Increased susceptibility of glutathione peroxidase-1 transgenic mice to kainic acid-related seizure activity and hippocampal neuronal cell death.

Glutathione peroxidase (GSHPx) has been demonstrated in several in vivo studies to reduce both the risk and severity of oxidatively-induced tissue damage. The seizure-inducing neurotoxin kainic acid (KA) has been suggested to elicit its toxic effects in part via generation of oxidative stress. In this study, we report that expression of elevated levels of murine GSHPx-1 in transgenic mice surprisingly results in increased rather than decreased KA susceptibility including increased seizure activity and neuronal hippocampal damage. Isolated transgenic primary hippocampal culture neurons also display increased susceptibility to KA treatment compared with those from wildtype animals. This could be due to alterations in the redox state of the glutathione system resulting in elevated glutathione disulfide (GSSG) levels which, in turn, may directly activate NMDA receptors or enhanced response of the NMDA receptor.

Changes in glutathione homeostasis during liver regeneration in the rat.

We have shown previously that plating primary cultures of rat hepatocytes under low density, which stimulates hepatocytes to shift from the G0 to the G1 phase of the cell cycle, resulted in increased levels of glutathione (GSH) and cysteine, and increased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu et al., Am. J. Physiol. 1992;263:C1181-C1189). In the current work we examined changes in GSH homeostasis after two-thirds partial hepatectomy (PH). Male Sprague-Dawley rats underwent two-thirds PH or sham operation. GSH, oxidized glutathione (GSSG), cysteine, GSH efflux, DNA synthesis, changes in GCS subunit messenger RNA (mRNA), and protein levels were measured 12 and 24 hours after PH. Both liver GSH and cysteine levels were doubled at 12 hours and remained elevated at 24 hours after PH. GSSG levels also increased, but the ratio of GSH to GSSG levels remained unchanged. The increase in GSH and cysteine levels preceded the increase in DNA synthesis. Sinusoidal GSH efflux was unchanged after two-thirds PH, but biliary GSH efflux decreased. However, total GSH efflux was minimally altered after two-thirds PH. The increase in GSH can be largely accounted for by the increase in both cysteine availability and the activity of GCS. The steady-state mRNA and protein levels of the GCS heavy subunit were increased at 12 hours after PH. The mRNA level of the GCS light subunit was unchanged. In summary, early in the course of liver regeneration the steady-state hepatic GSH levels double because of an increase in the biosynthesis of GSH.

Progressive defect in biliary GSH secretion in streptozotocin-induced diabetic rats.

This study examined the effect of streptozotocin-induced diabetes on biliary reduced glutathione (GSH) efflux. Biliary GSH efflux was measured before and after acivicin, an irreversible inhibitor of gamma-glutamyl transpeptidase (GGT). One week after streptozotocin treatment, liver GGT activity doubled in diabetic rats but was inhibited by approximately 90% after acivicin to levels comparable to controls. Despite maximal GGT inhibition, biliary GSH efflux in untreated diabetic rats decreased progressively to approximately 10% of control levels by week 4 and was partially restored by insulin. The mechanism for the decrease in biliary GSH efflux was not increased paracellular permeability. GSH transport kinetics, ATP-stimulated taurocholate, and oxidized glutathione (GSSG) transport in canalicular liver plasma membrane prepared from diabetic and control rats were similar. Inhibition of protein kinase C (PKC) with high-dose H-7 increased biliary GSH efflux in diabetic animals to near control basal levels. In conclusion, streptozotocin-induced diabetic rats exhibit a progressive impairment in biliary GSH transport. One of the responsible mechanisms is heightened PKC tone in diabetic animals.

Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats.

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady-state RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCS-HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR.

Evidence that interference with binding to hepatic cytosol binders can inhibit bile acid excretion in rats.

We previously identified that Y' bile acid binders (3alpha-hydroxysteroid dehydrogenases) interact with bile acids in intact rat hepatocytes using [3beta-3H, C24-14C]bile acids and that indomethacin, a competitive inhibitor of 3alpha-hydroxysteroid dehydrogenase, inhibits 3H-loss from the C3-position of bile acids as well as inhibits hepatic bile acid removal and excretion. To study the kinetics of these inhibitory effects, glycocholate transport was studied in the absence and presence of indomethacin in the single-pass perfused rat liver. Indomethacin decreased net hepatic glycocholate uptake in the perfused liver, which was confirmed in isolated hepatocytes and basolateral liver plasma membrane vesicles. However, indomethacin markedly increased the sinusoidal efflux and decreased the biliary excretion of glycocholate in the perfused liver. These observations indicate that the effect of indomethacin to delay biliary glycocholate excretion is related to either intracellular or canalicular glycocholate transport. The latter possibility seemed unlikely because indomethacin did not inhibit electrogenic or adenosine triphosphate (ATP)-dependent glycocholate uptake by canalicular liver plasma membrane vesicles. Thus, the current data support an important role for binding of bile acids to cytosolic proteins in overall hepatic transport and suggest that specific interference with cytosolic binding can interfere with the excretion of bile acids.

Toxicity of azathioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: the role of glutathione and relevance to hepatic venoocclusive disease.

The mechanisms leading to hepatic venoocclusive disease (HVOD) remain largely unknown. Azathioprine and monocrotaline were studied as part of a series of studies looking at a variety of toxins that induce HVOD to find common features that might be of pathogenic significance. In a previous study, dacarbazine showed selective in vitro toxicity to sinusoidal endothelial cells (SEC) compared with hepatocytes and a key role for SEC glutathione (GSH) was demonstrated. Murine SEC and hepatocytes were isolated and studied in culture. Azathioprine and monocrotaline were found to be selectively more toxic to SEC than to hepatocytes. The relative resistance of hepatocytes to azathioprine was due to enhanced GSH defense: hepatocytes exposed to azathioprine maintained intracellular GSH levels better than SEC, particularly when supplemental GSH precursors were added, and hepatocyte resistance was completely overcome by depletion of intracellular GSH. In contrast, monocrotaline toxicity in hepatocytes was largely unaffected by depletion of GSH, which suggests that selectivity of monocrotaline for SEC may be attributable to differences in metabolic activation. Both compounds are detoxified by GSH in SEC, as demonstrated by enhanced toxicity in the presence of buthionine sulfoximine (BSO) and attenuation of toxicity with exogenous GSH. SEC GSH levels were more than 70% to 80% depleted by monocrotaline and azathioprine, respectively, before cell death. Azathioprine and monocrotaline are selectively toxic to SEC; the mechanism of toxicity in the SEC may be caused by profound GSH depletion.

Specificity and directionality of thiol effects on sinusoidal glutathione transport in rat liver.

In rats the sinusoidal glutathione (GSH) carrier transports GSH bidirectionally, and its activity is influenced by the thiol-disulfide status; the Vmax of sinusoidal GSH efflux was increased by dithiothreitol (DTT) and decreased by cystine. In the present work we examined the specificity and directionality of the thiol effect. Using in situ perfused livers, we found that 1 mM DTT and other dithiols, including 1,2-ethanedithiol, 1,3-propanedithiol, and 1,4-butanedithiol, stimulated sinusoidal GSH efflux by 200-500% but dihydrothioctic acid, which is negatively charged, had no effect. Uncharged or positively charged monothiols (2 mM), such as dimercaprol, monothioglycerol, 2-mercaptoethanol, 3-mercapto-2-butanol, 1-mercapto-2-propanol, and cysteamine, also exerted a stimulatory effect on sinusoidal GSH efflux. In contrast, monothiols containing a negatively charged substituent, such as penicillamine, captopril, N-acetylcysteine, mercaptopropionylglycine, mercaptoethanesulfonic acid, mercaptoacetic acid, and mercaptopropionic acid, had no effect. The thiol moiety was essential for activity, inasmuch as ethanol, propanol, propanediol, and glycerol had no effect on sinusoidal GSH efflux. The effect of DTT or cystine pretreatment (2 mM or 0.5 mM, respectively, for 30 min) on GSH uptake was then examined using cultured rat hepatocytes. The linear rate of [35S]GSH uptake and the concentration dependence were measured after cells were pretreated with acivicin (0.5 mM, for 15 min) and buthionine sulfoximine (10 mM, 15 min), to prevent breakdown and resynthesis of GSH from precursors, respectively. Uptake buffer also contained 20 mM alpha-(methylamino)isobutyric acid and 20 mM threonine (inhibitors of amino acid transport systems A and ASC, respectively), to prevent uptake of cysteine. Pretreatment with DTT decreased the Vmax of GSH uptake by approximately 50% (control Vmax value, 24 nmol/10(6) cells/30 min), whereas the Km remained unaffected (approximately 8 mM). Cystine pretreatment had no influence on GSH uptake but inhibited efflux. In conclusion, the presence of at least one thiol group and the absence of negative charge are required to stimulate sinusoidal GSH efflux. The direction of GSH transport is modulated by the thiol-disulfide status, so that thiol reduction changes the GSH transporter from a bidirectional GSH transporter into a preferentially unidirectional (outward) transporter by inhibiting uptake while stimulating efflux and thiol oxidation favors inward transport by inhibiting only efflux.

Identification and purification of a human liver cytosolic tocopherol binding protein.

We recently purified rat tocopherol binding protein (TBP), a 32-kDa cytosol protein which specifically binds alpha-tocopherol, exists as two charge isoforms, and is expressed exclusively in liver and only in hepatocytes. For the present work, we sought to identify the human hepatic tocopherol binding protein from normal human livers harvested from organ donors. Gel filtration of hepatic cytosol identified a peak of [alpha-3H]-tocopherol binding in the 30- to 40-kDa fractions displaceable by excess unlabeled alpha-tocopherol. The fractions exhibiting this binding were pooled and run on Affi-Gel Blue affinity chromatography eluted with a salt gradient. A single major peak of tocopherol binding activity eluted at 22 mS/cm. This peak was further purified by FPLC chromatofocusing. A single protein peak of specific alpha-tocopherol binding eluted at pH 5.9. Finally, the peak from chromatofocusing was purified to apparent homogeneity by reversed-phase microbore HPLC chromatography. Two closely eluting protein peaks were separated and each was homogeneous, had identical migration on a SDS-PAGE (36 kDa), and had the same amino acid composition. The purified human TBP exhibited displaceable, specific alpha-tocopherol binding in the gel filtration assay of tocopherol binding. Laser desorption time-of-flight mass spectroscopy revealed a molecular weight of 36.6 kDa. Both forms of human TBP reacted in Western blot with polyclonal rabbit anti-rat TBP. Identification of the human tocopherol binding protein will allow future studies on its physiological function in human alpha-tocopherol metabolism.

Thiol-disulfide effects on hepatic glutathione transport. Studies in cultured rat hepatocytes and perfused livers.

In cultured rat hepatocytes, cystine led to an inhibition of GSH efflux by lowering the Vmax by approximately 35% without affecting the Km. The cystine-mediated inhibition of GSH efflux was rapid in onset (< 1 h), with near maximum effect at 0.1 mM. Inhibition was still observed when cystine uptake was prevented. Cystine and sulfobromophthalein-GSH, a selective inhibitor of sinusoidal transport of GSH, did not exhibit additive inhibitory effects on GSH efflux. Depletion of ATP or membrane depolarization after cystine treatment were excluded as potential mechanisms. DTT not only reversed the cystine-mediated inhibition of GSH efflux, it stimulated GSH efflux up to 400-500%. The DTT effect was immediate in onset, reaching maximum after 30 min, and was partially reversed by cystine, suggesting that the two share a common site(s) of action. DTT treatment did not alter cellular ATP levels or change the membrane potential. In cultured hepatocytes, DTT treatment increased the Vmax of GSH efflux by approximately 500% without affecting the Km. Inhibition of microtubular function and vesicular acidification did not affect basal or DTT stimulated efflux. Both cystine and DTT effects on sinusoidal GSH efflux were confirmed in perfused livers. In summary, the capacity of the sinusoidal GSH transporter is markedly influenced by thiol-disulfide status.

Transport of glutathione at blood-brain barrier of the rat: inhibition by glutathione analogs and age-dependence.

We showed previously that glutathione (GSH) may cross the blood-brain barrier intact by a saturable low affinity transport process (Km approximately 6 mM) (Kannan et al., J. Clin. Invest. 85: 2009-2013, 1990). In the present report, breakdown and resynthesis of GSH as the mechanism of apparent GSH uptake were excluded further because > 87% of injected 35S-cysteine taken up at the blood-brain barrier remained unchanged with negligible incorporation into GSH. In an effort to characterize further this GSH transport system, we have studied the influence of a number of potential inhibitors on brain uptake index (BUI) of GSH in rats pretreated with a gamma-glutamyl transpeptidase inhibitor, acivicin. The BUIs of tracer 35S-GSH uptake in the presence or absence of 1 to 20 mM cysteine, glutathione disulfide, gamma-glutamylglutamate, gamma-glutamyl-p-nitroanilide and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid did not differ significantly from each other. However, S-alkyl glutathiones (hexyl and octyl), sulfobromophthalein-glutathione, glutathione monoethyl ester, probenecid (5 mM) and ophthalmic acid (10 mM) inhibited GSH uptake significantly. Inhibition of GSH uptake by sulfobromophthalein-glutathione and GSH-monoethyl ester was concentration-dependent with apparent Ki approximately 0.016 and 0.083 mM, respectively. There was a decline in GSH-BUI as a function of age in both acivicin and nonacivicin-pretreated rats during the growth and developmental period from 25 to 135 days of age (100-500 g b.wt.). The decrease in BUI with age was specific for GSH; cysteine uptake did not change and no difference in diffusible (H2O) and nondiffusible (sucrose) components was found in this age range.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and glucocorticoid dependence of hepatic gamma-glutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo.

We reported that glucagon and phenylephrine decrease hepatocyte GSH by inhibiting gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu, S.C., J. Kuhlenkamp, C. Garcia-Ruiz, and N. Kaplowitz. 1991. J. Clin. Invest. 88:260-269). In contrast, we have found that insulin (In, 1 microgram/ml) and hydrocortisone (HC, 50 nM) increased GSH of cultured hepatocytes up to 50-70% (earliest significant change at 6 h) with either methionine or cystine alone as the sole sulfur amino acid in the medium. The effect of In occurred independent of glucose concentration in the medium. Changes in steady-state cellular cysteine levels, cell volume, GSH efflux, or expression of gamma-glutamyl transpeptidase were excluded as possible mechanisms. Both hormones are known to induce cystine/glutamate transport, but this was excluded as the predominant mechanism since the induction in cystine uptake required a lag period of greater than 6 h, and the increase in cell GSH still occurred when cystine uptake was blocked. Assay of GSH synthesis in extracts of detergent-treated cells revealed that In and HC increased the activity of GCS by 45-65% (earliest significant change at 4 h) but not GSH synthetase. In and HC treatment increased the Vmax of GCS by 31-43% with no change in Km. Both the hormone-mediated increase in cell GSH and GCS activity were blocked with either cycloheximide or actinomycin D. Finally, when studied in vivo, streptozotocin-treated diabetic and adrenalectomized rats exhibited lower hepatic GSH levels and GCS activities than respective controls. Both of these abnormalities were prevented with hormone replacement. Thus, both in vitro and in vivo, In and glucocorticoids are required for normal expression of GCS.

The relationship between biliary secretion of bilirubin and glutathione in the rat.

The relationship between biliary secretion of bilirubin and glutathione was investigated by infusing bilirubin solution (200 nmol/min/100 g body wt) into Sprague-Dawley rats and measuring bilirubin and glutathione in the bile. Hepatic glutathione level, when modified between the range of 1-10 mumols/g liver wt, did not affect biliary maximal secretory rate (Tm) of bilirubin (80 nmol/min/100 g body wt). However, when biliary secretion of bilirubin exceeded 10 mM or 30 nmol/min/100 g body wt, biliary secretion of glutathione was markedly impaired while the bile flow remained relatively constant. Thus, bilirubin impaired the biliary secretion of glutathione selectively compared to its effect on bile formation. The results indicate that the mechanisms of biliary secretion of the two physiological substances, bilirubin and glutathione, are closely related.

Neutrophil accumulation in ischemic reperfused rat liver: evidence for a role for superoxide free radicals.

Oxygen-derived free radicals and leukocytes have been implicated in the pathogenesis of ischemia-reperfusion injury. This study aimed at determining, by using biochemical and histochemical techniques, whether an accumulation of neutrophils occurs in the ischemic reperfused rat liver and whether superoxide free radicals play a role in mediating this neutrophil accumulation. Hepatic ischemia was induced by occluding blood supply to the left and median lobes, and reperfusion was reinstituted by releasing the occlusion. Myeloperoxidase activity of the liver was measured with a tetramethylbenzidine-H2O2 assay after removal of glutathione (by dialysis) and in the presence of 3-aminotriazole (catalase inhibitor). A modification of Graham and Karnovsky's method was used to stain neutrophils in liver frozen sections, and the number of neutrophils was counted. Results showed that ischemia-reperfusion of the liver produced a 4.4-fold increase in myeloperoxidase activity (from 0.073 +/- 0.009 to 0.320 +/- 0.017 units/mg liver, means +/- SE), which was proportional to the number of neutrophils (3.1-fold increase from 18 +/- 7 to 57 +/- 4 cells/mm2) in the liver tissue. Pretreatment with long-acting superoxide dismutase significantly attenuated the elevated myeloperoxidase activity and the number of neutrophils. These results indicate that reperfusion after a period of ischemia induces an accumulation of neutrophils in the liver, and superoxide anion free radicals are important mediators in the mechanism of this neutrophil accumulation.

Identification, purification, and immunochemical characterization of a tocopherol-binding protein in rat liver cytosol.

Tocopherol binding activity accompanying a rat liver cytosolic protein with molecular weight of 30-36 kDa has been demonstrated previously, although the isolation of the protein has not been reported. We now report the purification of an alpha-tocopherol-binding protein (TBP) from rat liver cytosol utilizing three chromatographic procedures: gel filtration, Affi-Gel Blue affinity chromatography, and chromatofocusing. Three peaks of specific alpha-tocopherol-binding activity were resolved on Affi-Gel Blue, referred to as AFB-1A, 1B, and 2. A 32-kDa homogeneous form was obtained after chromatofocusing of AFB-1B. D-alpha-[3H]tocopherol was displaced from homogeneous TBP in the presence of 500-fold excess of nonlabeled alpha-tocopherol, indicating the specificity of the binding. Anti-TBP rabbit antisera identified only one protein in rat hepatic cytosol on Western blotting. TBP immunoreactivity was found in the cytosol of rat liver and the lysate of fractionated hepatocytes, but not in the cytosol of other organs (including the heart, spleen, testes, and lung) nor in the lysate of fractioned Ito cells, endothelial cells, or Kupffer cells isolated from rat liver. Semi-quantitative ELISA demonstrated that rat liver cytosol contained approximately 2 mg TBP/g of cytosol protein. This immunoreactivity was associated with only the 30-36 kDa gel filtration fractions of rat liver cytosol and with both AFB-1A and -1B but not with AFB-2.

Effect of indomethacin on the uptake, metabolism and excretion of 3-oxocholic acid: studies in isolated hepatocytes and perfused rat liver.

3 alpha-Hydroxysteroid dehydrogenase catalyzes the reduction of 3-oxo-bile acids and binds 3 alpha-hydroxy bile acids. Indomethacin is a competitive inhibitor of the enzyme. In incubations of isolated rat hepatocytes, indomethacin delayed the intracellular reduction and the initial uptake of 3-oxocholic acid. Following a tracer dose of 3-oxocholic acid in perfused rat liver, rapid biliary excretion was observed mainly as taurocholic acid. Only 1.1% of the dose was recovered in the caval outflow and nearly all appeared in the first 5 min collection. When the tracer dose was given after initiating a constant infusion of indomethacin (50 microM), a dramatic decrease in biliary excretion was observed, still mainly as taurocholic acid, and 14% of the dose was recovered in the caval effluent: 10% in the first 5 min collection, mainly as 3-oxocholic acid, followed by a steady, slow release of mainly taurocholic acid. The increased intrahepatic retention of bile acids and slow release into perfusate and bile in response to indomethacin are consistent with displacement of bile acids from cytosolic protein.

Hormone-mediated down-regulation of hepatic glutathione synthesis in the rat.

Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.

Hormonal regulation of glutathione efflux.

The efflux of GSH has been shown previously to be a saturable process in both isolated rat hepatocytes and perfused liver, suggesting a carrier-mediated transport mechanism. The possibility in hormonal regulation of this process has been raised by recent reports. Our present work examined the role of hormones known to affect intracellular signal transduction mechanisms on GSH efflux in cultured rat hepatocytes and perfused rat livers. We found that cAMP-dependent factors, such as cholera toxin (CT), dibutyryl cAMP, forskolin, and glucagon all stimulated GSH efflux in cultured rat hepatocytes. The efflux kinetics were compared in cultured cells incubated with or without CT; the stimulation of GSH efflux was related to a near doubling of the Vmax while exhibiting no significant alteration of the Km. The increase in intracellular cAMP level associated with the threshold for this stimulatory effect was 25% above control. The stimulatory effect of CT could not be blocked by cyclohexamide pretreatment or reversed by colchicine treatment. The stimulatory effect of glucagon was abolished in the presence of ouabain but not in the presence of barium. On the other hand, hormones which act through Ca2+ and protein kinase C, such as phenylephrine and vasopressin, had no effect on GSH efflux in the cultured cells. In the perfused liver model, glucagon (10 nM) and dibutyryl cAMP (8 microM) stimulated sinusoidal GSH efflux to 130 and 144% of control values, respectively, and increased bile flow while not affecting biliary GSH efflux. Finally, the physiological significance of glucagon-mediated stimulation of sinusoidal GSH efflux was assessed by both plasma GSH and glucose levels in response to in vivo glucagon infusion. The threshold dose of glucagon for significant increase in plasma GSH (5.21 pmol/min) was lower than for glucose (15.61 pmol/min). At the highest glucagon infusion rate (261 pmol/min), plasma GSH level doubled while glucose level increased 80%. In conclusion, increased cAMP stimulates GSH efflux in cultured rat hepatocytes and perfused livers. The stimulatory effect of cAMP is exerted at the sinusoidal pole and appears to be mediated by hyperpolarization of hepatocytes by stimulation of Na(+)-K(+)-ATPase. In vivo studies confirmed the importance of cAMP-mediated stimulation of sinusoidal GSH efflux as it resulted in significant elevation of the plasma GSH level.

Evidence for carrier-mediated transport of glutathione across the blood-brain barrier in the rat.

Information on the origin of brain glutathione and the possibility of its transport from blood to brain is limited. We found a substantial uptake of 35S-labeled glutathione by the rat brain using the carotid artery injection technique. The brain uptake index of glutathione with and without an irreversible gamma-glutamyl transpeptidase inhibitor, acivicin, was similar. No significant differences in the regional uptake of labeled glutathione were found in rats pretreated with acivicin. The brain uptake index of tracer glutathione was similar to that of cysteine tracer and was lower than that of phenylalanine. The transport of oxidized glutathione (glutathione disfulfide) across the blood-brain barrier was not significantly different from that of sucrose, an impermeable marker. Brain radioactivity 15 s after carotid artery injection of labeled glutathione to rats pretreated with acivicin was predominantly in the form of glutathione. The in vivo glutathione uptake was saturable with an apparent Km of 5.84 mM. Amino acids, amino acid analogues, and other compounds [cysteine, phenylalanine, glutathione disulfide, gamma-glutamylglutamate, gamma-glutamyl p-nitroanilide, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)] did not affect glutathione transport. Our data suggest that glutathione is transported across the blood-brain barrier by a saturable and specific mechanism.

Role of glutathione status in protection against ethanol-induced gastric lesions.

The role of glutathione status in gastric mucosal cytoprotection has been a subject of controversy. Cysteamine, an exogenous sulfhydryl agent and diethyl maleate (DEM), an endogenous glutathione (GSH) depletor both appear to protect rats from ethanol-induced gastric lesions. In this study, we used various agents to alter gastric mucosal GSH levels and assessed the effects on susceptibility to ethanol injury. We found that DEM and buthionine sulfoximine both depleted gastric GSH but only DEM protected against ethanol-induced gastric lesions. L-Oxothiazolidine-4-carboxylate (OXT) and N-acetyl-L-cysteine (NAC) both potentiated ethanol-induced gastric lesions even though only NAC significantly raised the GSH level. The depletion of GSH by DEM was reversed by supplying cysteine in the form of OXT or NAC so that the net result was a GSH level close to normal control. The potentiation of ethanol injury by NAC and OXT was still apparent. These experiments show no relation between gastric GSH levels and susceptibility to ethanol injury.