Search

The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.

Glutamine is the most abundant free amino acid in the human body. With an arterial plasma concentration of 0.5–0.8 mM, the circulating pool of glutamine constitutes 20%–25% of the plasma amino acids. This chapter provides a brief overview of the interorgan metabolism of glutamine followed by a more detailed discussion of the biosynthesis, kinetic and biophysical properties, and the regulation of the various glutaminase isoforms. It discusses the role of glutaminase in the tissue-specific metabolism of glutamine and presents some insight for future research. Most of the ingested glutamine is rapidly catabolized within the epithelial cells of the small intestine. The exogenous glutamine that is absorbed intact is catabolized primarily in the liver. Therefore, the abundant pool of glutamine within the body is maintained largely through de novo synthesis, which is catalyzed by glutamine synthetase. In mammals, glutamine synthetase is a cytosolic enzyme.

Ammoniagenesis and gluconeogenesis are prominent metabolic features of the renal proximal convoluted tubule that contribute to maintenance of systemic acid-base homeostasis. Molecular analysis of the mechanisms that mediate the coordinate regulation of the two pathways required development of a cell line that recapitulates these features in vitro. By adapting porcine renal epithelial LLC-PK1cells to essentially glucose-free medium, a gluconeogenic subline, termed LLC-PK1-FBPase+cells, was isolated. LLC-PK1-FBPase+cells grow in the absence of hexoses and pentoses and exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase. The cells also express significant levels of the key gluconeogenic enzymes, fructose-1,6-bisphosphatase (FBPase) and phospho enolpyruvate carboxykinase (PEPCK). Thus the altered phenotype of LLC-PK1-FBPase+cells is pleiotropic. Most importantly, when transferred to medium that mimics a pronounced metabolic acidosis (9 mM HCO3−, pH 6.9), the LLC-PK1-FBPase+cells exhibit a gradual increase in NH4+ion production, accompanied by increases in glutaminase and cytosolic PEPCK mRNA levels and proteins. Therefore, the LLC-PK1-FBPase+cells retained in culture many of the metabolic pathways and pH-responsive adaptations characteristic of renal proximal tubules. The molecular mechanisms that mediate enhanced expression of the glutaminase and PEPCK in LLC-PK1-FBPase+cells have been extensively reviewed. The present review describes novel properties of this unique cell line and summarizes the molecular mechanisms that have been defined more recently using LLC-PK1-FBPase+cells to model the renal proximal tubule. It also identifies future studies that could be performed using these cells.

Metabolic acidosis is a common clinical condition that is caused by a decrease in blood pH and bicarbonate concentration. Increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis. A workflow was developed to characterize the mitochondrial proteome of the proximal convoluted tubule. Based upon the increase in specific activity of cytochrome c oxidase, the isolated mitochondria were enriched eightfold. Two-dimensional liquid chromatography coupled with mass spectrometry was utilized to compare mitochondrial-enriched samples from control and chronic acidotic rats. Proteomic analysis identified 901 proteins in the control and acidotic samples. Further analysis identified 37 peptides that contain an N-ε-acetyl-lysine; of these, 22 are novel sites. Spectral counting analysis revealed 33 proteins that are significantly altered in abundance in response to chronic metabolic acidosis. Western blot analysis was performed to validate the calculated changes in abundance. Thus the current study represents the first comprehensive analysis of the mitochondrial proteome of the rat renal proximal convoluted tubule and its response to metabolic acidosis.

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH4+and HCO3−ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMVPCT) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na+-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na+-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMVPCTisolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.

Human immunodeficiency virus (HIV) induces a neurological disease culminating in frank dementia referred to as HIV-associated dementia (HAD). Neurotoxins from HIV-1-infected and activated mononuclear phagocytes contribute to the neuropathogenesis of HAD. Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS) and functions through activation of multiple receptors. Excessive glutamate production by HIV-infected macrophages in HAD may contribute to neuronal injury. Our previous studies have suggested that mitochondrial glutaminase is responsible for the excessive production of glutamate. However, how HIV-1 infection regulates glutamate over-production remains unclear. In this study, we propose that HIV infection-induced oxidative stress contributes to mitochondrial glutaminase release, which results in the excessive production of glutamate and subsequent neuronal injury. We collected conditioned media from HIV-1 infected macrophages and analyzed glutamate concentration in the media by RP-HPLC, and found that the cyclosporine A (CsA), an inhibitor of HIV-1 replication and mitochondrial permeability transition pore, and N-acetylcysteine (NAC), a remover of reactive oxygen species (ROS), not only blocked the excessive glutamate production, but also decreased the glutamate-mediated neurotoxicity. In addition, HIV-infection-induced ROS generation was accompanied with the excessive glutamate production, suggesting that oxidative stress was involved in glutamate regulation. Using the isolated rat brain mitochondria as an ex vivo model and over-expressing GFP-glutaminase fusion protein in mammalian cells as a cell model, we confirm oxidative stress-mediated mitochondrial glutaminase release during HIV-1 infection contributes to glutamate over-production and the subsequent neurotoxicity. These results may provide insight into HAD pathogenesis and a therapeutic strategy for HAD treatment.

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK1-F+cells with an acidic medium (pH 6.9, 9 mM HCO3−). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO3−) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3′-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3′-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK1-F+cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.

The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMVCTX) and from purified proximal convoluted tubules (BBMVPCT). Both proteomic data and Western blot analysis indicate that the BBMVCTXcontain apical membrane proteins from cortical cells other than the proximal tubule. This heterogeneity was greatly reduced in the BBMVPCT. Proteomic analysis identified 193 proteins common to both samples, 21 proteins unique to BBMVCTX, and 57 proteins unique to BBMVPCT. Spectral counts were used to quantify relative differences in protein abundance. This analysis identified 42 and 50 proteins that are significantly enriched (p values ≤0.001) in the BBMVCTXand BBMVPCT, respectively. These data were validated by measurement of γ-glutamyltranspeptidase activity and by Western blot analysis. The combined results establish that BBMVPCTare primarily derived from the proximal convoluted tubule (S1 and S2 segments), whereas BBMVCTXinclude proteins from the proximal straight tubule (S3 segment). Analysis of functional annotations indicated that BBMVPCTare enriched in mitochondrial proteins and enzymes involved in glucose and organic acid metabolism. Thus the current study reports a detailed proteomic analysis of the brush-border membrane of the rat renal proximal convoluted tubule and provides a database for future hypothesis-driven research.

Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV-1 infection of cultured MP, a process dependent upon the glutamate-generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage-mediated neurotoxicity. Potential mechanisms of glutaminase-mediated excitotoxicity including enzyme up-regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV-infected human primary macrophage revealed up-regulation of the glutaminase isoform GAC, yet identified no changes in the kidney-type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

The renal response to metabolic acidosis is mediated, in part, by increased expression of the genes encoding key enzymes of glutamine catabolism and various ion transporters that contribute to the increased synthesis and excretion of ammonium ions and the net production and release of bicarbonate ions. The resulting adaptations facilitate the excretion of acid and partially restore systemic acid-base balance. Much of this response may be mediated by selective stabilization of the mRNAs that encode the responsive proteins. For example, the glutaminase mRNA contains a direct repeat of 8-nt AU sequences that function as a pH-response element (pHRE). This element is both necessary and sufficient to impart a pH-responsive stabilization to chimeric mRNAs. The pHRE also binds multiple RNA-binding proteins, including zeta-crystallin (zeta-cryst), AU-factor 1 (AUF1), and HuR. The onset of acidosis initiates an endoplasmic reticulum (ER)-stress response that leads to the formation of cytoplasmic stress granules. zeta-cryst is transiently recruited to the stress granules, and concurrently, HuR is translocated from the nucleus to the cytoplasm. On the basis of the cumulative data, a mechanism for the stabilization of selective mRNAs is proposed. This hypothesis suggests multiple experiments that should define better how cells in the kidney sense very slight changes in intracellular pH and mediate this essential adaptive response.

The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or γ-glutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a Ki of approx. 3 μM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.

Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic neuron.

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

Phospho enolpyruvate carboxykinase (PEPCK) catalyzes a rate-limiting step in hepatic and renal gluconeogenesis. In the kidney, PEPCK expression is enhanced during metabolic acidosis and in response to ANG II and parathyroid hormone. The effect of the latter hormone is mediated, in part, by cAMP. Treatment of subconfluent cultures of LLC-PK1-F+cells, a gluconeogenic line of porcine proximal tubule-like cells, with cAMP produces a pronounced increase in the level of PEPCK mRNA. The luciferase activity of pLuc/3′-PCK-1, a reporter construct that contains the 3′-UTR of the PEPCK mRNA, was increased three- to fourfold by coexpression of the catalytic subunit of protein kinase A (PKA). This result indicates that cAMP-dependent stabilization may contribute to the increased expression of PEPCK mRNA in LLC-PK1-F+cells. Various pLuc/3′ constructs containing different segments of the 3′-UTR of PEPCK mRNA were used to map the cAMP response to two segments that were previously shown to bind AUF1 and to function as instability elements. A tetracycline-responsive promoter system was used to quantify the effect of forskolin on the half-lives of chimeric β-globin-PEPCK (TβG-PCK) mRNAs. The half-life of the labile βG-PCK-1 mRNA was increased eightfold by addition of forskolin. In contrast, the half-lives of the constructs containing the individual instability elements were increased only twofold. Therefore, the multiple instability elements present within the 3′-UTR may function synergistically to mediate both the rapid degradation and the cAMP-induced stabilization of PEPCK mRNA. The latter process may result from a PKA-dependent phosphorylation of AUF1.

Metabolic acidosis is partially compensated by a pronounced increase in renal catabolism of glutamine. This adaptive response is sustained, in part, through increased expression of phosphoenolpyruvate carboxykinase (PEPCK). Previous inhibitor studies suggested that the pH-responsive increase in PEPCK mRNA in LLC-PK1-FBPase+ cells is mediated by a p38 mitogen-activated protein kinase (MAPK). These cells express high levels of the upstream kinase MAPK kinase (MKK) 3 but relatively low levels of the alternative upstream kinase MKK6. To firmly establish the role of the p38 MAPK signaling pathway, clonal lines of LLC-PK1-FBPase+ cells that express constitutively active (ca) and dominant negative (dn) forms of MKK3 and MKK6 from a tetracycline-responsive promoter were developed. Western blot analyses confirmed that 0.5 microg/ml doxycycline was sufficient to block transcription and that removal of doxycycline led to pronounced and sustained expression of the caMKKs and dnMKKs. Expression of caMKK6 (but not caMKK3) caused an increase in phosphorylation of p38 MAPK and an increase in the level of PEPCK mRNA that closely mimicked the effect of treatment with acidic medium (pH 6.9, 10 mm HCO3-). Only caMKK6 activated transcription of a PEPCK-luciferase reporter construct. Co-expression of both dnMKKs blocked the increases in phosphorylation of p38 MAPK and PEPCK mRNA. The latter effect closely mimicked that of the p38 MAPK inhibitor SB203580. The expression of either dnMKK3 or dnMKK6 was less effective than co-expression of both dnMKKs. Thus, the pH-responsive increase in PEPCK mRNA in the kidney is mediated by the p38 MAPK signaling pathway and involves activation of MKK3 and/or MKK6.

Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric beta-globin-PEPCK (betaG-PCK) mRNAs in LLC-PK -F(+) cells. The control betaG mRNA was extremely stable (t(1/2) = 5 days). However, betaG-PCK-1 mRNA, which contains the entire 3'-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the betaG-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3'-end of the 3'-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric betaG-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the betaG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The betaG-PCK-3 mRNA that contains the 3'-half of 3'-UTR was degraded with the same half-life. Surprisingly, the betaG-PCK-2 mRNA, containing the 5'-end of the 3'-UTR, was also degraded rapidly (t((1/2)) = 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3'-UTR that participate in the rapid turnover of the PEPCK mRNA.

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV-1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP-mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte-derived macrophage (MDM) supernatants by reverse phase high-performance liquid chromatography (RP-HPLC). Measurements were taken of supernatants from MDM infected with multiple HIV-1 strains including ADA and DJV (macrophage tropic, M-tropic), and 89.6 (dual tropic). High levels of glutamate were produced by MDM infected with M-tropic viruses. AZT, an inhibitor of HIV-1 replication, inhibited glutamate generation, demonstrating a linkage between HIV-1 infection and enhanced glutamate production. In our culture system, glutamate production was dependent upon the presence of glutamine and was inhibited by 6-diazo-5-oxo-L-norleucine, a glutaminase inhibitor. Supernatants collected from HIV-1-infected MP generated more glutamate following glutamine addition than supernatants isolated from uninfected MP. These findings implicate the involvement of a glutamate-generating enzyme, such as phosphate-activated mitochondrial glutaminase (PMG) in MP-mediated glutamate production.

LLC-PK1-FBPase+cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK1-FBPase+cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phospho enolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK1-FBPase+cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38α isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38α-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK1-FBPase+cells.

Phospho enolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK−2300Luc reporter constructs in LLC-PK1-F+cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK1-F+cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPβ but not for C/EBPα or C/EBPδ. Treatment of cells with cAMP did not affect the expression of C/EBPβ, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK−2300Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPβ, but not of C/EBPα or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPβ functions as a dominant negative inhibitor. Thus the binding of C/EBPβ to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.

During normal acid-base balance, the kidney extracts very little of the plasma glutamine. However, during metabolic acidosis, as much as one third of the plasma glutamine is extracted and metabolized in a single pass through this organ. The substantial increase in renal utilization occurs solely within the proximal convoluted tubule and is sustained by compensating adaptations in the intraorgan metabolism of glutamine. The primary pathway for renal glutamine metabolism involves its transport into mitochondria and its deamidation and deamination by glutaminase (GA) and glutamate dehydrogenase (GDH), respectively. The resulting ammonium ions are excreted predominantly in the urine where they function as expendable cations to facilitate the excretion of acids. The resulting alpha-ketoglutarate is further metabolized to phosphoenolpyruvate and subsequently to glucose or CO2. The intermediate steps yield two bicarbonate ions that are selectively transported into the venous blood to partially compensate the metabolic acidosis. In rat kidney, this adaptation is sustained in part by the cell-specific induction of the glutaminase that results primarily from stabilization of the GA mRNA. The 3'-nontranslated region of the GA mRNA contains a direct repeat of an 8-base AU-sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for zeta (z)-crystallin. The same protein binds to two separate, but homologous, 8-base AU-sequences within the 3'-nontranslated region of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs.

Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK(1)-FBPase(+) cells demonstrated that a single 8-base AU sequence is both necessary and sufficient to function as a pH response element (pH RE). A biotinylated oligoribonucleotide containing the direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with crude extracts and correlated with the elution of a 36-kDa protein. Microsequencing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta-crystallin/NADPH:quinone reductase. The purified protein contained eight tryptic peptides that were identical to sequences found in mouse zeta-crystallin and three peptides that differed by only a single amino acid. The observed differences may represent substitutions found in the rat homolog. A second protein purified by this protocol was identified as T-cell-restricted intracellular antigen-related protein (TIAR). However, the purified TIAR neither bound nor affected the binding of zeta-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to zeta-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus, zeta-crystallin/NADPH:quinone reductase is a pH response element-binding protein.

Phosph oenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3′-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3′-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3′-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

The pH-responsive stabilization of the rat renal glutaminase (GA) mRNA during metabolic acidosis is mediated by a pH-response element (pH-RE). The primary pH-RE within the GA mRNA consists of a direct repeat of an 8-base adenosine and uridine-rich sequence, which binds a specific cytosolic protein, the pH-response element binding protein (REBP). The functional analysis of this system was performed in LLC-PK(1)-F(+) cells, a pH-responsive line of porcine proximal tubule-like cells. Cytosolic extracts of LLC-PK(1)-F(+) cells also contain a protein that binds with high affinity to the rat GA mRNA pH-RE. The apparent binding of this protein is increased threefold in cytosolic extracts prepared from LLC-PK(1)-F(+) cells that were grown in acidic medium (pH = 6.9, HCO(3)- = 10 mM). Extracts prepared from the renal cortex of rats that were made acutely acidotic also exhibit a similar increase in binding to the RNA probe that contains the direct repeat of the pH-RE. The temporal increase in binding correlates with the temporal increase in GA mRNA. Scatchard analysis indicates that the increased binding is due to an increase in both the affinity and the maximal binding of the pH-REBP. Thus, increased binding of the pH-REBP to the GA mRNA may initiate its stabilization and increased expression during acidosis.

The Na+/K+/2Cl- cotransporter (BSC1/NKCC2) is the major transporter mediating sodium chloride and ammonium absorption in the medullary thick ascending limb. A loss-of-function mutation of BSC1 is responsible for Bartter's syndrome. We previously showed both in vivo and in vitro that acidosis increases the expression and activity of BSC1 and that acid pH enhances the stability of BSC1 mRNA by mechanisms involving its 3'-untranslated region (UTR). zeta-Crystallin is a pH response factor that protects the mitochondrial glutaminase mRNA by a specific interaction with AU-rich motifs. Here we identified the molecular determinant(s) within the 3'-UTR that are responsible for BSC1-mRNA expression and assessed the involvement of zeta-crystallin in this regulation. Deleting three out of six conserved AU-rich motifs drastically reduced the expression of BSC1-mRNA with maximal effect for motif 3 at position 870 of the 3'UTR. This motif was responsible for pH and zeta-crystallin-induced stability of BSC1 mRNA. The abundance of zeta-crystallin was increased by acid pH and its overexpression increased the stability of BSC1 mRNA, but its RNA silencing inhibited acid pH-induced BSC1 expression. Therefore the 3'UTR of BSC1-mRNA is a target for zeta-crystallin. The induction of zeta-crystallin by an acid pH plays an important role in preventing BSC1 mRNA decay, thus increasing its expression and activity.

Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.

The human kidneys produce approximately 160-170 L of ultrafiltrate per day. The proximal tubule contributes to fluid, electrolyte, and nutrient homeostasis by reabsorbing approximately 60%-70% of the water and NaCl, a greater proportion of the NaHCO3, and nearly all of the nutrients in the ultrafiltrate. The proximal tubule is also the site of active solute secretion, hormone production, and many of the metabolic functions of the kidney. This review discusses the transport of NaCl, NaHCO3, glucose, amino acids, and two clinically important anions, citrate and phosphate. NaCl and the accompanying water are reabsorbed in an isotonic fashion. The energy that drives this process is generated largely by the basolateral Na(+)/K(+)-ATPase, which creates an inward negative membrane potential and Na(+)-gradient. Various Na(+)-dependent countertransporters and cotransporters use the energy of this gradient to promote the uptake of HCO3 (-) and various solutes, respectively. A Na(+)-dependent cotransporter mediates the movement of HCO3 (-) across the basolateral membrane, whereas various Na(+)-independent passive transporters accomplish the export of various other solutes. To illustrate its homeostatic feat, the proximal tubule alters its metabolism and transport properties in response to metabolic acidosis. The uptake and catabolism of glutamine and citrate are increased during acidosis, whereas the recovery of phosphate from the ultrafiltrate is decreased. The increased catabolism of glutamine results in increased ammoniagenesis and gluconeogenesis. Excretion of the resulting ammonium ions facilitates the excretion of acid, whereas the combined pathways accomplish the net production of HCO3 (-) ions that are added to the plasma to partially restore acid-base balance.

The proximal convoluted tubule is the primary site of renal fluid, electrolyte and nutrient reabsorption, processes that consume large amounts of ATP. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule. Separation by liquid chromatography coupled with analysis by tandem mass spectrometry (LC-MS/MS) confidently identified 206 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N-ε-acetyl-lysine, 7 of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron. The mass spectrometry data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD000121.

Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.

The maintenance of systemic acid-base balance within relatively narrow limits is essential for life. However, the development of metabolic acidosis, which is characterized by a significant decrease in plasma pH and bicarbonate ions, is a common clinical condition. Metabolic acidosis is caused by the overproduction of acid due to a high protein diet, increased catabolism of endogenous proteins during prolonged fasting, sepsis or cachexia, and various alterations in metabolism such as diabetic ketoacidosis, lactic acidosis, or genetically determined acidurias. It also results from loss of base due to excessive diarrhea or renal defects in bicarbonate reabsorption. The onset of metabolic acidosis triggers an essential adaptive response in the kidney that is characterized by a pronounced increase in catabolism of plasma glutamine and an increased synthesis of bicarbonate and ammonium ions that occur predominantly within the proximal convoluted tubule. These adaptations are sustained, in part, by increased expression of the genes that encode a basolateral glutamine transporter, the mitochondrial glutaminase and glutamate dehydrogenase, and the cytoplasmic phospho enol pyruvatecarboxykinase and by activation of the mitochondrial glutamine transporter, the apical Na + /H + exchanger, and the basolateral Na + -3HCO 3 – co-transporter. The resulting increases in the corresponding activities facilitate the basolateral uptake of glutamine, an increased reabsorption of bicarbonate ions, the increased synthesis of ammonium and bicarbonate ions, and their vectoral transport across the apical and basolateral membranes, respectively. Nearly 80% of the generated ammonium ions are subsequently reabsorbed within the medullary thick ascending limb to produce a high interstitial concentration of ammonium ions within the renal medulla. This gradient provides the driving force for the final transport of ammonium ions into the urine that occurs via specific ammonia channels located within the basolateral and apical membranes of the collecting ducts. All of these steps are finely regulated to ensure that the levels of renal ammonium ion production and excretion are appropriate to sustain normal acid–base balance.

The onset of metabolic acidosis causes an increased transcription of the renal phosphoenolpyruvate carboxykinase (PCK) gene. When transgenic mice carrying a bovine growth hormone (bGH) gene driven by the -460 to +73 segment of the PCK promoter were made chronically acidotic, the bGH mRNA was increased twofold after 4 days. Confluent and well-differentiated cultures of LLC-PK1-F+ cells exhibit a 2.5-fold increase in PCK mRNA when transferred to acidic media (pH 6.9, 10 mM HCO3-) for 16 h. Confluent cultures transfected with PCK-490 CAT exhibit an increase (3.5-fold) in chloramphenicol acetyltransferase (CAT) activity when shifted to acidic medium for 48 h. Mutation or deletion of the P2 element causes a four- to fivefold decrease in basal CAT activity but does not affect the pH response. In contrast, mutations of the P3(II) element or the CRE-1 cAMP-response element have little effect on basal activity but cause a 50% decrease in the pH response. Other deletions or mutations have little effect on either activity. Thus changes in the activity or levels of the protein(s) in the renal proximal tubule that binds to the P3(II) and CRE-1 elements may mediate increased transcription of the PCK gene during metabolic acidosis.

LLC-PK1-F+ cells are porcine proximal tubule-like cells that have been used to model the renal ammoniagenic response to metabolic acidosis. A 3.2-kb porcine glutaminase (GA) cDNA (pGA201) containing 528 bp of coding sequence and 2.7 kb of 3'-untranslated region was cloned and sequenced. Probes derived from both porcine and rat GA cDNAs were used to characterize the expression of putative GA mRNAs in LLC-PK1-F+ cells. Two larger putative GA mRNAs (approximately 5.0 and 4.5 kb in length) were resolved and a smaller 2.5-kb species was also observed. The level of the 5.0-kb mRNA is detectable in freshly split LLC-PK1-F+ cells and increases as the cells reach confluence. In contrast, the amount of the 4.5-kb GA mRNA is greatest in freshly split cells and decreases gradually as the cells approach confluence. The levels of the 5.0- and 2.5-kb mRNAs are also affected by refeeding the cells, and the 2.5-kb mRNA accumulates to high levels if cells are retained in the same media for 4 days. Exposure to acidic media had little or no effect on the levels of GA mRNAs expressed in confluent or postconfluent cells, whereas, in growing and undifferentiated cells, this treatment did affect the level of the 4.5-kb mRNA. Thus the putative GA mRNA species are differentially expressed. Given this complexity, a careful assessment of GA mRNA species, of basal expression, and of growth conditions are essential for a meaningful analysis of GA mRNA levels in cultured cells.

Glutamine is synthesized primarily in skeletal muscle, lungs, and adipose tissue. Plasma glutamine plays an important role as a carrier of nitrogen, carbon, and energy between organs and is used for hepatic urea synthesis, for renal ammoniagenesis, for gluconeogenesis in both liver and kidney, and as a major respiratory fuel for many cells. The catabolism of glutamine is initiated by either of two isoforms of the mitochondrial glutaminase. Liver-type glutaminase is expressed only in periportal hepatocytes of the postnatal liver, where it effectively couples ammonia production with urea synthesis. Kidney­type glutaminase is abundant in kidney, brain, intestine, fetal liver, lymphocytes, and transformed cells, where the resulting ammonia is released without further metabolism. The two isoenzymes have different structural and kinetic properties that contribute to their function and short-term regulation. Although there is a high degree of identity in amino acid sequences, the two glutaminases are the products of different but related genes. The two isoenzymes are also subject to long-term regulation. Hepatic glutaminase is increased during starvation, diabetes, and feeding a high-protein diet, whereas kidney-type glutaminase is increased only in kidney in response to metabolic acidosis. The adaptations in hepatic glutaminase are mediated by changes in the rate of transcription, whereas kidney-type glutaminase is regulated at a posttranscriptional level.

HIV-1 associated neurocognitive disorders (HAND) develop during progressive HIV-1 infection and affect up to 50% of infected individuals. Activated microglia and macrophages are critical cell populations that are involved in the pathogenesis of HAND, which is specifically related to the production and release of various soluble neurotoxic factors including glutamate. In the central nervous system (CNS), glutamate is typically derived from glutamine by mitochondrial enzyme glutaminase. Our previous study has shown that glutaminase is upregulated in HIV-1 infected monocyte-derived-macrophages (MDM) and microglia. However, how HIV-1 leads to glutaminase upregulation, or how glutaminase expression is regulated in general, remains unclear. In this study, using a dual-luciferase reporter assay system, we demonstrated that interferon (IFN) α specifically activated the glutaminase 1 (GLS1) promoter. Furthermore, IFN-α treatment increased signal transducer and activator of transcription 1 (STAT1) phosphorylation and glutaminase mRNA and protein levels. IFN-α stimulation of GLS1 promoter activity correlated to STAT1 phosphorylation and was reduced by fludarabine, a chemical that inhibits STAT1 phosphorylation. Interestingly, STAT1 was found to directly bind to the GLS1 promoter in MDM, an effect that was dependent on STAT1 phosphorylation and significantly enhanced by IFN-α treatment. More importantly, HIV-1 infection increased STAT1 phosphorylation and STAT1 binding to the GLS1 promoter, which was associated with increased glutamate levels. The clinical relevance of these findings was further corroborated with investigation of post-mortem brain tissues. The glutaminase C (GAC, one isoform of GLS1) mRNA levels in HIV associated-dementia (HAD) individuals correlate with STAT1 (p

Microglia represent the main cellular targets of HIV-1 in the brain. Infected and/or activated microglia play a pathogenic role in HIV-associated neurocognitive disorders (HAND) by instigating primary dysfunction and subsequent death of neurons. Although microglia are known to secrete neurotoxins when infected with HIV-1, the detailed mechanism of neurotoxicity remains unclear. Using a human microglia primary culture system and macrophage-tropic HIV-1 strains, we have now demonstrated that HIV-1 infection of microglia resulted in a significant increase in extracellular glutamate concentrations and elevated levels of neurotoxicity. RNA and protein analysis revealed upregulation of the glutamate-generating enzyme glutaminase isoform glutaminase C in HIV-1-infected microglia. The clinical relevance of these findings was further corroborated with investigation of postmortem brain tissues. The glutaminase C levels in the brain tissues of HIV dementia individuals were significantly higher than HIV serum-negative control and correlated with elevated concentrations of glutamate. When glutaminase was subsequently inhibited by siRNA or by a small molecular inhibitor, the HIV-induced glutamate production and the neuronal loss was diminished. In conclusion, these findings support glutaminase as a potential component of the HAND pathogenic process as well as a novel therapeutic target in their treatment.

The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA(124-551)) that lacks either C-terminal sequence was expressed in E.Coli and purified. This construct exhibits a hyperbolic glutamine saturation profile (K(m) of 1.6 mM). BPTES, bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide, functions as a potent uncompetitive inhibitor of this construct (K(i) of 0.2 µM). The hGA(124-551) is inactive in the absence of phosphate, but exhibits a hyperbolic phosphate-dependent activation profile that is also inhibited by BPTES. Gel filtration studies indicate that hGA(124-551) forms a dimer in the absence or presence of 100 mM phosphate, whereas addition of BPTES causes the formation of an inactive tetramer. The combined data indicate that BPTES inhibits human glutaminase by a novel mechanism and that BPTES is a potential lead compound for development of an effective cancer chemotherapeutic agent.

The intent of this review is to provide a broad overview of the interorgan metabolism of glutamine and to discuss in more detail its role in acid-base balance. Muscle, adipose tissue, and the lungs are the primary sites of glutamine synthesis and release. During normal acid-base balance, the small intestine and the liver are the major sites of glutamine utilization. The periportal hepatocytes catabolize glutamine and convert ammonium and bicarbonate ions to urea. In contrast, the perivenous hepatocytes are capable of synthesizing glutamine. During metabolic acidosis, the kidney becomes the major site of glutamine extraction and catabolism. This process generates ammonium ions that are excreted in the urine to facilitate the excretion of acids and bicarbonate ions that are transported to the blood to partially compensate the acidosis. The increased renal extraction of glutamine is balanced by an increased release from muscle and liver and by a decreased utilization in the intestine. During chronic acidosis, this adaptation is sustained, in part, by increased renal expression of genes that encode various transport proteins and key enzymes of glutamine metabolism. The increased levels of phosphoenolpyruvate carboxykinase result from increased transcription, while the increase in glutaminase and glutamate dehydrogenase activities result from stabilization of their respective mRNAs. Where feasible, this review draws upon data obtained from studies in humans. Studies conducted in model animals are discussed where available data from humans is either lacking or not firmly established. Because there are quantitative differences in tissue utilization and synthesis of glutamine in different mammals, the review will focus more on common principles than on quantification.

In the rat, gamma-glutamyltranspeptidase (gamma GT) is transcribed into four unique mRNAs from a single gene by use of at least three different promoters and alternative splicing. For the first time, two distinct full-length cDNAs encoding the protein for rat renal gamma-glutamyltranspeptidase have now been isolated. Characterization by restriction enzyme mapping and nucleotide sequencing indicates that the two cDNAs, corresponding to transcripts I and II, differ only in the 5' noncoding region. However, transcription from promoter I, most proximal to the coding sequence, apparently began 20 bases upstream from the major transcription start previously reported. Since in vitro transcription and translation of these two new gamma GT cDNAs were found to produce a full-length peptide (M(r) approximately 62,000), both cDNAs were used to transfect LLC-PK1 (porcine) cells, a polarized cell line most representative of the renal proximal tubule. Rat gamma GT was expressed in transfected cells as judged by immunofluorescence analysis, direct immunoprecipitation after metabolic labeling with [35S]methionine, and an increase in gamma GT specific enzymatic activity (up to 5-fold). When clonal cell lines (I or II) were grown on Falcon filter inserts, the increased gamma GT activity was found only at the apical surface, consistent with polarized expression of the rat gamma GT. In contrast, transfection of the same cells with cDNA of human growth hormone resulted in both apical (70%) and basal lateral (30%) secretion of the expressed hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat kidney contains 3.5-kb and 2.8-kb mRNAs that encode for glutamate dehydrogenase (GDH). The levels of both mRNAs are increased gradually after onset of chronic metabolic acidosis and reach a maximum induction of 2.5-fold after 7 days. In contrast, during recovery from chronic acidosis, the levels of the GDH mRNAs are returned to normal within 1 day. The development of an acute metabolic acidosis causes a more rapid induction of GDH mRNA. This increase occurs after a 7-h lag and plateaus after 18 h at a level that is threefold greater than normal. A very similar profile was observed after the transfer of LLC-PK-F+ cells from normal medium to an acidic medium containing 10 mM bicarbonate and adjusted to pH 6.9. However, the transfer of cells from acidic to normal medium caused an immediate and rapid [half-life (t) = 1 h] decrease in GDH mRNA. The apparent half-lives of GDH mRNA were measured by treating cells grown in normal (t = 4 h) and acidic media (t = 12 h) with actinomycin D. Thus, increased stability may account for the induction of GDH mRNA that occurs during growth in response to acidosis. The levels of GDH mRNA are independently affected by changes in medium pH or bicarbonate concentration. The levels of GDH mRNA are also increased by treating cells with adenosine 3',5'-cyclic monophosphate, epinephrine, triiodothyronine, or retinoic acid, whereas treatment with angiotensin II, vasopressin, phorbol 12-myristate 13-acetate, or cycloheximide did not produce an increase. The inductive effect of dexamethasone, which is observed in vivo, is not reproduced in the LLC-PK-F+ cells.

A cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase (pGA) was generated by recombination of two cDNAs that were isolated from a random-primed rat brain lambda gt11 library. pGA encodes 674 amino acids which includes an N-terminal sequence of 16 residues that should form an amphipathic helix, typical of a mitochondrial targeting sequence. Residues 73-90 correspond to the N-terminal sequence of the more abundant 65-kDa glutaminase peptide. In vitro transcription and translation of pGA yields a 72-kDa peptide that is immunoprecipitated with glutaminase-specific antibodies. Incubation of the glutaminase precursor with isolated mitochondria yields the 68- and 65-kDa peptides that are characteristic of the mature glutaminase. Thus, the two mature glutaminase peptides are synthesized from a single precursor. The complete 3' nontranslated region of the GA mRNA was characterized by sequencing a GA cDNA (pGA12) that was isolated from an oligo(dT)-primed rat kidney lambda gt10 library. This segment contains numerous AU-rich regions, four potential stem-loop structures, and a 48 base pair repeat of CA dinucleotides. Such domains may contribute to the increased stability of the GA mRNA that occurs in response to metabolic acidosis.

Increased rat renal ammoniagenesis is sustained during chronic metabolic acidosis by the cell-specific induction of the regulatory enzymes of glutamine catabolism and of gluconeogenesis. A glutaminase-specific cDNA hybridizes to 6.0- and 3.4-kb mRNAs that are contained in total or poly(A)+ RNA isolated from rat kidney. When translated in a rabbit reticulocyte lysate, each of the fractionated mRNAs produces the 72-kDa precursor of the mitochondrial glutaminase. The levels of both mRNAs are increased 5-fold within 1 day following onset of chronic acidosis and reach a maximum (8-fold) after 5 days. During recovery from chronic acidosis, the levels of the glutaminase mRNAs are returned to normal within 1 day. The observed changes in mRNA levels correlate with equivalent changes in the relative levels of translatable glutaminase mRNA. Nuclear run-on assays indicate that the rate of transcription of the renal phosphoenolpyruvate carboxykinase gene is increased and decreased in response to onset and recovery from chronic acidosis, respectively. In contrast, the rates of transcription of the glutaminase and beta-actin genes are unaffected by alterations in acid-base balance. Thus, the increase in renal glutaminase activity during chronic acidosis results from an equivalent increase in the levels of total and translatable glutaminase mRNAs which apparently results from an increased stability of the glutaminase mRNA.

During chronic acidosis, the levels of the rat renal mRNAs that encode the mitochondrial glutaminase (GA) and cytosolic phosphoenolpyruvate carboxykinase (PCK) are increased 6-fold. Following acute recovery of chronic acidosis, the levels of the two mRNAs are rapidly and coordinately decreased, returning to normal within 13-17 h. In contrast, the increases in GA and PCK mRNAs during acute onset of acidosis occur with very different kinetics. The increase in PCK mRNA occurs rapidly and reaches a maximum within 7 h, whereas the GA mRNA is increased after a 4-7-h lag and then plateaus at 14-17 h. Treatment with dexamethasone or with cAMP analogs significantly increases the level of renal PCK mRNA but has no effect on the level of GA mRNA. Nuclear run-on experiments indicate that the acute induction of PCK mRNA is primarily due to an increased rate of transcription. However, transcription of GA mRNA is unaffected by acute acidosis. Therefore, the changes in the two mRNAs are temporally coordinated but occur through different mechanisms. Furthermore, the inductive effects of acidosis are not mediated solely through glucocorticoid or cAMP regulatory elements.

A gluconeogenic strain of renal epithelial cells (LLC-PK1-F+) was used to characterize the effect of pH and bicarbonate concentration on the levels of phosphoenolpyruvate carboxykinase (PCK) and glutaminase (GA) mRNAs. The levels of both mRNAs are markedly dependent upon medium glucose concentration. The level of PCK mRNA is increased with increasing glucose concentration from 0 to 40 mM, whereas the level of GA mRNA is maximal between 3 and 5 mM glucose. When LLC-PK1-F+ cells are grown with 5 mM glucose and then subjected to an acute decrease in pH (from 7.4 to 6.9) and bicarbonate concentration (from 25 to 10 mM), the level of PCK mRNA exhibits a biphasic response. The PCK mRNA is initially increased 4-fold within 3 h, then decreases slightly and subsequently increases between 10 and 20 h to a level that is 17-fold greater than normal. Only the initial increase parallels the changes observed in vivo. In contrast, after onset of acidosis, the level of GA mRNA initially remains unchanged, is then increased 8-fold between 10 and 16 h, and then decreases slightly. This response closely mimics the results obtained in vivo. A decrease in media pH at constant bicarbonate causes a marked increase in both mRNAs. However, the levels of the two mRNAs are also elevated by decreasing bicarbonate at a constant pH. Thus, both parameters independently affect the level of the two mRNAs. The use of actinomycin D to measure the half-lives of PCK and GA mRNAs at pH 7.4 and 6.9 indicates that stabilization may fully account for the induction of GA mRNA and contributes to the inductive effects of decreased pH and/or bicarbonate on PCK mRNA. Following recovery from acidic conditions, the two mRNAs exhibit a rapid and coordinate decrease (t1/2 approximately 20 min). Dexamethasone had no effect on the level of either mRNA, whereas cAMP increased only PCK mRNA. The latter effect was additive with the increase caused by decreased pH and/or bicarbonate and was reversed by incubating in alkalotic media. Thus, the induction of PCK and GA mRNAs during acidosis is initiated in direct response to a decrease in extracellular pH and/or bicarbonate.

The increase in intracellular pH (pHi) associated with various tumour cells triggers changes in gene expression. Similar adaptations also occur as part of the physiological response to changes in acid base balance. For example, during metabolic acidosis, increased renal ammoniagenesis and bicarbonate synthesis are sustained by the increased expression of various transport proteins and key enzymes of glutamine metabolism. In rat kidney, increased expression of the mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) results from stabilization of their respective mRNAs. The 3'-untranslated region (UTR) of the GA mRNA contains a direct repeat of an 8-base AU sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for z-crystallin. The same protein binds to two separate, but homologous, 8-base AU sequences within the 3'-UTR of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs. In contrast, induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene occurs at the transcriptional level. In LLC-PK1-FBPase+ kidney cells, a decrease in pHi leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of ATF-2. This transcription factor binds to the CRE-1 element within the promoter of the PEPCK gene to enhance transcription. Similar mechanisms may contribute to altered gene expression in tumour cells.

The amphipathic form of gamma-glutamyltransferase (EC 2.3.2.2) was reconstituted into unilamellar lecithin vesicles and labelled using the membrane-soluble reagent, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ( [125I]TID), which can be photoactivated. Label was incorporated exclusively into the large subunit of the transferase, but no label was found in the enzyme released from the vesicles by partial proteolysis with papain. Chromatography of the papain-treated vesicles on Sephadex LH-60 indicated that the [125I]TID was bound to two peptides of low relative molecular mass. The [125I]-TID-labelled peptides should constitute the hydrophobic membrane-binding domain. The transferase was also labelled by reductive methylation (Lys residues) or with immobilized galactose oxidase and NaB3H4 (Gal residues), incorporated into lecithin vesicles and treated with papain. Both procedures yielded a single [3H]-labelled hydrophobic peptide that remained associated with the vesicles. After chromatography on Sephadex LH-60, the peptide labelled by the latter two procedures corresponds to the larger of the two [125I]TID-labelled peptides. These results suggest that papain may cleave the hydrophobic domain into two peptides. The observed labelling is also consistent with the alternative possibility that the N-terminal hydrophobic domain is preceded by an initial sequence that contains both lysine and carbohydrate residues. The smaller of the two [125I]TID-labelled peptides could be generated by removal of the hydrophilic segment from the transferase molecules that are reconstituted with the N-terminus exposed on the external surface of the vesicles.