diacetylmonoxime and chelerythrine

Although protein kinase C (PKC) plays a pivotal role in ischemic preconditioning, it is not clear what the end effector is that protects the myocardium. In isolated, paced (1.25 Hz, 36-37 degrees C) adult rat cardiomyocytes, the effects of PKC preactivation by diacylglycerol on cell motion, intracellular Ca(2+) concentration ([Ca(2+)](i); indo 1), and intracellular pH (pH(i); seminaphthorhodafluor-1) during simulated ischemia-reperfusion (I/R) were investigated. The degree of reperfusion-induced contracture was significantly attenuated in the myocytes pretreated with 10 microM 1, 2-dioctanoyl-sn-glycerol (DOG; n = 19) compared with the untreated myocytes (n = 23, P < 0.02). There were no differences in twitch amplitude, end-diastolic [Ca(2+)](i), or peak-systolic [Ca(2+)](i) during I/R between the DOG-pretreated and untreated myocytes. Although there were no differences in pH(i) during ischemia, the pH(i) overshoot during reperfusion was significantly delayed in the DOG-pretreated myocytes compared with the untreated myocytes (n = 17 for each, P < 0.01). Chelerythrine completely abolished the favorable effects of DOG on the reperfusion-induced contracture and the pH(i) overshoot. These data suggest that diacylglycerol attenuates I/R injury in isolated, paced cardiomyocytes, which may be related to the slower pH(i) overshoot during reperfusion.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcium; Cardiac Pacing, Artificial; Cell Separation; Diacetyl; Diglycerides; Enzyme Inhibitors; Hydrogen; Hydrogen-Ion Concentration; Intracellular Membranes; Male; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Osmolar Concentration; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10-50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphatases; Adenosine Triphosphate; Alkaloids; Animals; Benzophenanthridines; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Adhesion; Cell Membrane Permeability; Cells, Cultured; Diacetyl; Enzyme Inhibitors; Genistein; Heart; Male; Myocardial Contraction; Myocardium; Myofibrils; Phenanthridines; Protein Kinases; Rats; Rats, Wistar; Uncoupling Agents

In this study, we explored the effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on glycine current (IGly) of murine ventromedial hypothalamic neurons. Co-application of 0.01 to 67 mM BDM increased IGly decay rate with little change of the peak amplitude. This effect was both rapid in onset and offset and required the presence of the agonist. Pretreatment with BDM alone did not alter-IGly decay. In addition, dialysis of neurons with 500 microM ATP-gamma-S did not alter the acute effect of BDM. Thus, this effect may result from open channel block rather than BDM-induced dephosphorylation of the receptor/channel protein. In contrast to the acute effect described above, relatively prolonged (i.e., greater than 80 s) pretreatment with BDM reduced peak IGly. The phorbol ester (PDBu), a protein kinase C (PKC) activator, mimicked this effect of BDM. Furthermore, chelerythrine, a specific PKC inhibitor, prevented this effect of BDM on peak IGly. Thus, activation of PKC may mediate this attenuating effect of BDM on IGly. For a sub-population of these pretreated neurons, there was a subsequent potentiation of IGly which followed the initial suppressant effect. This potentiation may be due to a phosphatase effect of BDM, since it was observed more frequently when neurons were also pretreated with the protein kinase inhibitors H7 or chelerythrine. These findings suggest that BDM alters protein kinase activity and acts as a phosphatase to regulate the activity of the glycine receptor/channel complex.

Topics: Alkaloids; Animals; Benzophenanthridines; Carcinogens; Cells, Cultured; Chloride Channels; Cholinesterase Reactivators; Diacetyl; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Glycine; Ion Channel Gating; Mice; Neurons; Phenanthridines; Phorbol 12,13-Dibutyrate; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinase C; Ventromedial Hypothalamic Nucleus