GlycineB antagonists as potential therapeutic agents

Summary It is not clear what therapeutic application is most likely for agents blocking glycine site of the NMDA receptors (glycine_B). Majority of the studies to date used either glycine_B antagonists with doubtful brain penetration or partial agonists. Following systemic administration to rats of our newly developed glycme_B antagonists (MRZ 2/570; 2/571 and 2/576) and L-701,324 (MSD) as a reference agent the following behavioural effects were observed: weak (if any) antiparkinsonian-like effects, lack of anxiolytic activity, inhibition of physical and motivational aspects of morphine dependence and neuroprotective activity in global ischaemia. The side effects include: sedation, ataxia, and myorelaxation. We detected neither vacuolisation in the cingulate cortex nor impairment of pre-pulse inhibition indicating lack of psychotomimetic potential.     

Modulation of NMDA receptors by glycine — introduction to some basic aspects and recent developments

Summary Glycine is a co-agonist at NMDA receptors and it's presence is a prerequisite for channel activation by glutamate or NMDA. Physiological concentrations reduce one form of NMDA receptor-desensitization. Interactions between the glycine_B site and other domains of the NMDA receptor are complex and include the glutamate, Mg⁺ and polyamines sites. Glycine shows different affinities at various NMDA receptor subtypes probably via to allosteric interactions between NMDA2 subunits and the glycine recognition site on the NMDAR1 subunit. There is still some debate whether the glycine_B site is saturatedin vivo but it seems likely that this depends on regional differences in receptor subtype expression, local glycine or D-serene concentrations and the expression of specific glycine transporters.Glycine_B antagonists and partial agonists have been reported to have good therapeutic indices as neuroprotective agents against focal ischaemia and trauma, anti-epileptics, anxiolytics, anti-psychotomimetics and in models of chronic pain. They clearly lack two potentially serious side effects classically associated with NMDA receptor blockade, namely neurodegenerative changes in the cingulate/retrosplenial cortex and psychotomimetic-like effects. This improved therapeutic profile may be partially due to the ability of full glycine_B antagonists to reveal Gycne-sensitive desensitization and possibly also via functional and/or regional NMDA receptor subtype selectivity.     

Effects of pretreatment with inducers of hepatic mixed function oxidases on DNA repair elicited by various compounds in hepatocytes from adult and neonatal rats

Studies were conducted to assess the effects of inducers of hepatic mixed function oxidases on DNA repair responses to 13 different genotoxic agents in hepatocytes from adult male mice. Phenobarbital pretreatment increased DNA repair elicited by diethylnitrosamine but had no effect on responses to the other compounds. Pretreatment with p,p-dichlorodiphenyltrichloroethane, 3-methylcholanthrene or -naphthoflavone induced the DNA repair responses to a variety of activation-dependent carcinogens. DNA repair responses to the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N-nitro-N-nitrosoguanidine were not increased by any of the pretreatments, which indicated that the pretreatment-related enhancement of responses to the other compounds was due to induction of their metabolic activation. Taken together, the findings suggest that Aroclor, or other pretreatments, may increase the sensitivity of the hepatocyte DNA repair assay for detecting the genotoxicity of certain compounds; however, the potential benefit may be limited due to specific features of the assay. In contrast, Aroclor pretreatment did not produce any enhancement of in vivo DNA repair elicited by dimethylnitrosamine, diethylnitrosamine, o-aminoazotoluene, 2-acetylaminofluorene, 3-methylcholanthrene or aflatoxin B₁, and thus does not appear to be useful for improving the sensitivity of the in vivo/in vitro assay.Whereas the amount of DNA repair produced by dimethylnitrosamine was not increased by classical inducers of liver microsomal enzymes, pretreatment with pyrazole greatly augmented in vitro and in vivo DNA repair responses to dimethylnitrosamine; responses to diethylnitrosamine were increased to a lesser degree by pyrazole pretreatment. The effects of lactational exposure to enzyme inducing agents on DNA repair in neonatal hepatocytes was also investigated.Abbreviations 2-AAF 2-acetylaminofluorene - 4-AB 4-aminobiphenyl - 6-AC 6-aminochrysene - AFB aflatoxin B₁ - ARO Aroclor 1254 - o-AT o-aminoazotoluene - B(a)P benzo[a]pyrene - B-NF beta-naphthoflavone - BZ benzidine - DDT p,p-dichlorodiphenyltrichloroethane - DDE p,p-dichlorodiphenyldichloroethylene - DEN diethylnitrosamine - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 3-MC 3-methylcholanthrene - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 2-NA 2-naphthylamine - NNG net nuclear grains - PB phenobarbital - PYR pyrazole     

Modulation of allo-immune responsesin vivo andin vitro by sperm specific lactate dehydrogenase-C4

The role of sperm specific lactate dehydrogenase-C₄ (LDH-C₄) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generationin vitro and local graft versus host (LGVH) reaction and allograft enhancementin vivo has been ascertained. LDH was purified from testes (LDH-C₄) and kidney (LDH-B₄) of C57 Bl/Ks mice. MLC and CTL were performed using C57 Bl/Ks-anti A/J lymphocytes in presence of 10^–3-1 g LDH-B₄ or LDH-C₄ per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C₄. Early MLC culture gave significantly low stimulation index at 10^–2–10^–1 g LDH-C₄ as compared to non-treated control cultures. However, the MLC response in presence of LDH-C₄ was not different from the LDH-B₄ treated one which showed a similar biphasic trend. On the other hand,⁵¹Cr release from YAC-222 target cells was practically abolished by LDH-C₄ at 10^–3–1^–1 g, and this was strikingly different from LDH-B₄ or non-treated cultures. LGVH reactivity as performed by using C57 Bl/Ks lymphocytes along with LDH-C₄ in (C57 Bl/Ks x A/J) F₁ hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C₄ or LDH-B₄) LGVH. Allograft enhancement of Sa I (A/J) in C57 Bl/Ks mice in presence of LDH-C₄, was delayed slightly but significantly during primary or secondary transplantation reaction. The reaction of LDH-C₄ in the modulation of allo-immune responses was more specificin vivo thanin vitro since the B₄ isozyme did not modify LGVH and Sa1 allograft rejection. Resultsper se suggest that LDH-C₄ is immunosuppressive for cell mediated allo-immune responsesin vivo andin vitro.     

Metabolism of leukotrienes

Summary The in vitro metabolism of leukotriene B₄ is initiated by -hydroxylation. This reaction is followed by oxidation of the -hydroxyl group to a carboxyl group. In vivo extensive -oxidation occurs and the main excreted products after administration of leukotriene B₄ are water and carbon dioxide.Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal y-glutamyl residue, catalyzed by the enzyme -glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E₄, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C₄. In rats, the two major fecal metabolities of leukotriene C₄4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E₄. These compounds are formed in reactions between leukotriene E₄ or 11-trans leukotriene E₄ and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.     

The Role of GlycineB Binding Site and Glycine Transporter (GlyT1) in the Regulation of [3H]GABA and [3H]Glycine Release in the Rat Brain

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [³H]-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [³H]GABA overflow with an EC₅₀ value of 0.09 mM. The [³H]GABA releasing effect of NMDA was an external Ca²⁺-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [³H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycine_B site, decreased the [³H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [³H]GABA outflow. These data suggest that glycine_B binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [³H]glycine (IC₅₀ 33 and 16 M) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [³H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [³H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [³H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na⁺ for superfusion of hippocampal slices produced an increased [³H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [³H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [³H]glycine efflux even when buffer with low Na⁺ concentration was applied.     

Selective induction of a giant puff in Drosophila hydei by vitamin B6 and derivatives

Vitamin B₆ induces in vivo as well as in vitro the appearance of a puff at region 2–48C in Drosophila hydei. At concentrations of 10^–2 M or lower, region 2–48C is the only region responding to vitamin B₆ provided that oligomycin is present in the incubation medium. Pyridoxal phosphate and pyridoxamine phosphate supplied to medium containing oligomycin induce upon incubation of salivary glands a larger puff at 2–48C. — Puff 2–48C produces large quantities of a unique RNP-product; globular 140–220 Å particles which aggregate to stable complexes of 0.1–0.2 in diameter. Upon continuous in vitro incubation with vitamin B₆, puff 2–48C becomes loaded with these aggregates which have never been observed in any other puff of Drosophila hydei.     

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B₄) but no trace of LDH-5 (A₄) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonale Entwicklung and Differenzierung.     

Comparison of high-affinity binding of [3H]GABA to subcellular particles of rat brain and liver

The binding of [³H]GABA and retention of [¹⁴C]sucrose have been studied in freshly prepared synaptosomal-mitochondrial (P₂) fractions of rat cerebral cortex and liver using bicarbonate-buffered medium (containing 147 mEq/liter of N⁺), and in frozen/thawed crude membrane fractions of rat whole brain and liver using Na⁺-free Tris HCl medium. GABA-sensitive sites (GSS) and bicucul-line-methiodide-(BMI-) sensitive sites (BMI-SS) were defined as those amounts of [³H]GABA that were sensitive to the displacement by 10^–3 M unlabeled GABA or BMI. In the presence of added Na⁺, two high-affinity GABA-binding processes were detected in the P₂fraction of cerebral cortex. The lower-affinity process (likely related mainly to uptake sites) hadK _B10^–5 M,B _max for GSS3 nmol/mg protein, andB _max for BMI-SS0.5 nmol/mg protein, whereas the higher-affinity process (likely related to synaptic GABA receptors) hadK _B10^–7 M,B _max for GSS43 pmol/mg protein, andB _max for BMI-SS2 pmol/mg protein. Only the higher-affinity process was detected in the liver P₂ fraction and it hadK _B3.7×10^–8 M,B _max for GSS0.48 pmol/mg protein, andB _max for BMI-SS0.1 pmol/mg protein (i.e., about 1/100 and 1/20 the receptiveB _max values of cerebral cortex). This binding process of the liver P₂ fraction could represent sites involved in mitochondrial GABA transport. In Na⁺-free Tris HCl medium, high-affinity [³H]GABA binding appeared to exist in frozen/thawed membrane preparations of both brain and liver when data were expressed on a protein basis. However, this binding to liver membranes was not displaceable by 10^–3 M unlabeled GABA, and when these data were expressed on a weight basis and corrected for [³H]GABA present in trapped supernatant fluid of the pellets, no [³H]GABA binding was detected in the liver preparation.     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

The influence of NMDA, a potent agonist of glutamate receptor, on behavioral activity of rats with experimental hyperammonemia evoked by liver failure

Summary. The study was designed to investigate the effects of NMDA receptor agonist on the behavioral activity in rats with experimental hyperammonemia. The experiments were performed on adult male Wistar rats. Experimental hyperammonemia was induced by intraperitoneal injections of tioacetamide (TAA, 200mg/kg) for three consecutive days. Rats treated with saline (0.9%) served as control. Stimulation of the NMDA glutamatergic receptor was evoked by ip. injection of agonist N-methyl-D-aspartate acid (NMDA) in a dose of 30mg/kg thirty minutes before experiments. Memory motivated affectively was evaluated in the passive avoidance responses. The speculative influence of the treatment on anxiety and motor activity was tested in elevated plus-maze and in open field respectively.To show change of NMDA receptor function after various doses of agonist, the seizures evoked by N-methyl-D-aspartate acid was carried out. This experiment showed that with rise of dose of NMDA time to appear of convulsions was contracted in rats with hyperammonemia as well as in control rats. Dose of NMDA caused convulsions was three times as less in rats with hyperammonemia than dose in control. Time of duration of convulsions was proportional to applied dose of NMDA and it lengthened with rise of agonists dose in both groups of studied animals.Furthermore, we observed that NMDA increased motor activity of control rats in open field test, but not in rats with hyperammonemia (treated tioacetamide). Hyperammonemia did not have significant influence on motor activity and on a passive avoidance latency. The NMDA given in control and in hyperammonemia, increased acquisition, consolidation and recall of a passive avoidance responses. Moreover, NMDA had anxiogenic-like profile in elevated plus-maze.In rats with hyperammonemia NMDA had no influence on locomotor activity but it significantly increased memory in a passive avoidance responses. Furthermore, we observed that reactivity of NMDA glutamate receptor in rats with hyperammonemia was higher than in control rats.     

Arsenic‐induced NFκβ transactivation through Erks‐ and JNKs‐dependent pathways in mouse epidermal JB6 cells

Carnosine, a alanylLhistidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe²⁺, H₂O₂)generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fenitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

N-Methyl-d-aspartate Receptor- and Calpain-mediated Proteolytic Cleavage of K+-Cl? Cotransporter-2 Impairs Spinal Chloride Homeostasis in Neuropathic Pain

Loss of synaptic inhibition by γ-aminobutyric acid and glycine due to potassium chloride cotransporter-2 (KCC2) down-regulation in the spinal cord is a critical mechanism of synaptic plasticity in neuropathic pain. Here we present novel evidence that peripheral nerve injury diminishes glycine-mediated inhibition and induces a depolarizing shift in the reversal potential of glycine-mediated currents (E_glycine) in spinal dorsal horn neurons. Blocking glutamate N-methyl-d-aspartate (NMDA) receptors normalizes synaptic inhibition, E_glycine, and KCC2 by nerve injury. Strikingly, nerve injury increases calcium-dependent calpain activity in the spinal cord that in turn causes KCC2 cleavage at the C terminus. Inhibiting calpain blocks KCC2 cleavage induced by nerve injury and NMDA, thereby normalizing E_glycine. Furthermore, calpain inhibition or silencing of μ-calpain at the spinal level reduces neuropathic pain. Thus, nerve injury promotes proteolytic cleavage of KCC2 through NMDA receptor-calpain activation, resulting in disruption of chloride homeostasis and diminished synaptic inhibition in the spinal cord. Targeting calpain may represent a new strategy for restoring KCC2 levels and tonic synaptic inhibition and for treating chronic neuropathic pain.     

In vitro and in vivo studies to assess the effectiveness of cholestyramine as a binding agent for fumonisins

Several adsorbent materials were tested at 1 mg/ml for their in vitrocapacity to adsorb fumonisin B₁ (FB₁) from aqueous solutions. Cholestyramine showed the best adsorption capacity (85% from a solution containing 200 g/ml FB₁) followed by activated carbon (62% FB₁). Bentonite adsorbed only 12% of the toxin from a solution containing 13 g/ml FB₁, while celite was not effective even at the lowest tested FB₁ concentration (3.2 g/ml). Cholestyramine was tested in vivoto evaluate its capacity to reduce the bioavailability of fumonisins (FBs) in rats fed diet contaminated with toxigenic Fusarium verticillioidesculture material. Rats were exposed for one week to FBs-free diet, FBs-contaminated diet containing 6 or 20 g/g FB₁ + FB₂ and the same FBs-contaminated diet added of 20 mg/g cholestyramine. The increase of sphinganine/sphingosine (SA/SO) ratio in urine and kidney of treated rats was used as specific and sensitive biomarker of fumonisin exposure.The addition of cholestyramine to the FBs-contaminated diets consistently reduced the effect of FBs by reducing significantly (P < 0.05) both urinary and renal SA/SO ratios.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of some metals and related metal-orgaic compounds on ALA-dehydratase activity of corn

Summary The activity of ALA-dehydratase from corn seedlings is affected by Mn⁺⁺, Fe⁺⁺, Pb⁺⁺, Cu⁺⁺, Zn⁺⁺ and Sn⁺⁴ ions, in vivo Mn⁺⁺ and Fe⁺⁺ are ativators while Pb⁺⁺ and Sn⁺⁴ are inhibitors; in vitro Cu⁺⁺ and Zn⁺⁺ are inhibitors. The kinetic parameters (V_max and K_M) support the hypothesis that Mn, Fe, Sn and Pb ions act on the biosynthesis of the enzyme and Zn and Cu ions on the enzyme-substrate affinity. Some related metal-organic compounds interrere in vivo on the ALA-dehydratase activity modifying the kinetic parameters, therefore the enzyme biogenesis and/or enzyme-sustrat affinity are affected.     

Suppression of β-oxidation restores pyruvate inhibition of pyruvate dehydrogenase kinase in starved rat heart

Kinetic behaviour of rat heart pyruvate dehydrogenase kinase (PDHK) was studied in the multi-enzyme complex (PDC) contained in two preparations: mitochondria (mPDC) and a high speed pellet of Triton-extracted tissue (hPDC). Two parameters were evaluated: Va_av, related to V_max, and Fractional Pyruvate Inhibition (FPI). Starvation of rats for 48 h led to a rise in V_av and a fall in FPI. Injection into starved rats of agents which reduce -oxidation of fatty acids restored, in succession, FPI and then V_av, of hPDC, to levels found in hPDC from fed animals. In vitro incubation at 30°C of hPDC from starved animals restored FPI, but not V_av, to fed values; both were restored during in vitro incubation of mPDC from starved animals within the same time frame as in the in vivo experiments. A sharp increase of FPI, but not V_av, of hPDC from both fed and starved rats was observed in later experiments. This could have been due to differential selection of the two genes for isoenzymes of PDHKa proposed by other workers.     

Stimulation of σ1 Receptors Alleviates Reperfusion-Induced Myocardial Stunning

In vivo or in vitro administration of a ₁ receptor agonist d-SKF 10.047 (1 mg/kg intravenously or 10 mg/l in vitro) promoted an increase in the resistance of isolated perfused rat heart to ischemia/reperfusion injury. Both in vivo and in vitro stimulation of receptors prevents the development of reperfusion contracture and creatine kinase release and increases the developed pressure, double product, +dP/dt, and –dP/dt in the left ventricle. Activation of receptors has no significant effect on the occurrence of reperfusion arrhythmias ex vivo. Stimulation of cardiac sigma receptors is proposed to prevent myocardial stunning.     

Phosphorylation of neuronal microtubule proteins

Summary Phosphorylation of microtubule protein was tested during differentiation in neuroblastoma cells. Two microtubule proteins were modified, -tubulin and MAP-1 B. In the first case less than one mol of phosphate was incorporated per mol of protein, whereas several residues were phosphorylated in MAP-1 B. The localization of the phosphorylated residue of -tubulin indicated that it is present in an isoform, at its carboxy-terminal region, and probably correspond to the serine 444. When comparing thein vivo phosphorylation of tubulin with that produced by casein kinase IIin vitro, a similar pattern was obtained. A similar result was found upon the comparison of the phosphorylation pattern of MAP-1 B after phosphorylationin vivo andin vitro using casein kinase II. These results suggest a role for casein kinase II in the phosphorylation of microtubule proteins in neuroblastoma cells. A result similar to that found for neuroblastoma cells was found after injection of [³²P]phosphate into the brain of seven-day-old rats; however, a more complex pattern was found for the phosphorylationin vivo in adult rats.