Biomedical subjects

S C Lu

Publications and source records attributed to S C Lu.

At least 73 records · Page 4Linked to original sources

PubMed1996

A high cholesterol, (n-3) polyunsaturated fatty acid diet induces hypercholesterolemia more than a high cholesterol (n-6) polyunsaturated fatty acid diet in hamsters.

This study was designed to study the effects of (n-3) polyunsaturated fatty acids (PUFA) on plasma and liver lipids, particularly lipoprotein cholesterol concentrations, in hamsters. Diets rich in (n-3) PUFA (21 g/100 g fatty acid) or (n-6) PUFA (37.4 g/ 100 g fatty acid) with or without 5 g/kg cholesterol (C) supplements were given for 4 wk to male hamsters weighing 70-90 g. The VLDL- and (IDL + LDL)-cholesterol concentrations were 114 and 128% higher in hamsters fed the (n-3) PUFA + C diet than in those fed the (n-6) PUFA + C diet. However, these differences were not observed when cholesterol was not supplemented. Hamsters fed the (n-3) PUFA diet had significantly lower plasma and hepatic triglyceride concentrations than those fed the (n-6) PUFA diet. Concentrations were comparable in hamsters fed (n-6) PUFA + C and (n-3) PUFA + C. Hepatic cholesteryl esters were significantly lower, while hepatic microsomal acyl-CoA:cholesterol acyltransferase activity and VLDL cholesteryl esters were significantly higher in hamsters fed the (n-3) PUFA + C diet than in those fed the (n-6) PUFA + C diet. Our results demonstrate that elevation of VLDL- and (IDL + LDL)-cholesterol in hamsters by (n-3) PUFA, compared with (n-6) PUFA, is dependent on dietary cholesterol supplementation and may be due to decreased catabolism of these lipoproteins.

Acetyl-CoA C-Acyltransferase↗

PubMed1995

Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes.

Cylindrospermopsin (CY) is a naturally occurring alkaloid produced by the cyanobacterium Cylindrospermopsis raciborskii, which has been linked to an outbreak of hepatoenteritis in humans. We previously showed that CY is cytotoxic to primary cultures of rat hepatocytes and that CY lowers cell reduced glutathione (GSH) at nontoxic doses. Lower cell GSH also potentiates CY-induced cytotoxicity (Runnegar et al., Biochem Biophys Res Commun 201: 235-241, 1994). Our current work examined the mechanism of the fall in cell GSH induced by CY. We excluded several possible explanations for the loss in GSH, namely increased formation of oxidized glutathione (GSSG), increased GSH efflux, hidden forms of GSH, decreased GSH precursor availability, or decreased cellular ATP level. To address whether the fall in GSH was due to decreased GSH synthesis or increased GSH consumption, we examined the rate of fall in total GSH after 5 mM buthionine sulfoximine (BSO, an irreversible inhibitor of GSH synthesis) treatment. The rates of fall in total GSH (nmol/10(6) cells/hr) were 8.2 +/- 2.5, 6.0 +/- 1.7 and 5.9 +/- 1.3 for control, 2.5 microM and 5 microM CY-pretreated cells, respectively. This suggests that the fall in GSH induced by CY was due to the inhibition of GSH synthesis rather than increased consumption, because in the latter case the rate of fall in GSH would have been accelerated by CY pretreatment. Furthermore, excess GSH precursor (20 mM N-acetylcysteine), which supported GSH synthesis in control cells, did not prevent the fall in GSH or toxicity induced by CY. Treatment of cells with the cytochrome P450 inhibitor alpha-naphthoflavone protected partially from CY-mediated toxicity and from the fall in cell GSH. Thus, it is likely that cytochrome P450 is involved in the metabolism of CY, and the metabolite(s) that is generated may be more toxic and/or potent in inhibiting GSH synthesis. Inhibition of GSH synthesis is most likely an important factor in the cytotoxicity of CY.

Alkaloids↗

PubMed1995

Bidirectional glutathione transport by cultured human retinal pigment epithelial cells.

PURPOSE: To characterize glutathione (GSH) transport by cultured human retinal pigment epithelial (HRPE) cells. METHODS: Cultured HRPE cells were pretreated with acivicin for GSH efflux and with buthionine sulfoximine for GSH uptake to prevent the breakdown and resynthesis of GSH. Efflux was measured by the linear rate of accumulation of GSH in the supernatant; uptake was measured using [35S] GSH plus varying concentrations of GSH. Molecular forms were verified by high-performance liquid chromatography. HRPE cell mRNA was probed for the presence of the two recently cloned rat sinusoidal and canalicular GSH transporters, (RsGshT and RcGshT), by Northern blot analysis. RESULTS: Glutathione efflux was temperature dependent (undetectable at 4 degrees C), and its averaged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH effluxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflux was not influenced by dithiothreitol or sulfobromophthalein-reduced GSH adduct, agents known to affect liver sinusoidal GSH transport. Glutathione uptake was linear up to 45 minutes and was temperature dependent. The difference between 37 degrees C and 4 degrees C uptake values represented true uptake. Glutathione uptake (2 microCi/ml + 1 mM mass) was Na independent and was inhibited significantly by phenol-3,6-dibromphthalein disulfonate. The kinetics of GSH uptake was assessed by measuring uptake with 35S-GSH and 0.05 to 40 mM extracellular GSH for 30 minutes. Uptake was saturable with Vmax = 18.7 +/- 1.7 nmol/10(6) cells/30 minutes, Km = 12.1 +/- 1.9 mM, n (binding site) = 1. On Northern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGshT. CONCLUSIONS: The similarities in functional characteristics of GSH transport and the presence of RcGshT-like mRNA suggest GSH transport in HRPE cells is mediated by a RcGshT homolog. Although the transporter can operate bidirectionally, it is expected to be a net efflux pump under normal physiologic conditions because the intracellular GSH concentration is much higher.

Biological Transport↗

PubMed1995

Hormonal and cell density regulation of hepatic gamma-glutamylcysteine synthetase gene expression.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, is under both hormonal and cell density regulation in cultured rat hepatocytes. Specifically, the addition of insulin or hydrocortisone to culture media or the lowering of the initial plating cell density increased cell GSH by increasing the activity of GCS. In the present study, we examined the molecular mechanism of these effects. To determine whether the increase in GCS activity is associated with an increase in GCS heavy subunit (GCS-HS) mRNA expression, the steady state mRNA levels of GCS-HS were examined with the use of Northern blots. After 24-hr treatment of high density (0.6 x 10(5) cells/cm2) cultured rat hepatocytes with insulin (1 micrograms/ml) or hydrocortisone (50 nM), the steady state GCS-HS mRNA level increased by approximately 1-2 fold. When the plating density was decreased to 0.1 x 10(5) cells/cm2, the steady state GCS-HS mRNA level also increased by 1-2 fold 24 hr later. An increase in the steady state GCS-HS mRNA level was found within 4 hr of either hormonal treatment or cell density manipulation. The increase in steady state GCS-HS mRNA level resulted from increased gene transcription, as the transcriptional rates of GCS-HS after hormonal or cell density manipulation were increased by 2-3-fold, whereas the rates of GCS-HS mRNA degradation remained unchanged. Western blotting confirmed the increase in GCS-HS protein level after hormone treatment or lowering of plating cell density. When examined in vivo, the steady state GCS-HS mRNA level decreased by 50% in a rat in which diabetes had been induced with streptozotocin for 1 week; this was prevented with insulin replacement. In summary, GCS-HS gene expression is under both hormonal and cell density regulation.

Amino Acid Sequence↗

PubMed1995

Lipoprotein responses to fish, coconut and soybean oil diets with and without cholesterol in the Syrian hamster.

Thirty-six young male Syrian hamsters were fed with test diets containing coconut oil, soybean oil or fish oil with and without 0.5% cholesterol for 6 weeks. Without dietary cholesterol supplementation, animals on the fish oil diet had significantly lower plasma total triglyceride (TG) and total cholesterol than those on the coconut oil or soybean oil diet. The decrease of TG was seen mainly in the very low density lipoprotein (VLDL) fraction. The degree of decrease in cholesterol was similar in all of the lipoprotein fractions. With 0.5% dietary cholesterol supplementation, there was no significant difference in plasma TG level among the three dietary groups. However, the fish oil group had significantly higher plasma cholesterol than the coconut oil and soybean oil groups. The increase of cholesterol was mainly in the VLDL and low density lipoprotein (LDL) fractions. In contrast to the plasma cholesterol level, the hepatic cholesteryl ester content was significantly lower in the cholesterol-supplemented fish oil group than in the coconut oil and soybean oil counterparts. The cholesterol-supplemented fish oil group showed higher liver microsomal acyl-coenzyme A:cholesterol acyltransferase activity than the other two groups, while there was no significant difference in the excretion of fecal neutral and acidic sterols among the three dietary groups.

Animals↗

PubMed1995

[Disseminated Strongyloides stercoralis infection mimicking pneumonia].

Strongyloides stercoralis is an intestinal nematode. In an immunocompetent host, Strongyloides infections usually produce only mild gastrointestinal symptoms. However, in an immunocompromised host, widespread dissemination of larvae to the extra-intestinal organs may occur. If unrecognized, the mortality rate is high. Here we report a case of disseminated strongyloidiasis in a chronic obstructive pulmonary disease (COPD) subject whose chest radiograph demonstrated multiple pneumonic patches and interstitial infiltrates. Strongyloides larvae were found in stool, sputum, and urine, and embryonated eggs were also found in sputum. The patient was treated successfully with mebendazole and alben albendazole. In conclusion, although high mortality rate is noted in disseminated strongyloidiasis, it is still a curable disease when early diagnosis and treatment could be made.

Animals↗

PubMed1995

Effects of 3-morpholinosydnonimine-N-ethylcarbamide on hypoxia-induced mechanical and electric responses of isolated pig coronary artery.

AIM: To study effects of 3-morpholinosydnonimine-N -ethylcarbamide (SIN-1) on hypoxia-induced mechanical and electric activities of the isolated pig coronary artery. METHODS: Mechanical tension and membrane potential were measured simultaneously. RESULTS: Hypoxia initially caused a transient vascular smooth muscle cell membrane hyperpolarization followed by a membrane depolarization in isolated pig coronary artery. Subsequent addition of SIN-1 100 mumol . L-1 or verapamil (Ver) 10 mumol . L-1 led to membrane repolarization and relaxation of the vascular smooth muscle. Nitro-L-arginine (NLA) 0.2 mmol . L-1 and KCI 40 mmol . L-1 also induced membrane depolarization and vasoconstriction, which were similarly suppressed by SIN-1 or Ver. CONCLUSION: Hypoxic contractile response in isolated pig coronary artery is mediated by an increased Ca2+ influx via suppression of nitric oxide release.

Animals↗

PubMed1994

The role of glutathione in the toxicity of a novel cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes.

Cylindrospermopsin (CY) is a newly isolated alkaloid produced by the cyanobacterium Cylindrospermopsis raciborskii, which has been linked to an outbreak of hepatoenteritis in man. The current work examined the suitability of primary cultures of rat hepatocytes as an in vitro model for studying the cytotoxicity of CY. We found that CY (3.3-5.0 microM) caused significant cell death (40-67% of cells by LDH release) in cultured hepatocytes after 18 hr incubation. While investigating possible mechanisms for CY toxicity, we found that lower, nontoxic doses of CY (1.6-2.5 microM) decreased cell glutathione (GSH) to about 50% of control. For toxic doses (5 microM), the loss of GSH preceded the onset of toxicity by six hr. Lowering cell GSH predisposed cells to CY toxicity. In conclusion, cultured hepatocytes are a suitable model for studies of CY cytotoxicity and GSH is involved in the detoxification of CY.

Alkaloids↗

PubMed1994

Comparison of sulfur amino acid utilization for GSH synthesis between HepG2 cells and cultured rat hepatocytes.

HepG2 cells are widely used as a model of human hepatocytes for studies of drug metabolism and toxicity. However, GSH metabolism in HepG2 cells is poorly characterized. This report describes the utilization of sulfur amino acids for GSH synthesis in HepG2 cells. In contrast to primary cultures of rat hepatocytes, which rely mostly on methionine for GSH synthesis, HepG2 cells use cystine. Their inability to utilize methionine for GSH synthesis was not due to lack of methionine uptake or low cellular ATP levels, but rather to the lack of S-adenosyl-methionine synthetase activity. When HepG2 cells were cultured overnight in medium containing cystine as the only sulfur amino acid, addition of glutamate or acivicin had minimal to no effect on cell GSH; however, addition of threonine significantly depleted cell GSH. When cystine (0.18 mM) uptake was measured, glutamate (2.5 mM), which inhibited cystine uptake in cultured rat hepatocytes, had a minimal effect in HepG2 cells. Instead, threonine (20 mM) strongly inhibited the apparent uptake of cystine by HepG2 cells. Strong inhibition by threonine of apparent cystine uptake was actually due to inhibition of cysteine uptake, which resulted from GSH-cystine mixed disulfide exchange. Radio-HPLC confirmed this. After incubating cells with [35S]cystine (0.18 mM) for 10 min, the total counts inside the cell matched the counts in the uptake medium in the form of GSH-cysteine mixed disulfide. Finally, HepG2 cells took up cysteine by both Na(+)-dependent and -independent mechanisms. The former exhibited high affinity and low capacity, whereas the latter exhibited the opposite. At a physiologic concentration of cysteine (10 microM), 68% of cysteine uptake occurred via the Na(+)-dependent system and 32% via system L1.

Adenosine Triphosphate↗

PubMed1994

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.

Analysis of Variance↗

PubMed1993

Identification of the 5' regulatory elements of avian lipoprotein lipase gene: synergistic effect of multiple factors.

The organization of cis-acting regulatory elements of the chicken lipoprotein lipase gene was investigated in 5.4 kb of 5' flanking sequences. Various lengths of 5' flanking sequence were linked to the bacterial chloramphenicol acyltransferase (CAT) gene and transfected into primary cultures of chicken adipocytes by DEAE-dextran transfection method. Negative elements are present between -1947 and -139 of the 5' flanking sequence. Removal of these sequences revealed the presence of positive elements located within 138 bp upstream of the major transcription start site. Sequence analysis showed that the region from the major transcription start site to -138 contains an inverted GC box (ACCACGCCCC), a CCAAT element and two direct repeats of the octamer motif, ATTTGCAT. DNase I footprinting assays using a probe extending from -175 to +191, identified three sites protected by nuclear factors. Site I (-126 to -123), a C-rich sequence, GCCC, was identified only on the coding strand. Site II covered the sequence from -95 to -68 and includes the GC box. Site III, from -53 to -26, contained two octamer repeats. Site I is the 5' portion of a 10 bp sequence (CCCTCCCCCC; -126/-116) which is perfectly conserved in the avian and the human promoter. Single or multiple copies of a 37 bp DNA fragment (-138/-102) containing the 10 bp conserved sequence were cloned into LPLCAT-51, upstream or downstream of the major transcription start site and in both orientations; transfection and CAT activity assays with these constructs indicate that the -138/-102 fragment has an enhancer like activity. Additional 5' and internal deletions of LPLCAT-138 suggest that the factors binding to the C-rich element, the GC box and the two octamer repeats have a synergistic effect on promoter activity.

Adipocytes↗

PubMed1993

Restoration of gap-junctional intercellular communication in a communication-deficient rat liver cell mutant by transfection with connexin 43 cDNA.

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.

Animals↗

PubMed1993

Bidirectional membrane transport of intact glutathione in Hep G2 cells.

Rat hepatocytes exhibit bidirectional carrier-mediated transport of reduced glutathione (GSH) across the plasma membrane. Transport of GSH has not been well characterized in human-derived cells. We examined Hep G2 cells as a possible human liver model for GSH homeostasis. Hep G2 cell GSH averaged 25.9 +/- 1.4 nmol/10(6) cells. When Hep G2 cells were incubated in buffer, no GSH appeared in the medium over 2 h. However, after pretreatment with acivicin to inhibit gamma-glutamyl transpeptidase activity, GSH efflux was unmasked and measured 30 +/- 4 pmol x 10(6) cells-1 x min-1, which is comparable to rat hepatocytes. GSH efflux was inhibited by sulfobromophthalein GSH adduct (BSP-GSH) and cystathionine, agents that inhibit sinusoidal efflux in the rat, and was stimulated by adenosine 3',5'-cyclic monophosphate-dependent agents. GSH uptake was measured after cells were pretreated with acivicin and buthionine sulfoximine to prevent breakdown of GSH and resynthesis of GSH from precursors, respectively. In the presence of 4 microCi/ml of [35S]GSH and 10 mM unlabeled GSH, GSH uptake was linear up to 45 min and did not require Na+ or Cl-. GSH uptake exhibited saturability with a maximal velocity of 4.15 +/- 0.23 nmol.mg-1 x 30 min-1, a Michaelis constant of 2.36 +/- 0.26 mM, and two interactive transport sites. BSP-GSH cis-inhibited GSH uptake in a dose-dependent manner with an inhibitory constant of 0.46 +/- 0.05 mM. Inhibition by BSP-GSH (1 mM) of GSH uptake was through a single inhibitor site and was overcome at > 10 mM GSH, which is consistent with competitive inhibition. Similar to the rat, 10 mM extracellular GSH trans-stimulated GSH efflux. These findings may be important in gaining better insights into GSH homeostasis in human liver cells.

Biological Transport↗

PubMed1993

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.

Adenosine Triphosphate↗

PubMed1992

The structure and complete nucleotide sequence of the avian lipoprotein lipase gene.

The entire gene for chicken lipoprotein lipase (LPL) has been isolated and characterized by primer extension and sequence analysis. The gene is 17 kilobase pairs long and comprises 10 exons and 9 introns. As determined by primer extension analysis the start sites of transcription map 176, 204 and 218 nucleotides upstream of the initiator methionine codon. The 1947 base pairs of 5' flanking sequence contains several putative regulatory elements including two adjacent Oct I binding elements, four glucocorticoid regulatory elements and a sequence very homologous to the previously described fat specific element at--1402 nt. The first intron is very large (6433 bp) and contains four consensus SpI binding-site sequences. Five polyadenylation signals are found in the 3' untranslated region, the last three of which give predicted mRNA species identical in size to those determined by Northern blot. The 5' flanking sequences of the LPL, pancreatic lipase and hepatic lipase genes do not show homology, however. This may account for the homologous amino acid sequences but dissimilar gene expression of these enzymes.

Amino Acid Sequence↗

PubMed1992

Loss of suppression of GSH synthesis at low cell density in primary cultures of rat hepatocytes.

Primary cultures of adult rat hepatocytes shift into the growth phase when plated at low density (LD). We used this model to examine changes in glutathione (GSH) metabolism, since cells undergoing active growth may be more susceptible to environmental toxins. When primary cultures of adult rat hepatocytes were plated on collagen or Matrigel-precoated dishes, cell number and GSH varied inversely. This density effect on cell GSH occurred as early as 2 h after plating, when the media contained 1 mM methionine, but was delayed until 20 h if the media contained only 0.5 mM cystine. The density effect on GSH synthesis occurred in the absence of serum, hormones, changes in cell volume, GSH efflux, ATP levels, and uptake of methionine or cystine and was blocked by cycloheximide or actinomycin D. When methionine was available, the cellular cysteine level was 65% higher at LD than at high density (HD). gamma-Glutamylcysteine synthetase (GCS) activity was 64% higher at LD than at HD. GSH synthetase activity was unaffected by density. Both the increase in cellular cysteine levels and GCS activity were blocked by cycloheximide and actinomycin D. When cells were cocultured using cluster plates and Transwell inserts for 4 h, cell GSH of HD cells was unaffected by the density of cocultured cells; however, LD cells exhibited significantly lower GSH and GCS activity when cocultured with HD cells than when cocultured with LD cells. Cysteine levels were elevated in the LD cells regardless of the density of cocultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

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.

Adrenalectomy↗

PubMed1991

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.

Animals↗