) channels) with glybenclamide abolished the antiarrhythmic effect of kappa-opioid receptor activation. Selective inhibitor of sarcolemmal K(ATP) channels did not modulate the kappa-opioid receptor-mediated increase in cardiac electrical stability. Our results suggest that protein kinase C and mitochondrial K(ATP) channels play an important role in the antiarrhythmic effect associated with activation of peripheral kappa-opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Alkaloids; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Benzophenanthridines; Glyburide; Male; Myocardial Reperfusion Injury; Naltrexone; Oxymorphone; Potassium Channels; Protein Kinase C; Rats; Rats, Wistar; Receptors, Opioid, kappa

Previous work has shown that enkephalins target N-type calcium (Ca2+) channels in striatal and globus pallidus (GP) neurons, principally through activation of mu-like receptors. Here, we examined the effects of selective mu, delta, and kappa agonists on Ca2+ currents in striatal and GP neurons isolated from either control or reserpine-treated rats. In cells from control rats DAMGO and dynorphin (DYN) inhibited high-voltage-activated (HVA) Ca2+ currents preferentially in "medium-to-small" GP cells (likely to correspond to parvalbumin-negative cells). The kappa response was elicited by several agonists (DYN 17, DYN 13, BRL, U50-488-H), U50-488-H being the most effective (>30% maximal inhibition). U50-488-H affected both omega-CgTxGVIA-sensitive and nimodipine-sensitive Ca2+ conductances. The kappa-mediated effect (but not the mu response) was slow and blocked by chelerythrine, supporting the involvement of protein kinase C. In neurons from reserpinized rats we observed modest changes in the mu-inhibited fraction in small GP cells and a dramatic reduction of the kappa-sensitive fraction in principal striatal cells. These data imply that aminergic depletion alters opiate transmission differentially in the indirect and direct pathways. The suppression of the kappa response only in striatum reinforces the notion of an imbalance of endogenous opiates as relevant in extrapyramidal motor dysfunctions.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adrenergic Uptake Inhibitors; Alkaloids; Analgesics, Non-Narcotic; Analgesics, Opioid; Analysis of Variance; Animals; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels; Cell Size; Cells, Cultured; Corpus Striatum; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enzyme Inhibitors; Male; Membrane Potentials; Naltrexone; Narcotic Antagonists; Neural Inhibition; Neurons; omega-Conotoxin GVIA; Patch-Clamp Techniques; Phenanthridines; Rats; Rats, Wistar; Receptors, Opioid, kappa; Reserpine

The role of protein kinase C-epsilon (PKC-epsilon) in the development of kappa-opioid receptor (kappa-OR) tolerance to the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) (U50,488H), the selective agonist of kappa-OR, was determined in rat ventricular myocytes. Incubation of ventricular myocytes with 1 microM U50,488H for 24 h significantly attenuated the inhibitory effects of 30 microM U50,488H on the electrically-induced [Ca(2+)](i) transient and forskolin-stimulated cyclic AMP accumulation, indicating the development of tolerance to the kappa-OR agonist. Chronic treatment of ventricular myocytes with U50,488H also induced translocation of PKC-epsilon to the particulate fraction. On the other hand, administration of 30 microM U50,488H for 10 min induced translocation of PKC-alpha to the particulate fraction in naïve ventricular myocytes, but not in cells pretreated with 1 microM U50,488H for 24 h. In ventricular myocytes incubated for 24 h with 1 microM U50,488H together with 1 microM chelerythrine or 1 microM GF109203X, PKC inhibitors, or 0.1 microM epsilonV1-2 peptide, a selective inhibitor of PKC-epsilon, 30 microM U50,488H still produced the inhibitory effect on the electrically-induced [Ca(2+)](i) transient as it did in naïve ventricular myocytes. Chronic treatment of ventricular myocytes with U50,488H and chelerythrine also attenuated the development of tolerance to acute U50,488H on cyclic AMP accumulation. Cells exposed to chelerythrine, GF109203X, or epsilonV1-2 peptide alone did not show an altered [Ca(2+)](i) response to U50,488H. These results indicate that activation of PKC-epsilon is a critical step in the development of tolerance in the kappa-OR.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Alkaloids; Analgesics, Non-Narcotic; Animals; Benzophenanthridines; Biological Transport; Calcium; Colforsin; Cyclic AMP; Drug Interactions; Drug Tolerance; Electric Stimulation; Enzyme Inhibitors; Heart Ventricles; Immunoblotting; In Vitro Techniques; Indoles; Isoenzymes; Male; Maleimides; Myocardium; Peptide Fragments; Phenanthridines; Platelet Aggregation Inhibitors; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa

This study determined whether phosphodiesterase (PDE) was activated by protein kinase C (PKC) upon kappa-receptor stimulation, and if so, to identify the isozyme. We first studied the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective kappa-opioid receptor (OR) agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, on cAMP accumulation and PDE activity in rat ventricular myocytes when PKC and PDE were inhibited by respective inhibitors. Like PMA, U50,488H decreased the forskolin-stimulated cAMP accumulation and dose-dependently stimulated the PDE activity, which were antagonized by 10(-6) M chelerythrine and bisindolylmaleimide I, selective PKC antagonists. In addition, 3-isobutyl-1-methylxanthine, a PDE inhibitor, dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and abolished the stimulation on PDE activity by U50,488H and PMA. The observations suggest that PKC may enhance cAMP degradation through activating PDE upon kappa-OR stimulation. To identify the isozyme(s) mediating the effect of PKC upon kappa-OR stimulation, selective inhibitors were used. We found that 10(-5) M Ro-20-1724, a selective cAMP-specific PDE (PDE-IV) inhibitor, abolished the inhibitory effects of U50,488H and PMA, whereas 8-methoxymethyl-3-isobutyl-1-methylxanthine, erythro-9-(2-hydroxy-3-nonyl) adenine, cilostamide, and zaprinast, selective inhibitors of Ca(2+)/calmodulin-dependent PDE (PDE-I), cGMP-stimulated PDE (PDE-II), cGMP-inhibited PDE (PDE-III), and cGMP-specific PDE (PDE-V), respectively, had no effect. Moreover, rolipram, another selective PDE-IV inhibitor, also dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and stimulation on PDE activity by U50,488H and PMA. In conclusion, this study has provided evidence for the first time that PKC and PDE-IV mediate the action of kappa-OR.

Topics: 1-Methyl-3-isobutylxanthine; 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Alkaloids; Animals; Benzophenanthridines; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Male; Myocardium; Phenanthridines; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Tetradecanoylphorbol Acetate