Joseph B. Warshaw

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.

A Comparison of Early-Onset Group B Streptococcal Neonatal Infection and the Respiratory-Distress Syndrome of the Newborn

In attempting to differentiate early-onset Group B streptococcal infection from hyaline-membrane disease we found features of severe Group B infection to be rupture of the membranes for more than 12 hours before delivery (four or eight versus one of nine), gram-positive cocci in the gastric aspirate (four or four versus none of one), apnea and shock in the first 24 hours of life (seven of eight versus none of nine), and the generation of lower peak inspiratory pressures on avolume-cycled respirator (mean of 36.5 +/- 2.8 versus 63.9 +/- 6.2 cm of water; P = 0.005). In eight fatal cases of Group B infection, four patients had radiographic features indistinguishable from hyaline-membrane disease whereas the other cases were consistent with neonatal pneumonia. Seven of the eight infected infants had no histologic evidence of coexisting hyaline-membrane disease. Microscopical features of Group B infection included cocci in unevenly distributed hyaline membranes and minimal atelectasis. Group B streptococcal infection differs clinically and pathologically from hyaline-membrane disease. Differentiating clinical features include early apnea and shock and lower inspiratory pressures on mechanical ventilation.

Amelioration of bronchopulmonary dysplasia after vitamin E administration. A preliminary report.

We studied the effect of vitamin E on the development of bronchopulmonary dysplasis in neonates with respiratory-distress syndrome. Twenty infants received vitamin E administered intramuscularly during the acute phase of the syndrome, and 20 infants served as controls. Administration of vitamin E significantly increased the serum vitamin E concentration. Nine vitamin-treated and 13 control patients required supplemental oxygen for longer than 250 hours; all were treated with positive-pressure ventilation and endotracheal continuous distending airway pressure. Six of those 13 controls had x-ray changes consistent with bronchopulmonary dysplasia, and four died. None of the nine vitamin-treated patients had changes characteristic of bronchopulmonary dysplasia (P = 0.046), and all survived. Administration of vitamin E during the acute phase of the respiratory-distress syndrome appears to modify the development of bronchopulmonary dysplasis.

Cardiac hypertrophy in rats with iron and copper deficiency: quantitative contribution of mitochondrial enlargement.

ExtractQuantitative studies of the ultrastructure of heart muscle in iron- and copper-depleted rats show an increased mitochondrial area that contributes to cardiac hypertrophy in both conditions. The mean ratios of mitochondrial/myofibrillar areas are 1.73 and 1.69, respectively, in the deficient groups compared with 0.70 in control animals. The markedly enlarged mitochondria appear to displace and distort the myofibrils. After iron-deficient rats are provided with iron, the reversal of the abnormal mitochondrial/myofibrillar ratio and of cardiac hypertrophy requires about 16 days or approximately twice as long as the complete repair of anemia.In heart muscle from iron-deficient animals, the mitochondrial cytochromes, which all contain iron, remain essentially normal in concentration. In the copper-deficient rats, in contrast, cytochrome a+a3, which contains copper, is depressed to less than one-half the normal concentration. Isolated mitochondria from heart and liver of all animals deficient in iron and copper function normally with respect to respiration and phosphorylation. Thus, a correlation between abnormality of mitochondrial structure, composition, and function is not as yet apparent.The mitochondrial contribution to the cardiac hypertrophy of iron and copper deficiency cannot be attributed entirely to increased work load secondary to anemia, particularly in copper-deficient rats whose cardiac enlargement precedes the development of anemia. The morphologic changes are distinct from those observed in experimental work hypertrophy and can represent a response to the lack of essential precursors required for the cytochromes or other mitochondrial constituents.Speculation: Cardiac hypertrophy in iron and copper deficiency is in part attributable to enlargement of the mitochondrial compartment. This results from the lack of trace metals required for the production of cytochromes or other mitochondrial constituents.

Influence of epidermal growth factor on fetal rat lung development in vitro.

ABSTRACT. Epidermal growth factor (EGF) has been shown to enhance cell multiplication or differentiation in a number of developing tissues. We have examined the effects of this growth factor on the biochemical development of explants of fetal rat lung, cultured in serum-free medium for 48 h. EGF enhanced the rate of choline incorporation into phosphatidylcholine and disaturated phosphatidylcholine in a dose dependent fashion. Half maximal stimulation occurred at a concentration of 1.0 nM, similar to the Kd for EGF binding to rat lung cell membranes. There was also significant stimulation of acetate incorporation into all phospholipids, particularly phosphatidylglycerol (539%), and increased distribution of radioactivity from acetate in this phospholipid fraction. Exposure to EGF stimulated PC synthesis in 18- and 19-day explants (term is 22 days) whereas maximal enhancement of DNA synthesis occurred after this time. This sequence differs from that observed during early embryonic development when EGF initially enhances cell multiplication. An additive interaction with regard to enhancement of PC synthesis was observed with EGF and thyroid hormone, but not EGF and dexamethasone. EGF had no effect on the activity of the enzymes of the choline incorporation pathway of phosphatidylcholine synthesis or on the activity of enzymes involved with acidic phospholipid synthesis. Fetal lung EGF content and EGF binding capacity were not increased by glucocorticoid treatment and similarly glucocorticoid binding capacity was not increased by EGF. These data indicate that EGF enhances fetal rat lung phospholipid synthesis in a dose-dependent manner and suggest that this is a direct effect on the lung tissue mediated by specific receptors.

The Responses of Glutathione and Antioxidant Enzymes to Hyperoxia in Developing Lung

ABSTRACT: Total glutathione levels and the activity of enzymes associated with antioxidant protection in neonatal lung are increased in response to hyperoxia. Glutathione levels in developing rat lung decreased from 24 nmol/mg protein on day 19 of gestation to approximately 12 nmol/mg protein at birth. The initial decrease in glutathione may be due to emergence of other antioxidant systems. Newborn rats placed in 100% oxygen showed a rapid and sustained increase in total glutathione levels which was primarily due to an increase in reduced glutathione. Explants obtained from 16-wk gestation human fetal lung or from 17- to 18-day fetal rat lung also showed increased total and reduced glutathione when cultured in 95% oxygen, 5% CO2 as compared with explants cultured in room air. Type II cells isolated from neonatal rats maintained in oxygen for 6 days also showed glutathione levels twice those found in cells isolated from animals in room air. The activity of antioxidant enzymes (glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase) was increased in lungs of newborn rats exposed to 100% oxygen either at birth or 2 days of age. Antioxidant enzyme activity of lung explants cultured in 95% oxygen, 5% CO2 was also higher than in explants maintained in room air. These results suggest that the increases in glutathione and of antioxidant enzymes in vivo and in vitro are a direct effect of oxygen exposure in lung and that the increase of both glutathione and antioxidant enzyme activity is intrinsic to the lung cell itself. It is likely that increases in glutathione in lung represent an important protective mechanism against oxidant injury.

Characterization of multiple cysteine and cystine transporters in rat alveolar type II cells

Cysteine availability is rate limiting for the synthesis of glutathione, an important antioxidant in the lung. We used rat alveolar epithelial type II cells to study the mechanism of cysteine and cystine uptake. Consistent with carrier-mediated transport, each uptake process was saturable with Michaelis-Menten kinetics and was inhibited at 4°C and by micromolar levels of amino acids or analogs known to be substrates for a specific transporter. A unique system XAG was found that transports cysteine and cystine (as well as glutamate and aspartate, the only substrates previously described for system XAG). We also identified a second Na+-dependent cysteine transporter system, system ASC, and two Na+-independent transporter systems, system xc for cystine and system L for cysteine. In the presence of glutathione at levels measured in rat plasma and alveolar lining fluid, cystine was reduced to cysteine and was transported on systems ASC and XAG, doubling the transport rate. Cysteinylglycine, released from glutathione at the cell surface by γ-glutamyl transpeptidase, also stimulated uptake after reduction of cystine. These findings suggest that, under physiological conditions, cysteine and cystine transport is influenced by the extracellular redox state.

The Phagocytosis-Associated Respiratory Burst in Human Monocytes Is Associated with Increased Uptake of Glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20–30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by γ-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 μM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.

Thyrotropin-Releasing Hormone Increases the Amount of Surfactant in Lung Lavage from Fetal Rabbits

Summary: Administration of thyrotropin-releasing hormone (TRH) to pregnant rabbits at 25 and 26 days of gestation results in increased pulmonary surfactant production by the fetus at 27 days (full term is 31 days). There was 60% more total phospholipid and 150% more phosphatidylcholine (the major component of surfactant) in the lung lavage from the fetuses in the treated group than in that from the controls. Lung lavage from the fetuses in the treated litters contained 13.4 ± 1.6 μg of total phospholipid phosphorus/g lung dry wt and 5.6 ± 1.1 μg of phosphatidylcholine phosphorus while that from the fetuses in the control litters contained only 8.2 ± 1.1 μg and 2.2 ± 0.4 μg, respectively. The phosphatidylcholine/sphingomyelin ratio increased from 1.0 in the lavage from the controls to 2.2 in that from the treated group. These changes in lung lavage phospholipid content and composition are in the direction of increased lung maturation. TRH administration had no effect on the incorporation of choline into phosphatidylcholine in fetal lung slices. These data suggest that TRH stimulates surfactant release rather than synthesis.Speculation: TRH has a physiologic role in fetal lung maturation and surfactant production. It may potentially be used in the prevention of the respiratory distress syndrome in humans.

Effect of chronic hypoxia on glucose transporters in heart and skeletal muscle of immature and adult rats

Glucose transporter (GLUT) modulation can be an important mechanism that contributes to adaptation to hypoxic stress, but little is known about GLUT modulation in heart and skeletal muscle with prolonged hypoxia. In this work, the effect of chronic hypoxia on GLUT-4 and GLUT-1 mRNA and protein was studied in these two tissues in the adult and during development. Hypoxia (fractional inspired O2 = 9 ± 0.5%) was administered to two groups, i.e., an immature group exposed from 3 to 30 days of age and an adult group exposed from 90 to 120 days of age. Rats were then killed and their heart and skeletal muscles were sampled for measurements of GLUT mRNA and protein with Northern and Western blots. In the adult, chronic hypoxia significantly decreased cardiac GLUT mRNA level by >25% of control ( P < 0.05), but had little effect on GLUT protein. A very different hypoxic effect was seen in the immature rat heart with a major increase in protein and no appreciable change in mRNA density. Adult skeletal muscle had no change in GLUT mRNA level but GLUT protein increased (15-20%, P < 0.05) while both GLUT mRNA and protein were significantly increased in the immature skeletal muscles (60-90% over control). We conclude that during chronic O2 deprivation, GLUT-1 and GLUT-4 expressions show a similar pattern but greatly depend on tissue type and age. These differences in GLUT regulation may be due to different strategies for coping with prolonged O2 deprivation in both immature and adult animals.Glucose transporter (GLUT) modulation can be an important mechanism that contributes to adaptation to hypoxic stress, but little is known about GLUT modulation in heart and skeletal muscle with prolonged hypoxia. In this work, the effect of chronic hypoxia on GLUT-4 and GLUT-1 mRNA and protein was studied in these two tissues in the adult and during development. Hypoxia (fractional inspired O2 = 9 +/- 0.5%) was administered to two groups, i.e., an immature group exposed from 3 to 30 days of age and an adult group exposed from 90 to 120 days of age. Rats were then killed and their heart and skeletal muscles were sampled for measurements of GLUT mRNA and protein with Northern and Western blots. In the adult, chronic hypoxia significantly decreased cardiac GLUT mRNA level by > 25% of control (P < 0.05), but had little effect on GLUT protein. A very different hypoxic effect was seen in the immature rat heart with a major increase in protein and no appreciable change in mRNA density. Adult skeletal muscle had no change in GLUT mRNA level but GLUT protein increased (15-20%, P < 0.05) while both GLUT mRNA and protein were significantly increased in the immature skeletal muscles (60-90% over control). We conclude that during chronic O2 deprivation, GLUT-1 and GLUT-4 expressions show a similar pattern but greatly depend on tissue type and age. These differences in GLUT regulation may be due to different strategies for coping with prolonged O2 deprivation in both immature and adult animals.