diamide and 2-cyclohexen-1-one

Topics: Anaerobiosis; Antioxidants; Ascorbic Acid; Carboxypeptidases; Chediak-Higashi Syndrome; Cyclohexanones; Diamide; Granulomatous Disease, Chronic; Humans; Methylene Blue; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxidation-Reduction; Peptide Synthases; Protein Processing, Post-Translational; Tubulin; Tyrosine

Freshly isolated adult rat heart myocytes contain total glutathione and reduced glutathione (GSH) at levels quite comparable to those in intact rat heart. Total glutathione can be depleted from 11 to 1 nmol/mg protein or less by treatment with cyclohex-2-ene-1-one without effect on either cellular ATP, rod-cell morphology or the integrity of the sarcolemma. Glutathione levels and redox state are not altered significantly when the Ca-tolerant, quiescent cells are subjected to a period of anoxia followed by reoxygenation. This oxygen paradox protocol results in irreversible hypercontracture of the contractile elements into an amorphous mass in the bulk of the cells, but little loss of sarcolemmal integrity. When the myocytes are subjected to an externally applied oxidant stress by the addition of either diamide or t-butylhydroperoxide, GSH is rapidly depleted with accumulation of oxidized glutathione (GSSG. On continued aerobic incubation both of these reagents promote a slower depletion of cellular ATP and a parallel hypercontracture. Cells treated with t-butylhydroperoxide, but not those with diamide, also generate increasing amounts of thiobarbituric acid reactive species as an indication of lipid peroxidation and show a parallel loss of sarcolemmal integrity. It is concluded that respiring myocytes and those subjected to the oxygen paradox do not produce oxygen radicals in sufficient amounts to displace the GSH/GSSG redox poise and depletion of myocyte glutathione per se is not detrimental to the short term survival of the cells. In addition, aerobic myocytes subjected to external oxidant stress can be damaged irreversibly by two pathways, a hypercontracture that correlates with depletion of ATP and a loss of sarcolemmal integrity that correlates with lipid peroxidation.

Topics: Animals; Coronary Disease; Cyclohexanones; Diamide; Glutathione; Heart; Hypoxia; Myocardium; Oxidation-Reduction; Oxygen; Peroxides; Rats; Reperfusion Injury; tert-Butylhydroperoxide

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.

Topics: Aminopyrine; Animals; Carmustine; Cyclohexanones; Diamide; Dithiothreitol; Dogs; Dose-Response Relationship, Drug; Gastric Acid; Glutathione; Maleates; Parietal Cells, Gastric; Peroxides; Sulfhydryl Reagents; tert-Butylhydroperoxide