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Glutaminase (GLS) enzyme activity in the L4 DRG at 7 days AIA in the right hindpaw. GLS activity from the DRG of AIA rats (2.83 ± 0.30 moles/kg/hr) was elevated (*P < 0.05) over control values (2.20 ± 0.18 moles/kg/hr).
Source publication
Following inflammation, primary sensory neurons in the dorsal root ganglion (DRG) alter the production of several proteins. Most DRG neurons are glutamatergic, using glutaminase as the enzyme for glutamate production, but little is known about glutaminase following inflammation. In the present study, adjuvant-induced arthritis (AIA) was produced in...
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Glutamate is a neurotransmitter used at both the peripheral and central terminals of nociceptive primary sensory neurons, yet little is known concerning regulation of glutamate metabolism during peripheral inflammation. Glutaminase (GLS) is an enzyme of the glutamate-glutamine cycle that converts glutamine into glutamate for neurotransmission and i...
Citations
... This GLS is activated regionally by calcium and phosphate and, hence, also known as phosphate-activated glutaminase (PAG) [18]. We previously reported that the level of GLS is elevated significantly in DRG cell bodies during chronic inflammation, and peripheral inhibition of GLS reduces sensitization and pain associated with inflammation [15,[19][20][21]. ...
... The levels of GLS were evaluated after 48 h in the L4 and L5 DRGs of rats treated with CFA and selective TrkA inhibitor (GW441756). Almost all the DRG neurons were immunoreactive for GLS, as reported in the previous studies [15,21,24,25]. The GLS-ir was elevated in the animals treated with CFA as compared to naïve rats. ...
... The peripheral inhibition of Rab7GTPase during CFA-induced inflammation decreased the GLS-ir in the L4 and L5 DRGs. The quantitative western blot data show the elevation of GLS after peripheral inflammation, confirming previous findings [19,21,24,26]. After the Rab7 inhibition, the GLS levels were attenuated significantly in the DRGs of AIA rats ( Figure 5). ...
Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.
... This GLS is activated regionally by calcium and phosphate and hence also known as Phosphate-activated Glutaminase (PAG) [17]. We previously reported that the level of GLS is elevated significantly in DRG cell bodies during chronic inflammation, and peripheral inhibition of GLS reduces sensitization and pain associated with inflammation [14,[18][19][20]. ...
... The levels of GLS were evaluated after 48 hours in the L4 and L5 DRG's of rat treated with CFA and selective TrkA inhibitor (GW441756). Almost all the DRG neurons were immunoreactive for GLS, 4 as reported in the previous studies [14,20,23,24]. The GLS-ir was elevated in the animals treated with CFA as compared to naïve rats. ...
... The peripheral inhibition of Rab7GTPase during CFA-induced inflammation decreased the GLSir in the L4 and L5 DRG's. The quantitative western blot data show the elevation of GLS after peripheral inflammation confirming previous findings [18,20,23,25]. After the Rab7 inhibition, the GLS levels were attenuated significantly in the DRG's of AIA rats ( Figure 5). ...
Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. These results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioural data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.
... Ammonia can be produced from urea catalyzed by the enzyme urease, an enzyme presents in some bacteria species, such as Helicobacter pylori, described in human gastric ulcers [28], although in the case of horses, it seems that these bacteria are not involved in EGUS. However, ammonia can also be produced without the need for bacteria and urease by increased activity of the enzyme glutaminase, known to increase with inflammation [29]. Glutamine deamination, produced by the activity of glutaminase, has a central role in the immune pathogenesis of celiac disease and has been found to be implicated in autoimmune responses caused by various toxic substances and different pathogens [30]. ...
Ammonia (NH3) and bicarbonate (HCO3) have been related to gastric ulcers in humans. Ammonia is considered a possible cause of gastric ulcers, whereas bicarbonate has a protective function. The presence of ulcers in the stomach of horses is defined as Equine Gastric Ulcer Syndrome (EGUS), which is a frequent disease in this species, and it has been associated with changes in saliva composition, such as in analytes related to inflammation, immune system and oxidative stress. The objectives of this study were (1) to perform an analytical validation of two automated spectrophotometric assays, one for ammonia and one for bicarbonate, in the horses’ saliva and (2) to evaluate their possible variations with EGUS. Analytical validation of the automated assays for ammonia and bicarbonate in the saliva of horses showed that both assays were precise and accurate. In addition, significantly higher values of ammonia and lower values of bicarbonate were found in the saliva of horses with EGUS compared to healthy horses. It can be concluded that ammonia and bicarbonate can be measured in the saliva of horses and that ammonia increases and bicarbonate decreases in this sample type could be related to the presence of EGUS in this species.
... Thermal stability of EcA constructs was determined by performing asparaginase activity assay at a temperature range from 55 • C to 70 • C. Kinetic properties measured by varying substrate concentrations of AHA in the range of 0-5 mM using the same indooxine assay [28]. Glutaminase activity was measured using a two-step discontinuous coupled enzymatic reaction as described previously [29,30] (Supplemental methods). ...
Introduction
Escherichia coli l-asparaginase (EcA), an integral part of multi-agent chemotherapy protocols of acute lymphoblastic leukemia (ALL), is constrained by safety concerns and the development of anti-asparaginase antibodies. Novel variants with better pharmacological properties are desirable.
Methods
Thousands of novel EcA variants were constructed using protein engineering approach. After preliminary screening, two mutants, KHY-17 and KHYW-17 were selected for further development. The variants were characterized for asparaginase activity, glutaminase activity, cytotoxicity and antigenicity in vitro. Immunogenicity, pharmacokinetics, safety and efficacy were tested in vivo. Binding of the variants to pre-existing antibodies in primary and relapsed ALL patients’ samples was evaluated.
Results
Both variants showed similar asparaginase activity but approximately 24-fold reduced glutaminase activity compared to wild-type EcA (WT). Cytotoxicity against Reh cells was significantly higher with the mutants, although not toxic to human PBMCs than WT. The mutants showed approximately 3-fold lower IgG and IgM production compared to WT. Pharmacokinetic study in BALB/c mice showed longer half-life of the mutants (KHY-17- 267.28±9.74; KHYW-17- 167.41±14.4) compared to WT (103.24±18). Single and repeat-doses showed no toxicity up to 2000 IU/kg and 1600 IU/kg respectively. Efficacy in ALL xenograft mouse model showed 80–90 % reduction of leukemic cells with mutants compared to 40 % with WT. Consequently, survival was 90 % in each mutant group compared to 10 % with WT. KHYW-17 showed over 2-fold lower binding to pre-existing anti-asparaginase antibodies from ALL patients treated with l-asparaginase.
Conclusion
EcA variants demonstrated better pharmacological properties compared to WT that makes them good candidates for further development.
... The cytoplasmic expression pattern of NeuN IR is important for accurate algorithmic processing, accordingly, the potential range of NeuN IR expression in DRG neuronal cytoplasm was evaluated during naïve conditions and at a peak swelling timepoint of inflammation, 48-hours, in the adjuvant induced arthritis model (AIA) [1,3,11,12]. Complete Freund's adjuvant (CFA) (Sigma, F5881) was emulsified in a 1:1 ratio with phosphate buffered saline (PBS) and injected into the right hind paw of experimental animals to establish unilateral adjuvant-induced arthritis. ...
Immunohistochemistry (IHC) is a valuable tool in clinical and biological research for evaluating proteins and other antigens in spatially bound tissue. In neuroinflammatory pain research, primary afferent neurons of the dorsal root ganglion (DRG) are studied to understand molecular signaling mechanisms involved in nociception (pain) and inflammation. Measuring IHC (immunofluorescence) in DRG neurons requires manual hand tracing of nuclear and somatic boundaries, which is laborious, error-prone, and may require several weeks to collect the appropriate sample size with a mouse or pen-input display monitor. To overcome these limitations and increase standardization of sampling and measurement, we employed a reliable neuronal cytoplasmic reporter, exclusive to DRG neuronal soma, in a semi-automated algorithm-based approach of Image Cytometry in rat DRG (IC-DRG). The resulting output images are binary nuclear and somatic masks of DRG neurons, defining boundaries of measurement for CellProfiler and manually scored at 94% accurate. Herein, we successfully show a novel approach of automated image analysis for DRG neurons using a robust ImageJ/FIJI script, overcoming morphological variability and imaging artifacts native to imaging frozen tissue sections processed with immunofluorescence.
... Serum ammonia was measured using enzyme-linked immunosorbant assay at Assaygate, Ijamsville, MD. Small-intestinal glutaminase activity was determined using the methods published in Miller et al., 18 whereas cecal glutamine content and cecal content amino-acid metabolomics were defined using GC-MS at UC Davis after false-discovery rate-adjusted analysis of variance for the named metabolites. 18,19 Cecal contents were used given its role as a major producer of microbially produced metabolites. ...
... Small-intestinal glutaminase activity was determined using the methods published in Miller et al., 18 whereas cecal glutamine content and cecal content amino-acid metabolomics were defined using GC-MS at UC Davis after false-discovery rate-adjusted analysis of variance for the named metabolites. 18,19 Cecal contents were used given its role as a major producer of microbially produced metabolites. Inflammatory expression and intestinal barrier study. ...
Objectives
Rifaximin has clinical benefits in minimal hepatic encephalopathy (MHE) but the mechanism of action is unclear. The antibiotic-dependent and -independent effects of rifaximin need to be elucidated in the setting of MHE-associated microbiota. To assess the action of rifaximin on intestinal barrier, inflammatory milieu and ammonia generation independent of microbiota using rifaximin.
Methods
Four germ-free (GF) mice groups were used (1) GF, (2) GF+rifaximin, (3) Humanized with stools from an MHE patient, and (4) Humanized+rifaximin. Mice were followed for 30 days while rifaximin was administered in chow at 100 mg/kg from days 16–30. We tested for ammonia generation (small-intestinal glutaminase, serum ammonia, and cecal glutamine/amino-acid moieties), systemic inflammation (serum IL-1β, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1β, IL-6, MCP-1, e-cadherin and zonulin) along with microbiota composition (colonic and fecal multi-tagged sequencing) and function (endotoxemia, fecal bile acid deconjugation and de-hydroxylation).
Results
All mice survived until day 30. In the GF setting, rifaximin decreased intestinal ammonia generation (lower serum ammonia, increased small-intestinal glutaminase, and cecal glutamine content) without changing inflammation or intestinal barrier function. Humanized microbiota increased systemic/intestinal inflammation and endotoxemia without hyperammonemia. Rifaximin therapy significantly ameliorated these inflammatory cytokines. Rifaximin also favorably impacted microbiota function (reduced endotoxin and decreased deconjugation and formation of potentially toxic secondary bile acids), but not microbial composition in humanized mice.
Conclusions
Rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression independent of gut microbiota. MHE-associated fecal colonization results in intestinal and systemic inflammation in GF mice, which is also ameliorated with rifaximin.
... These findings indicate that the production and release of glutamate are altered in response to pain, most likely due to modified flux control and local changes in the GA-mediated glutamate-glutamine cycle [84]. In support of this latter notion, persistent inflammation, which was experimentally induced by complete Freund's adjuvant in a rat model of arthritis, was shown to increase GA expression and enzymatic activity in DRG neurons [85]. It was hypothesized that elevated GA in primary sensory neurons could increase the production of glutamate in spinal primary afferent terminals, thereby either directly contributing to central or peripheral sensitization [85]. ...
... In support of this latter notion, persistent inflammation, which was experimentally induced by complete Freund's adjuvant in a rat model of arthritis, was shown to increase GA expression and enzymatic activity in DRG neurons [85]. It was hypothesized that elevated GA in primary sensory neurons could increase the production of glutamate in spinal primary afferent terminals, thereby either directly contributing to central or peripheral sensitization [85]. In an animal model of MS, GA was found to be highly expressed and correlated with axonal damage in macrophages and microglial cells associated with active lesions [59]. ...
Background
Chronic pain is a major symptom that develops in cancer patients, most commonly emerging during advanced stages of the disease. The nature of cancer-induced pain is complex, and the efficacy of current therapeutic interventions is restricted by the dose-limiting side-effects that accompany common centrally targeted analgesics.
Methods
This review focuses on how up-regulated glutamate production and export by the tumour converge at peripheral afferent nerve terminals to transmit nociceptive signals through the transient receptor cation channel, TRPV1, thereby initiating central sensitization in response to peripheral disease-mediated stimuli.
Results
Cancer cells undergo numerous metabolic changes that include increased glutamine catabolism and over-expression of enzymes involved in glutaminolysis, including glutaminase. This mitochondrial enzyme mediates glutaminolysis, producing large pools of intracellular glutamate. Up-regulation of the plasma membrane cystine/glutamate antiporter, system xc-, promotes aberrant glutamate release from cancer cells. Increased levels of extracellular glutamate have been associated with the progression of cancer-induced pain and we discuss how this can be mediated by activation of TRPV1.
Conclusion
With a growing population of patients receiving inadequate treatment for intractable pain, new targets need to be considered to better address this largely unmet clinical need for improving their quality of life. A better understanding of the mechanisms that underlie the unique qualities of cancer pain will help to identify novel targets that are able to limit the initiation of pain from a peripheral source–the tumour.
... GLS-ir was present in many rat ICG neurons as puncta found throughout the cytoplasm ( Figure 1A,C; Hoffman et al., 2010;Miller et al., 2012;Crosby et al., 2015;Hoffman et al., 2016). Among 1318 ICG neurons analyzed (n=5 rats), the cell soma area ranged from 53.0 to 867.9µm 2 ( Figure 3A). ...
... Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (Fremeau et al., 2004, Wojcik et al., 2004 and is used by primary sensory neurons in TG (Yang et al., 2014) and DRG (Brumovsky et al., 2007 of the peripheral nervous system. In primary sensory neurons in DRG, neurotransmitter glutamate production utilizing the glutamate-glutamine cycle is important for sensory function (Miller et al., 1993, Miller et al., 2002, Miller et al., 2012. Phosphate-activated GLS, regulated by calcium (Ca 2+ ) and inorganic phosphate (P i ), is the synthetic enzyme for conversion of glutamine into glutamate for use as a neurotransmitter (Erecinska et al., 1990. ...
... GLS, VGLUT1, and VGLUT2 immunoreactivity occurred in many neurons of the rat ICG. The antisera used for GLS, VGLUT1, and VGLUT2 have been employed previously in our laboratory for rat DRG neurons (Miller et al., 2012, Crosby H., 2015, Bolt, 2016, Hoffman et al., 2016 and pre-absorption control studies in ICG and other rat tissues (colon, skin, cornea, sciatic nerve, DRG, TG, spinal cord) support the specificity of the three antibodies. Furthermore, quantitative image analysis allowed us to categorize immunoreactive neurons into moderate and high classes and exclude neurons that were non-immunoreactive. ...
The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside intrinsic cardiac ganglia. In the present study, rat intrinsic cardiac ganglia neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) intrinsic cardiac ganglia contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial intrinsic cardiac ganglia contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT. (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac nervous system.
... Glutamic acid (Glu) in vivo is produced from glutamine deamination (Erickson and Cerione 2010;List et al. 2012;Marquez et al. 2013). Glu is the most important excitatory amino acid in the excitatory neurotransmitter system, which plays a key role in learning, memory and central pain sensory conduction (de la Rosa et al. 2009;Marquez et al. 2006;Miller et al. 2012). Previous studies have revealed that Glu is one of the most important neurotransmitters to mediate pain information transfer (Odai et al. 2001;Toth et al. 1992;Kazemi and Hoop 1991). ...
Chronic post-surgical pain (CPSP) is a normal and significant symptom in clinical surgery, such as breast operation, biliary tract operation, cesarean operation, uterectomy and thoracic operation. Severe chronic post-surgical pain could increase post-surgical complications, including myocardial ischemia, respiratory insufficiency, pneumonia and thromboembolism. However, the underlying mechanism is still unknown. Herein, a rat CPSP model was produced via thoracotomy. After surgery, in an initial study, 5 out of 12 rats after surgery showed a significant decrease in mechanical withdrawal threshold and/or increase in the number of acetone-evoked responses, and therefore classified as the CPSP group. The remaining seven animals were classified as non-CPSP. Subsequently, open-chest operation was performed on another 30 rats and divided into CPSP and non-CPSP groups after 21-day observation. Protein expression levels in the dorsal spinal cord tissue were determined by 12.5 % SDS-PAGE. Finally, differently expressed proteins were identified by LC MS/MS and analyzed by MASCOT software, followed by Gene Ontology cluster analysis using PANTHER software. Compared with the non-CPSP group, 24 proteins were only expressed in the CPSP group and another 23 proteins expressed differentially between CPSP and non-CPSP group. Western blot further confirmed that the expression of glutaminase 1 (GLS1) was significantly higher in the CPSP than in the non-CPSP group. This study provided a new strategy to identify the spinal proteins, which may contribute to the development of chronic pain using differential proteomics, and suggested that GLS1 may serve as a potential biomarker for CPSP.
... Despite glutamate being released from the same neurons that release neuropeptides [13] and the co-localization of GLS within peptidergic neurons [14], very little is known about glutamate metabolism in DRG neurons during inflammation. One study indicates that GLS is elevated in small and medium diameter lumbar 4 (L4) DRG neurons after seven ys of unilateral adjuvant-induced arthritis (AIA) [15]. ...
... Many of the proteins necessary for the glutamate-glutamine cycle are present in DRG neurons and their glia [7,[17][18][19][20][21]. Acute increase in the production of glutamate available for release in DRG neurons could involve an increase in flux through the glutamate-glutamine cycle near the sites of glutamate release. Long-term responses may require regulating the expression of glutamate-glutamine cycle proteins at the cell body in the DRG and then transporting them to the peripheral and central terminals [7,15]. To address this issue, we hypothesize that hind paw inflammation increases GLS production in rat DRG neurons. ...
... Images of L4 DRG tissue sections were acquired on a BX51 epifluorescence microscope (Olympus; Center Valley, PA, USA) using a SPOT RT740 camera (Diagnostic Instruments; Sterling Heights, MI, USA). An exposure and gain combination was determined empirically for each of the four slide sets in which the dimmest regions of tissue could be discerned visually for tracing, but the brightest regions were not oversaturated [15,18,25,26]. Three fields of view were captured randomly from each section of each DRG. ...
Glutamate is a neurotransmitter used at both the peripheral and central terminals of nociceptive primary sensory neurons, yet little is known concerning regulation of glutamate metabolism during peripheral inflammation. Glutaminase (GLS) is an enzyme of the glutamate-glutamine cycle that converts glutamine into glutamate for neurotransmission and is implicated in producing elevated levels of glutamate in central and peripheral terminals. A potential mechanism for increased levels of glutamate is an elevation in GLS expression. We assessed GLS expression after unilateral hind paw inflammation by measuring GLS immunoreactivity (ir) with quantitative image analysis of L4 dorsal root ganglion (DRG) neurons after one, two, four, and eight days of adjuvant-induced arthritis (AIA) compared to saline injected controls. No significant elevation in GLS-ir occurred in the DRG ipsilateral to the inflamed hind paw after one or two days of AIA. After four days AIA, GLS-ir was elevated significantly in all sizes of DRG neurons. After eight days AIA, GLS-ir remained elevated in small (<400 µm²), presumably nociceptive neurons. Western blot analysis of the L4 DRG at day four AIA confirmed the elevated GLS-ir. The present study indicates that GLS expression is increased in the chronic stage of inflammation and may be a target for chronic pain therapy.
