Exploring the Role of Asp1116 in Selective Drug Targeting of CREBcAMP- Responsive Element-binding Protein Implicated in Prostate Cancer.

BACKGROUND: The selective targeting of CREB-cAMP-responsive element-binding protein (CBP) has recently evolved as a vital therapeutic approach for curtailing its aberrant upregulation associated with the development of prostate cancer. Inhibition of CBP has been discovered to be an important therapeutic option in androgen receptor signalling pathway mediated prostate cancer. Y08197, a novel selective inhibitor of CBP, has shown promising therapeutic outcome in prostate carcinogenesis over non-selective analogues such as CPI-637. METHODS/RESULTS: Herein, we used molecular dynamics simulation to gain insights into the mechanistic and selective targeting of Y08197 at the bromodomain active site. Molecular Mechanics/ Poisson-Boltzmann Surface Area (MM/PBSA) analysis revealed a similar inhibitory effect between Y08197 and CPI-637. Furthermore, in exploring the selective affinity of Y08197 towards CBP in combination with Bromodomain and PHD finger-containing protein 1(BRPF1), our findings highlighted Asp1116 as the 'culprit' residue responsible for this selective targeting. Upon binding, Asp1116 assumed a conformation that altered the architecture of the bromodomain active site, thereby orienting the helices around the active site in a more compacted position. In addition to some specific structural perturbations mediated by Asp1116 on the dynamics of CBP, our study revealed that the strong hydrogen bond interaction (N-H...O) elicited in CBP-Y08197 sequestered Y08197 tightly into the CBP bromodomain active site. CONCLUSION: Conclusively, the inhibition and selective pattern of Y08197 can be replicated in future structure-based CBP inhibitors and other bromodomain implicated in carcinogenesis.

A delta opioid receptor agonist, KNT-127, in the prelimbic medial prefrontal cortex attenuates glial glutamate transporter blocker-induced anxiety-like behavior in mice.

We previously reported that systemic administration of a delta opioid receptor (DOP) agonist, KNT-127, produced a potent anxiolytic-like effect in rats. Interestingly, DOPs are highly distributed in the prelimbic medial prefrontal cortex (PL-PFC). In the present study, we investigated the effect of KNT-127 co-perfusion in the PL-PFC on anxiety-like behavior in mice, induced by a glial glutamate transporter inhibitor, (3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA). Extracellular glutamate levels were measured in male C57BL/6N mice by in vivo microdialysis high-performance liquid chromatography/electrochemical detection, with behavior simultaneously assessed in the open field test. As expected, extracellular glutamate levels were significantly increased, and anxiety-like behavior was induced after local perfusion of TFB-TBOA in the PL-PFC. Uniquely, co-perfusion of KNT-127 in the PL-PFC diminished anxiety-like behavior induced by TFB-TBOA without affecting extracellular glutamate levels. Further, the effect of KNT-127 on anxiety-like behavior was antagonized by a selective DOP antagonist, naltrindole, suggesting that KNT-127 acts via DOPs. These findings do not support our preconceived hypothesis that KNT-127 in PL-PFC produces an anxiolytic-like effect via suppression of glutamatergic transmission. Hence, further studies are necessary to understand the mechanisms of DOP agonist-induced anxiolytic-like effects in the PL-PFC.

Antiepileptic activity of total triterpenes isolated from Poria cocos is mediated by suppression of aspartic and glutamic acids in the brain.

CONTEXT: Triterpenes from Poria cocos Wolf (Polyporaceae) have been used to treat various diseases in traditional Chinese medicine. However, the antiepileptic effects and mechanism are not fully understood. OBJECTIVE: The objective of this study is to investigate the antiepileptic properties of total triterpenes (TTP) from the whole P. cocos. MATERIALS AND METHODS: The ethanol extract TTP was identified by HPLC fingerprint analysis. Male ICR mice were gavaged (i.g.) with TTP (5, 20, 80 or 160 mg/kg) or reference drugs twice a day for 7 d. Antiepileptic activities of TTP were evaluated by maximal electroshock (MES)- and pentylenetetrazole (PTZ)-induced seizures in mice for 30 and 60 min, respectively. Locomotor activity and Rota-rod tests were performed for 60 min and 5 min, respectively. The levels of glutamic acid (Glu), aspartic acid (Asp), γ-aminobutyric acid (GABA) and glycine (Gly) in convulsive mice were estimated. The chronic epileptic model of Wistar rats was built to measure expressions of glutamate decarboxylase 65 (GAD65) and GABAA in rat brain after TTP treatment. RESULTS: The LC50 of TTP (i.g.) was above 6 g/kg. TTP (5-160 mg/kg) protected mice against MES- and PTZ-induced convulsions at 65.0% and 62.5%, respectively, but have no effect on rota-rod treadmill; TTP (20-160 mg/kg) significantly reduced the locomotor activities, shortened the onset of pentobarbital sodium-induced sleep; TTP decreased Glu and Asp levels in convulsive mice, but increased the GAD65 and GABAA expressions in chronic epileptic rats at doses usage. DISCUSSION AND CONCLUSION: TTP extracted from P. cocos possessed potential antiepileptic properties and is a candidate for further antiepileptic drug development.

Identification and characterization of natural microbial products that alter the free d-aspartate content of mammalian cells.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

Postsynaptic mGluR mediated excitation of neurons in midbrain periaqueductal grey.

Metabotropic glutamate (mGlu) receptors modulate pain from within the midbrain periaqueductal grey (PAG). In the present study, the postsynaptic mGlu receptor mediated effects on rat PAG neurons were examined using whole-cell patch-clamp recordings in brain slices. The selective group I agonist DHPG (10 µM) produced an inward current in all PAG neurons tested which was associated with a near parallel shift in the current-voltage relationship. By contrast, the group II and III mGlu receptor agonists DCG-IV (1 µM) and l-AP4 (3 µM) produced an outward current in only 10-20% of PAG neurons tested. The DHPG induced current was concentration dependent (EC(50) = 1.4 µM), was reduced by the mGlu1 antagonist CPCCOEt (100 µM), and was further reduced by CPCCOEt in combination with the mGlu5 antagonist MPEP (10 µM). The glutamate transport blocker TBOA (30 µM) also produced an inward current, however, this was largely abolished by CNQX (10 µM) plus AP5 (25 µM). Slow EPSCs were evoked following train, but not single shock stimulation, which were enhanced by TBOA (30 µM). The TBOA enhancement of slow EPSCs was abolished by MPEP plus CPCCOEt. These findings indicate that endogenously released glutamate, under conditions in which neurotransmitter spill-over is enhanced, activates group I mGlu receptors to produce excitatory currents within PAG. Thus, postsynaptic group I mGlu receptors have the potential to directly modulate the analgesic, behavioural and autonomic functions of the PAG. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Effects of fatty acids on cardioprotection by pre-ischaemic inhibition of the malate-aspartate shuttle.

1. The malate-aspartate shuttle (MAS) is the main pathway for balancing extra- and intramitochondrial glucose metabolism. Pre-ischaemic shutdown of the MAS by aminooxyacetate (AOA) mimics ischaemic preconditioning (IPC) in rat glucose-perfused hearts. The aim of the present study was to determine the effects of fatty acids (FA) on cardioprotection by pre-ischaemic inhibition of the MAS. 2. Isolated rat hearts were divided into four groups (control; pre-ischaemic AOA (0.2 mmol/L); IPC; and AOA + IPC) and were perfused with 11 mmol/L glucose, 3% bovine serum albumin plus 0, 0.4 or 1.2 mmol/L FA. The perfusion protocol included 30 min global no-flow ischaemia and 120 min reperfusion. Infarct size (IS), haemodynamic recovery, glucose oxidation and lactate release were evaluated in all four groups. 3. Pre-ischaemic AOA reduced the IS of the left ventricle in hearts perfused with 0, 0.4 and 1.2 mmol/L FA compared with that in control hearts (26 ± 2% vs 53 ± 4%, 29 ± 3% vs 53 ± 4% and 61 ± 4% vs 81 ± 3%, respectively; P < 0.01 for all). After 2 h reperfusion, AOA improved haemodynamic recovery in the absence (52 ± 2 vs 27 ± 3 mmHg in the AOA and control groups, respectively; P < 0.001) but not in the presence, of FA. Both IPC and AOA + IPC reduced IS and improved haemodynamic recovery regardless of FA levels. Postischaemic glucose oxidation was suppressed by FA and did not differ significantly between the different groups. 4. In conclusion, the reduction in IS induced by pre-ischaemic MAS shutdown is not compromised by physiological FA concentrations. Transient MAS shutdown may be involved in IPC, but is not sufficient on its own as the underlying mechanism for IPC.

Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn.

Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.

Xenon augmented hypothermia reduces early lactate/N-acetylaspartate and cell death in perinatal asphyxia.

OBJECTIVE: Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia-ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry. METHODS: Thirty-six newborn piglets were randomized (all groups n = 9), with intervention from 2 to 26 hours, to: (1) normothermia; (2) normothermia + 24 hours 50% inhaled xenon; (3) 24 hours hypothermia (33.5°C); or (4) 24 hours hypothermia (33.5°C) + 24 hours 50% inhaled xenon. Serial MRS was acquired before, during, and up to 48 hours after hypoxia-ischemia. RESULTS: Mean arterial blood pressure was lower in all treatment groups compared with normothermia (p < 0.01) (although >40mmHg); the combined therapy group required more fluid boluses (p < 0.05) and inotropes (p < 0.001). Compared with no intervention, both hypothermia and xenon-augmented hypothermia reduced the temporal regression slope magnitudes for phosphorus-MRS inorganic phosphate/exchangeable phosphate pool (EPP) and phosphocreatine/EPP (both p < 0.05); for lactate/N-acetylaspartate (NAA), only xenon-augmented hypothermia reduced the slope (p < 0.01). Xenon-augmented hypothermia also reduced transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)(+) nuclei and caspase 3 immunoreactive cells in parasagittal cortex and putamen and increased microglial ramification in midtemporal cortex compared with the no treatment group (p < 0.05). Compared with hypothermia, however, combination treatment did not reach statistical significance for any measure. Lactate/NAA showed a strong positive correlation with TUNEL; nucleotide triphosphate/EPP showed a strong negative correlation with microglial ramification (both p < 0.01). INTERPRETATION: Compared with no treatment, xenon-augmented hypothermia reduced cerebral MRS abnormalities and cell death markers in some brain regions. Compared with hypothermia, xenon-augmented hypothermia did not reach statistical significance for any measure. The safety and possible improved efficacy support phase II trials.

Impact of gestational and lactational phthalate exposure on hypothalamic content of amino acid neurotransmitters and FSH secretion in peripubertal male rats.

This study investigated the effect of the pre- and perinatal exposure to di-(2-ethylhexyl) phthalate (DEHP) on the neuroendocrine parameters that regulate reproduction in peripubertal male rats. DEHP at dose of 3 and 30mg/kg bw/day was administered orally to female rat since pregnancy onset until weaning. The male litters were sacrificed at 30 days of age to determine gonadotropin serum level and the hypothalamic contents of the amino acids aspartate and gamma-aminobutyric acid. No changes in gonadotropin, aspartate and gamma-aminobutyric acid levels were detected at the low dose. DEHP 30mg/kg bw/day reduced testis weight and serum FSH, in correlation with a significant increase in the inhibitory GABAergic tone and a reduction in the stimulatory effect of aspartate on gonadotropin level. This study provides unknown data regarding changes in the hypothalamic contents of the amino acid neurotransmitters, which are involved in the neuroendocrine regulation of reproductive axis, in peripubertal male rat offspring from dams exposed to DEHP during gestational and lactational periods. This could be related with the gonadotropin modifications also here described.

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine (KIN59).

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11A from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V(max)/K(m) of mutant enzyme approximately 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.

Genome-scale analysis of anti-metabolite directed strain engineering.

Classic strain engineering methods have previously been limited by the low-throughput of conventional sequencing technology. Here, we applied a new genomics technology, scalar analysis of library enrichments (SCALEs), to measure >3 million Escherichia coli genomic library clone enrichment patterns resulting from growth selections employing three aspartic-acid anti-metabolites. Our objective was to assess the extent to which access to genome-scale enrichment patterns would provide strain-engineering insights not reasonably accessible through the use of conventional sequencing. We determined that the SCALEs method identified a surprisingly large range of anti-metabolite tolerance regions (423, 865, or 909 regions for each of the three anti-metabolites) when compared to the number of regions (1-3 regions) indicated by conventional sequencing. Genome-scale methods uniquely enable the calculation of clone fitness values by providing concentration data for all clones within a genomic library before and after a period of selection. We observed that clone fitness values differ substantially from clone concentration values and that this is due to differences in overall clone fitness distributions for each selection. Finally, we show that many of the clones of highest fitness overlapped across all selections, suggesting that inhibition of aspartate metabolism, as opposed to specific inhibited enzymes, dominated each selection. Our follow up studies confirmed our observed growth phenotypes and showed that intracellular amino-acid levels were also altered in several of the identified clones. These results demonstrate that genome-scale methods, such as SCALEs, can be used to dramatically improve understanding of classic strain engineering approaches.

Study of hypothalamic metabolism in cluster headache by proton MR spectroscopy.

The authors used 1H-MRS to investigate hypothalamic metabolism in 26 patients with cluster headache (CH) and 12 healthy subjects. Hypothalamic N-acetylaspartate/creatine was reduced in patients with CH vs controls (p < 0.01). Dividing the patients into episodic CH outside- and in-cluster periods and chronic CH, the hypothalamic N-acetylaspartate/creatine in all three subgroups of patients was reduced. The reduction of the neuronal marker N-acetylaspartate is consistent with hypothalamic neuronal dysfunction in patients with CH.

Electrical and pharmacological properties of the slow depolarization induced by Schaffer collateral stimulation in astrocytes of the rat hippocampal CA1 region.

Intracellular recordings were made from astrocytes of the hippocampal CA1 region in rat slice preparations. A single stimulus at the Schaffer collaterals with high intensities (>/=10 V), but not low intensities (<10 V), induced a slow depolarization similar to the evoked postsynaptic potentials observed in hippocampal CA1 neurons. The slow depolarization was almost abolished in the presence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM) and N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonopentanoic acid (100 microM). Tetrodotoxin (0.5 microM) or Co(2+) (2 mM) also abolished this slow depolarization. The slow depolarization reversed its polarity at -8 mV in the presence of tetraethylammonium (20 mM). These results suggest that the synaptically released glutamate (Glu) induces depolarization, which is mainly mediated by ionotropic Glu receptors, in astrocytes in the hippocampal CA1 region.

Investigation on acetylcholine, aspartate, glutamate and GABA extracellular levels from ventral hippocampus during repeated exploratory activity in the rat.

The extracellular levels of aspartate, glutamate, gamma-aminobutyric acid (GABA), and acetylcholine (ACh) were investigated by microdialysis, coupled with HPLC, in the ventral hippocampus of rats during two 30-min exploration periods. Motor activity was monitored. During exploration I, an increase in motor activity associated with a 315% increase in aspartate, 181% in glutamate, and 264% in ACh levels, occurred during the first 10 min. The increase in GABA level reached a maximum of 257% during the second 10 min. The neurotransmitter levels returned to basal values within 40 min. During exploration II, 1 h later, a smaller increase in neurotransmitter levels and motor activity was observed. In both explorations, the increase in neurotransmitter levels was completely abolished by 1 and 3 microM TTX. A statistically significant relationship was found between neurotransmitter extracellular levels and motor activity, for aspartate and glutamate in exploration I, and for ACh in exploration I and II. In conclusion, exploratory activity is associated with or depends on the activation of neuronal systems in the ventral hippocampus releasing aspartate, glutamate, GABA, and ACh. The activation is dampened by habituation.

Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties.

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.

A cellular mechanism that reversibly inactivates pancaspase inhibitor zAsp-CH(2)-DCB: a potential pitfall causing discrepancy between in vitro and in vivo caspase assays.

Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.

A pharmacological analysis of the contractile effects of glutamate on rat and human isolated gut smooth muscle strips.

Although the contractile effects of glutamate and related excitatory amino acids on gut smooth muscle strips have been demonstrated, the mechanisms, and particularly the physiological importance of that action, remain unknown. In this study, glutamate, aspartate, AMPA, quisqualate, cis-ACPD and (2R,4R)-APDC evoked concentration-dependent contraction of isolated adult rat gastric fundus, with EC50 values of 210 microM, 150 microM, 20 microM, 33 microM, and 2.7 microM and 7.9 microM, respectively. L-SOP (0.1 microM-1.9 mM) did not change the basal tone of the preparations. The maximal contractions evoked by glutamate (20 mM) were 38.83% compared with those elicited by acetylcholine (20 microM). The glutamate-evoked contractions were not affected by atropine, verapamil and nicardipine, blocked by CNQX (0.01 microM), or potentiated by Mg2+ (0.01-100 microM), ketamine (0.01-100 microM) and DL-AP5 (0.1-100 microM), as well as L-trans-2,4-PDC (1-100 microM). Analysis of glutamate's action on rat rectum (EC50 = 44 microM) could only be carried out at the early stages, as half of the preparations were not affected by glutamate. Only 5 out of 26 human longitudinal and circular smooth muscle preparations taken from the stomach and three segments of the large intestine were very slightly contracted by glutamate, excluding further analysis. The contractile effects of glutamate on rat gut smooth muscles were mediated by multiple GluR (non-NMDA > NMDA > group I/II mGluRs) located primarily on smooth muscle cells but functional GluRs on neurons and/or nerve fibers of myenteric nervous plexuses could not be excluded. To fully understand the physiological significance of glutamate-evoked contractions in the gut, more research is required, most likely using many different methodological approaches.

D-aspartate disposition in neuronal and endocrine tissues: ontogeny, biosynthesis and release.

High levels of D-aspartate occur in the brain and endocrine glands, such as pineal, adrenal and pituitary. In the brain, D-aspartate levels are highest in embryonic and early postnatal stages. Notably high levels occur in the early postnatal cortical plate and subventricular zone of the cerebral cortical cultures, implying a role in development. In embryonic neuronal primary culture cells, we detected high levels of endogenous D-aspartate and demonstrated biosynthesis of [14C]D-aspartate using [14C]L-aspartate as precursor. Synthesis of D-aspartate in cell cultures is inhibited by amino-oxyacetic acid, an inhibitor of pyridoxal phosphate-dependent enzymes. In the rat adrenal medulla, D-aspartate is depleted by treatment of the animals with intraperitoneal nicotine injections. In adrenal slices, D-aspartate is released by depolarization with KCl or acetylcholine, implying physiological release by activation of the cholinergic innervation of the adrenal. Our characterization of D-aspartate ontogeny, biosynthesis and depolarization-induced release implies specific physiological roles for this amino acid.

Deramciclane inhibits N-methyl-D-aspartate receptor function.

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca(2+) ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 microM) significantly inhibited [(3)H]D-aspartate release and transmembrane Ca(2+) flux to N-methyl-D-aspartate in the absence of Mg(2+). By contrast, inhibition of [(3)H]D-aspartate release and transmembrane Ca(2+) flux evoked by 0.1 mM (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg(2+) and 10 microM cyclothiazide by 10 microM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 microM) did not inhibit [(3)H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.

Apoptosis induced by inhibitors of nucleotide synthesis in deoxyadenosine-resistant leukemia L1210 cells that lack p53 expression.

An L1210 cell line (Y8) selected for resistance to deoxyadenosine contains ribonucleotide reductase that is not subject to inhibition by dATP. In addition, the Y8 cells have other phenotypic expressions that include increased sensitivity to apoptosis induced by various agents such as radiation, doxorubicin, anisomycin and roscovitine. The Y8 cells were found to be more sensitive to apoptosis induced by methotrexate (MTX), tiazofurin (TZ), deoxyguanosine (dGuo) and N-(phosphonoacetyl)-L-aspartate (PALA). Deoxyguanosine, at concentrations that did not cause apoptosis in the Y8 cells, prevented the apoptotic response of the Y8 cells to MTX and TZ. Deoxycytidine had no effect. Since caspase-3 activation is involved in apoptotic pathways, the effects of the caspase-3 inhibitor, Ac-DEVD-CHO, were studied on the dGuo-, MTX- or TZ-induced apoptosis in the Y8 cells. Ac-DEVD-CHO caused a marked decrease in the fraction of cells in the early phase of apoptosis. However, there was a corresponding increase in the fraction of cells in the late apoptotic/necrotic stages of cell death. This is in marked contrast to the dGuo-induced decrease in apoptosis seen in the MTX- and TZ-treated Y8 cells in which there were no increases in the late apoptotic/necrotic fraction of cells. These data show that alterations of nucleotide pools in the Y8 cells cause marked increases in the apoptotic response which may indicate that the Y8 cells are much more susceptible to the effects of misincorporation of nucleotides into DNA than are the parental WT L1210 cells.