Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism.

Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (TH17) cells towards induced regulatory T (iTreg) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating TH17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards TH17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of TH17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iTreg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between TH17 and iTreg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against TH17-mediated autoimmune diseases.

H2S biogenesis by cystathionine beta-synthase: mechanism of inhibition by aminooxyacetic acid and unexpected role of serine.

Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.

Overproduction of H2S, generated by CBS, inhibits mitochondrial Complex IV and suppresses oxidative phosphorylation in Down syndrome.

Down syndrome (DS) is associated with significant perturbances in mitochondrial function. Here we tested the hypothesis that the suppression of mitochondrial electron transport in DS cells is due to high expression of cystathionine-ß-synthase (CBS) and subsequent overproduction of the gaseous transmitter hydrogen sulfide (H2S). Fibroblasts from DS individuals showed higher CBS expression than control cells; CBS localization was both cytosolic and mitochondrial. DS cells produced significantly more H2S and polysulfide and exhibited a profound suppression of mitochondrial electron transport, oxygen consumption, and ATP generation. DS cells also exhibited slower proliferation rates. In DS cells, pharmacological inhibition of CBS activity with aminooxyacetate or siRNA-mediated silencing of CBS normalized cellular H2S levels, restored Complex IV activity, improved mitochondrial electron transport and ATP synthesis, and restored cell proliferation. Thus, CBS-derived H2S is responsible for the suppression of mitochondrial function in DS cells. When H2S overproduction is corrected, the tonic suppression of Complex IV is lifted, and mitochondrial electron transport is restored. CBS inhibition offers a potential approach for the pharmacological correction of DS-associated mitochondrial dysfunction.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate trichloroethylene reproductive toxicity via metabolism in Wistar rats.

Exposure to the industrial solvent trichloroethylene (TCE) has been associated with adverse pregnancy outcomes in humans and decreased fetal weight in rats. TCE kidney toxicity can occur through formation of reactive metabolites via its glutathione (GSH) conjugation metabolic pathway, largely unstudied in the context of pregnancy. To investigate the contribution of the GSH conjugation pathway and oxidative stress to TCE toxicity during pregnancy, we exposed rats orally to 480 mg TCE/kg/day from gestational day (GD) 6 to GD 16 with and without N-acetyl-L-cysteine (NAC) at 200 mg/kg/day or aminooxyacetic acid (AOAA) at 20 mg/kg/day as pre/co-treatments from GD 5-16. NAC is a reactive oxygen species scavenger that modifies the GSH conjugation pathway, and AOAA is an inhibitor of cysteine conjugate ß-lyase (CCBL) in the GSH conjugation pathway. TCE decreased fetal weight, and this was prevented by AOAA but not NAC pre/co-treatment to TCE. Although AOAA inhibited CCBL activity in maternal kidney, it did not inhibit CCBL activity in maternal liver and placenta, suggesting that AOAA prevention of TCE-induced decreased fetal weight was due to CCBL activity inhibition in the kidneys but not liver or placenta. Unexpectedly, NAC pre/co-treatment with TCE, relative to TCE treatment alone, altered placental morphology consistent with delayed developmental phenotype. Immunohistochemical staining revealed that the decidua basale, relative to basal and labyrinth zones, expressed the highest abundance of CCBL1, flavin-containing monooxygenase 3, and cleaved caspase-3. Together, the findings show the differential effects of NAC and AOAA on TCE-induced pregnancy outcomes are likely attributable to TCE metabolism modulation.

Genomic and metabolomic analysis of step-wise malignant transformation in human skin fibroblasts.

Metabolic changes accompanying a step-wise malignant transformation was investigated using a syngeneic lineage of human fibroblasts. Cell immortalization was associated with minor alterations in metabolism. Consecutive loss of cell cycle inhibition in immortalized cells resulted in increased levels of oxidative phosphorylation (OXPHOS). Overexpression of the H-Ras oncoprotein produced cells forming sarcomas in athymic mice. These transformed cells exhibited increased glucose consumption, glycolysis and a further increase in OXPHOS. Because of the markedly increased OXPHOS in transformed cells, the impact of a transaminase inhibitor, aminooxyacetic acid (AOA), which decreases glutamine influx to the tricarboxylic acid (TCA) cycle, was tested. Indeed, AOA significantly decreased proliferation of malignantly transformed fibroblasts and fibrosarcoma-derived cells in vitro and in vivo. AOA also decreased proliferation of cells susceptible to malignant transformation. Metabolomic studies in normal and transformed cells indicated that, in addition to the anticipated effect on the TCA cycle, AOA decreased production of nucleotides adenosine triphosphate (ATP) and uridine monophosphate. Exogenous nucleotides partially rescued decreased proliferation of the malignant cells treated with AOA. Our data indicate that AOA blocks several metabolic pathways essential for growth of malignant cells. Therefore, OXPHOS may provide important therapeutic targets for treatment of sarcoma.

Aminooxyacetic acid attenuates post-infarct cardiac dysfunction by balancing macrophage polarization through modulating macrophage metabolism in mice.

Excessive activation of pro-inflammatory M1 macrophages following acute myocardial infarction (MI) aggravates adverse cardiac remodelling and heart dysfunction. There are two break points in the tricarboxylic acid cycle of M1 macrophages, and aspartate-arginosuccinate shunt compensates them. Aminooxyacetic acid (AOAA) is an inhibitor of aspartate aminotransferase in the aspartate-arginosuccinate shunt. Previous studies showed that manipulating macrophage metabolism may control macrophage polarization and inflammatory response. In this study, we aimed to clarify the effects of AOAA on macrophage metabolism and polarization and heart function after MI. In vitro, AOAA inhibited lactic acid and glycolysis and enhanced ATP levels in classically activated M1 macrophages. Besides, AOAA restrained pro-inflammatory M1 macrophages and promoted anti-inflammatory M2 phenotype. In vivo, MI mice were treated with AOAA or saline for three consecutive days. Remarkably, AOAA administration effectively inhibited the proportion of M1 macrophages and boosted M2-like phenotype, which subsequently attenuated infarct size as well as improved post-MI cardiac function. Additionally, AOAA attenuated NLRP3-Caspase1/IL-1ß activation and decreased the release of IL-6 and TNF-α pro-inflammatory cytokines and reciprocally increased IL-10 anti-inflammatory cytokine level in both ischaemic myocardium and M1 macrophages. In conclusion, short-term AOAA treatment significantly improves cardiac function in mice with MI by balancing macrophage polarization through modulating macrophage metabolism and inhibiting NLRP3-Caspase1/IL-1ß pathway.

Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondiiin vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Altered dynamics in the circadian oscillation of clock genes in serum-shocked NIH-3T3 cells by the treatment of GYY4137 or AOAA.

BACKGROUND AND PURPOSE: Several members of the core clock mechanism are equipped with a Per-Arnt-Sim (PAS) domain through which they can bind haem [Fe(II)]. Haem is a ligand for the orphan receptors REV-ERBα/ß (NR1D1/2), which regulate circadian rhythm and metabolism. The ability to bind haem sensitises these clock components to the action of small molecule gases, including NO, CO and H2S. Studies conducted with European hamsters revealed that during winter sleep, key clock genes stop oscillating. At the same time, H2S, when administered at subtoxic concentrations, can induce a hypometabolic state in the cell. We suppose that core clock components, including the nuclear receptors REV-ERBs, neuronal PAS domain protein 2 (nPAS2) and PER2, can be H2S targets. The general objective of this study was to investigate the effect of the H2S system on the expression profile of the core clock genes in cells in vitro. EXPERIMENTAL APPROACH: We analysed the expression of Per1, Per2, Per3, Bmal1, Cry1, Cry2, Nr1d1, Nfil-3 and Dbp messenger RNA (mRNA) in serum-shocked NIH-3T3 cells treated with a slow-releasing H2S donor (GYY4137) or the cystathionine beta-synthase (CBS) inhibitor (AOAA) cultured under constant darkness and collected during 3 days in 3 h interval. KEY RESULTS AND CONCLUSIONS AND IMPLICATIONS: We found that pharmacological CBS inhibition increased the general expression and dynamics of several clock genes. On the other hand, increased H2S decreased Per2 expression. These data suggest that CBS can affect circadian clock and effect on clock-controlled transcription output.

Hydrogen sulfide upregulates renal AQP-2 protein expression and promotes urine concentration.

Increasing evidence supports the important role of H2S in renal physiology and the pathogenesis of kidney injury. Whether H2S regulates water metabolism in the kidney and the potential mechanism are still unknown. The present study was conducted to determine the role of H2S in urine concentration. Inhibition of both cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS), 2 major enzymes for endogenous H2S production, with propargylglycine (PPG) and amino-oxyacetate (AOAA), respectively, caused increased urine output and reduced urine osmolality in mice that was associated with decreased expression of aquaporin (AQP)-2 in the renal inner medulla. Mice treated with both PPG and AOAA developed a urine concentration defect in response to dehydration that was accompanied by reduced AQP-2 protein expression. Inhibition of CSE alone was associated with a mild decrease in AQP-2 protein level in the renal medulla of heterozygous CBS mice. GYY4137, a slow H2S donor, markedly improved urine concentration and prevented the down-regulation of renal AQP-2 protein expression in mice with lithium-induced nephrogenic diabetes insipidus (NDI). GYY4137 significantly increased cAMP levels in cell lysates prepared from inner medullary collecting duct (IMCD) suspensions. AQP-2 protein expression was also upregulated, but was significantly inhibited by the adenyl cyclase inhibitor MDL12330A or the PKA inhibitor H89, but not the vasopressin 2 receptor (V2R) antagonist tolvaptan. Inhibition of endogenous H2S production impaired urine concentration in mice, whereas an exogenous H2S donor improved urine concentration in lithium-induced NDI by increasing AQP-2 expression in the collecting duct principal cells. H2S upregulated AQP-2 protein expression, probably via the cAMP-PKA pathway.-Luo, R., Hu, S., Liu, Q., Han, M., Wang, F., Qiu, M., Li, S., Li, X., Yang, T., Fu, X., Wang, W., Li, C. Hydrogen sulfide upregulates renal AQP-2 protein expression and promotes urine concentration.

Brain hydrogen sulfide suppresses the micturition reflex via brain GABA receptors in rats.

We recently reported that hydrogen sulfide (H2S) is a possible relaxation factor in the rat bladder. However, there is no available information about the roles of central H2S in the micturition reflex, so we investigated the effects of centrally administered GYY4137 (H2S donor) and AOAA (H2S synthesis inhibitor) on the micturition reflex in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. Cystometry was performed before and after the administration of GYY4137 (3 or 10 nmol/rat, icv) or AOAA (30 or 100 µg/rat, icv). In some rats, SR95531 (GABAA receptor antagonist, 0.1 nmol/rat, icv) or SCH50911 (GABAB receptor antagonist, 0.1 nmol/rat, icv) was administered 30 min before GYY4137 administration (10 nmol/rat, icv). Centrally administered GYY4137 dose-dependently prolonged the intercontraction intervals (ICI) without altering maximum voiding pressure (MVP). On the other hand, centrally administered AOAA dose-dependently shortened ICI without altering MVP. The AOAA (30 µg/rat, icv)-induced ICI shortening was reversed in the central presence of GYY4137 (10 nmol/rat, icv). Centrally pretreated SR95531 or SCH50911 significantly attenuated the GYY4137 (10 nmol/rat, icv)-induced prolongation of ICI, respectively. These findings suggest that endogenous brain H2S can inhibit the rat micturition reflex via both GABAA and GABAB receptors in the brain.

Effects of aminooxyacetic acid on hippocampal mitochondria in rats with chronic alcoholism: the analysis of learning and memory-related genes.

The incidence of chronic alcoholism leading to central and peripheral nervous system damage has been increasing year-to-year. The purpose of this study is to explore the effects of aminooxyacetic acid on hippocampus mitochondria in rats with chronic alcoholism and analyze learning and memory-related genes. Sixty male Sprague Dawley rats were randomly divided into three groups. Except for the control group, each group was fed with the water containing (v/v) 6% alcohol for 28 days. After 14 days, rats in the treatment group were intraperitoneally injected daily for 14 days with aminooxyacetic acid. High throughput sequencing was combined and tested for learning and memory abilities, Hydrogen sulfide content, catalase activity in mitochondria, and the expression of F-actin in the hippocampus of the rats in each group. Compared with the control group, the learning and memory abilities of rats with chronic alcoholism were significantly impaired, mitochondria contained vacuoles, hydrogen sulfide increased, but catalase activity and F-actin content were significantly decreased, After treatment with aminooxyacetic acid, mitochondrial morphology improved, hydrogen sulfide content was decreased, while catalase activity and F-actin expression of in hippocampus were increased. This indicates that aminooxyacetic acid may improve learning and memory in rats with chronic alcoholism, and the mechanism is related to decreased hydrogen sulfide content and an increase of both catalase activity and F-actin level in the hippocampus, thereby reducing the damage of alcohol to mitochondria and neurons.

Impact of Pharmacological Inhibition of Hydrogen Sulphide Production in the SOD1G93A-ALS Mouse Model.

A number of factors can trigger amyotrophic lateral sclerosis (ALS), although its precise pathogenesis is still uncertain. In a previous study done by us, poisonous liquoral levels of hydrogen sulphide (H2S) in sporadic ALS patients were reported. In the same study very high concentrations of H2S in the cerebral tissues of the familial ALS (fALS) model of the SOD1G93A mouse, were measured. The objective of this study was to test whether decreasing the levels of H2S in the fALS mouse could be beneficial. Amino-oxyacetic acid (AOA)-a systemic dual inhibitor of cystathionine-ß-synthase and cystathionine-γ lyase (two key enzymes in the production of H2S)-was administered to fALS mice. AOA treatment decreased the content of H2S in the cerebral tissues, and the lifespan of female mice increased by approximately ten days, while disease progression in male mice was not affected. The histological evaluation of the spinal cord of the females revealed a significant increase in GFAP positivity and a significant decrease in IBA1 positivity. In conclusion, the results of the study indicate that, in the animal model, the inhibition of H2S production is more effective in females. The findings reinforce the need to adequately consider sex as a relevant factor in ALS.

Endogenous H2S sensitizes PAR4-induced bladder pain.

Bladder pain is a prominent symptom of interstitial cystitis/painful bladder syndrome. Hydrogen sulfide (H2S) generated by cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) facilitates bladder hypersensitivity. We assessed involvement of the H2S pathway in protease-activated receptor 4 (PAR4)-induced bladder pain. A bladder pain model was induced by intravesical instillation of PAR4-activating peptide in mice. The role of H2S in this model was evaluated by intraperitoneal preadministration of d,l-propargylglycine (PAG), aminooxyacetic acid (AOAA), or S-adenosylmethionine or the preintravesical administration of NaHS. SV-HUC-1 cells were treated in similar manners. Assessments of CBS, CSE, and macrophage migration inhibitory factor (MIF) expression, bladder voiding function, bladder inflammation, H2S production, and referred bladder pain were performed. The CSE and CBS pathways existed in both mouse bladders and SV-HUC-1 cells. H2S signaling was upregulated in PAR4-induced bladder pain models, and H2S-generating enzyme activity was upregulated in human bladders, mouse bladders, and SV-HUC-1 cells. Pretreatment with AOAA or NaHS inhibited or promoted PAR4-induced mechanical hyperalgesia, respectively; however, PAG only partially inhibited PAR4-induced bladder pain. Treatment with PAG or AOAA decreased H2S production in both mouse bladders and SV-HUC-1 cells. Pretreatment with AOAA increased MIF protein levels in bladder tissues and cells, whereas pretreatment with NaHS lowered MIF protein levels. Bladder pain triggered by the H2S pathway was not accompanied by inflammation or altered micturition behavior. Thus endogenous H2S generated by CBS or CSE caused referred hyperalgesia mediated through MIF in mice with PAR4-induced bladder pain, without causing bladder injury or altering micturition behavior.

l-Cysteine suppresses hypoxia-ischemia injury in neonatal mice by reducing glial activation, promoting autophagic flux and mediating synaptic modification via H2S formation.

We previously reported that l-Cysteine, an H2S donor, significantly alleviated brain injury after hypoxia-ischemic (HI) injury in neonatal mice. However, the mechanisms underlying this neuroprotective effect of l-Cysteine against HI insult remain unknown. In the present study, we tested the hypothesis that the protective effects of l-Cysteine are associated with glial responses and autophagy, and l-Cysteine attenuates synaptic injury as well as behavioral deficits resulting from HI. Consistent with our previous findings, we found that treatment with l-Cysteine after HI reduced early brain injury, improved behavioral deficits and synaptic damage, effects which were associated with an up-regulation of synaptophysin and postsynaptic density protein 95 expression in the lesioned cortex. l-Cysteine attenuated the accumulation of CD11b+/CD45high cells, activation of microglia and astrocytes and diminished HI-induced increases in reactive oxygen species and malondialdehyde within the lesioned cortex. In addition, l-Cysteine increased microtubule associated protein 1 light chain 3-II and Beclin1 expression, decreased p62 expression and phosphor-mammalian target of rapamycin and phosphor-signal transducer and activator of transcription 3. Further support for a critical role of l-Cysteine was revealed from results demonstrating that treatment with an inhibitor of the H2S-producing enzyme, amino-oxyacetic acid, reversed the beneficial effects of l-Cysteine described above. These results demonstrate that l-Cysteine effectively alleviates HI injury and improves behavioral outcomes by inhibiting reactive glial responses and synaptic damage and an accompanying triggering of autophagic flux. Accordingly, l-Cysteine may provide a new a therapeutic approach for the treatment of HI via the formation of H2S.

Pre-ischaemic mitochondrial substrate constraint by inhibition of malate-aspartate shuttle preserves mitochondrial function after ischaemia-reperfusion.

KEY POINTS: Pre-ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against ischaemia-reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion. The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC). However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR-injured heart. ABSTRACT: Mitochondrial dysfunction plays a central role in ischaemia-reperfusion (IR) injury. Pre-ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR-injured heart to the same extent as ischaemic preconditioning (IPC), which is a well-validated cardioprotective strategy against IR injury. In the present study, we show that pre-ischaemic administration of AOA preserved mitochondrial complex I-linked state 3 respiration and fatty acid oxidation during late reperfusion in IR-injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I-linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR-injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)-cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR-injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.

Versatile Alkylation of (Hetero)Aryl Iodides with Ketones via ß-C(sp3)-H Activation.

We report Pd(II)-catalyzed ß-C(sp3)-H (hetero)arylation of a variety of ketones using a commercially available 2,2-dimethyl aminooxyacetic acid auxiliary. Facile installation and removal of the auxiliary as well as its superior scope for both ketones and (hetero)aryl iodides overcome the significant limitations of the previously reported ß-C(sp3)-H arylation of ketones. The ready availability of ketones renders this reaction a broadly useful method for alkyl-(hetero)aryl coupling involving both primary and secondary alkyls.

The role of eNOS on the compensatory regulation of vascular tonus by H2S in mouse carotid arteries.

The gasotransmitter nitric oxide (NO) has an important role in vascular function and a decrease in its bioavailability is accepted as a main pathological mechanism for cardiovascular diseases. However, other gasotransmitters such as hydrogen sulfide (H2S) are also generated by the endothelium and can also affect vascular tone and a crosstalk may exist between H2S and NO. We therefore investigated the consequences of deficiency, replacement or overexpression of endothelial nitric oxide synthase (eNOS) on H2S-induced vascular responses in murine carotid arteries. In pre-contracted carotid arteries from wild-type (WT) mice, l-cysteine elicited relaxation that was inhibited by the H2S synthesis inhibitor amino-oxyacetic acid (AOAA). Genetic deletion of eNOS increased l-cysteine-induced relaxation compared to WT, but the replacement of eNOS by adenoviral transfection or H2S synthesis inhibition by AOAA reversed it. Furthermore, eNOS deletion did not alter NaHS-induced relaxation in carotid arteries while eNOS overexpression/replacement increased NaHS-induced relaxation responses in carotid arteries from WT or eNOS-/-. We suggest that, endogenously produced H2S can compensate for impaired vasodilatory responses in the absence of NO to maintain vascular patency; while, eNOS abundance can limit endogenous H2S-induced vascular responses in mice carotid arteries. Our result suggests that endogenous vs. exogenous H2S-induced relaxation are reciprocally regulated by NO in mice carotid arteries.

The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum.

The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10-6-10-3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10-6-10-3 M) and acetylcholine (10-9-10-4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10-5 M), selective M1 mAChR antagonist pirenzepine (5 × 10-5 M) and selective M3 mAChR antagonist 4-DAMP (10-7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10-6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10-2 M) and cystathionine-ß-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10-3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded.

Assessment of the low inhibitory specificity of oxamate, aminooxyacetate and dichloroacetate on cancer energy metabolism.

BACKGROUND: Exceedingly high therapeutic/experimental doses of metabolic drugs such as oxamate, aminooxyacetate (AOA) and dichloroacetate (DCA) are required to diminish growth, glycolysis and oxidative phosphorylation (OxPhos) of different cancer cells. To identify the mechanisms of action of these drugs on cancer energy metabolism, a systematic analysis of their specificities was undertaken. METHODS: Hepatocarcinoma AS-30D cells were treated with the inhibitors and glycolysis and OxPhos enzyme activities, metabolites and fluxes were analyzed. Kinetic modeling of glycolysis was used to identify the regulatory mechanisms. RESULTS: Oxamate (i) not only inhibited LDH, but also PYK and ENO activities inducing an increase in the cytosolic NAD(P)H, Fru1,6BP and DHAP levels in AS-30D cells; (ii) it slightly inhibited HPI, ALD and Glc6PDH; and (iii) it inhibited pyruvate-driven OxPhos in isolated heart mitochondria. AOA (i) strongly inhibited both AAT and AlaT, and 2-OGDH and glutamate-driven OxPhos; and (ii) moderately affected GAPDH and TPI. DCA slightly affected pyruvate-driven OxPhos and Glc6PDH. Kinetic modeling of cancer glycolysis revealed that oxamate inhibition of LDH, PYK and ENO was insufficient to achieve glycolysis flux inhibition. To do so, HK, HPI, TPI and GAPDH have to be also inhibited by the accumulated Fru1,6BP and DHAP induced by oxamate. CONCLUSION: Oxamate, AOA, and DCA are not specific drugs since they inhibit several enzymes/transporters of the glycolytic and OxPhos pathways through direct interaction or indirect mechanisms. GENERAL SIGNIFICANCE: These data explain why oxamate or AOA, through their multisite inhibitory actions on glycolysis or OxPhos, may be able to decrease the proliferation of cancer cells.

The functional and molecular studies on involvement of hydrogen sulphide in myometrial activity of non-pregnant buffaloes (Bubalus bubalis).

BACKGROUND: Hydrogen sulphide (H2S), a member of the gasotransmitters family, is known to play patho-physiological role in different body systems including during pregnancy. But its involvement in myometrial spontaneity and associated signalling pathways in uterus in non-pregnant animals is yet to be studied. Present study describes the effect of L-cysteine, an endogenous H2S donor, on isolated myometrial strips of non-pregnant buffaloes and the underlying signaling mechanism(s). RESULTS: L-cysteine (10 nM-30 mM) produced concentration-dependent contractile effect on buffalo myometrium which was extracellular Ca2+ and L-type calcium channels-dependent. Significant rightward shift of dose-response curve of L-cysteine was observed with significant decrease in maxima in the presence of amino-oxyacetic acid (AOAA; 100 µM) and d, l-propargylglycine (PAG; 100 µM), the specific blockers of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. Existence of CBS enzyme of 63 kDa and CSE of 45 kDa molecular weights was confirmed by western blot using specific antibodies and also by immunohistochemistry. CONCLUSIONS: Endogenous H2S along with its biosynthetic enzymes (CBS and CSE) is evidently present in uteri of non-pregnant buffaloes and it regulates spontaneity in uteri of non-pregnant buffaloes and this effect is dependent on extracellular Ca2+ influx through nifedipine-sensitive L-type calcium channels. Thus H2S-signalling pathway may be a potential target to alter the uterine activities in physiology and patho-physiolgical states.