2-2-dimethyl-5-hydroxy-1-pyrrolidinyloxy and Myocardial-Ischemia

The reactive oxygen species (ROS) are known to be generated upon post-ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide-induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto-oxidation-resistant pro-vitamin C, the 2-O-phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen-activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide-treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R-induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2-O-alpha-glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R-operated hearts was moderately inhibited by pro-vitamin C, but restored nearly to the normal non-operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non-phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro-vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS-related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R-operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin-trapping DMPO-based ESR method.

Topics: Aldehyde Dehydrogenase; Animals; Cell Division; Cells, Cultured; Cyclic N-Oxides; Cytoprotection; Electron Spin Resonance Spectroscopy; Heart; Hydrogen Peroxide; Male; Mitochondria; Mitogen-Activated Protein Kinases; Myoblasts; Myocardial Ischemia; Myocardial Reperfusion Injury; Oxidants; Oxidative Stress; Phosphorylation; Rats; Rats, Wistar; Reactive Oxygen Species; Spin Trapping; Subcellular Fractions; Sugar Acids

Hemodynamic and electron spin resonance analyses were used to assess the in vivo and in vitro cardioprotective and antioxidant effects of therapeutically relevant doses of Ginkgo biloba extract (EGb 761) and its terpenoid constituents (ginkgolides A and B, bilobalide) in the rat. Significant anti-ischemic effects, indicating improved myocardial functional recovery, were observed after repeated (15-day) oral treatments with both EGb 761 (60 mg/kg/day) and ginkgolide A (4 mg/kg/day), as compared to placebo. In vitro pre- and post-ischemic perfusion of hearts in the presence of the ginkgolides A and B (both at 0.05 microgram/ml) or bilobalide (0.15 microgram/ml), but not EGb 761 (5 micrograms/ml), significantly improved all hemodynamic parameters. Post-ischemic levels of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/hydroxyl radical spin-adduct (DMPO-OH) in coronary effluents were significantly decreased after in vivo oral treatments or after in vitro perfusion with EGb 761 or the terpenes, the most effective compound being ginkgolide A. As the presence of the terpenes did not influence the formation of the superoxide/DMPO adduct or DMPO-OH in acellular tests with superoxide and hydroxyl radical generators, their cardioprotective effects appear to involve an inhibition of free radical formation rather than direct free radical scavenging.

Topics: Animals; Antioxidants; Cyclic N-Oxides; Cyclopentanes; Diterpenes; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Fibrinolytic Agents; Free Radical Scavengers; Furans; Ginkgo biloba; Ginkgolides; Heart; Hemodynamics; Lactones; Male; Myocardial Ischemia; Perfusion; Plant Extracts; Rats; Rats, Wistar; Terpenes