Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver

The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction.     

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.     

Endothelin, a potent peptide agonist in the liver

Endothelin, a peptide mediator produced by vascular endothelial cells, caused sustained vasoconstriction of the portal vasculature in the perfused rat liver. The vasoactive effect of endothelin was accompanied by increased glycogenolysis and alterations in hepatic oxygen consumption. The endothelin-induced increase in the portal pressure was concentration-dependent with an EC50 of 1 nM. Endothelin-induced hepatic glycogenolysis was dose-dependent but exhibited a different EC50 than for the vasoconstrictive effects of endothelin. Hepatic vasoconstriction and glycogenolysis following endothelin infusion were inhibited when Ca2+ was removed from the perfusion medium. The endothelin-induced responses in the liver were not altered by prior infusion of phenylephrine (alpha-adrenergic agonist), isoproterenol (beta-adrenergic agonist), angiotensin II, glucagon, platelet-activating factor, or the platelet-activating factor antagonist, BN52021. However, repeated infusion of endothelin resulted in desensitization of the glycogenolytic response but was without a significant effect on hepatic vasoconstriction. Endothelin also stimulated metabolism of inositol phospholipids in isolated hepatocytes and Kupffer cells in primary culture. The present experiments demonstrate, for the first time, that endothelin is a very potent agonist in the liver eliciting both a sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output.     

Beta 2-adrenergic agonist regulation of immune aggregate- and platelet-activating factor-stimulated hepatic metabolism

Vasoconstriction and subsequent glycogenolysis stimulated by immune complex infusion into perfused rat livers was inhibited by prior infusion of isoproterenol. Similarly, isoproterenol inhibited the biosynthesis of bioactive lipid autacoids such as platelet-activating factor, prostaglandin E2, and thromboxane B2 which was stimulated by immune aggregates. The adrenergic receptor specificity of these effects was determined through the use of specific adrenergic subtype-specific agonists and antagonists to be mediated by beta 2-adrenergic receptors. Indirect evidence for the differential expression of hepatic sinusoidal and parenchymal beta-adrenergic receptors in the male rat during ontogeny suggested that inhibition of immune aggregate-stimulated autacoid biosynthesis, vasoconstriction, and glycogenolysis by isoproterenol occurs at a sinusoidal locus, most likely Kupffer cells. In contrast with the ability of beta 2-adrenergic agonists to inhibit immune aggregate- and platelet-activating factor-stimulated hepatic metabolism, dibutyryl cyclic AMP did not mimic these sinusoidal beta 2-adrenergic effects, despite stimulating hepatic parenchymal cell glycogenolysis as effectively as isoproterenol. These observations suggest a role for cyclic AMP-independent mechanisms in the regulation of heterologous stimulus-response coupling by hepatic sinusoidal beta 2-adrenergic receptors.     

Effects of thiol agents on the accumulation of cadmium by rat liver parenchymal and Kupffer cells

The rate of Cd accumulation by adult rat liver parenchymal cells in serum free primary culture in the presence of 100 μM CdCl₂ was 10 times greater than that by non-parenchymal Kupffer cells. Addition of the monothiol chelating agents, cysteine and penicillamine, decreased Cd uptake in both cell types, the effect becoming more pronounced as the monothiol concentration was increased from 0.1 to 1.0 mM. These monothiols thus appear to reduce the availability of Cd for transport across the cell membrane. In contrast 1–10 molar excesses of the dithiol agents 2,3-dimercaptopropanol (BAL) or dithiothreitol (DTT) stimulated to variable extents the rate of Cd accumulation 2–10-fold in parenchymal cells and by over 100-fold in Kupffer cells. Supplementation of the media with 3% serum had little effect on the Cd accumulation in the presence of monothiols but substantially depressed Cd uptake in the presence of dithiols. Intravenous injection of Cd (0.05 mg/kg CdCl₂) with up to a 10-fold molar excess of cysteine or penicillamine had little effect on the hepatocellular Cd distribution. However Cd uptake by non-parenchymal cells was increased markedly by the simultaneous administration of BAL or DTT in 2 or 10 molar excess. Evidence is provided that these results may be partially explained by the endocytosis, particularly in Kupffer cells, of colloidal complexes of Cd which are formed with the dithiols but not the monothiols. These observations demonstrate that the physicochemical form of Cd determines its hepatocellular distribution which may be an important factor in the manifestation of Cd toxicity after thiol treatment.     

Evidence for the involvement of vicinal sulfhydryl groups in the insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cells

Phenylarsine oxide (PhAsO), a dithiol reagent that blocks insulin stimulation of glucose transport in 3T3 L1 cells, also altered insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cultured cells. PhAsO (2 M) similarly inhibited the insulin-induced glycogen and lipid syntheses without modifying the basal level of these processes, cell viability or the ATP content. Prior incubation of the cells with PhAsO did not prevent insulin binding to the cells, or activation of the receptor tyrosine kinase, while it minimally (16%) altered receptor internalization. These results indicate that cellular dithiols located at a post-receptor step are involved in the transduction of the insulin signal to intracellular glucose metabolism.     

Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants

The effects of tamoxifen (TAM) were studied on the mitochondrial permeability transition (MPT) induced by the prooxidant tert-butyl hydroperoxide (t-BuOOH) or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence of Ca2+, in order to clarify the mechanisms involved in the MPT inhibition by this drug. The combination of Ca2+ with t-BuOOH or PhAsO induces mitochondrial swelling and depolarization of membrane potential (deltapsi). These events are inhibited by cyclosporine A (CyA), suggesting the inhibition of the MPT. The pre-incubation of mitochondria with TAM also prevents those events and induces a time-dependent reversal of deltapsi depolarization following MPT induction, similarly to CyA. Moreover, TAM inhibits the Ca2+ release and the oxidation of NAD(P)H and protein thiol (-SH) groups promoted by t-BuOOH plus Ca2+. On the other hand, the MPT induced by PhAsO plus Ca2+ does not induce -SH groups oxidation, supporting the notion that MPT induction by this compound is not mediated by the oxidation of specific membrane proteins groups. However, TAM also inhibits the PhAsO induced MPT, suggesting that this drug may inhibit this phenomenon by inhibiting PhAsO binding to -SH vicinal groups, implicated in the MPT induction. These data indicate that the MPT inhibition by TAM may be related to its antioxidant capacity in preventing the oxidation of NAD(P)H and -SH groups or by blocking these groups, since the oxidation of these groups increases the sensitivity of mitochondria to the MPT induction. Additionally, they suggest an MPT-independent pathway for TAM-induced apoptosis and a potential ER-independent mechanism for the effectiveness of this drug in the cancer therapy and prevention.     

Stimulation of glycogenolysis and vasoconstriction in the perfused rat liver by the thromboxane A2 analogue U-46619

Infusion of the thromboxane A2 analogue U-46619 into isolated perfused rat livers resulted in dose-dependent increases in glucose output and portal vein pressure, indicative of constriction of the hepatic vasculature. At low concentrations, e.g. less than or equal to 42 ng/ml, glucose output occurred only during agonist infusion; whereas at concentrations greater than or equal to 63 ng/ml, a peak of glucose output also was observed upon termination of agonist infusion coincident with relief of hepatic vasoconstriction. Effluent perfusate lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios increased significantly in response to U-46619 infusion. Hepatic oxygen consumption increased at low U-46619 concentrations (less than or equal to 20 ng/ml) and became biphasic with a transient spike of increased consumption followed by a prolonged decrease in consumption at higher concentrations. Increased glucose output in response to 42 ng/ml U-46619 was associated with a rapid activation of glycogen phosphorylase, slight increases in tissue ADP levels, and no increase in cAMP. At 1000 ng/ml, U-46619 activation of glycogen phosphorylase was accompanied by significant increases in tissue levels of AMP and ADP, decreases in ATP, and slight increases in cAMP. In isolated hepatocytes, U-46619 did not stimulate glucose output or activate glycogen phosphorylase. Reducing the perfusate calcium concentration from 1.25 to 0.05 mM resulted in a marked reduction of the glycogenolytic response to U-46619 (42 ng/ml) with no efflux of calcium from the liver. U-46619-induced glucose output and vasoconstriction displayed a similar dose dependence upon the perfusate calcium concentration. Thus, U-46619 exerts a potent agonist effect on glycogenolysis and vasoconstriction in the perfused rat liver. The present findings support the concept that U-46619 stimulates hepatic glycogenolysis indirectly via vasoconstriction-induced hypoxia within the liver.     

Platelet-activating factor-stimulated hepatic glycogenolysis is not mediated through cyclooxygenase-derived metabolites of arachidonic acid

Platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)) is a potent lipid mediator which stimulates hepatic glycogenolysis, causes hepatic vasoconstriction, and stimulates the production of cyclooxygenase-derived metabolites of arachidonic acid, primarily prostaglandin (PG) D2 in the perfused liver. Following infusion of platelet-activating factor (1 nM) in the perfused rat liver the production of PGD2, measured in the effluent perfusate, increased 4-fold after only 2 min. Infusion of the cyclooxygenase inhibitor, ibuprofen (50 microM), abolished the stimulated production of PGD2 and thromboxane B2 in response to AGEPC without significantly affecting the hepatic glycogenolytic or vasoconstrictive responses to AGEPC. Contrary to previous reports, these observations do not support the suggestion that cyclooxygenase-derived metabolites mediate directly either the glycogenolytic or the vasoactive effects of AGEPC in the perfused rat liver.     

Stimulation of hepatic glycogenolysis by phorbol 12-myristate 13-acetate.

In isolated perfused rat livers, infusion of phorbol 12-myristate 13-acetate (PMA) (150 nM) resulted in a 3-fold stimulation of the rate of glucose production. This response was maximal at a perfusate PMA concentration of 150 nM, and was significantly diminished at higher concentrations of PMA (e.g. 300 nM). Stimulation of glycogenolysis by PMA was greatly decreased in livers perfused with Ca2+-free medium. PMA infusion into livers perfused in the absence of Ca2+ did not result in Ca2+ efflux from the livers. Additionally, in hepatocytes isolated from livers of fed rats, neither PMA nor 1-oleoyl-2-acetyl-rac-glycerol stimulated the rate of glucose production. Although indomethacin has been demonstrated to block PMA-stimulated hepatic glycogenolysis [Garcia-Sainz & Hernandez-Sotomayor (1985) Biochem. Biophys. Res. Commun. 132, 204-209], infusion of PMA into perfused rat livers did not alter the rates of production of either prostaglandin E2 or 6-oxo-prostaglandin F1 alpha in the livers. These data, along with the observed increases in the perfusion pressure and decrease in O2 consumption in isolated perfused livers suggest that phorbol-ester-stimulated glycogenolysis is not a consequence of a direct effect of phorbol ester on liver parenchymal cells.     

Quantification and identification of mitochondrial proteins containing vicinal dithiols

Vicinal dithiols may play a role in mitochondrial antioxidant defences and in redox signalling. We quantified protein vicinal dithiols within mammalian mitochondria using the vicinal dithiol-specific reagent phenylarsine oxide (PAO). We found 5-15% of thiols exposed on mitochondrial proteins were vicinal dithiols and that these thiols were particularly sensitive to oxidation by hydrogen peroxide. To visualise these proteins we used PAO to block vicinal dithiols, followed by alkylation of other thiols with N-ethylmaleimide (NEM). The PAO was then removed with 2,3-dimercapto-1-propanesulfonic acid (DMPS) and the exposed vicinal dithiols were labelled with iodoacetamide-biotin. To identify these proteins, we developed a selective proteomic methodology, based on Redox difference in gel electrophoresis (Redox-DIGE). Vicinal dithiol proteins were selectively labelled with a red fluorescent thiol-reactive Cy5 maleimide and mixed with Cy3 maleimide labelled protein in which vicinal dithiols remained untagged. Individual proteins were resolved by 2D gel electrophoresis and fluorescent scanning revealed vicinal dithiol proteins by the increase in Cy5 red fluorescence. These proteins were identified by peptide mass fingerprinting and mass spectrometry. These findings are consistent with roles for mitochondrial vicinal dithiol proteins in antioxidant defence and redox signalling and these methodologies will enable these roles to be explored.     

Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.     

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of D-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 microM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 microM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexose transport by PhAsO. The data presented suggest a direct interaction between the D-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.     

Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.     

Melanocortin-independent effects of leptin on hepatic glucose fluxes

Leptin and insulin share some hypothalamic signaling molecules, but their central administration induces different effects on hepatic glucose fluxes. Acute insulin infusion in the third cerebral ventricle inhibits endogenous glucose production (GP), whereas acute leptin infusion stimulates gluconeogenesis but does not alter GP because of a compensatory decrease in glycogenolysis. Because melanocortin agonists also stimulate hepatic gluconeogenesis, here we examined whether central melanocortin blockade modifies the acute effects of leptin on GP, on gluconeogenesis, on glycogenolysis, and/or on the hepatic expression of the gluconeogenic enzymes glucose-6-phosphatase (Glc-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Systemic or central administration of leptin alone did not alter GP, despite increasing both the rate of gluconeogenesis and the expression of Glc-6-Pase and PEPCK. When activation of the central melanocortin pathway was prevented, the effects of leptin on gluconeogenesis, Glc-6-Pase, and PEPCK were abolished, and a marked suppression of glycogenolysis resulted in decreased GP. We conclude that leptin regulates hepatic glucose fluxes through a melanocortin-dependent pathway leading to stimulation of gluconeogenesis and a melanocortin-independent pathway causing inhibition of GP and glycogenolysis.     

Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function

Low concentrations of arsenite, but not arsenate, and Cd2+ blocked steroid binding to the glucocorticoid receptors of HTC cells. Inhibition by arsenite was faster and occurred at lower concentrations than for Cd2+. Half-maximal inhibition of [3H]dexamethasone binding was seen after a 30-min preincubation with approximately 7 microM arsenite. The effect of arsenite and of Cd2+ appears to be mediated by a reaction with vicinal dithiols of the receptor as shown by (a) the reversal of arsenite inhibition by much lower concentrations of dithiothreitol (approximately 0.1 mM) than of beta-mercaptoethanol (approximately 10 mM); (b) the ability of both arsenite and Cd2+ to block [3H]dexamethasone 21-mesylate labeling of receptors but not of other thiol-containing proteins; and (c) the known selectivity of arsenite and of Cd2+ for reactions with vicinal dithiols. Arsenite forms a tight complex with these vicinal dithiols since the removal of loosely associated arsenite by gel exclusion chromatography did not reverse the inhibition of steroid binding. The effect of other ions on steroid binding was also examined. Half-maximal inhibition of binding occurred with approximately 5 microM selenite, whereas up to 300 microM Zn2+ was without effect. Much higher concentrations of arsenite were required for effects on unactivated and activated complexes. Arsenite slowly induced a loss of unactivated complexes but rapidly inhibited a portion of the DNA binding of activated complexes. Any effect on activation occurred at arsenite concentrations equal to or higher than those that inhibited DNA binding. In contrast, Cd2+ concentrations similar to those that block steroid binding caused a biphasic loss of unactivated complexes and a marginal loss of activated complexes. This is the first report of effects of arsenite on glucocorticoid receptors. These results confirm directly our earlier hypothesis that steroid binding to rat glucocorticoid receptors involves a vicinal dithiol (Miller, N. R., and Simons, S. S., Jr. (1988) J. Biol. Chem. 263, 15217-15225) and show that arsenite is a potent new reagent for probing receptor structure and function.     

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

     

Regulation of Cellular Thiols in Human Lymphocytes by α-Lipoic Acid: A Flow Cytometric Analysis

Modulation of cellular thiols is an effective therapeutic strategy, particularly in the treatment of AIDS. Lipoic acid, a metabolic antioxidant, functions as a redox modulator and has proven clinically beneficial effects. It is also used as a dietary supplement. We utilized the specific capabilities of N-ethylmaleimide to block total cellular thiols, phenylarsine oxide to block vicinal dithiols, and buthionine sulfoximine to deplete cellular GSH to flow cytometrically investigate how these thiol pools are influenced by exogenous lipoate treatment. Low concentrations of lipoate and its analogue lipoamide increased Jurkat cell GSH in a dose-dependent manner between 10 (25 μM for lipoamide) to 100 μM. This was also observed in mitogenically stimulated peripheral blood lymphocytes (PBL). Studies with Jurkat cells and its Wurzburg subclone showed that lipoate dependent increase in cellular GSH was similar in CD4+ and − cells. Chronic (16 week) exposure of cells to lipoate resulted in further increase of total cellular thiols, vicinal dithiols, and GSH. High concentration (2 and 5 mM) of lipoate exhibited cell shrinkage, thiol depletion, and DNA fragmentation effects. Based on similar effects of octanoic acid, the cytotoxic effects of lipoate at high concentration could be attributed to its fatty acid structure. In certain diseases such as AIDS and cancer, elevated plasma glutamate lowers cellular GSH by inhibiting cystine uptake. Low concentrations of lipoate and lipoamide were able to bypass the adverse effect of elevated extracellular glutamate. A heterogeneity in the thiol status of PBL was observed. Lipoate, lipoamide, or N-acetylcysteine corrected the deficient thiol status of cell subpopulations. Hence, the favorable effects of low concentrations of lipoate treatment appears clinically relevant. © 1997 Elsevier Science Inc.     

Inhibition of coupling factor B activity by cadmium ion, arsenite-2,3-dimercaptopropanol, and phenylarsine oxide, and preferential reactivation by dithiols

Coupling factor B activity was measured by the stimulation of the ATP-driven NAD+ reduction by succinate or the 32Pi-ATP exchange activity of Factor B-depleted submitochondrial particles. Half-maximal coupling activity was inhibited by 30 microM cadmium, 5 microM phenylarsine oxide, or 0.3 mM arsenite-2,3-dimercaptopropanol. The inhibition was relieved by slight excess of dithiol but not by a 10-fold molar excess of 2-mercaptoethanol. Inhibition of coupling activity by phenylarsine oxide or cadmium was not due to interference in binding of Factor B to depleted particles. Isolated Factor B binds phenylarsine oxide resulting in loss of ability to stimulate depleted submitochondrial particles. The inhibition was largely overcome by dithiol but not by monothiols. The residual coupling activity of depleted submitochondrial particles was highly resistant to cadmium or arsenical. Moreover, binding of arsenical to the depleted particles per se, did not result in inhibition of Factor B-stimulated activity. Furthermore, the addition of phenylarsine oxide to H+-ATPase resulted in loss of Pi-ATP exchange and stimulation of oligomycin-sensitive ATPase activities. Both effects were further potentiated by 2-mercaptoethanol and reversed by dithiols. These effects parallel uncoupling of oxidative phosphorylation in mitochondria by these inhibitors and point to Factor B as the probable component sensitive to these inhibitors.     

Methylisobutylxanthine blocks insulin antagonism of cAMP-stimulated glycogenolysis at a site distinct from phosphodiesterase. Evidence favoring an insulin-insensitive calcium release mechanism

The widely used phosphodiesterase inhibitor MIX (1-methyl 3-isobutyl xanthine) blocked insulin antagonism of cAMP-stimulated glycogenolysis in rat hepatocytes but other phosphodiesterase inhibitors including Ro 20-1724 had no effect. Dose-response curves for MIX potentiation of cAMP-stimulated glycogenolysis and for MIX inhibition of the effects of insulin on cAMP-stimulated glycogenolysis suggested that at higher concentrations (250 microM) MIX may act at a site other than phosphodiesterase inhibition. MIX, at 250 microM, attenuated the insulin antagonism of glucose release stimulated by 8-bromo-cAMP, an extremely poor substrate for phosphodiesterase; other phosphodiesterase inhibitors did not. The possibility that MIX acts as an adenosine antagonist interfering with a postulated role for adenosine in insulin action was examined using N6-phenylisopropyladenosine (PIA), an Ra adenosine receptor agonist which increases hepatic cAMP levels. MIX inhibited insulin antagonism of PIA-stimulated glycogenolysis under conditions where it did not act as an adenosine antagonist (MIX and Ro 20-1724 both increased the response to PIA equally). The effect of concanavalin A on cAMP-stimulated glycogenolysis was antagonized by MIX, suggesting a post-receptor site of action for MIX. MIX paradoxically increased lactate production in the presence of 8-bromo-cAMP, reminiscent of the reported actions of calcium mobilizing hormones on lactate formation in fed hepatocytes. Cytosolic free Ca2+, as measured in Quin 2-loaded cells, was increased by MIX. In cells depleted of calcium, MIX no longer blocked insulin antagonism of 8-bromo-cAMP-stimulated glucose release, suggesting that MIX may function through an insulin-insensitive release of calcium. MIX greatly potentiated the stimulation of glycogenolysis by phenylephrine but did not alter the response to vasopressin. The relationship of this effect of MIX to the mechanism of insulin action and the ability of insulin to antagonize only alpha-adrenergic responses and not those of vasopressin is discussed.