Oxidation of galactose by galactose-1-phosphate uridyltransferase-deficient lymphoblasts.

The ability of EB virus-transformed lymphoblasts with undetectable galactose-1-phosphate uridyltransferase (GALT) from 15 galactosaemic patients to oxidize [1-(14)C]galactose to 14CO2 was compared to that of cells from 7 normal subjects. The oxidation of galactose but not of glucose was markedly diminished by cells from Q188R homozygous galactosaemic patients but was not absent. After 2.5 h these cells liberated 14CO2 at nearly 3% and at 5 h up to 9% of normal. Cells from patients homozygous for the S135L mutation produced much larger amounts of 14CO2 (15-17% of normal) and were distinguishable from the Q188R homozygous cells. A cell line with a homozygous deletion of the GALT gene oxidized galactose at 7% of the normal rate, suggesting that pathways(s) other than GALT exist in these cells as well as Q188R homozygous cells for oxidation of galactose to CO2. Concentration dependence studies are consistent with the presence of a pathway that is unsaturable or has a very high Km The ability of 10(7) lymphoblasts with the S135L genotype to oxidize more than 7% of the sugar to 14CO2 in 5 h suggests the presence of residual GALT despite the inability to detect the activity by enzymatic analysis.

Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli.

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord.

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP.

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs.

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

Endovascular suture occlusion of the middle cerebral artery in rats: effect of suture insertion distance on cerebral blood flow, infarct distribution and infarct volume.

Sprague-Dawley rats anesthetized with isoflurane, underwent MCA occlusion by intraluminal 3-0 suture insertion, either 22 mm (n = 8) or 18 mm (n = 6) beyond the CCA bifurcation or were sham-operated as controls (n = 3) for autoradiographic analysis of cerebral blood flow. Infarct volume was measured 24 hours after the onset of ischemia (22 mm, n = 11; 18 mm, n = 10); neurological examinations were performed at 6 and 24 hours. Cerebral blood flow in the MCA distribution was significantly lower in the 22 mm suture insertion group than in the 18 mm group (p < 0.05). The total infarct volume was significantly larger (197 +/- 15 versus 135 +/- 19 mm3, p < 0.05) and the coefficient of variance was significantly smaller (23.8% versus 43.9%, p < 0.05) in the 22 mm group. Border zone regions of medial caudoputamen and dorsolateral cortex were often spared in the 18 mm group but never in the 22 mm group. The neurological deficit was more severe in the 22 mm group at 24 hours (p < 0.05), but not at 6 hours. The greater blood flow reduction and the less variable histological damage in dorsolateral cortex (a watershed area between the middle and anterior cerebral arteries) and the greater histological damage in medial caudate in the 22 mm group further characterizes this focal ischemia model for two potential applications: 22 mm insertion for studies requiring extensive and reproducible infarcts; 18 mm insertion for studies requiring less severe and more variable lesions after permanent MCA occlusion.

Expression of zinc finger immediate early genes in rat brain after permanent middle cerebral artery occlusion.

The prolonged expression of the leucine zipper fos/jun immediate early genes (IEG) has been correlated with neuronal death after cerebral ischemia. In this study, the expression of six zinc finger IEG was examined using in situ hybridization in adult rats after middle cerebral artery occlusion (MCAO) with the suture model. NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, and Nurr1 mRNA were all induced throughout the ipsilateral cortex at 1 hour to 12 hours after MCAO. The cortical induction for most of the genes was greatest in the anterior cingulate and the anterior cerebral artery (ACA) and middle cerebral artery (MCA) transition zone. All of the zinc finger IEG were induced at 1 hour in all regions of hippocampus. NGFI-A and NGFI-B were induced in ipsilateral thalamus. Within areas of infarction, the basal IEG mRNA expression, and expression of the housekeeping gene cyclophilin A mRNA, decreased below control levels by 12 hours after the ischemia. Immediate early gene expression outside areas of infarction returned to control levels in most brain regions by 24 hours except for egr-3, which continued to be induced in the MCA/ ACA transition zone for 24 hours, and NGFI-A, which continued to be expressed in specific regions of the thalamus for 72 hours. The induction of these IEG in the cortex is likely caused by ischemia-induced cortical spreading depression, with the hippocampal and thalamic IEG induction being caused by activation of efferent cortical pathways to these regions. The prominent induction of NGFI-B, NGFI-C, egr-2, and egr-3 in the anterior cingulate cortex, the ACA/MCA transition zone, and medial striatum could reflect the ischemic regions around MCA infarcts. The prolonged NGFI-A expression observed in thalamus in this study, and in CA1 of hippocampus after global ischemia in the gerbil in a previous study, suggests that the prolonged NGFI-A, expression could be the result of or the cause of the delayed cell death. Prolonged NGFI-A expression, like c-fos and c-jun, seems to provide a marker for slowly dying neurons.

DNA fragmentation and HSP70 protein induction in hippocampus and cortex occurs in separate neurons following permanent middle cerebral artery occlusions.

DNA nick end-labeling (TUNEL) and heat shock protein (HSP)70 immunocytochemistry were performed on the same brain sections 1 (n = 6), 3 (n = 12), and 7 (n = 7) days following permanent middle cerebral artery (MCA) occlusions produced in adult rats using the endovascular carotid suture method. In the cortex at 1 and 3 days following MCA occlusions, HSP70 immunoreactive neurons were located outside areas of infarction and showed little evidence of DNA fragmentation. HSP70-stained cortical neurons were intermingled with TUNEL cells near the infarct, but extended for greater distances away from the infarct. DNA fragmentation occurred in CA1 hippocampal neurons in 39% of the animals at 1 and 3 days following ipsilateral MCA occlusion. Bilateral DNA fragmentation occurred in CA1 neurons in one subject. HSP70 protein was expressed in CA1 hippocampal neurons in nine of 18 (50%) animals at 1 and 3 days following MCA occlusions, including all animals that exhibited hippocampal DNA fragmentation. Three animals had bilateral expression of HSP70 in CA1 neurons. Cells that stained for either HSP70 protein or DNA fragmentation existed in close proximity to one another. Approximately 5-7% of HSP70-stained cells were TUNEL stained and 3% of TUNEL-positive cells also stained for HSP70. There was no HSP70 staining or DNA fragmentation in the brains of sham-operated controls (n = 4) or in the brains of animals 7 days following MCA occlusions. These data suggest that ischemic cells capable of translating HSP70 protein generally do not undergo DNA fragmentation. These data support the concept that most HSP70 protein-containing neurons in the cortical "penumbra" and hippocampus survive ischemic injury and are "reversibly injured." It is shown that CA1 hippocampal pyramidal neurons die or are reversibly injured in approximately 50% of animals following permanent MCA occlusions. Although the mechanism of this hippocampal injury is unknown, it could relate to transynaptic activation of N-methyl-D-aspartate (NMDA) receptors that mediate induction of early genes in hippocampus.

Maternal insulin manipulations in rats organize body weight and noradrenergic innervation of the hypothalamus in gonadally intact male offspring.

In previous work it has been shown that adult male, but not female, offspring of rats that have been injected with protamine zinc insulin (6 IU/kg) on days 15-20 of gestation, develop significant obesity beginning about 50 days of age. This obesity is accompanied by elevated medial hypothalamic extracellular norepinephrine levels. To examine whether the expression of obesity in male offspring is mediated by perinatal testosterone levels, male offspring of insulin-treated or control dams were either castrated or received sham surgery on postnatal day 1. Castrated male offspring of insulin-treated dams did not become obese like their gonadally intact male littermates. This suggests that perinatal testosterone levels may interact with developmental processes mediating the obesity in male offspring of insulin-treated dams. A second question addressed was whether the elevated hypothalamic extracellular norepinephrine levels observed in our earlier work are evident as morphological changes in norepinephrine-containing systems in the medial hypothalamus and locus coeruleus. We found a significant enhancement of dopamine-beta-hydroxylase immunoreactivity in fibers innervating the paraventricular nucleus of the hypothalamus in 121-day-old, gonadally intact male offspring of insulin-injected dams. This suggests that the impact of maternal insulin injections on offspring obesity may be mediated through its organizing action on feeding-related fibers in the paraventricular nucleus.

Subarachnoid injections of lysed blood induce the hsp70 stress gene and produce DNA fragmentation in focal areas of the rat brain.

BACKGROUND AND PURPOSE: Most experimental studies of subarachnoid hemorrhage have demonstrated little histological evidence of injury. In the present study we examined both the expression of the hsp70 heat-shock gene, a molecular marker of reversible neuronal injury, and DNA fragmentation, a marker of irreversible cell injury and death. METHODS: Lysed blood, whole blood, oxyhemoglobin, bovine serum albumin, and saline were injected into the cisterna magna of adult rats. The induction of hsp70 mRNA and HSP70 heat-shock protein was assessed with the use of in situ hybridization and immunocytochemistry, respectively. Fragmentation of genomic DNA was studied by DNA nick end- labeling with the use of terminal deoxynucleotidyl transferase and biotinylated dATP. RESULTS: Expression of the hsp70 gene was not induced in the brains of rats injected with whole blood, oxyhemoglobin, bovine serum albumin, or saline. Lysed blood injections, however, induced hsp70 mRNA at 6 and 24 hours in the cerebellar hemispheres and in focal regions of the basal forebrain. HSP70 protein was induced by 24 hours and persisted for at least 4 days in the same regions. HSP70 protein was localized to patches of glial cells and occasional neurons in the forebrain. In the cerebellum HSP70 was localized to Bergmann glial cells, granule cells, molecular layer stellate cells, and occasional Purkinje cells. DNA nick end-labeling showed patches of labeled cells in the basal forebrain that occurred in the same regions that hsp70 mRNA was induced. CONCLUSIONS: The results demonstrate focal stress gene induction and DNA fragmentation after subarachnoid hemorrhage. It is hypothesized that the focal areas of hsp70 induction may reflect ischemic injury due to vasospasm produced by lysed blood and/or injury mediated by direct toxic effects of the lysed blood. The hsp70 induction and DNA nick end-labeling in the same regions suggests that lysed blood produces a spectrum of injury from HSP70 protein-labeled, reversibly injured cells to dead cells with fragmented DNA. Induction of the hsp70 stress gene and DNA nick end-labeling may be useful for evaluating the causes of injury, the spectrum of injury, and potential pharmacological therapies in experimental models of subarachnoid hemorrhage.

Hormonal regulation of glutamine metabolism by OK cells.

The precise mechanism(s) of action of PTH, insulin or glucagon in the regulation of renal glutamine and ammonia metabolism is unknown. Our aim was to delineate the effects and the site(s) of action of these hormones on renal glutamine metabolism. Experiments were carried out using OK cells as a model system. Cell cultures were incubated for three hours in a bicarbonate buffer of pH 7.4 supplemented with either 1 mM [2-15N] or [5-15N] glutamine and 10(-7) M PTH, insulin or glucagon. Comparative studies were performed at pH 6.8, 7.4 or 7.6 without hormone. PTH and acute acidosis significantly stimulated glutamine metabolism via both the phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GLDH) pathways. The opposite was observed at pH 7.6. Insulin augmented flux via PDG with little effect on the GLDH pathway. Glucagon had insignificant effects on either PDG or GLDH pathways. Intracellular [15N] glutamate formed from [2-15N] glutamine was removed partially by transamination to alanine, aspartate and serine and partially by translocation to an extracellular compartment. Acidosis, PTH and insulin enhanced the formation of [15N] alanine with little effect on [15N] aspartate. PTH, insulin and glucagon significantly stimulated the production of [15N]serine, whereas acidosis had little effect. The translocation of intracellular glutamate was significantly increased by acidosis, PTH and insulin and decreased by acute alkalosis. The data indicate that: (a) PTH mimicks the effect of acute acidosis on renal glutamine metabolism, that is, augmented glutamine metabolism through both PDG and GLDH pathways and stimulated the output of intracellular glutamate. This effect might be mediated via decreased activity of the Na(+)-H+ exchanger associated with cellular acidification and/or through a second messenger; (b) insulin, but not glucagon, increased glutamine uptake and metabolism, and simultaneously enhanced output of intracellular glutamate sufficiently to stimulate the PDG pathway; and (c) overall, glucagon had little effect on glutamine metabolism by OK cells compared with either PTH or insulin.

Uridine diphosphate hexoses in leukocytes and fibroblasts of classic galactosemics and patients with other metabolic diseases.

To examine uridine diphosphate hexose (UDPhexose) content of cells that have more complete metabolic patterns than erythrocytes, which have been commonly used in the study of galactosemia, the concentrations of uridine diphosphate galactose (UDPgalactose) and uridine diphosphate glucose (UDPglucose) were determined in white blood cells (WBC) and fibroblasts cultured from skin biopsies. Leukocyte UDPgalactose and UDPglucose values were determined in 60 normal individuals, 14 classic galactosemics, and 18 patients with other metabolic diseases on protein-restricted and low-lactose diets. There was no difference in the average concentration of these compounds between any of these groups. There was no relationship between age and WBC UDPhexose content or correlation of WBC and erythrocyte UDPhexose levels in the same blood specimens. WBC from galactosemic individuals differ from their red blood cells because the former do not show the low average UDPgalactose levels and abnormal UDPglucose to UDPgalactose ratio previously reported for erythrocytes from galactose-1-phosphate uridyltransferase-deficient individuals. Fibroblast cell lines from 10 normal and 10 galactosemic individuals, cultured and grown to confluence in glucose medium, also showed no difference in nucleotide sugar concentrations. Thus far, of the cell types easily available, red blood cells appear to be unique in showing an abnormality in nucleotide sugar metabolism. The fact that galactosemic fibroblasts demonstrate no abnormality in the concentration of these compounds suggests that the defective galactosylation that has been observed in galactosemic fibroblasts is not due to unavailability of UDPgalactose.

The effect of glucose and galactose toxicity on myo-inositol transport and metabolism in human skin fibroblasts in culture.

Myo-inositol transport and metabolism were studied in cultured human skin fibroblasts exposed to potentially toxic levels of glucose or galactose. Although variable among 11 different cell lines, the myo-inositol level in confluent cells, ranging from 10-50 nmol/mg protein, was constant with passage. A high-affinity transport system for myo-inositol had an apparent Kt of 55 microM and Vmax of 16 pmol/min/mg protein. No obvious relationship existed between cellular levels and transport capacity. Dependency on sodium was complex. When medium sodium was lowered to 23 mM, myo-inositol uptake ceased after about 1 h. However, the initial rate of myo-inositol uptake only showed a sodium dependence at low myo-inositol concentrations. Both phloretin and phloridzin inhibited myo-inositol uptake. Phloridzin had a Ki of 60 microM, and phloretin was either a noncompetitive or uncompetitive inhibitor. Glucose and galactose were only weak competitive inhibitors, with a Ki of 30 mM and 65 mM, respectively. After 24 h of incubation with myo-[2-3H]inositol, only 10% of the total cell label was incorporated into phospholipid. Compared with control media with 5 mM glucose, the incubation of confluent cells in media with 20 mM glucose had little effect on intracellular glucose and sorbitol, whereas cells incubated in control media supplemented with 5 mM galactose showed a large increase in galactose and polyol levels. In media with more than 200 microM of myo-inositol, neither treatment had an effect on myo-inositol levels after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glutamine on heat-shock-induced mRNA and stress proteins.

Our aim was to delineate the effect of glutamine on the level of heat shock-inducible mRNA and synthesis of stress protein(s) in cultured kidney cells. Experiments were carried out using opossum kidney (OK) cells. The induction of HSP70 mRNA as well as the synthesis of 72,73 kDa stress proteins was evaluated in cell monolayers exposed to 45 degrees C for 15 minutes followed by a recovery period at 37 degrees C for 3 hours. Incubations were performed in Krebs buffer supplemented with 0, 2, 5, or 10 mM glutamine. A separate series of experiments was performed in the presence of glutamine metabolites, such as NH4Cl, glutamate, or aspartate. Glutamine without preincubation at 37 degrees C remarkably increased the steady-state level of HSP70 mRNA as well as the production of 72,73 kDa stress proteins in a dose-dependent manner. The production of stress protein(s) in the presence of glutamine was associated with decreased percent LDH efflux, suggesting cytoprotective action of glutamine in cultured kidney cells. However, when OK cells were preincubated for 1 hour at 37 degrees C with 10 mM glutamine, there was an approximately fourfold decline in level of HSP70 mRNA compared with experiments in the presence of 10 mM glutamine without preincubation. In addition, metabolites of glutamine, i.e., ammonia and glutamate decreased the level of heat-inducible HSP70 mRNA. Furthermore, aspartate or NH4Cl had little effect on LDH release compared with heat shock experiments, without addition of amino acids. These observations suggest that metabolites of glutamine may blunt the steady-state level of glutamate or HSP70 mRNA. The decreased level of HSP70 mRNA in the presence of NH4Cl may explain the role of ammonia in renal injury and brain toxicity, as well as glutamate excitotoxicity.

myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells.

The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system. Uptake was competitively inhibited by phlorhizin, with a Ki of 50 microM; phloretin was a non-competitive inhibitor, with half-maximal inhibition between 0.2 and 0.5 mM. Glucose was a weak competitive inhibitor, with a Ki of 37 mM; galactose failed to inhibit uptake. A weak dependence on Na+ for the initial rate of uptake was observed at 11 microM myo-inositol. When fetal-bovine-serum (FBS)-supplemented medium, which contained 225 microM myo-inositol, was used, the cells contained about 200 nmol of myo-inositol/mg of DNA. With adult-bovine-serum (ABS)-supplemented medium, which contained 13 microM myo-inositol, the cells contained about 110 nmol/mg of DNA. Transport of 11 microM myo-[2-3]inositol was 18 and 125 pmol/min per mg of DNA for cells grown in FBS and ABS respectively. Kinetic analysis showed that for the cells grown in FBS the Vmax. of the high-affinity system was decreased by 64%, whereas the Kt remained essentially unchanged. Increased cell myo-inositol levels were not associated with an increased rate of phosphatidylinositol synthesis. After prolonged exposure of fetal endothelial cells to a myo-inositol concentration which approximated to a high fetal as opposed to a low adult blood level, cell myo-inositol levels doubled and high-affinity transport underwent down-regulation.

A mechanism of glycine and alanine cytoprotective action: stimulation of stress-induced HSP70 mRNA.

Studies done both in vitro and in vivo have shown that glycine and alanine protect kidney cells from stress injury. However, the mechanism(s) of this cytoprotection is unknown. Our aim was to test the hypothesis that the cytoprotective action is in part due to stimulation of gene(s) expression encoding stress protein synthesis. Experiments were carried out using heat shock as a model for stress in the opossum kidney cell line (OK cells). The induction of HSP70 mRNA was evaluated in cell monolayers exposed to 45 degrees C for 15 minutes followed by a recovery period at 37 degrees C for either 0.5, 1, 2, 3, 4, 6 or 24 hours. The results demonstrate that the maximum level of HSP70 mRNA occurred at approximately three hours after heat treatment. Although the mRNA levels declined thereafter, appreciable amounts were still seen even 24 hours after heat-shock. To examine the effect of glycine or alanine on HSP70 mRNA levels and on the synthesis of stress protein, cultures were preincubated for 30 minutes with Krebs-Henseleit buffer, pH 7.4, supplemented with either 1, 2, 5 or 10 mM glycine or alanine, or with no added amino acids. Comparative studies were performed with 10 mM glutamate, aspartate, arginine or leucine. Following preincubation, cultures were heat-shocked (45 degrees C for 15 min) and then reincubated at 37 degrees C for three hours. Both glycine and alanine enhanced the level of HSP70 mRNA and the synthesis of 72, 73 kDa stress proteins, but neither amino acid induced HSP70 mRNA without concomitant heat treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences between OK and LLC-PK1 cells: cystine handling.

Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.

Analysis and physiological implications of renal 2-oxoglutaramate metabolism.

The relative significance of the flux through the glutamine aminotransferase (glutaminase II) pathway to renal ammoniagenesis is poorly understood. A basic and unresolved question is whether 2-oxoglutaramate (2-OGM), a product of the glutaminase II reaction, is deamidated to yield 2-oxoglutarate and NH3, or whether 2-OGM accumulates as an unreactive lactam, depending on the environmental pH. In the current studies we utilized 13C n.m.r. as well as 15N n.m.r. as well as 15N n.m.r. to demonstrate that 2-OGM occurs as a lactam, i.e. 5-hydroxypyroglutamate, regardless of the environmental pH. Our additional aims were to determine whether human kidney cells (HK cells) in culture can produce 2-OGM and to ascertain a pH-dependent relationship between NH3 and 2-OGM production from glutamine. We therefore developed an isotope dilution assay for 2-OGM utilizing 5-hydroxy[4-13C,1-15N]pyroglutamate as the labelled species. Incubations of HK cells in minimal essential medium supplemented with 1 mM-[2-15N]glutamine demonstrated significantly higher production of 2-OGM at pH 6.8 and lower production at pH 7.6 compared with pH 7.4. Similarly both 15NH3 and [15N]alanine formation were significantly higher in acute acidosis (pH 6.8) and lower in acute alkalosis (pH 7.6) compared with that at physiological pH. Addition of 1 mM-amino-oxyacetate to the incubation medium at pH 7.4 significantly diminished [15N]alanine and 2-OGM production, but the production of 15NH3 via the glutamate dehydrogenase pathway was significantly stimulated. The current observations indicate that the glutaminase II pathway plays a minor role and that flux through glutamate dehydrogenase is the predominant site for regulation of ammoniagenesis in human kidney.

Cystine and dibasic amino acid uptake by opossum kidney cells.

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.