CATECHOLAMINE INACTIVATION OF MYOCARDIAL LIPOAMIDE DEHYDROGENASE - PREVENTION BY CAPTOPRIL AND OTHER THIOL COMPOUNDS

Inactivation of lipoamide dehydrogenase (LipDH) by the Cu(II)/H2O2 Fen ton system (SF-Cu(II): (5.0 mu M Cu(II), 3.0 mM H2O2) was enhanced by catecholamines (CAs), namely, epinephrine, levoDOPA (DOPA), DOPAMINE, 6-hydroxyDOPAMINE (OH-DOPAMINE) and related compounds (DOPAC, CATECHOL , etc.). After 5 min incubation with the Cu(II)/H2O2/CA system (0,4 mM CA), the enzyme activity decayed as indicated by the following percen tage values (mean +/- S.D.; in parenthesis, number of determinations): SF-Cu(II) alone, 43 +/- 10 (18); SF-Cu(II) + epinephrine, 80 +/- 9 (5 ); SF-Cu(II) + DOPA, 78 +/- 2 (4); SF + Cu(II) + DOPAMINE, 88 +/- 7 (5 ); SF-Cu(II) + OH-DOPAMINE 87 +/- 6 (7); SF-Cu(II) + DOPAC, 88 +/- 3 ( 6); SF-Cu(II) + catechol, 85 +/- 6 (5). In all cases P < 0,05, with re spect to the SF-Cu(II) control sample. CAs effect was concentration-de pendent and at the 0-100 mu M concentration range, it varied with the CA structure. Above the 100 mu M concentration, CAs were equally effec tive and produced 90-100% enzyme, inactivation (Figure 2). In the abse nce of oxy-radical generation, the enzyme specific activity (mean +/- S.D.) was 149 +/- 10 (24) mu mol NADH/min/mg protein. Assay of HO . pr oduction by the Cu(II)/H2O2/CA system in the presence of deoxyribose ( TBA assay) yielded values much greater than those obtained omitting CA . Hydroxyl radical production depended on the presence of Cu(II) and H 2O2 and significant HO . values were obtained with OH-DOPAMINE, DOPAC, epinephrine, DOPAMINE, DOPA and catecol supplemented systems (Table 2 ). LipDH (1.0 mu M) inhibited 50-80% deoxyribose oxidation, the inhibi tion depending on the CA structure (Table 2), Native catalase (20 mu g /ml) and bovine serum albumin (40 ugiml) effectively prevented LipDH i nactivation by the Cu(II)/H2O2/CA system; denaturated catalase, SOD, 0 ,3 M mannitol, 6,0 mM ethanol and 0,2 M benzoate were less effective o r did not protect LipDH (Table 3). Incubation of CAs with the Cu(II)/H 2O2 system produced a time and Cu(II)-dependent destruction of CAs, th e corresponding oquinone, production as illustrated with epinephrine ( figures 6 and 7), as illustrated with epinephrine and DOPAMINE (Table 4). These results support LipDH inactivation by (a) reduction of Cu(II ) to Cu(I) by CAs followed by Cu-catalyzed production of HO . from H2O 2; (b) CA oxidation followed by the corresponding o-guinone interactio n with LipDH. CAPTOPRIL, N-acetylcysteine, mercaptopropionylglycine an d penicillamine prevented to various degree LipDH inactivation by the Cu(II)/H2O2/CA systems (Table 1). The former was the most effective an d 0,4 mM CAPTOPRIL prevented about 95-100% the effect of Cu(II)/H2O2/C A systems supplemented with epinephrine, DOPAMINE and OH-DOPAMINE (Fig ures 3 and Table 1). LipDH increased and CAPTOPRIL inhibited epinephri ne oxidation by Cu(II)/H2O2 (Figures 4 and 5). Since un-physiological concentrations of CAs and Cu(II) may be released in the myocardium aft er ischemia-reperfusion, the summarized observations may contribute to explain myocardial damage in that condition.