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N Kaplowitz

Publications and source records attributed to N Kaplowitz.

At least 91 records · Page 5Linked to original sources
PubMed1990

The use of monochlorobimane to determine hepatic GSH levels and synthesis.

We have used the specific reaction of monochlorobimane (mBCI) with GSH to analyze hepatic GSH, mBCI, itself nonfluorescent, forms a stable, fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases (GST). When hepatocytes were labeled with mBC1 (100 microM) in Krebs-Henseleit buffer, the fluorescent signal recorded over time was directly proportional to the concentration of GSH. The HPLC analyses of hepatocytes that were preloaded with the dye indicated that GSH was the only thiol labeled. When the technique was applied to freshly isolated intact hepatocytes that contained different levels of GSH, a close correlation between the levels of GSH measured by the present method (mBC1) and the standard enzymatic recycling method was found. A similar agreement for the cytosolic and mitochondrial pools of GSH determined by the two methods was established. The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell. In addition, we have applied this technique to determine directly the rate of synthesis of GSH in both cell-free conditions and in cell suspensions by monitoring the increase in fluorescent adduct when mBC1 is present in excess in the incubation.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Calcium compartmentation and exchange rates in primary hepatocyte culture.

We utilized a technique, previously used to study myocardial cells (G. A. Langer, J. S. Frank, and L. M. Nudd, 1979, Amer. J. Physiol. 237, H239-H246), to study 45Ca2+ isotope exchange kinetics in hepatocyte monolayers, cultured on scintillation disks, and perfused in a flow-through chamber. Isolated rat hepatocytes were plated directly on Primaria-coated disks impregnated with scintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium (45Ca2+), to apparent asymptote, the washout of 45Ca2+ from the cells was measured. A large very fast turnover compartment, as well as small fast and slow turnover compartments, were identified in each experiment. Surface calcium (Ca2+) was determined by its displacement with 1 mM La3+ after asymptote had been reached during 45Ca2+ labeling (1.59 mmol Ca2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (greater than 3.4 min-1 with a t1/2 less than 12 s). The rate constants for the fast and slow exchangeable compartments were 0.11 min-1 (t1/2 = 6.5 min) and 0.013 min-1 (t1/2 = 56 min), respectively. The fast compartment contained 0.40 mmol Ca2+/kg dry wt and the slow compartment contained 0.27 mmol Ca2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca2+ in response to 100 microM phenylephrine, 10 nM angiotensin II, and 100-microM 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca2+/kg dry cell wt, respectively. These agents had an effect only during the washout of the fast compartment. In conclusion, this novel technique of on-line measurement of 45Ca2+ exchange in hepatocyte monolayers identified three exchangeable compartments: (1) a very rapidly exchangeable surface compartment, (2) a fast "microsomal" hormone-releasable compartment, and (3) a slow, non-hormone-releasable compartment.

Angiotensin II↗
PubMed1990

Oxidation and reduction of bile acid precursors by rat hepatic 3 alpha-hydroxysteroid dehydrogenase and inhibition by bile acids and indomethacin.

Enzyme kinetics of purified rat hepatic 3 alpha-hydroxysteroid dehydrogenase for bile acid precursors and effects of bile acids and indomethacin on those activities were studied. This enzyme catalyzed the oxidoreduction of the C3 position of bile acid precursors. Km for 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one (1.6 microM) was markedly lower than Km for 7 alpha-hydroxy-5 beta-cholestan-3-one (28 microM) but Vmax was similar. Km for 3 alpha, 7 alpha-dihydroxy-5 beta-cholestane (12 microM) was lower than Km for 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestane (150 microM) although Vmax/Km values were similar for both compounds. Bile acids and indomethacin inhibited the reduction of 3-oxo bile acid precursors. NADPH inhibited the binding of lithocholic acid (3 alpha-hydroxy-5 beta-cholanic acid) by 3 alpha-hydroxysteroid dehydrogenase. These data suggest that intrahepatic bile acid concentrations may affect the reduction of 3-oxo-bile acid precursors and intrahepatic redox conditions may affect intracellular bile acid transfer.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Relationship between the newly identified bile acid binder and bile acid oxidoreductases in human liver.

Gel filtration fractions of human hepatic cytosol obtained from an autopsy liver were examined for elution of bile acid oxidoreductases. Several enzymes including 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 3-ketosteroid reductase, and dihydrodiol dehydrogenase eluted mainly in the 30,000-40,000 Mr fractions known to contain the newly identified bile acid binder (Stolz, A., Sugiyama, Y., Kuhlenkamp, J., and Kaplowitz, N. (1984) FEBS Lett. 177, 31-35). These enzyme activities could be resolved into six peaks of dihydrodiol dehydrogenase activity on chromatofocusing, some of which also had oxidoreductase activity with bile acids. Using equilibrium dialysis, the major lithocholate-binding activity was found to coelute with 3 beta-HSD, completely separate from 3 alpha-HSD. Reexamination of the surgical liver specimen originally used to purify the bile acid binder confirmed these results. The peak fraction from chromatofocusing, which exhibited the bulk of binding activity with bile acids, had 3 beta-HSD activity, whereas other fractions had 3 alpha-HSD. Anti-serum to the previously purified binder identified a single 36-kDa protein in both liver specimens and exclusively in the chromatofocusing fractions containing both the binding and 3 beta-HSD activity. However, upon further purification of the binder from this fraction, 3 beta-HSD activity was separated from the binder, but the homogeneous protein retained dihydrodiol dehydrogenase activity. Thus, in contrast to the rat in which the major bile acid binder is identical to 3 alpha-HSD, in human liver the bile acid binder is distinct from 3 alpha-HSD and copurifies with a different oxidoreductase that has dihydrodiol dehydrogenase activity but no activity with bile acids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Inhibition of GSH efflux from rat liver by methionine: effects of GSH synthesis in cells and perfused organ.

The inhibition of efflux of intracellular reduced glutathione (GSH) by methionine was determined in isolated rat hepatocytes suspended either in Krebs-Henseleit buffer or in modified Fisher's medium. Methionine (1 mM) added to Krebs-Henseleit suspensions of isolated rat hepatocytes inhibited GSH efflux, with greater retention of GSH in the cells compared with control. Results were similar with methionine and 0.3 mM propargylglycine cystathionase inhibitor), suggesting no net synthesis of GSH from methionine. In Fisher's medium, the inhibitory effect of methionine on GSH efflux was masked due to increasing cellular GSH; however, the inhibitory effect of methionine was unmasked by propargylglycine, which prevented the utilization of methionine for GSH synthesis. The addition of serine (0.1 mM) to methionine in Krebs-Henseleit buffer raised cellular GSH, overcoming the inhibition of GSH efflux. In the perfused liver, infusion of 1 and 5 mM methionine initially inhibited GSH efflux, but the inhibition was reversed with continued methionine infusion. After removal of methionine, GSH efflux increased immediately. The reversal and rebound were blocked by propargylglycine, revealing concentration-dependent inhibition of sinusoidal GSH efflux by methionine. Thus, when methionine is utilized to promote GSH synthesis, its inhibitory effect on GSH efflux tends to be overcome.

Alkynes↗
PubMed1990

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.

Animals↗
PubMed1989

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.

Acetylcysteine↗
PubMed1989

Effects of chronic ethanol feeding on rat hepatocytic glutathione. Relationship of cytosolic glutathione to efflux and mitochondrial sequestration.

Chronic ethanol feeding to rats increases the sinusoidal component of hepatic glutathione (GSH) efflux, despite a lower steady-state GSH pool size. In the present studies, no increase of biliary GSH efflux in vivo was found in chronic ethanol-fed cells. Studies were performed on ethanol-fed and pair-fed cells to identify the kinetic parameters of cellular GSH concentration-dependent efflux. The relationship between cytosolic GSH and the rate of efflux was modeled by the Hill equation, revealing a similar Vmax, 0.22 +/- 0.013 vs. 0.20 +/- 0.014 nmol/min per 10(6) cells for ethanol-fed and pair-fed cells, respectively, whereas the Km was significantly decreased (25.3 +/- 2.3 vs. 33.5 +/- 1.4 nmol/10(6) cells) in ethanol-fed cells. The difference in Km was larger when the data were corrected for the increased water content in ethanol-fed cells. We found a direct correlation between mitochondria and cytosolic GSH, revealing that mitochondria from ethanol-fed cells have less GSH at all cytosolic GSH values. The rate of resynthesis in depleted ethanol-fed cells in the presence of methionine and serine was similar to control cells and gamma-glutamylcysteine synthetase remained unaffected by chronic ethanol. However, the reaccumulation of mitochondrial GSH as the cytosolic pool increased was impaired in the ethanol cells. The earliest time change in GSH regulation was a 50% decrease in the mitochondrial GSH at 2 wk.

Alcoholism↗
PubMed1988

Comparison of the effects of bile acids and GSH on the fluorescence of bound 1-anilino-8-naphthalene sulfonate and the enzymatic activity of cationic and neutral human hepatic GSH S-transferases.

Three cationic (C1, C2, A1) and a neutral (N1) glutathione (GSH) S-transferase were purified to homogeneity from human liver, as we have previously reported. GSH had no effect on the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS) bound by transferase C1 and N1, but markedly enhanced the fluorescence with C2 and A1 without changing the affinity for ANS. This effect of GSH was saturable and with C2 was intermediate between A1 and C1. Bile acids inhibited the fluorescence of ANS bound to C1 and C2. GSH in the presence of bile acids further decreased the fluorescence of ANS bound to C1 and increased the fluorescence with C2. Transferase A1 showed decreased fluorescence in the presence of lithocholic acid and increased fluorescence in the presence of cholic acid; both changes were reversed by GSH. Transferase N1 showed increased fluorescence of bound ANS in the presence of various bile acids and this effect was diminished in the presence of GSH. Enzyme activity of the transferase was inhibited by bile acids with the exception of transferase A1. All the proteins bound lithocholic acid. The inhibition of C1 and N1 was greater at pH 6.5 than 7.4 and the order of addition of substrates and inhibitor made no difference.

Anilino Naphthalenesulfonates↗
PubMed1988

Effect of membrane potential and cellular ATP on glutathione efflux from isolated rat hepatocytes.

total glutathione (GSH) efflux was studied in isolated rat hepatocyte suspensions at repleted GSH content (45-55 nmol/10(6) cells). The increase in concentrations of medium K+ in place of Na+ caused a parallel fall in membrane potential and total GSH efflux. Ouabain (1 mM) and replacement of Na+ with choline caused a gradual fall in membrane potential and GSH efflux. Hyperpolarization of hepatocytes with lipophilic anions, thiocyanate, and nitrate was associated with significantly increased efflux. Total GSH efflux was inhibited by increasing concentrations of fructose, antimycin A, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and there was a direct relationship between the rate of efflux and cellular ATP. Changes in total GSH efflux were paralleled by changes in GSH determined by high-performance liquid chromatography. Vanadate markedly inhibited efflux but caused only a modest decrease in cellular ATP. Fructose, antimycin A, and vanadate did not affect membrane potential or cell volume under the conditions at which efflux was inhibited. These results suggest independent requirements for both membrane potential and ATP in the transport of GSH.

Adenosine Triphosphate↗
PubMed1988

Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.

Animals↗
PubMed1988

Comparison of the binding sites of GSH S-transferases of the Ya- and Yb-subunit classes: effect of glutathione on the binding of bile acids.

We have previously observed that the Ya subunit-containing glutathione (GSH) S-transferases from rat liver exhibit a common high affinity binding site for lithocholic acid, bilirubin, and sulfobromophthalein (BSP) (1984. J. Lipid Res. 25: 1177-1183). Subsequently we found that cholic acid and its amidates bound to a site on the Ya subunit separate for the lithocholic acid/bilirubin site (1986. J. Lipid Res. 27: 955-966). We now have extended this work by showing that amidates of lithocholic acid as well as chenodeoxycholic acid and its amidates competitively displace [14C]lithocholic acid from the Ya subunit. GSH did not inhibit binding of any of the ligands to the high affinity Ya site, but did inhibit binding to the cholic acid site on the Ya subunit. We have also defined the binding sites and effects of GSH on the Yb class of subunits. Lithocholic, chenodeoxycholic, and cholic acids (and amidates) shared a common site on the Yb or Y'b subunit, whereas BSP and bilirubin were bound at a different site. Both the bile acid and organic anion sites on the Yb subunit were inhibited by GSH. The inhibition by GSH in all cases (Ya cholic acid site or Yb bile acid or bilirubin sites) was saturable, of the competitive type, and incomplete at maximal GSH concentrations, suggesting that when GSH binds to its distinct substrate site, it induces a conformational change in the proteins affecting the other binding sites.

Animals↗