Mettl3 Mediated m6A Methylation Involved in Epithelial-Mesenchymal Transition by Targeting SOCS3/STAT3/SNAI1 in Cigarette Smoking-Induced COPD.

Purpose: Persistent inflammation and epithelial-mesenchymal transition are essential pathophysiological processes in chronic obstructive pulmonary disease (COPD) and involve airway remodeling. m6A methylation modification was discovered to play an important role in various diseases. Nevertheless, the regulatory role of m6A methylation has not yet been investigated in cigarette smoking-induced COPD. The study aims to explore the regulatory role of m6A methylation in cigarette smoking-induced COPD. Patients and Methods: In this study, two Gene Expression Omnibus (GEO) datasets were first utilized to analyze the expression profiles of m6A RNA methylation regulators in COPD. We then established a cell model of COPD by exposing human bronchial epithelial cells (HBECs) to cigarette smoke extract (CSE) in vitro and detected the expression of m6A writer Mettl3 and EMT phenotype markers. RNA interference, cycloleucine, RT-qPCR, western blot, MeRIP-sequencing, and cell migration assay were performed to investigate the potential effect of Mettl3 on the EMT process in CSE-induced HBECs. Results: Our results showed that Mettl3 expression was significantly elevated in cigarette smoking-induced COPD patients and in a cellular model of COPD. Furthermore, Mettl3 silence and cycloleucine treatment inhibited the EMT process of HBECs caused by CSE. Mechanically, Mettl3 silence weakens the m6A methylation of SOCS3 mRNA to enhance the protein expression of SOCS3, inhibiting CSE-induced SOCS3/STAT3/SNAI1 signaling and EMT processes in HBECs. Conclusion: Our study inferred that Mettl3-mediated m6A RNA methylation modification modulates CSE-induced EMT by targeting SOCS3 mRNA and ultimately serves as a crucial regulator in the emergence of COPD. This conclusion reinforces the regulatory role of m6A methylation in COPD.

Effects of exogenous glycine betaine and cycloleucine on photosynthetic capacity, amino acid composition, and hormone metabolism in Solanum melongena L.

Although exogenous glycine betaine (GB) and cycloleucine (Cyc) have been reported to affect animal cell metabolism, their effects on plant growth and development have not been studied extensively. Different concentrations of exogenous glycine betaine (20, 40, and 60 mmol L-1) and cycloleucine (10, 20, and 40 mmol L-1), with 0 mmol L-1 as control, were used to investigate the effects of foliar spraying of betaine and cycloleucine on growth, photosynthesis, chlorophyll fluorescence, Calvin cycle pathway, abaxial leaf burr morphology, endogenous hormones, and amino acid content in eggplant. We found that 40 mmol L-1 glycine betaine had the best effect on plant growth and development; it increased the fresh and dry weight of plants, increased the density of abaxial leaf hairs, increased the net photosynthetic rate and Calvin cycle key enzyme activity of leaves, had an elevating effect on chlorophyll fluorescence parameters, increased endogenous indoleacetic acid (IAA) content and decreased abscisic acid (ABA) content, and increased glutamate, serine, aspartate, and phenylalanine contents. However, cycloleucine significantly inhibited plant growth; plant apical dominance disappeared, plant height and dry and fresh weights decreased significantly, the development of abaxial leaf hairs was hindered, the net photosynthetic rate and Calvin cycle key enzyme activities were inhibited, the endogenous hormones IAA and ABA content decreased, and the conversion and utilization of glutamate, arginine, threonine, and glycine were affected. Combined with the experimental results and plant growth phenotypes, 20 mmol L-1 cycloleucine significantly inhibited plant growth. In conclusion, 40 mmol L-1 glycine betaine and 20 mmol L-1 cycloleucine had different regulatory effects on plant growth and development.

Acetaminophen changes the RNA m6A levels and m6A-related proteins expression in IL-1ß-treated chondrocyte cells.

BACKGROUND: Acetaminophen is commonly recommended for the early analgesia of osteoarthritis. However, the molecular mechanism by which it acts remains unknown. The aim of this study is to investigate the effect of acetaminophen on inflammation and extracellular matrix degradation in human chondrocytes, and the possible molecular mechanisms involved in its effect. METHODS: The normal chondrocyte cell line C28/I2 was treated with interleukin-1ß to mimic the inflammatory state. Acetaminophen and the methylation inhibitor (cycloleucine) were used to treat interleukin-1ß-induced C28/I2 cells. The expression of RNA N6-methyladenosine -related proteins was detected by RT-qPCR and western blot. The total RNA N6-methyladenosine level was measured by dot blot analysis and enzyme linked immunosorbent assay. The levels of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α were measured by enzyme linked immunosorbent assay. The extracellular matrix synthesis and degradation were examined by western blot. RESULTS: After interleukin-1ß stimulated C28/I2 cells, the intracellular RNA N6-methyladenosine level increased, and the expression of regulatory proteins also changed, mainly including the increased expression of methyltransferase like 3 and the downregulated expression of AlkB family member 5. The use of cycloleucine inhibited interleukin-1ß-induced inflammation and extracellular matrix degradation by inhibiting RNA N6-methyladenosine modification. In contrast, acetaminophen treatment counteracted interleukin-1ß-induced changes in RNA N6-methyladenosine levels and regulatory protein expression. Furthermore, acetaminophen treatment of interleukin-1ß-induced C28/I2 cells inhibited the secretion of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α, down-regulated the expression of matrix metalloproteinase-13 and Collagen X, and up-regulated the expression of collagen II and aggrecan. In addition, AlkB family member 5 overexpression activated interleukin-1ß-induced chondrocyte viability and suppressed inflammation and extracellular matrix degradation. CONCLUSION: Acetaminophen affects inflammatory factors secretion and extracellular matrix synthesis of human chondrocytes by regulating RNA N6-methyladenosine level and N6-methyladenosine-related protein expression. Stimulation of the normal chondrocyte cell line C28/I2 with the cytokine IL-1ß (10 µM) mimics the inflammatory state in vitro. Acetaminophen (Ace, 50 µg/mL) changes the m6A related proteins expression and the total RNA m6A levels in IL-1ß-treated chondrocyte cells. Furthermore, regulation of RNA m6A levels (by methylation inhibitor Cyc and/or Ace) affects IL-1ß-induced inflammatory cytokines secretion and extracellular matrix synthesis in C28/I2 cells.

Transcriptome analysis of the inhibitory effect of cycloleucine on myogenesis.

N6-Methyladenosine (m6A) has been reported to involve and play an important role in various biological activities but seldom in poultry myogenesis. Cycloleucine usually functions as a nucleic acid methylation inhibitor, the inhibition efficiency of cycloleucine at the m6A level and corresponding dynamic changes of poultry muscle cells remain unknown. In this study, we aim to find out the effect of cycloleucine on the total N6-Methyladenosine level and its molecular mechanism for regulating myogenesis. A total of 745 differentially expressed genes (DEGs) were obtained by 10 mM, 20 mM, and 30 mM of cycloleucine treatment compared with 0 mM treatment. DEGs in 10 mM cycloleucine were significantly enriched in the biological process of skeletal muscle and satellite cell proliferation and differentiation, DEGs in 20 and 30 mM cycloleucine were enriched in some metabolic and biosynthetic processes. The trend analysis showed that 85% of all DEGs were significantly clustered into 4 files, among them 59% DEGs were dose-dependent and 26% were dose-independent, 52% DEGs were in downtrend and 33% DEGs were in uptrend. Also, the cycloleucine treatment could trigger cell cycle arrest in the G1 phase and depress myoblast cell proliferation and inhibit myotube formation. In conclusion, cycloleucine could continuously reduce the m6A level of myoblast cells, depress myoblast cell proliferation and inhibit myotube formation.

Triple-Methyl Blockade With Recombinant Methioninase, Cycloleucine, and Azacitidine Arrests a Pancreatic Cancer Patient-Derived Orthotopic Xenograft Model.

OBJECTIVES: Methionine addiction is a fundamental and general hallmark of cancer caused by enhanced methyl flux. In the present study, we effected a novel methionine-methylation blockade to target a patient-derived orthotopic xenograft model of pancreatic cancer. METHODS: The pancreatic cancer patient-derived orthotopic xenograft mouse models were randomized into 6 groups of 8 mice each and treated for 2 weeks: untreated control; azacitidine; oral recombinant methioninase (o-rMETase); o-rMETase plus cycloleucine; o-rMETase plus cycloleucine plus azacitidine (triple-methyl blockade therapy); and gemcitabine (positive control). RESULTS: Triple-methyl blockade therapy arrested tumor growth (mean relative tumor volume, 1.03 [standard deviation, 0.36]) and was significantly more effective compared with azacitidine (P = 0.0001); o-rMETase (P = 0.007); or o-rMETase plus cycloleucine (P = 0.04). Gemcitabine alone also inhibited but did not arrest tumor growth (mean relative tumor volume, 1.50 [standard deviation, 0.30]). The percentage of cancer cells that were negative for 5-methylcytosine staining in immunohistochemistry, indicating reduction of DNA methylation, increased with triple-methyl blockade therapy (37.5%), compared with gemcitabine (1.8%); o-rMETase (2.8%); azacitidine (9.0%); or o-rMETase plus cycloleucine (10.6%). CONCLUSIONS: This new concept of triple-methyl blockade therapy has clinical potential for pancreatic cancer, which is currently a recalcitrant disease.

Effects of Cycloleucine in the Nucleus Accumbens Septi on the Elevated plus Maze Test in Rats.

INTRODUCTION: In recent years, an important number of studies have emphasized the psychopharmacological actions of cycloleucine (1-aminocyclopentanecarboxylic acid) acting on the NR1 subunit (glycine allosteric site) of NMDA (N-methyl-D-aspartic acid) receptor. We studied the effects of its injection in an anxiety test. METHODS: The elevated plus maze test was used. Male rats bilaterally cannulated into the nucleus accumbens septi (NAS) were employed. Rats were divided into 5 groups that received either 1 µL injections of saline or cycloleucine (0.5, 1, 2, or 4 µg) 15 min before testing. RESULTS: Time spent in the open arm was significantly increased by cycloleucine treatment with all doses (1 and 2 µg, p < 0.05; 0.5 and 4 µg, p < 0.01), like number of extreme arrivals (0.5 and 1 µg, p < 0.05; 2 µg, p < 0.01; and 4 µg, p < 0.001). Open arm entries were increased by the highest dose only (4 µg, p < 0.01). DISCUSSION/CONCLUSION: Present results show no difference between all doses in the time spent in the open arm, suggesting an indirect, noncompetitive action of the drug. The increase in extreme arrivals and open arm entries suggests a dose influence in these parameters. We conclude that cycloleucine influence on the NMDA receptors within NAS leads to anxiolytic-like effects and behavioral disinhibition, which once more confirms the involvement of NAS in anxiety processing.

Ablation of TRPV1 Elevates Nocturnal Blood Pressure in Western Diet-fed Mice.

BACKGROUND: This study tested the hypothesis that genetically ablation of transient receptor potential vanilloid type 1 (TRPV1) exacerbates impairment of baroreflex in mice fed a western diet (WD) and leads to distinct diurnal and nocturnal blood pressure patterns. METHODS: TRPV1 gene knockout (TRPV1-/-) and wild-type (WT) mice were given a WD or normal diet (CON) for 4 months. RESULTS: Capsaicin, a selective TRPV1 agonist, increased ipsilateral afferent renal nerve activity in WT but not TRPV1-/- mice. The sensitivity of renal sympathetic nerve activity and heart rate responses to baroreflex were reduced in TRPV1-/--CON and WT-WD and further decreased in TRPV1-/--WD compared to the WT-CON group. Urinary norepinephrine and serum insulin and leptin at day and night were increased in WT-WD and TRPV1-/--WD, with further elevation at night in TRPV1-/--WD. WD intake increased leptin, IL-6, and TNF-α in adipose tissue, and TNF-α antagonist III, R-7050, decreased leptin in TRPV1-/--WD. The urinary albumin level was higher in TRPV1-/--WD than WT-WD. Blood pressure was not different during daytime among all groups, but increased at night in the TRPV1-/--WD group compared with other groups. CONCLUSIONS: TRPV1 ablation leads to elevated nocturnal but not diurnal blood pressure, which is probably attributed to further enhancement of sympathetic drives at night.

Reduced nucleic acid methylation impairs meiotic maturation and developmental potency of pig oocytes.

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.

FTO reduces mitochondria and promotes hepatic fat accumulation through RNA demethylation.

Fat mass and obesity-associated protein (FTO) is a RNA demethylase, whether FTO regulates fat metabolism through its demethylation is unclear. The results of this study confirmed that N6-methyladenosine (m6 A) is associated with fat accumulation both in vivo and in vitro. The data showed that FTO down-regulated m6 A levels, decreased mitochondrial content, and increased triglyceride (TG) deposition. However, an FTO (R316A) mutant lacking demethylation activity could not regulate mitochondria and TG content, indicating that FTO affects mitochondrial content and fat metabolism by modulating m6 A levels in hepatocytes. In addition, the regulatory roles of cycloleucine (methylation inhibitor) and betaine (methyl donor) could regulate m6 A levels and fat deposition. This work clarified that the demethylation function of FTO plays an essential role in the fat metabolism of hepatocytes and links the epigenetic modification of RNA with fat deposition, thereby providing a new target (m6 A) for regulation of hepatic fat metabolism.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Serine alleviates oxidative stress via supporting glutathione synthesis and methionine cycle in mice.

SCOPE: Serine lies at the central node linking biosynthetic flux from glycolysis to glutathione synthesis and one-carbon metabolic cycle which are closely related to antioxidant capacity. The present study was conducted to determine the effects of serine supplementation on oxidative stress and its relative mechanisms. METHODS AND RESULTS: Diquat treatment was performed to induce oxidative stress in mice and primary hepatocytes. The results showed that hepatic glutathione anti-oxidant systems were impaired and reactive oxygen species and homocysteine were increased in diquat-induced mice and hepatocytes, while such disadvantageous changes were diminished by serine supplementation both in vivo and in vitro. However, when cystathionine ß-synthase expression was inhibited by interference RNA in hepatocytes, the effects of serine supplementation on the improvement of glutathione synthesis and the alleviation of oxidative stress were diminished. Moreover, when hepatocytes were treated with cycloleucine, an inhibitor of methionine adenosyltransferase, the effects of serine supplementation on the improvement of methionine cycle and the alleviation of DNA hypomethylation and oxidative stress were also diminished. CONCLUSION: Our results indicated that serine supplementation alleviated oxidative stress via supporting glutathione synthesis and methionine cycle, mostly by condensing with homocysteine to synthesize cysteine and providing one-carbon units for homocysteine remethylation.

A Critical Role for Astrocytes in Hypercapnic Vasodilation in Brain.

Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO2, arterial O2, and brain activity and is largely constant in the awake state. Although small changes in arterial CO2 are particularly potent to change CBF (1 mmHg variation in arterial CO2 changes CBF by 3%-4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E2 (PgE2) plays a key role for cerebrovascular CO2 reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE2 synthesis. We demonstrate that hypercapnia (increased CO2) evokes an increase in astrocyte [Ca2+]i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE2 (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE2 is decreased and vasodilation triggered by increased astrocyte [Ca2+]iin vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO2 Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage.SIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO2, which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE2 We demonstrate that hypercapnia (increased CO2) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE2 production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE2 is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO2-evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage.

The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca(2+)]i.

Calcium-imaging techniques were used to determine if mouse retinal astrocytes in situ respond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA) and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG; trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD) glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration ([Ca(2+)]i) increased in response to these glutamate agonists. The one exception was AMPA that increased [Ca(2+)]i in some, but not all, mouse retinal astrocytes in situ. However, AMPA did not increase [Ca(2+)]i in mouse retinal astrocytes in vitro, suggesting that the effect of AMPA in situ may be indirect.

Spinal D1-like dopamine receptors modulate NMDA receptor-induced hyperexcitability and NR1 subunit phosphorylation at serine 889.

Activation of the N-methyl-d-aspartate receptor (NMDAR) in dorsal horn neurons is recognized as a fundamental mechanism of central sensitization and pathologic pain. This study assessed the influence of dopaminergic, D1-like receptor-mediated input to the spinal dorsal horn on NMDAR function. Spinal superfusion with selective NMDAR agonist cis-ACPD significantly increased C-fiber-evoked field potentials in rats subjected to spinal nerve ligation (SNL), but not in sham-operated rats. Simultaneous application of D1LR antagonist SCH 23390 dramatically reduced hyperexcitability induced by cis-ACPD. Furthermore, cis-ACPD-induced hyperexcitability seen in nerve-ligated rats could be mimicked in unin-jured rats during stimulation of D1LRs by agonist SKF 38393 at subthreshold concentration. Phosphorylation of NMDAR subunit NR1 at serine 889 at postsynaptic sites was found to be increased in dorsal horn neurons 90 min after SNL, as assessed by increased co-localization with postsynaptic marker PSD-95. Increased NR1 phosphorylation was attenuated in the presence of SCH 23390 in the spinal superfusate. The present results support that D1LRs regulate most basic determinants of NMDAR function in dorsal horn neurons, suggesting a potential mechanism whereby dopaminergic input to the dorsal horn can modulate central sensitization and pathologic pain.

mRNA m6A methylation downregulates adipogenesis in porcine adipocytes.

Fat Mass and Obesity-associated protein (FTO), associated with obesity, is proved to demethylate N6-methyladenosine (m(6)A), which raises questions regarding whether m(6)A plays vital roles in adipogenesis. To prove this, overexpression and knockdown of FTO and METTL3, as well as the chemical treatment in procine adipocytes were conducted. The results showed FTO negatively regulated m(6)A levels and positively regulated adipogenesis, while METTL3 positively correlated with m(6)A levels and negatively with adipogenesis. To remove the potential effect of FTO and METTL3 gene, chemical reagents of methylation inhibitor cycloleucine and methyl donor betaine were used to test the regulation effect of m(6)A on adipogenesis. The results showed the inverse effect of m(6)A on lipid accumulation in porcine adipocytes. These findings provide compelling evidence that m(6)A plays a critical role in the regulation of adipogenesis.

Activation of lateral hypothalamic mGlu1 and mGlu5 receptors elicits feeding in rats.

Metabotropic glutamate receptors (mGluRs) have been popular drug targets for a variety of central nervous system (CNS) disease models, ranging from seizures to schizophrenia. The current study aimed to determine whether mGluRs participate in lateral hypothalamic (LH) stimulation of feeding. To this end, we used satiated adult male Sprague-Dawley rats stereotaxically implanted with indwelling bilateral LH guide cannulas to determine if injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a broad mGluR group I and II agonist, would elicit feeding. Administration of 100 nmol ACPD induced feeding with a short latency. Similarly, unilateral LH injection of the selective mGluR group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elicited significant feeding beginning 60 min postinjection and continuing until 4 h postinjection. Administration of the mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) produced a smaller delayed feeding response. These delayed but prolonged eating responses suggest that activation of LH mGluR1 and/or mGluR5 might be sufficient to elicit feeding. To determine which subtypes were involved, LH DHPG injections were preceded by LH injection of either the group I antagonist n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), the mGluR1 antagonist 6-amino-n-cyclohexyl-n,3-dimethylthiazolo[3,2-a]benzimi dazole-2-carboxamide hydrochloride (YM-298198) or the mGluR5 antagonist 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP), and food intake was measured. PHCCC blocked DHPG-elicited feeding, and each of the other antagonists produced significant feeding suppression. These findings suggest roles for mGluR1 and/or mGluR5 in lateral hypothalamic circuits capable of stimulating feeding behavior.

[Modulation of GABA- and kainate-activated currents by metabotropic receptors in isolated rat cortical neurons].

Whole-cell patch-clamp recordings from isolated neurons from rat prefrontal cortex have been made to study GABAb and mGluR receptor modulation of currents induced by applications of GABA and kainate. The GABAb-receptor antagonist CGP-55845 (5 microM) enhanced the peak by 26 +/- 13% (n = 6) but had no effect on the steady-state of GABA-activated current. Bath application of GABAb-receptor agonist baclofen (50 microM) enhanced the GABAa currents by 9 +/- 2% (n = 8). Kainate-activated currents were not affected by baclofen. Both GABA-activated currents and kainate-activated currents were not affected by trans-ACPD (MGluR agonist). These results suggest that in cortex postsynaptic response of GABAa-receptors can be modulated by GABAb-receptors.

In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase.

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knock-out (KO) mice, in which the primary mechanism of astrocytic calcium increase-the release of calcium from intracellular stores following activation of an IP3-dependent pathway-is lacking. Further, our results in wild-type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.

Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase A2 alpha regulate arteriole responses in mouse neocortical brain slices.

Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)α) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.

Investigation of the molecular response to folate metabolism inhibition.

We investigated the molecular response to folate metabolism inhibition by exposing human lymphoblast cell lines to the methionine adenosyltransferase inhibitor cycloleucine. We carried out microarray analysis on replicate control and exposed cells by examining 47,000 transcripts on the Affymetrix HG U133 plus 2.0 arrays. We identified 13 genes that we considered reliable responders to cycloleucine treatment: chemokine receptor 3 (CXCR3), prostaglandin-endoperoxide synthase 2, growth arrest-specific 7, reduced folate carrier, klotho beta, early growth response 1, diaphanous homolog 3, prostaglandin D2 synthase (PGDS), butyrophilin-like 9, low-density lipoprotein receptor-related protein 11, chromosome 21 orf15, G-protein-coupled receptor 98 (GPR98) and cystathionine-beta-synthase (CBS). We further demonstrated that four of these genes, CXCR3, PGDS, GPR98 and CBS, consistently responded to cycloleucine treatment in additional experiments over a range of concentrations. We carried out gene-specific DNA methylation analysis on five genes, including CBS, and found no evidence that DNA methylation changes were mediating the gene expression changes observed. Pathway analysis of the microarray data identified four pathways of relevance for response to cycloleucine; the immune response NF-AT signaling pathway was the most statistically significant. Comparison with other gene expression studies focusing on folate deficiency revealed that gene products related to immune cells or the immune response is a common theme. This indicates that apart from their role in the immune response, it is likely that these gene products may also have a role to play in the cellular response to folate status.