Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1ß and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Asparagine sustains cellular proliferation and c­Myc expression in glutamine­starved cancer cells.

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c­Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c­Myc protein expression, leading to a reduction in the levels of the canonical c­Myc isoform and an increase in the expression of c­Myc 1, a c­Myc isoform translated from an in­frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c­Myc expression as in glutamine­fed cells, with high levels of canonical c­Myc and c­Myc 1 almost undetectable. In asparagine­fed cells, global protein translation was higher than in glutamine­starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine­starved asparagine­fed cells, the inhibition of c­Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c­Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.

Hydrogen enriched saline alleviates morphine tolerance via inhibiting neuroinflammation, GLT-1, GS nitration and NMDA receptor trafficking and functioning in the spinal cord of rats.

The development and maintenance of morphine tolerance showed association with neuroinflammation and dysfunction of central glutamatergic system (such as nitration of glutamate transporter). Recent evidence indicated that hydrogen could reduce the levels of neuroinflammation and oxidative stress, but its role in morphine tolerance has not been studied. The rats were intrathecally administered with morphine (10 µg/10 µL each time, twice/day for 5 days). Hydrogen enriched saline (HS) or saline was given intraperitoneally at 1, 3 and 10 mL/kg for 10 min before each dose of morphine administration. The tail-flick latency, mechanical threshold and thermal latency were assessed one day (baseline) before and daily for up to 5 days during morphine injection. The pro-inflammatory cytokine expressions [tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6)] (by western blotting), astrocyte activation (by immunofluorescence and western blotting), and nitration of glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) (by immunoprecipitation), membrane and total expression of N-methyl-d-aspartic acid (NMDA) receptor NR1 and NR2B subunits were carried out in the spinal dorsal horns. Chronic morphine administration induced antinociceptive tolerance, and together led to increased TNF-α, IL-1ß and IL-6 expression, astrocyte activation, GLT-1 and GS nitration, increased membrane and total NR1, NR2B expression. Injection of HS attenuated morphine tolerance in a dose-dependent manner, decreased proinflammatory cytokine expression, inhibited astrocyte activation, decreased GLT-1 and GS nitration, and inhibited membrane trafficking of NMDA receptor. Our result showed that hydrogen pretreatment prevented morphine tolerance by reducing neuroinflammation, GLT-1, GS nitration, NMDA receptor trafficking in the spinal dorsal horn. Pretreatment with hydrogen might be considered as a novel therapeutic strategy for the prevention of morphine tolerance.

Glutamine Synthetase as a Therapeutic Target for Cancer Treatment.

The significance of glutamine in cancer metabolism has been extensively studied. Cancer cells consume an excessive amount of glutamine to facilitate rapid proliferation. Thus, glutamine depletion occurs in various cancer types, especially in poorly vascularized cancers. This makes glutamine synthetase (GS), the only enzyme responsible for de novo synthesizing glutamine, essential in cancer metabolism. In cancer, GS exhibits pro-tumoral features by synthesizing glutamine, supporting nucleotide synthesis. Furthermore, GS is highly expressed in the tumor microenvironment (TME) and provides glutamine to cancer cells, allowing cancer cells to maintain sufficient glutamine level for glutamine catabolism. Glutamine catabolism, the opposite reaction of glutamine synthesis by GS, is well known for supporting cancer cell proliferation via contributing biosynthesis of various essential molecules and energy production. Either glutamine anabolism or catabolism has a critical function in cancer metabolism depending on the complex nature and microenvironment of cancers. In this review, we focus on the role of GS in a variety of cancer types and microenvironments and highlight the mechanism of GS at the transcriptional and post-translational levels. Lastly, we discuss the therapeutic implications of targeting GS in cancer.

Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells.

BACKGROUND: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance. METHODS: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance. CONCLUSIONS: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Macrophage-derived glutamine boosts satellite cells and muscle regeneration.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1-4. Macrophages and satellite cells communicate in different ways1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.

Semisynthesis of the Organoarsenical Antibiotic Arsinothricin.

Arsinothricin [AST (1)], a new broad-spectrum organoarsenical antibiotic, is a nonproteinogenic analogue of glutamate that effectively inhibits glutamine synthetase. We report the chemical synthesis of an intermediate in the pathway to 1, hydroxyarsinothricin [AST-OH (2)], which can be converted to 1 by enzymatic methylation catalyzed by the ArsM As(III) S-adenosylmethionine methyltransferase. This is the first report of semisynthesis of 1, providing a source of this novel antibiotic that will be required for future clinical trials.

Mechanism of Fully Reversible, pH-Sensitive Inhibition of Human Glutamine Synthetase by Tyrosine Nitration.

Glutamine synthetase (GS) catalyzes an ATP-dependent condensation of glutamate and ammonia to form glutamine. This reaction-and therefore GS-are indispensable for the hepatic nitrogen metabolism. Nitration of tyrosine 336 (Y336) inhibits human GS activity. GS nitration and the consequent loss of GS function are associated with a broad range of neurological diseases. The mechanism by which Y336 nitration inhibits GS, however, is not understood. Here, we show by means of unbiased MD simulations, binding, and configurational free energy computations that Y336 nitration hampers ATP binding but only in the deprotonated and negatively charged state of residue 336. By contrast, for the protonated and neutral state, our computations indicate an increased binding affinity for ATP. pKa computations of nitrated Y336 within GS predict a pKa of ∼5.3. Thus, at physiological pH, nitrated Y336 exists almost exclusively in the deprotonated and negatively charged state. In vitro experiments confirm these predictions, in that, the catalytic activity of nitrated GS is decreased at pH 7 and 6 but not at pH 4. These results indicate a novel, fully reversible, pH-sensitive mechanism for the regulation of GS activity by tyrosine nitration.

NF-YA overexpression protects from glutamine deprivation.

The heterotrimeric transcription factor NF-Y binds to CCAAT boxes of genes of glutamine metabolism. We set out to study the role of the regulatory NF-YA subunit in this pathway. We produced U2OS and A549 clones stably overexpressing -OE- the two splicing isoforms of NF-YA. NF-YA OE cells show normal growth and colony formation rates, but they become resistant to cell death upon glutamine deprivation. Increased mRNA and protein expression of the key biosynthetic enzyme GLUL in U2OS entails increased production of endogenous glutamine upon deprivation. The use of GLUL inhibitors dampens the NF-YA-mediated effect. NF-YA OE prevents activation of the pro-apoptotic transcription factor CHOP/DDIT3. Elevated basal levels of SERCA1/2, coding for the molecular target of Thapsigargin, correlate with resistance of NF-YA OE cells to the drug. The work represents a proof-of-principle that elevated levels of NF-YA, as found in some tumor types, helps altering cancer metabolic pathways.

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

Targeting of glutamine transporter ASCT2 and glutamine synthetase suppresses gastric cancer cell growth.

PURPOSE: Glutamine (Gln) is essential for the proliferation of most cancer cells, making it an appealing target for cancer therapy. However, the role of Gln in gastric cancer (GC) metabolism is unknown and Gln-targeted therapy against GC remains scarce. The aim of this study was to investigate the relevance of Gln in GC growth and targeting. METHODS: Expression of Gln transporter ASCT2 and glutamine synthetase (GS) in the parental and molecularly engineered GC cells or in human GC specimens was determined by RT-PCR and western blot analysis or immunohistochemistry. Cell proliferation and survival was assessed by CCK-8 assay and colony formation assay. Intracellular Gln content was measured by a HPLC system. Effects of ASCT2 and/or GS inhibitor on tumor growth were investigated in xenograft models. RESULTS: A significant heterogeneity of GC cells was observed with respect to their response to the treatment of ASCT2 inhibitor benzylserine (BenSer). Gln deprivation did not affect the BenSer-resistant cell growth due to endogenous GS expression, whose inhibition remarkably reduced cell proliferation. The differential in vitro sensitivity correlated with overall intracellular Gln content. Combined therapy with both ASCT2 and GS inhibitors produced a greater therapeutic efficacy than the treatment of either inhibitor alone. Furthermore, 77% human GC tissues were found to express moderate and high levels of ASCT2, 12% of which also co-expressed relatively high levels of GS. CONCLUSION: Gln mediates GC growth and the therapeutic efficacy of Gln-targeted treatment relies on distinct ASCT2 and GS expression pattern in specific gastric cancer groups.

Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-ß-lactam.

Tabtoxinine-ß-lactam (TßL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TßL 3-(S)-hydroxy-ß-lactam (3-HßL) warhead. Phosphorylation of TßL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TßL ß-lactam ring remains intact during enzyme inhibition, making TßL mechanistically distinct from traditional ß-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HßL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement.

A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

Effects of nitrogen metabolism on growth and aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxins (AFs), produced mainly by Aspergillus flavus and Aspergillus parasiticus, are strongly toxic and carcinogenic. Here, we showed that glutamine is the optimal nitrogen source for AF-production in A. flavus grown in Czapek Dox medium. Additionally, 4mM glutamine was the threshold for high production of aflatoxin B1. However, no significant impact of glutamine synthetase inhibitor was detected for on AF biosynthesis. In contrast, rapamycin could significantly suppress the glutamine inducing effect on AFs production, simultaneously inhibiting the fungal growth and conidiation. To identify the genes and regulatory networks involved in AFs biosynthesis, especially concerning the nitrogen source metabolism pathway and the target of rapamycin (TOR) signaling pathway, we obtained transcriptomes for A. flavus under treatment of three nitrogen sources by RNA-sequencing. We identified 1429 differentially expressed genes. Through GO and KEGG pathway analyses, the relationship between nitrogen metabolism and AFs biosynthesis was revealed, and the effects of TOR inhibitor were confirmed. Additionally, the quantitative real-time PCR results verified the credibility and reliability of the RNA-seq data, and were consistent with the other experimental results. Our research laid the foundation for a primary study on the involvement of the nitrogen regulatory network and TOR signaling pathway in AF biosynthesis.

GLUL Promotes Cell Proliferation in Breast Cancer.

Glutamate-ammonia ligase (GLUL) belongs to the glutamine synthetase family. It catalyzes the synthesis of glutamine from glutamate and ammonia in an ATP-dependent reaction. Here, we found higher expression of GLUL in the breast cancer patients was associated with larger tumor size and higher level of HER2 expression. In addition, GLUL was heterogeneously expressed in various breast cancer cells. The mRNA and protein expression levels of GLUL in SK-BR-3 cells were obviously higher than that in the other types of breast cancer cells. Results showed GLUL knockdown in SK-BR-3 cells could significantly decrease the proliferation ability. Furthermore, GLUL knockdown markedly inhibited the p38 MAPK and ERK1/ERK2 signaling pathways in SK-BR-3 cells. Thus, GLUL may represent a novel target for selectively inhibiting p38 MAPK and ERK1/ERK2 signaling pathways and the proliferation potential of breast cancer cells. J. Cell. Biochem. 118: 2018-2025, 2017. © 2016 Wiley Periodicals, Inc.

Pathogenesis of hepatic encephalopathy in cirrhosis: the concept of synergism revisited.

The concept of synergistic mechanisms as the pathophysiologic basis of hepatic encephalopathy started with the pioneering work of Les Zieve in Minneapolis some 60 years ago where synergistic actions of the liver-derived toxins ammonia, methanethiol, and octanoic acid were described. More recently, synergistic actions of ammonia and manganese, a toxic metal that is normally eliminated via the hepatobiliary route and shown to accumulate in brain in liver failure, on the glutamatergic neurotransmitter system were described. The current upsurge of interest in brain inflammation (neuroinflammation) in relation to the CNS complications of liver failure has added a third dimension to the synergy debate. The combined actions of ammonia, manganese and pro-inflammatory cytokines in brain in liver failure result in oxidative/nitrosative stress resulting from activation of glutamate (NMDA) receptors and consequent nitration of key brain proteins. One such protein, glutamine synthetase, the sole enzyme responsible for brain ammonia removal is nitrated and inactivated in brain in liver failure. Consequently, brain ammonia levels increase disproportionately resulting in alterations of brain excitability, impaired brain energy metabolism, encephalopathy and brain swelling. Experimental therapeutic approaches for which proof-of-principle has been established include the NMDA receptor antagonist memantine, N-acetyl cysteine (recently shown to have antioxidant properties at both hepatic and cerebral levels) and probiotics.

Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation.

c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

L-NAME combats excitotoxicity and recuperates neurological deficits in MCAO/R rats.

PURPOSE OF RESEARCH: Since, transient focal cerebral ischaemia exhibits detrimental effect not only during the course of ischaemia but also after the onset of reperfusion, the current study is focussed on identifying the appropriate therapeutic time point at which NG-nitro-l-arginine methyl ester (l-NAME) exerts better neuroprotection. PRINCIPAL RESULTS: Pre-ischaemic administration of l-NAME ameliorated neurological deficits much better than the during ischaemic and post-ischaemic groups. Pre-ischaemic l-NAME has also mitigated glutamate excitotoxicity, increased glutamine synthetase activity, ATP and NAD levels, decreased nitrate/nitrite content, down regulated TNF-α and upregulated IL-10 expressions and reduced the cerebral infarction significantly than the during ischaemic and post-ischaemic groups. MAJOR CONCLUSION: Current study revealed that l-NAME improved neurological deficit at the pre-ischaemic state in transient focal cerebral ischaemia and has also significantly ameliorated glutamate excitotoxicity. Though l-NAME showed neuroprotective effects when administered at during and post-ischaemia (during reperfusion), it exerts considerable neuroprotection when administered pre-ischaemically.