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The Ang II (Angiotensin II)-Angiotensin-(1-7) axis of the Renin Angiotensin System encompasses 3 enzymes that form Angiotensin-(1-7) [Ang-(1-7)] directly from Ang II: ACE2 (angiotensin-converting enzyme 2), PRCP (prolylcarboxypeptidase), and POP (prolyloligopeptidase). We investigated their relative contribution to Ang-(1-7) formation in vivo and also ex vivo in serum, lungs, and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In wild-type (WT) mice, infusion of Ang II resulted in a rapid increase of plasma Ang-(1-7). In ACE2 −/− / PRCP −/− mice, Ang II infusion resulted in a similar increase in Ang-(1-7) as in WT (563±48 versus 537±70 fmol/mL, respectively), showing that the bulk of Ang-(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a POP inhibitor, Z-Pro-Prolinal reduced the rise in plasma Ang-(1-7) after infusing Ang II to control WT mice. In POP −/− mice, the increase in Ang-(1-7) was also blunted as compared with WT mice (309±46 and 472±28 fmol/mL, respectively P =0.01), and moreover, the rate of recovery from acute Ang II-induced hypertension was delayed ( P =0.016). In ex vivo studies, POP inhibition with ZZP reduced Ang-(1-7) formation from Ang II markedly in serum and in lung lysates. By contrast, in kidney lysates, the absence of ACE2, but not POP, obliterated Ang-(1-7) formation from added Ang II. We conclude that POP is the main enzyme responsible for Ang II conversion to Ang-(1-7) in the circulation and in the lungs, whereas Ang-(1-7) formation in the kidney is mainly ACE2-dependent.

Metabotropic glutamate (mGlu) receptors impact learning and memory. Although some evidence indicates the importance of these receptors in conditioned taste aversion (CTA), the subtype-specific involvement of mGlu receptors in this associative learning task remains to be determined. These experiments examined the effects of (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), a selective group II mGlu receptor agonist, cis-2-[[(3,5-dichlorophenyl)amino]carbonyl]cyclohexanecarboxylic acid (VU0155041), a mGlu4 positive allosteric modulator, N,N'-dibenzhydrylethane-1,2-diamine (AMN082), a mGlu7 allosteric agonist, and 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), a mGlu7 negative allosteric modulator, on the acquisition of CTA using male Sprague-Dawley rats. Systemic injections of LY379268, AMN082, and MMPIP prior to conditioning decreased the acquisition of CTA, revealing that mGlu2/3 and mGlu7 are involved in CTA learning.

Metabotropic glutamate receptor 5 (mGlu5) has been implicated in a variety of learning and memory processes and is important for avoidance learning. The present studies used an mGlu5 receptor positive allosteric modulator, 3-cyano-N-(1,3 diphenyl-1H-hyrazol-5-yl)benzamide (CDPPB), to characterize the importance of mGlu5 receptors in aversively- and appetitively-motivated spatial learning tasks (tasks in which the instrumental contingency involves discriminative cues that differ in spatial location). C57Bl/6 male mice were initially trained in the Barnes maze in the absence of drug. Subsequently, CDPPB (30 mg/kg, i.p.), administered 20 min prior to each of 3 daily reversal learning training sessions in the Barnes maze, significantly enhanced performance compared to vehicle-treated controls and had a significant effect on search strategy. Mice treated with CDPPB also displayed significantly less perseverative behavior than control-treated animals. In a second experiment, male Sprague–Dawley rats were trained in an appetitively-motivated, delayed alternation version of a T-maze. 30 mg/kg CDPPB (s.c.), delivered 20 min prior to each of 5 daily training sessions, enhanced the delay rats were able to withstand between the sample and choice portions of each T-maze trial. The present results emphasize the role of mGlu5 receptors in spatial learning tasks and support previous studies which report mGlu5 positive allosteric modulators can enhance learning in some tasks and may have potential as nootropic drugs.

Metabotropic glutamate receptor 5 (mGlu5) has been implicated in a variety of learning processes and is important for inhibitory avoidance and conditioned taste aversion learning. MGlu5 receptors are physically connected with NMDA receptors and they interact with, and modulate, the function of one another in several brain regions. The present studies used systemic co-administration of an mGlu5 receptor positive allosteric modulator, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and an NMDA receptor antagonist dizocilpine maleate (MK-801) to characterize the interactions of these receptors in two aversive learning tasks. Male Sprague–Dawley rats were trained in a single-trial step-down inhibitory avoidance or conditioned taste aversion task. CDPPB (3 or 10 mg/kg, s.c.), delivered by itself prior to the conditioning trial, did not have any effect on performance in either task 48 h after training. However, CDPPB (at 3 mg/kg) attenuated the MK-801 (0.2 mg/kg, i.p.) induced learning deficit in both tasks. CDPPB also reduced MK-801-induced hyperactivity. These results underlie the importance of mGlu5 and NMDA receptor interactions in modulating memory processing, and are consistent with findings showing the efficacy of positive allosteric modulators of mGlu5 receptors in reversing the negative effects of NMDA receptor antagonists on other behaviors such as stereotypy, sensorimotor gating, or working, spatial and recognition memory.

In conditioned taste aversion (CTA), animals learn to avoid a flavored solution (conditioned stimulus, CS) previously paired with internal malaise (unconditioned stimulus, US). Metabotropic glutamate receptor 5 (mGlu5) has been implicated in learning and memory processes and is necessary for CTA. In the present study, local microinjections of a mGlu5-selective antagonist, 3-[2-methyl-1,3-thiazol-4yl)ethynyl]pyridine (MTEP, 0, 1 or 5 μg) into the insular cortex and basolateral amygdala were used in male, Sprague-Dawley rats to examine the role of mGlu5 receptors in the encoding of taste memory. MTEP was infused 20 min before saccharin intake during CTA conditioning. MTEP injection into the basolateral amygdala resulted in robust CTA, similar to the vehicle-treated animals but slowed extinction; that is, MTEP enhanced CTA. MTEP injection into the insular cortex resulted in an increased saccharin intake on the conditioning trial, which potentially influenced the performance on the test trials; MTEP had no effect on CTA learning when controlled access to saccharin was used on the conditioning trial. These results indicate that mGlu5 receptors are involved in taste memories in a region-specific manner.

Metabotropic glutamate receptors (mGluRs) have been implicated in several types of cognitive and associative learning. Although recent evidence indicates an influence of mGluRs in conditioned taste aversion (CTA), the subtype-specific involvement of mGluRs in this learning paradigm remained to be determined. The aim of this study was to examine the role of Group I mGluR subtypes in CTA using a selective mGluR5 antagonist (2-methyl-6-(phenylethynyl)-pyridine, MPEP) and a selective mGluR1 antagonist (1-aminoindan-1,5-dicarboxylic acid, AIDA). Male, water-deprived, Sprague–Dawley rats were injected i.p. with 6 or 12 mg/kg MPEP or saline. Twenty-five minutes later, all rats received 15-min access to a 0.1% saccharin solution (Sac) immediately followed by an injection of 0.15 M LiCl at 1.33% body weight. The animals were tested with 15-min access to Sac on each of four test days. MPEP-treated animals consumed more Sac on the test trials than saline-treated rats. In another experiment, controlled access to Sac was used by infusing the solution on the conditioning trial. Consistent with the above results, MPEP attenuated the degree of CTA. Similar experiments using the mGluR1 antagonist AIDA, have found no effect on CTA learning. These results suggest that the two subtypes of Group I mGluRs are differentially involved in taste aversion learning.

Alzheimer's disease (AD) is a progressive, age-dependent neurodegenerative disorder affecting specific brain regions that control memory and cognitive functions. Epidemiological studies suggest that exercise and dietary antioxidants are beneficial in reducing AD risk. To date, botanical flavonoids are consistently associated with the prevention of age-related diseases. The present study investigated the effects of 4 months of wheel-running exercise, initiated at 2-months of age, in conjunction with the effects of the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) administered orally in the drinking water (50 mg/kg daily) on: (1) behavioral measures: learning and memory performance in the Barnes maze, nest building, open-field, anxiety in the light-dark box; and (2) soluble amyloid-β (Aβ) levels in the cortex and hippocampus in TgCRND8 (Tg) mice. Untreated Tg mice showed hyperactivity, relatively poor nest building behaviors, and deficits in spatial learning in the Barnes maze. Both EGCG and voluntary exercise, separately and in combination, were able to attenuate nest building and Barnes maze performance deficits. Additionally, these interventions lowered soluble Aβ1-42 levels in the cortex and hippocampus. These results, together with epidemiological and clinical studies in humans, suggest that dietary polyphenols and exercise may have beneficial effects on brain health and slow the progression of AD.

The O2-dependent oxidation of urate catalyzed by urate oxidase has been examined in order to identify the immediate product of the enzymatic reaction. Specifically labeled [13C]urates were utilized as substrates, and the time courses were monitored by 13C NMR. On the basis of chemical shift values and 18O labeling, the product of the reaction was identified as 5-hydroxyisourate. This identification was substantiated by calculation of the 13C NMR spectrum of 5-hydroxyisourate using ab initio density functional theory methods. The predominant tautomers of urate and allantoin in aqueous solution were identified from 13C NMR titration data; the ionization behavior of urate and 5-hydroxyisourate were also examined by computational methods. The nonenzymatic pathway for production of allantoin from 5-hydroxyisourate was delineated; the reaction proceeds by the hydrolysis of the N1−C6 bond, followed by an unusual 1,2-carboxylate shift and decarboxylation to form allantoin.

The substrate specificity of tartrate dehydrogenase has been probed using a series of alternative substrates to identify the molecular interactions which determine whether a particular substrate undergoes enzyme-catalyzed decarboxylation or not. A series of 3-substituted malate analogs, in which F, Cl, Br, I, SH, or NH2 substituents were placed at the 3R- or 3S-position, was prepared, and the product resulting from the action of tartrate dehydrogenase on each compound was identified. All of the halomalates and both diastereomers of aminomalate underwent oxidative decarboxylation; both diastereomers of 3-thiomalate underwent net nonoxidative decarboxylation. The results were interpreted in terms of a model in which decarboxylation is conformationally controlled. The data are not consistent with a model which suggests that substrates assume the conformation that is necessary to avoid steric crowding between the enzyme and the substituent at the 3-position of the substrate. These data are consistent with a model in which the course of the reaction with (+)-tartrate and meso-tartrate is dictated by the coordination of the substrate hydroxyls to the active site Mn2+. However, the observed reactivities of the 3-methyltartrate diastereomers are not consistent with this model, either: (2R,3R)-3-methyltartrate undergoes oxidative decarboxylation, and (2R,3S)-3-methyltartrate undergoes simple oxidation. These results suggest that for these compounds the conformation is dictated by the positioning of the hydrophobic substituent in a specific binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

l-Glutamate-mediated transmembrane influx of 22Na+ into a native membrane vesicle preparation from rat hippocampus was resolved into components due to glutamate receptor and Na+-glutamate symport, by kinetic analysis and pharmacological specificity. Measurements made with a quench-flow technique showed that receptor-mediated cation exchange proceeded in two phases, the faster phase progressively attenuated by a desensitization process with a half time of approximately 90 ms. The influx of 22Na+ continued in the second phase with a half time of 6 s. Receptor-mediated 22Na+ influx was replicated with the glutamate mimetics, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate but not with N-methyl-d-aspartate and was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione but not by 2-amino-5-phosphonovaleric acid. Receptor-mediated 22Na+ influx did not require glycine and was pH dependent; influx was inhibited at pH values less than pH 5.0. Thus, the receptor-mediated activity was of the γ-amino-3-hydroxy-5-methyl-4-isoxazole propionate-kainate subtype. The N-methyl-d-aspartate receptor inhibitor 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine or MK801), increased receptor-mediated transmembrane 22Na+ flux in both phases, but did not accelerate receptor desensitization. Receptor-mediated radiotracer exchange was the major contribution to 22Na+ influx in times less than 5 s and the receptor-mediated responses could be measured with a small correction for sodium-glutamate symport or in the presence of a glutamate-uptake inhibitor. A third phase of 22Na+ influx accessed a different internal volume and proceeded with the same first-order rate constant as [3H]glutamate uptake when measured simultaneously. This was shown to be mediated by the glutamate uptake mechanism, with a half time of 20–40 s varying with the preparation. The Na+ -glutamate symport became the largest cause of 22Na+ influx after at least 5 s, the final magnitude being, on average, twofold larger than the receptor-mediated 22Na+ influx.

Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA-mediated influx of 36Cl-. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 microM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10-fold at 0.3 microM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 microM GABA, was unaltered. The half-response concentration was reduced from 80 microM to 50 microM. CDPX alone caused no measurable 36Cl- exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel opening mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.

We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules approximately 100-fold to apparent homogeneity with a final specific activity of 10 micromol/min/mg. The enzyme exhibited a native molecular mass of approximately 68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 +/- 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and the K(m) for 5-hydroxyisourate was determined to be 15 microM. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.

Group I metabotropic glutamate receptors (mGlu1 and 5) have been implicated in synaptic plasticity and learning and memory. However, much of our understanding of how these receptors in different brain regions contribute to distinct memory stages in different learning tasks remains incomplete. The present study investigated the effects of the mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and mGlu1 receptor antagonist, ( S )-(+)-α-amino-4-carboxy-2-methylbenzene-acetic acid (LY 367385) in the dorsal hippocampus on the consolidation and extinction of memory for inhibitory avoidance learning. Male, Sprague–Dawley rats were trained in a single-trial step-down inhibitory avoidance task. MPEP, LY 367385 or saline were infused bilaterally into the CA1 region immediately after training or immediately after the first retention test which was given 24 h after training. Rats receiving MPEP (1.5 or 5.0 μg/side) or LY 367385 (0.7 or 2.0 μg/side) infusion exhibited a dose-dependent decrease in retention when tested 24 h later. MPEP was ineffective while LY 367385 significantly attenuated extinction when injected after the first retention test using an extinction procedure. These findings indicate a selective participation of hippocampal group I mGlu receptors in memory processing in this task.

Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed.

Latent inhibition (LI) is a phenomenon by which pre-exposure of a conditioned stimulus (CS) prior to the CS-unconditioned stimulus (US) pairings retards conditioned responding (CR). LI has been demonstrated in a variety of learning tasks including conditioned taste aversion (CTA). Earlier work has shown that systemic administration of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective metabotropic glutamate receptor 5 (mGlu5) antagonist, is able to disrupt classical conditioning in CTA. The present study investigated the involvement of mGlu5 receptors in LI using a CTA procedure. In the first experiment, rats received either water (non-pre-exposed, NPE) or a saccharin solution (pre-exposed, PE) on 2 consecutive days. The animals then received conditioning in which a fixed amount of saccharin was paired with lithium chloride and then the CR to the taste was tested. Either MPEP (3, 6, 12 mg/kg) or vehicle was injected intraperitoneally prior to taste pre-exposure or testing. Animals in the vehicle control groups displayed LI. MPEP injections before pre-exposure trials attenuated LI but also reduced consumption during pre-exposure, which obscured interpretation of the LI effect. The second experiment used four pre-exposure trials and controlled access to fixed amount of the solutions during the pre-exposure as well as the conditioning trials. Rats were injected before pre-exposure trials but not before the test trial. The results found that MPEP attenuates latent inhibition suggesting that the mGlu5 receptor exerts an influence on the processes that underlie the effects of taste pre-exposure on conditioning.