Main

GSH (L-γ-glutamyl-L-cysteinylglycine) is an intracellular tripeptide present in most eukaryotic cells. It is involved in various biologic phenomena including DNA, protein, and leukotriene synthesis as well as in detoxification of xenobiotics and reactive oxygen species (1). It is synthesized from cysteine, glutamate, and glycine by consecutive actions of two ATP-dependent enzymes, GCS (EC 6.3.2.2) and GSH synthetase (EC 6.3.2.3) (2). The former is the rate-limiting enzyme, and it is feedback inhibited by GSH. The synthesis of GSH may also be limited by the availability of cysteine (3). The mammalian GCS holoenzyme is a heterodimer with 28-kD GCSl and 73-kD GCSh subunits (4). The catalytic activity of the enzyme resides in GCSh, whereas GCSl regulates the activity (5).

GSH is present in millimolar concentrations within the cell. It is a substrate of GSH peroxidases, which remove hydrogen peroxides and organic peroxides. High GSH concentrations are found in alveolar epithelial lining fluid, which suggests that it contributes to the defense against oxygen toxicity in the lung (6).

The concentration of GSH in leukocytes from tracheal aspirates from newborn infants has been reported to be lower in preterm babies than in full-term infants (7). Furthermore, preterm neonates have markedly lower GSH concentrations in bronchoalveolar lavage fluid compared with adults (8, 9). These results suggest that either GSH synthesis is decreased or its consumption exceeds the synthetic capacity of the lung in preterm infants.

Little is known about GSH synthesis during development. In human fetal erythrocytes (10), leukocytes (11), and liver (12), GCS activities were in the same range as in adults. However, nothing is known about the developmental expression of GCS in lung or kidney, yet these organs undergo major changes during birth with respect to the amount of oxygen they are exposed to. Therefore, the aim of our studies was to investigate mRNA expression and activity of GCS in liver, lung, and kidney during human development.

METHODS

Tissue samples.

Fetal lung, liver, and kidney samples (15–19 gestational wk;n = 6) were obtained from legal abortions. The delay from delivery room to laboratory was approximately 1 h. The samples between 26–42 gestational wk were obtained from neonatal autopsies performed within 12 h of death. The causes of death were RDS (n = 2), congenital heart disease (n = 1), respiratory failure and hydronephrosis (n = 1), and meconium aspiration (n = 1). Adult lung tissue samples (n = 3) were obtained from macroscopically normal tissues of patients with lung cancer undergoing lung surgery and from donor lungs of single lung transplantations. Adult liver tissue (n = 4) was obtained from partial liver transplantations. Kidney tissue (n = 5) was obtained from renal biopsies or from cadaver donors.

The tissues were frozen in liquid nitrogen and stored at −80°C until analyzed. The study protocol was approved by the Ethical Committees of the Hospital for Children and Adolescents, and Department of Thoracic and Cardiovascular Surgery, University of Helsinki, Finland.

RNA analysis.

Total RNA was extracted from tissues using the cesium chloride-guanidinium method (13). Samples with signs of degradation of ribosomal RNA on visual inspection after agarose gel electrophoresis were discarded. RNA was quantified using the ribonuclease protection assay (RPA II, Ambion Inc., Austin, TX). 32P-radiolabeled antisense RNA probes were transcribed from the Pst I-Nco I fragment of GCSh (nucleotides 1375–1628) (14), and the Hin dII fragment of GCSl cDNA (nucleotides 583–888) (15), and hybridized with 10 μg (liver and kidney) or 20 μg (lung) of total RNA at 42°C overnight. To normalize for RNA content, the samples were hybridized with RNA probes transcribed from human β-actin cDNA (pTRI-Actin-Human, Ambion Inc.). After RNase A+T1 digestion, the protected fragments were separated on 5% polyacrylamide/8 M urea gels and exposed to Kodak BioMax MR autoradiography film (Eastman Kodak Co, Rochester, NY). The x-ray films were scanned (Microtek ScanMaker, Microtek International, Inc., Hsinchu, Taiwan) and analyzed with the National Institutes of Health Image (1.61) analysis software (Bethesda, MD).

GCS activity.

The activity of GCS was measured as described by Nardi et al. (16), with modifications. Frozen samples were homogenized in 100 mM Tris-HCl buffer (pH 8.2) containing 50 mM KCl, 20 mM MgCl2, and 2 mM EDTA. The homogenates were centrifuged at 14 000 ×g for 30 min at 4°C. The liver and lung homogenates were filtered through P-10 gel filtration columns (Pharmacia, Uppsala, Sweden) and kidney homogenates through Micro Bio-Spin 6 Column (Bio-Rad, Hercules, CA) to remove endogenous inhibitors and amino acids. The specificity of the assay was confirmed using buthionine sulfoximine, a specific inhibitor of GCS. The mixture (final volume, 100 μL) containing 10 mM ATP, 6 mM DTT, 3 mM L-cysteine, and 15 mM L-glutamic acid in 100 mM Tris-HCl buffer (pH 8.2) was preincubated at 37°C for 15 min to ensure complete reduction of thiols. The reaction was initiated by addition of the sample (70 μL). The liver samples were incubated for 15 min and lung and kidney samples for 30 min at 37°C. After the incubation, 50 μL of the mixture was added to 50 μL of 30 mM monobromobimane (Thiolyte, Calbiochem, La Jolla, CA) in 50 mM N-ethylmorpholine (pH 8.4) and incubated at room temperature for 5 min. The reaction was stopped by addition of 10 μL of 100% trichloroacetic acid, and the precipitated protein was pelleted at 14 000 ×g for 5 min. Five microliters of supernatant was injected into a Waters Novapak C-18 HPLC column (4 μm, 3.9 × 150 mm) running an isocratic mobile phase consisting of 4% acetonitrile, 0.25% acetic acid, and 0.25% perchloric acid, pH 3.7, and the fluorescent product was detected using a spectrofluorometer (model RF-10AxL, Shimadzu Corporation, Kyoto, Japan) with excitation and emission wavelengths of 394 nm and 480 nm, respectively. The product was quantified by comparison to γ-glutamylcysteine (Bachem, Bubendorf, Switzerland) standards derivatized and analyzed as above. For specific enzyme activity, the protein concentration was determined from filtered homogenates using Bio-Rad DC protein assay kit.

Statistical analysis.

For statistical analysis of results, the Mann-Whitney U test was used with a level of p ≤ 0.05 chosen to indicate significant differences. Adult samples were compared with fetal and neonatal samples. The Spearman rank correlation test was used to assess the correlation between enzyme activity and mRNA concentrations.

RESULTS

Liver.

In the liver, the GCS mRNA species were detectable using Northern blotting (results not shown), whereas in other tissues, only the ribonuclease protection assay was sensitive enough to detect GCSl and GCSh. The mRNA expression of GCSl and GCSh as well as the GCS activity in the neonatal and fetal liver samples did not differ significantly from adult samples (Fig. 1). The mean activity of GCS was 3 to 4 times higher in the liver than in other tissues studied.

Figure 1
figure 1

A, mRNA concentrations of GCS in human liver. GCSl (white bars) and GCSh (gray bars) mRNA concentrations were quantified relative to β-actin signal. Numbers indicate age in gestational weeks of fetuses and neonates. The original ribonuclease protection assay autoradiographs of GCSl, GCSh and β-actin are shown above each lane. B, Activity of GCS in human liver. Specific activity is expressed as nmol·min−1·mg−1 of protein.

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Lung.

In the lung, the mRNA expression of GCSh was in the same range in fetal, neonatal, and adult groups, whereas GCSl expression was higher in the samples from neonates than in those from fetuses and adults (p = 0.03) (Fig. 2A). The mRNA expression of GCSl was highest in two neonatal patients (gestational age, 26 wk) who died of RDS. Also, the highest specific activity (1.9 nmol·min−1·mg−1 of protein) in the lung was measured from one of the RDS patients. There was no statistically significant difference in the GCS activity in fetal, neonatal, and adult groups (Fig. 2B).

Figure 2
figure 2

A, mRNA concentrations of GCS in human lung. GCSl (white bars) and GCSh (gray bars) mRNA concentrations were quantified relative to β-actin signal. Numbers indicate age in gestational weeks of fetuses and neonates. The original ribonuclease protection assay autoradiographs of GCSl, GCSh and β-actin are shown above each lane. B, Activity of GCS in human lung. Specific activity is expressed as nmol·min−1·mg−1 of protein.

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Kidney.

The mRNA expression of GCSh was in the same concentration in all groups (Fig. 3A). The GCSl expression was significantly lower in the samples from fetuses than in those from neonates and adults (p = 0.02), but GCSl mRNA expression did not correlate with enzyme activity (r = −0.03). GCS activity (Fig. 3B) in the samples from adults was statistically significantly higher than in those from fetuses and neonates (p = 0.03), but this may be accounted for by the variation in the small number of samples.

Figure 3
figure 3

A, mRNA concentrations of GCS in human kidney. GCSl (white bars) and GCSh (gray bars) mRNA concentrations were quantified relative to β-actin signal. Numbers indicate age in gestational weeks of fetuses and neonates. The original ribonuclease protection assay autoradiographs of GCSl, GCSh and β-actin are shown above each lane. B, Activity of GCS in human kidney. Specific activity is expressed as nmol·min−1·mg−1 of protein.

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DISCUSSION

It has been proposed that GSH plays an important role in cell proliferation (17), and GSH depletion in embryonic cells has been associated with increased frequency of malformations (18). This study, as well as earlier studies from human liver (12) and erythrocytes (10), supports the notion that GSH synthetic capacity is mature in early developmental stages. However, there may be organ-specific differences in the maturation of GSH synthesis. For instance, the low concentration of GSH in the epithelial lining fluid of premature newborn infants (8) may reflect the immaturity of the lung to synthesize GSH efficiently.

In this study, the highest GCS activity was measured in the liver. The activities in the lung and kidney were somewhat lower. In the rat, the activity in the kidney is an order of magnitude higher than in the liver (19). However, in late fetal kidney of the rat, the GCS activity is almost absent, but increases abruptly in the postnatal period (20). A similar trend has also been reported in mice (21). In our study, there was no such dramatic increase in enzyme activity. These differences in tissue-specific expression of GCS between humans and rodents may reflect differences in interorgan GSH metabolism.

In this study, there was large variation in the enzyme activity and mRNA concentrations of samples obtained from different individuals. This may in part be related to the fact that the period between the time of death and autopsy was variable. All infants were treated in intensive care units for up to 5 d before death, and all but one received oxygen therapy. Because both GCS mRNA and activity levels have been shown to be induced on oxidative stress in lung epithelial cells (22, 23), the up-regulation of GCSl in the lungs of infants at gestational age 26 wk may well be caused by oxygen therapy. Thus, all the changes observed in mRNA concentrations do not necessarily reflect alterations of gene expression during development, but may be caused by pathologic changes or therapeutic interventions.

The mRNAs of GCSh and GCSl are coded by different genes (14, 15). Although transcripts of both subunits are ubiquitously expressed in human tissues (15), the expression varies considerably, and there is no correlation between the steady-state mRNA concentrations of the two subunits in tissues. This indicates that the expression of the two GCS subunit genes is differentially regulated. Although GCSh possesses all the catalytic activity of GCS, GCSl has an important regulatory function (5). When the light subunit is present, the Km for L-glutamate is reduced from 18 mM to 1.4 mM and the ki for GSH is increased from 1.8 mM to 8.2 mM in rat kidney holoenzyme. The kinetics of the recombinant human enzyme are similar (24). Because intracellular concentrations of L-glutamate and GSH are 1–3 mM and 1–10 mM, respectively, it is likely that the presence of the light subunit is needed for full activity of GCS in vivo (5, 25). Given the complexity of GCS regulation at the transcriptional and posttranscriptional levels (26), it is not surprising that no correlation was found between mRNA concentrations and enzyme activity.

In conclusion, we have shown that GCS is expressed and active in human liver, lung, and kidney already in the second trimester. This suggests that GCS is fully operative also in premature infants in the tissues studied. Other factors, such as limited availability of the GSH precursor cysteine (27), or increased GSH consumption because of increased oxidative stress in neonates requiring ventilatory support, may account for the lower concentrations of GSH found in preterm infants.