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Mature circulating red blood cells (RBCs) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO production via the RBC-specific nitric oxide synthase isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBCs were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semiquantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its serine

Chronic hyperglycemia in type-1 diabetes mellitus is associated with oxidative stress (OS) and sudden death. Mechanistic links remain unclear. We investigated changes in electrophysiological (EP) properties in a model of chronic hyperglycemia before and after challenge with OS by GSH oxidation and tested reversibility of EP remodeling by insulin. Guinea pigs survived for 1 mo following streptozotocin (STZ) or saline (sham) injection. A treatment group received daily insulin for 2 wk to reverse STZ-induced hyperglycemia (STZ + Ins). EP properties were measured using high-resolution optical action potential mapping before and after challenge of hearts with diamide. Despite elevation of glucose levels in STZ compared with sham-operated ( P = 0.004) and STZ + Ins ( P = 0.002) animals, average action potential duration (APD) and arrhythmia propensity were not altered at baseline. Diamide promoted early (

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca2+oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca2+oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]idynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N, N′-bis(2-chloroethyl)- N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+release from IP3-sensitive internal Ca2+stores and elevates basal [Ca2+]iin the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+pump, respectively.

Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO2)-mediated oxidation, diazene dicarboxylic acid bis- N, N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO2 > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO2 and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.

The integration of proteolytic pathways with metabolism was investigated in perfused rat myocardium. After a 10-min incorporation period, the minute-to-minute release of [3H]leucine from myocardial proteins was measured in nonrecirculating effluent perfusate. The nontoxic pro-oxidant probe diamide (100 μM) or a supraphysiological concentration of the endogenous oxidative metabolite dehydroascorbic acid (200 μM) reversibly inhibited 75% of myocardial proteolysis consisting of several known subcomponents (redox dependent); however, 25% of proteolysis was diamide insensitive (redox independent). Decrease in extracellular glucose concentration from 10 to 0.1 mM strongly increased the potencies of minimally effective concentrations of diamide (10 μM) or dehydroascorbic acid (15 μM) by ∼10-fold to the respective potencies maximally inhibiting proteolysis. The reversal of diamide action was also strongly dependent on the perfusate glucose concentration observed at 0.1, 0.2, 1.0 and 10 mM glucose. Proteolytic inhibition caused by diamide (100 μM) was not accompanied by change in basal tissue ATP content of 5 μmol/g wet wt. Conversely, nearly lethal 60% ATP depletion caused by sodium azide (0.4 mM) was not accompanied by change in total [3H]leucine release. Results indicate that a large proteolytic subcomponent (75%) is maintained by redox chains fed by glucose; however, there is no apparent linkage of this proteolysis to short-term ATP fluctuations. A distinct major proteolytic subcomponent (25%) does not vary in response to experimental intervention in either ATP content or redox chains.

Oxidative stress induces the formation of protein-mixed disulfides with low-molecular-weight thiols, especially glutathione. We analyzed this process, termed S-thiolation, in cultured gastric mucosal cells from guinea pigs by gel electrophoresis and autoradiography after radiolabeling of the intracellular glutathione pool with 35S. Hydrogen peroxide (H2O2) or diamide initiated rapid and reversible S-thiolation of specific proteins with molecular masses of 42, 30, 29, 28, and 22 kDa. Diamide caused particularly prominent S-thiolation of the 42 kDa protein. This protein was identified as actin by immunoblot analysis and actin-myosin precipitation. Fluorescence microscopy revealed that diamide caused a disappearance of normal stress fibers and a concomitant increase in actin polymerization in association with contraction of the cells. These morphological changes were completely reversible within minutes. With cells depleted of glutathione by incubation with DL-buthionine-[S,R]-sulfoximine, diamide caused severe contraction and rounding, and the cells detached from the culture plates. S-thiolation of actin could help protect gastric mucosal cells against irreversible organization of microfilaments by preserving microfilament dynamics under oxidative stress.

The granulocyte-derived oxidant, monochloramine (NH2Cl), is known to stimulate chloride ion secretion in rat distal colonic mucosa mounted in Ussing chambers, through mechanisms that are sensitive and insensitive to tetrodotoxin (TTX). The possible role of intracellular thiols, in the mechanism of action of NH2Cl as a secretagogue, was evaluated with the thiol-oxidizing agent diamide and by measuring tissue sulfhydryl levels in response to NH2Cl. Serosal exposure to the antioxidant glutathione (0.25 mM), 5 min before NH2Cl (50 microM) addition, decreased the maximal effect of 50 microM NH2Cl on short-circuit current (Isc). The NH2Cl-stimulated increase in Isc was not affected by mucosal amiloride (5 microM). Pretreatment with 0.1 mM diamide shortened the lag period before the increase in Isc in response to NH2Cl, but it did not affect the maximal increase in Isc. Although TTX (0.5 microM) increased the lag time for achievement of the maximal Isc response to NH2Cl, the neurotoxin did not inhibit the effect of diamide, suggesting that diamide acts primarily on the nonneural component of NH2Cl-stimulated secretion. Incubation of colonic mucosa with NH2Cl, with or without diamide, decreased cellular acid-soluble sulfhydryl concentrations. Taken together, the results support a role for epithelial cell thiols in NH2Cl-stimulated electrolyte secretion by the rat colon.

The availability of intracellular reduced thiols, such as L-cysteine or glutathione (GSH), may be critically important for the biosynthesis of endothelium-derived relaxing factor (EDRF). We have, therefore, investigated the effects of various sulfhydryl (SH) reagents, such as 1-chloro-2,4-dinitrobenzene (CDNB), diamide, 2,2'-dithiodipyridine (DTDP), or N-ethyl-maleimide (NEM), on the release of EDRF from cultured endothelial cells. None of the SH reagents tested affected the flow-induced EDRF release, but DTDP and NEM inhibited the release of EDRF stimulated by ADP, ionomycin, or poly-L-lysine. In contrast, NG-nitro-L-arginine methyl ester, an inhibitor of EDRF biosynthesis, blocked both the flow- and agonist-induced release of EDRF. Although NEM substantially potentiated the flow-induced release of prostacyclin (PGI2), probably due to a blockade of the reacylation of arachidonic acid, it inhibited the stimulated release of PGI2, whereas diamide did not significantly affect either release. Like CDNB or diamide, NEM, but not DTDP, caused a significant decrease in endothelial GSH. In contrast, both NEM and DTDP, but not CDNB or diamide, inhibited the ADP-induced mobilization of intracellular calcium, suggesting that they act on specific target proteins involved in endothelial cell calcium homeostasis rather than intracellular free SH groups. Moreover, the selective inhibition by these two SH reagents of the stimulated release of EDRF implies that a fundamental regulatory difference exists between agonist- and flow-induced EDRF biosynthesis.

Hydrogen peroxide (H2O2), arachidonic acid (AA), and U-44069, a thromboxane analogue, all induced vaso- and bronchoconstriction in the isolated perfused rat lung. The role of protein sulfhydryl modifications in these processes was investigated. The thiol oxidizing agent diamide inhibited both vaso- and bronchoconstriction induced by H2O2, AA, or U-44069. Diamide had only a marginal effect on glutathione and protein thiol levels and no effect on lung mechanics. The diamide inhibition was reversible, and H2O2-induced vaso- and bronchoconstriction was almost maximal after 10 min of perfusion with buffer. The recovery was more rapid if dithiothreitol, a thiol reducing agent, was used in the buffer. H2O2- and AA-induced vaso- and bronchoconstriction is caused by thromboxane release. Diamide did not influence H2O2- or AA-dependent thromboxane formation, indicating that neither AA release nor AA metabolism to thromboxane is sensitive to thiol oxidation. Thus our results indicate that the site of diamide-induced thiol oxidation is the thromboxane receptor or its signal transduction.

Brush-border membrane vesicles (BBMV) were prepared from rat and human renal cortical tissue by magnesium aggregation and differential centrifugation, and the uptake of L-cystine, L-cysteine, and L-cysteine-D-penicillamine were assessed by a rapid-filtration technique. L-Cystine uptake was relatively sodium independent and associated with membrane binding. Sodium-stimulated uptake was sensitive to a cation but not anion diffusion potential. Both sodium-independent and sodium-stimulated uptake rates were inhibited by the cationic L-amino acids and by some neutral L-amino acids. The uptake rates of L-cysteine and L-cysteine-D-penicillamine were more sodium dependent, and sodium-stimulated uptake rates were more sensitive to cation and anion diffusion potentials. Neither the sodium-independent nor the sodium-stimulated uptake rates of L-cysteine or L-cysteine-D-penicillamine were inhibited by the cationic L-amino acids. L-Cysteine-D-penicillamine showed relatively little membrane binding. It is concluded that L-cystine is transported into renal cortical BBMV by pathways distinct from those concerned with the transport of L-cysteine and L-cysteine-D-penicillamine, and it is postulated that these differences may account for some of the effects of D-penicillamine in cystinuria.

Calcium influx can be stimulated in dog red blood cells by preexposure to diamide under certain conditions. Diamide-activated calcium influx resembles swelling-induced Ca2+-Na+ exchange in several respects. These include saturation of calcium influx at external calcium levels greater than 0.5 mM, suppression of calcium influx by external sodium, and inhibition by quinidine. The ability of diamide to stimulate this transport pathway depends critically on the ionic composition of the medium in which the cells are bathed at the time of diamide exposure. The effect is greatest if the diamide preincubation is conducted in a hypotonic lithium chloride medium containing at least 1 microM calcium. Stimulation of Ca2+-Na+ exchange is seen at diamide concentrations (0.10-0.33 mM) that are lower than those reported to cause major spectrin cross-linking, glutathione depletion, Ca2+-ATPase inhibition, or ion channel formation. The results suggest that dog red cells have a large latent capacity for Ca2+-Na+ exchange.