The Bindings of [3H]Muscimol and [3H]Flunitrazapam Are Elevated in Discrete Brain Regions of Butorphanol-Withdrawal Rats

We have investigated the effects of continuous infusion of butorphanol on the modulation of GABA_A receptor binding. Butorphanol was infused continuously into intracerebroventricle (ICV) at a constant rate of 26 nmol/l/h for 3 days, and the withdrawal from opioid was rendered 7 h after the cessation of infusion. The GABA_A receptor bindings in rat brain slices were analyzed by quantitative autoradiography using [³H]muscimol and [³H]flunitrazepam. In the rats withdrawn from butorphanol, the levels of [³H]muscimol binding were significantly elevated in cortex, thalamus, and part of the hippocampus. The levels of [³H]flunitrazepam binding were elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, brainstem, and cerebellum in the rats withdrawn from butorphanol. The levels of binding of either [³H]muscimol or [³H]flunitrazepam were not changed in the rats tolerant to butorphanol. However, the activity of GABAergic neuron was not found to have been modulated by butorphanol withdrawal, because the level of glutamic acid decarboxylase was not changed markedly either in rats that were tolerant to or withdrawn from butorphanol by Western blot and immunohistochemical data. These results suggest that the withdrawal from butorphanol infusion markedly elevates the binding of [³H]muscimol and [³H]flunitrazepam throughout the brain in a region-specific manner, and that the regulatory mechanisms in butorphanol tolerance and withdrawal may be different.     

Binding characteristics of [3H]flunitrazepam in pentobarbital-withdrawal rats

The effects of chronic pentobarbital (PB) treatment on the binding characteristics of [³H]flunitrazepam (FLU) in rat brain were examined. Saline or sodium PB (500 g/10l/hr) was infused into the lateral cerebral ventricles of rats for 6 days using osmotic pumps. Immediately before withdrawal, there were no significant differences in [³H]FLU binding constants (K_D and B_max) between saline and PB groups. However, 24 hr withdrawal caused an increase in B_max with no changes in K_D. The enhancement of [³H]FLU binding by in vitro addition of chloride ions and PB was not affected after the PB infusion. The PB enhancement of [³H]FLU binding was inhibited by the convulsant, picrotoxicin. PB withdrawal did not cause significant differences in the binding constants of [³H]Ro 15-1788, a benzodiazepine (BZ) antagonist, between the saline and PB groups. Pretreatment of membranes with 0.02 mM of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent, caused decreases in both K_D and B_max in FLU binding in PB-withdrawal membrane, but not in the saline-treated membrane. The enhancement of [³H]FLU binding by chloride ions and PB was not affected by the CHAPS treatment. These results suggest that the change in BZ receptors induced by PB withdrawal is functionally linked to the GABA-BZ-barbiturate receptor complex and that PB withdrawal induces some conformational changes in BZ receptors.     

Changes of [<Superscript>3</Superscript>H]Muscimol, [<Superscript>3</Superscript>H]Flunitrazepam and [<Superscript>3</Superscript>H]MK-801 Binding in Rat Brain by Prolonged Ventricular Infusion of 7-Nitroindazole

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), to examine modulation of NMDA and GABA_A receptor binding in rat brain. The duration of sleeping time was significantly increased by the pre-treatment with 7-NI (100 mg/kg) 30 min before pentobarbital (40 mg/kg) treatment in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of 7-NI into cerebroventricle for 7 days. We have investigated the effect of NOS inhibitor on NMDA and GABA_A receptor binding characteristics in discrete areas of brain regions by using autoradiographic techniques. The GABA_A receptors were analyzed by quantitative autoradiography using [³H]muscimol and [³H]flunitrazepam binding, and NMDA receptor binding was analyzed by using [³H]MK-801 binding in rat brain slices. Rats were infused with 7-NI (500 pmol/10 l/ h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [³H]muscimol were markedly elevated in cortex, caudate putamen, and thalamus while the levels of [³H]flunitrazepam binding were only elevated in cerebellum by NOS inhibitor. However, there was no change in the level of [³H]MK-801 binding except decreasing in the thalamus. These results show that the prolonged inhibition of NOS by 7-NI-infusion highly elevates [³H]muscimol binding in a region-specific manner and decreases the pentobarbital-induced sleep.     

Changes of [3H]Muscimol Binding and GABAA Receptor β2-Subunit mRNA Level by Tolerance to and Withdrawal from Pentobarbital in Rats

Effects of continuous pentobarbital administration on binding characteristics of [³H]muscimol were examined by autoradiography, and levels of GABA_A receptor 2-subunit mRNA were investigated by in situ hybridization histochemistry in the rat brain. In order to eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. An experimental model of barbiturate tolerance and withdrawal was developed using i.c.v. infusion of pentobarbital (300 g/10 l/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [³H]muscimol binding were elevated in cingulate of frontal cortex (46%) and granule layer of cerebellum (32%) of rats 24-hr after withdrawal from pentobarbital, while it was only elevated in cingulate (58%) of tolerant rats. The GABA_A receptor 2-subunit mRNA was increased in the withdrawal rats only: in the cortex (9–14%), hippocampus (15–21%), inferior colliculus (21%), and granule layer of cerebellum (24%). These results show the involvement of GABA_A receptor and its 2-subunit up-regulations in pentobarbital withdrawal rats, and suggest that the levels of [³H]muscimol binding and GABA_A receptor 2-subunit mRNA are altered in a region-specific manner during pentobarbital withdrawal.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Role of NMDA Receptors in Pentobarbital Tolerance/Dependence

Effects of continuous pentobarbital administration on binding characteristics of [³H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300g/10l/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [³H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10l/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [³⁵S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam Binding in Rat Brain by the Prolonged Ventricular Infusion of Transformed Ginsenosides

Ameliorating effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. Ginseng saponins are transformed by intestinal microflora and the transformants would be absorbed from intestine. In the present study, we have investigated the effects of transformed ginsenoside Rg3, Rh2 and compound K on the modulation of NMDA receptor and GABA_A receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using [³H]MK-801 binding, and GABA_A receptor bindings were analyzed by using [³H]muscimol and [³H]flunitrazepam binding in rat brain slices. Ginsenoside Rg3, Rh2 and compound K were infused (10 g/10 l/h) into rat brain lateral ventricle for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]MK-801 binding were highly decreased in almost all regions of frontal cortex and hippocampus by ginsenoside Rh2 and compound K. The levels of [³H]muscimol binding were elevated in part of frontal cortex and granule layer of cerebellum by the treatment of ginsenoside Rh2 and compound K. However, the [³H]flunitrazepam binding was not modulated by any tested ginsenosides. Ginsenoside Rh2 and compound K induced the downregulation of the [³H]MK-801 binding as well as upregulation of the and [³H]muscimol binding in a region-specific manner after prolonged infusion into lateral ventricle. However, ginsenoside Rg3 did not show the significant changes of ligand bindings. In addition, ginsenoside Rh2 decreased the expression of nNOS in the hippocampus although Rg3 decreased the expression in the cortex. These results suggest that biotransformed ginsenoside Rh2 and compound K could play an important role in the biological activities in the central nervous systems and neurodegenerative disease.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Phorbol Ester Differentiates the Levels of [<Superscript>3</Superscript>H]MK-801 Binding in Rats Lines Selected for Differential Sensitivity to the Hypnotic Effects of Ethanol

These studies addressed the possible involvement between sensitivity to the hypnotic action of ethanol and function of the NMDA receptor. The studies were carried out using high-alcohol sensitive (HAS) and low-alcohol sensitive (LAS) rats, two rats having differential sensitivity to the acute hypnotic action of ethanol. The animal models were developed by a selective breeding experiment. Using a quantitative autoradiograph technique, it was demonstrated that [³H]MK-801 binding to the NMDA receptor was highest in hippocampus in both HAS and LAS rats, but significant [³H]MK-801 binding was also detected in cortex, caudate-putamen, and thalamus of HAS and LAS rats. The density of [³H]MK-801 binding was lower only in cerebellar granule layers of untreated HAS rats as compared to the same brain area in untreated LAS rats. Activation of protein kinase C (PKC) by 100 nM PDBu, increased [³H]MK-801 binding in cortex, caudate-putamen, thalamus, central gray, and cerebellum of HAS rats but activation of PKC did not influence [³H]MK-801 binding in LAS rats. These activation of PKC differentiates between [³H]MK-801 binding of HAS and LAS rats in frontal cortex (layer II-IV and cingulate), caudate-putamen, and ventral lateral thalamic nuclei. The basal level of PKC- mRNA was higher in HAS rats than that of LAS rats. These results suggest that the activation of PKC potentiates NMDA receptor function of the rat line which is more sensitive to alcohol (HAS) but does not affect [³H]MK-801 binding of alcohol resistant (LAS) rats.     

Differential inhibition of brain specific [3H]flunitrazepam binding by several types of dyes

Several dyes, representing different structural classes, inhibit [³H]flunitrazepam binding to brain specific receptors in the rat with 50% inhibition in the 1 to 100 M range. Crystal Violet and Methyl Violet 2B inhibited more potently in the forebrain than in the cerebellum. Congo Red yielded a Hill number near 2.3, probably reflecting positive cooperativity between interacting binding sites in benzodiazepine receptor complexes. Toluidine Blue 0 was the most potent of the dyes tested (IC₅₀=1 M in cerebellum) and inhibited more potently in cerebellum than in forebrain.     

Modulation of NMDA Receptor Subunit mRNA in Butorphanol-Tolerant and —Withdrawing Rats

The NMDA receptor has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of NMDA receptor subunit NR1, NR2A, NR2B, and NR2C gene expression were investigated by using in situ hybridization technique. Continuous intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/l/h) resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels. The level of NR1 mRNA was significantly decreased in the cerebral cortex, thalamus, and CA1 area of hippocampus in butorphanol tolerant and withdrawal (7 h after stopping the infusion) rats. The NR2A mRNA was significantly decreased in the CA1 and CA3 of hippocampus in tolerant rats and increased in the cerebral cortex and dentate gyrus in butorphanol withdrawal rats. NR2B subunit mRNA was decreased in the cerebral cortex, caudate putamen, thalamus, CA3 of hippocampus in butorphanol withdrawal rats. No changes of NR1, NR2A, NR2C subunit mRNA in the cerebellar granule cell layer were observed in either butorphanol tolerant or withdrawal rats. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased significantly in all brain regions except in the thalamus and hippocampus, at the 7 hr after stopping the butorphanol infusion. These results suggest that region-specific changes of NMDA receptor subunit mRNA (NR 1 and NR2) as well as NMDA receptor binding ([³H]MK-801) are involved in the development of tolerance to and withdrawal from butorphanol.     

Changes in NMDAR2 Subunit mRNA Levels During Pentobarbital Tolerance/Withdrawal in the Rat Brain: An In Situ Hybridization Study

Little is known about the functional modulation of NMDA receptor subunits at the molecular level. Therefore, a series of experiments were conducted to elucidate more fully the role of NMDA receptor subtypes in pentobarbital tolerance and withdrawal. We investigated the influence of centrally administered pentobarbital on the regulation of mRNA levels of the family of NMDA receptor 2 (NR2) subtypes (NR2A, NR2B, and NR2C) by in situ hybridization histochemistry in rat brain. Animals were rendered tolerant by continuous intracerebroventricular (i.c.v.) infusion with pentobarbital (300 g/10 l/hr for 6 days) through pre-implanted cannulae connected to osmotic mini-pumps, and dependent, by abrupt withdrawal from pentobarbital. The NR2A subunit mRNA was increased in cortical areas in pentobarbital tolerant and withdrawal rats. In contrast, the NR2B mRNA was decreased in parietal cortex and hippocampus in both tolerance and withdrawal rats. The level of NR2C mRNA was increased in withdrawal rats, while there was no change in tolerant rats. These results indicate that continuous i.c.v. infusion with pentobarbital alters NR2 subunit mRNA expression in the rat brain, suggesting that NR2 subunits may play an important role in the development of tolerance to and withdrawal from pentobarbital.     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

Spermine Inhibits [3H]Glycine Binding at the NMDA Receptors from Plexiform Layers of Chick Retina

Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [³H]glycine binding to membranes from synaptosomal fractions from the outer (P₁) and the inner (P₂) plexiform layers of 1–3 day-old chick retinas in a dose-dependent manner with an IC₅₀ = 35 M for the P₁ fraction and 32 M for the P₂ fraction. Kinetic experiments and non-linear regression analysis of [³H]glycine-specific binding showed a K_d ~ 100–150 nM in both fractions, and a higher B_max (4.11 ± 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (B_max = 2.76 ± 0.25 pmol/mg protein). Strychnine-insensitive [³H]glycine binding was inhibited by 100 M spermine, due to a reduction in B_max (P₁ = 0.84 ± 0.16 pmol/mg protein; P₂ = 0.81 ± 0.16 pmol/mg protein) without affecting the K_d. Association and dissociation constants in the absence and presence of 50 M spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina.     

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Upregulation of (+)-7-Hydroxy-N,N-di-n-[3H]Propyl-2-Aminotetralin Binding Following Intracerebroventricular Administration of a Nitric Oxide Generator

Nitric oxide modulation of dopamine D₂ and D₃ receptor binding was examined using [¹²⁵I]epidepride (D₂) and (+)7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin([³H](+)-7-OH-DPAT, D₃). Nitric oxide, generated by i.c.v. injection of S-nitroso-N-acetyl-penicillamine (SNAP; 5 g or 10 g), significantly increased the density of [³H](+)-7-OH-DPAT binding sites (39% and 134%, respectively) in the striatum 24 hours post-injection in the absence of Gpp(NH)p, representing an upregulation of either D₃, receptors or high affinity D₂ receptors. In the presence of 10 M Gpp(NH)p, D₃ receptor upregulation was maintained in both the 5 g (increased 35%) and 10 g SNAP (increased 44%) groups. [³H](+)-7-OH-DPAT binding was reduced in both striatum and nucleus accumbens in the presence of 10 M Gpp(NH)p compared to binding in the absence of Gpp(NH)p, suggesting an upregulation of D₃ receptors. Administration of SNAP did not alter total specific [¹²⁵I]epidepride binding in either brain region. These data suggest that; 1) D₃ receptor density is modified following nitric oxide generation, and 2) the density of high affinity D₂ receptors identified by [³H](+)-7-OH-DPAT increases in the striatum, but decreases in the nucleus accumbens.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Effect of dithiol chelating agents on [3H]MK-801 and [3H]glutamate binding to synaptic plasma membranes

2,3-Dimercaptopropanol (BAL- British Anti-Lewesite) is a dithiol chelating agent used for the treatment of heavy metal poisoning, however, BAL can produce neurotoxic effects in a variety of situations. Based on the low therapeutic efficiency of BAL other dithiols were developed and DMSA (meso-2,3-dimercaptosuccinic acid) and DMPS (2,3-dimercaptopropane-1-sulfonic acid) are becoming used for treatments of humans exposed to heavy metals. In the present investigation the effect of dithiols in the glutamatergic system was examined. The results showed that BAL inhibited [³H]MK-801 and [³H]glutamate binding in a concentration-dependent manner. At 100 M BAL and DMSA caused a significantly inhibition of [³H]MK-801 binding to brain membranes (p < 0.05 by Duncan's multiple range test). BAL at 100 M caused an inhibition of 40% on [³H]glutamate binding. DMPS and DMSA had no significant effect on [³H]glutamate binding. Dithiotreitol (DTT), abolished the inhibitory effect of BAL on [³H]MK-801 binding. The protection exerted by DTT suggests that BAL inhibit [³H]MK-801 binding by interacting with cysteinyl residues that are important for redox modulation of receptor responses. ZnCl₂ inhibited [³H]glutamate and [³H]MK-801 binding to brain synaptic membrane; nevertheless, the inhibitory effect was slight more accentuated for [³H]MK-801 than [³H]glutamate binding (p < 0.05). The inhibition caused by 10 M ZnCl₂ on [³H]MK-801 binding was attenuated by BAL. The findings present in this study may provide the evidence that BAL affect the glutamatergic system and these effects can contributed to explain, at least in part, why BAL, in contrast to DMPS and DMSA is neurotoxic.     

Characterization of divalent cation-induced [3H]Acetylcholine release from EGTA-treated rat hippocampal synaptosomes

Calcium-naive synaptosomes were used to assess the effects of divalent cations on [³H]acetylcholine release from rat hippocampal homogenates. Following equilibration with calcium-free buffer (containing 10M EGTA), calcium reversibly increased [³H]acetylcholine efflux (up to five-fold) while causing no measurable efflux of lactate dehydrogenase. When substituted for calcium, strongtium and barium behaved similarly although barium exhibited three-fold greater efficacy. In the presence of elevated potassium, 4-aminopyridine or tetraethylammonium, the secretagogue efficacy of calcium (but not barium) was markedly increased. The release-promoting effects of both cations were inhibited by lanthanum, magnesium, cadmium, and -conotoxin but were insensitive to nifedipine and cobalt (both 10 M). In addition, stimulation of muscarnic cholinergic autoreceptors substantially inhibited both calcium and barium-evoked [³H]acetylcholine release. Taken together, these results indicate that cation-evoked transmitter release from calcium-naive synaptosomes is subject to normal neuroregulatory mechanisms and therefore should be useful for investigating presynaptic modulation of neuronal exocytosis.     

The effect of pretreatment with pentobarbital on the extent of [14C] incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofrucktokinase

In the present investigation we monitored the incorporation of [¹⁴C] from [U-¹⁴C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30 and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of¹⁴C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mol/min/g and 0.56 mol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of¹⁴C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose, 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.