Effects of low- and high-dose tranylcypromine on [3H]tryptamine binding sites in the rat hippocampus and striatum

Chronic studies were initiated in rats to determine the effects of high- and low-dose tranylcypromine (TCP) on [³H]tryptamine (³H-T) binding sites. Male Sprague-Dawley rats were administered TCP (0.5 or 2.5 mg/kg/day) or vehicle (distilled water) for 4, 10 or 28 days via Alzet minipumps. After decapitation, the hippocampus and striatum were used to prepare membrane fragments for single point³H-T binding. Hippocampal³H-T binding was reduced after 10 and 28 days with the low dose and after 4, 10 and 28 days with the high dose. Striatal³H-T binding was reduced by both doses at all time intervals. The high dose resulted in a significantly greater reduction in striatal³H-T binding than did the low-dose after 4, 10, and 28 days. These results suggest that a more rapid reduction of³H-T binding in the hippocampus and/or a greater reduction of³H-T binding in the striatum by high-dose TCP than by low-dose TCP may be contributing factors in the reported efficacy of the former in refractory depression.     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

Characteristics of 3H-cis-flupenthixol binding in rat striatum

Characteristics of membrane receptor binding by 3H-cis-flupenthixol were examined in rat striatum. Using modifications of standard dopamine receptor binding techniques, it was possible to obtain 70% specific binding with 3H-cis-flupenthixol. Association and dissociation rates were very rapid, with equilibrium reached in 2 min and half-time of dissociation being 1 min. Analysis of saturation and competition studies using cis-flupenthixol and spiroperidol indicated that cis-flupenthixol binds to two striatal receptors with apparent KD's of 0.7 and 4.8 nM. It is suggested these represent D1 and D2 dopamine receptors respectively. The further characterization of the properties of cis-flupenthixol binding presented here should enable more detailed studies of multiple dopamine receptors to be designed.     

Temperature dependence and GABA modulation of [3H]triazolam binding in the rat brain

The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Our major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of [3H]TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (Kd = 0.27 +/- 08 nM at 0 degree C; Kd = 1.96 +/- 0.85 nM at 37 degrees C) while the Bmax values remained unchanged (1220 +/- 176 fmoles/mg protein at 0 degree C and 1160 +/- 383 fmoles/mg protein at 37 degrees C). Saturation studies of [3H]TZ binding in the presence or absence of GABA (100 microM) showed a GABA-shift. At 0 degrees C the Kd values were (Kd = 0.24 +/- 0.03 nM/-GABA; Kd = 0.16 +/- 0.04/+GABA) and at 37 degrees C the Kd values were (Kd = 1.84 +/- 0.44 nM/-GABA; Kd = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, our findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists.     

Negative allosteric modulators of AMPA-preferring receptors inhibit [(3)H]GABA release in rat striatum

The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective glutamate receptor agonist, on the release of previously incorporated [(3)H]GABA was examined in superfused striatal slices of the rat. The slices were loaded with [(3)H]GABA in the presence of beta-alanine (1 mM) and superfused with Krebs-bicarbonate buffer containing nipecotic acid (0.1 mM) and aminooxyacetic acid (0.1 mM) to inhibit GABA uptake and metabolism. AMPA (0.01 to 3 mM) increased basal [(3)H]GABA outflow and nipecotic acid potentiated this effect. The [(3)H]GABA releasing effect of AMPA was an external Ca(2+)-dependent process in the absence but not in the presence of nipecotic acid. Cyclothiazide (0.03 mM), a positive modulator of AMPA receptors, failed to evoke [(3)H]GABA release by itself, but it dose-dependently potentiated the [(3)H]GABA releasing effect of AMPA. The AMPA (0.3 mM)-induced [(3)H]GABA release was antagonized by NBQX (0.01 mM) in a competitive fashion (pA(2) 5.08). The negative modulator of AMPA receptors, GYKI-53784 (0.01 mM) reversed the AMPA-induced [(3)H]GABA release by a non-competitive manner (pD'(2) 5.44). GYKI-53784 (0. 01-0.1 mM) also decreased striatal [(3)H]GABA outflow on its own right, this effect was stereoselective and was not influenced by concomitant administration of 0.03 mM cyclothiazide. GYKI-52466 (0. 03-0.3 mM), another negative modulator at AMPA receptors, also inhibited basal [(3)H]GABA efflux whereas NBQX (0.1 mM) by itself was ineffective in alteration of [(3)H]GABA outflow.The present data indicate that AMPA evokes GABA release from the vesicular pool in neostriatal GABAergic neurons. They also confirm that multiple interactions may exist between the agonist binding sites and the positive and negative modulatory sites but no such interaction was detected between the positive and negative allosteric modulators. Since GYKI-53784, but not NBQX, inhibited [(3)H]GABA release by itself, AMPA receptors located on striatal GABAergic neurons may be in sensitized state and phasically controlled by endogenous glutamate. It is also postulated that these AMPA receptors are located extrasynaptically on GABAergic striatal neurons.     

Preincubation of homogenates from rat brain thalamus and striatum affects the rate of [3H]GABA uptake

The effect of preincubation of brain homogenates on the subsequent uptake of [³H]GABA was studied in rat thalamus and corpus striatum. The results show that in both brain regions preincubation causes a decrease in the initial rate of [³H]GABA influx, the phenomenon being most evident in the thalamus.     

Properties of H-diazepam binding to benzodiazepine receptors in rat cerebral cortex

The recently discovered specific binding sites for benzodiazepines in the central nervous system have been characterized further. Specific ³H-diazepam binding to synaptosomal preparations from rat cerebral cortex is saturable with the dissociation constant for diazepam K_D = 3.6 ± 0.1 nM. The maximal amount of specifically bound ³H-diazepam is 0.88 ± 0.05 pmole per mg protein. In the binding assay equilibrium between the specific binding sites and ³H-diazepam is reached within 15 min. Specific ³H-diazepam binding shows a linear dependency on protein up to 3.0 mg protein per assay and a sharp pH-optimum between pH 7.0 to pH 7.4 ³H-diazepam binding is temperature dependent with the highest amount bound at 4° C. Specific ³H-diazepam binding is altered by treating the synaptosomal preparations with proteolytic enzymes: trypsin and -chymotrypsin lead to the complete disappearance of specific ³H-diazepam binding; carboxypeptidase A or B decrease specific ³H-diazepam binding by 36 %, while leucineaminopeptidase was without effect.     

Evidence for two types of binding of 3H-gaba and 3H-muscimol in rat cerebral cortex and cerebellum.

The binding of n$\text{M}$ concentrations of ³HGABA and ³Hmuscimol to synaptosomal membrane preparations from different areas of rat brain were studied by a radioreceptor assay. The characteristics of binding, with respect to kinetic parameters and inhibition of binding by nonradioactive GABA, before and after detergent treatment, suggest the presence of at least two types of binding at putative GABA receptor sites.     

Effects of GABA receptor stimulation on the release of [3H]GABA from slices of rat neostriatum.

Slices of rat neostriatum were incubated in Krebs-Henseleit medium. Modulation of [3H]GABA release by GABA agonists and antagonists was investigated. The GABAA receptor agonists muscimol (0.1 microM) and isoguvacine (5 microM) enhanced the stimulated release of [3H]GABA. The antagonists picrotoxin (1 microM) and bicuculline (50 microM) prevented the effects of the agonists. In the presence of naloxone (1 microM), which blocked the effects of enkephalinergic neurons within the slice preparation, muscimol (1 microM) no longer affected the release of [3H]GABA.     

Enhancement of the binding of 3H-diazepam to rat brain membranes in vitro by SQ 20009, A novel anxiolytic, gamma-aminobutyric acid (GABA) and muscimol.

SQ 20009, a compound with anxiolytic-like activity, was found to cause an enhancement of the binding of ³H-diazepam to rat brain membranes in a concentration-dependent manner with inhibition of binding occuring at 10^?4 M. Both GABA and the GABA agonist, muscimol, were also found to enhance ³H-diazepam binding while the GABA antagonist (+)-bicuculline decreased binding. The effects of SQ 20009, the GABA agonists and (+)-bicuculline were reflected as a change in the affinity rather than number of binding sites. The effects of SQ 20009 and GABA and muscimol are discussed in terms of an interaction with an endogenous diazepam-like factor.     

Effects of unilateral 6-OHDA lesions on [3H]-N-propylnorapomorphine binding in striatum ex vivo and vulnerability to amphetamine-evoked dopamine release in rat

It has been argued that agonist ligands for dopamine D(2/3) receptors recognize a privileged subset of the receptors in living striatum, those which are functionally coupled to intracellular G-proteins. In support of this claim, the D(2/3) agonist [(3)H]-N-propylnorapomorphine ([(3)H]NPA) proved to be more vulnerable to competition from endogenous dopamine than was the antagonist ligand [(11)C]raclopride, measured ex vivo in mouse striatum, and subsequently in multi-tracer PET studies of analogous design. Based on these results, we predicted that prolonged dopamine depletion would result in a preferential increase in agonist binding, and a lesser competition from residual dopamine to the agonist binding. To test this hypothesis we used autoradiography to measure [(3)H]NPA and [(3)H]raclopride binding sites in hemi-parkinsonian rats with unilateral 6-OHDA lesions, with and without amphetamine challenge. Unilateral lesions were associated with a more distinct increase in [(3)H]NPA binding ex vivo than was seen for [(3)H]raclopride binding in vitro. Furthermore, this preferential asymmetry in [(3)H]NPA binding was more pronounced in amphetamine treated rats. We consequently predict that agonist ligands should likewise be fitter than antagonists for detecting responses to denervation in positron emission tomography studies of idiopathic Parkinson's disease. Agonist binding increases in vivo are likely to reflect the composite of a sensitization-like phenomenon, and relatively less competition from endogenous dopamine, as seen in the lesioned side of 6-OHDA induced hemi-parkinsonism.     

[3H]GABA binding in developing rabbit retina

We have studied the developmental sequence of the GABA system in the rabbit retina using an in vitro binding assay to monitor developmental changes in the post-synaptic receptor. A variety of tissue treatments including perchlorate and Triton X-100 were employed to optimize binding and remove endogenous factors which inhibit binding. Pre-treatment of the tissue with 0.05% Triton X-100 revealed high affinity binding for [³H]GABA which increased in a sigmoidal fashion with the post-natal age of the animal. A constant level of binding, at about 16% of adult levels, was noted until day 8, at which time a rapid increase occurred. At 16 days post-natal, the amount of specific binding reached a plateau near adult levels. Kinetic analysis of the GABA receptor showed an increase in the number of receptors (B_max) with little or no change in the apparent affinity (K_D). Our results suggest that the onset of post-synaptic receptor activity is delayed approximately 1 to 2 days, relative to the pre-synaptic components, and the period of rapid increase in GABA receptor binding coincides with the period of maximum increase in retinal synaptic density.     

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.     

Methylene blue prevents methylmalonate-induced seizures and oxidative damage in rat striatum

Methylene blue (MB) is a thiazine dye with cationic and lipophilic properties that acts as an electron transfer mediator in the mitochondria. Due to this metabolic improving activity and free radicals scavenging effects, MB has been used in the treatment of methemoglobinemia and ifosfamide-induced encephalopathy. Considering that methylmalonic acidemia consists of a group of inherited metabolic disorders biochemically characterized by impaired mitochondrial oxidative metabolism and reactive species production, we decided to investigate whether MB, protects against the behavioral and neurochemical alterations elicited by the intrastriatal injection of methylmalonate (MMA). In the present study we showed that intrastriatal injection of MB (0.015-1.5nmol/0.5microl) protected against seizures (evidenced by electrographic recording), protein carbonylation and Na(+),K(+)-ATPase inhibition ex vivo induced by MMA (4.5micromol/1.5microl). Furthermore, we investigated whether convulsions elicited by intrastriatal MMA administration are accompanied by striatal protein carbonyl content increase and changes in Na(+),K(+)-ATPase activity in rat striatum. The effect of MB (0.015-1.5nmol/0.5microl) and MMA (4.5micromol/0.5microl) on striatal NO(x) (NO(2) plus NO(3)) content was also evaluated. Statistical analysis revealed that the MMA-induced NO(x) content increase was attenuated by intrastriatal injection of MB and the duration of convulsive episodes correlated with Na(+),K(+)-ATPase inhibition, but not with MMA-induced total protein carbonylation. In view of that MB decreases MMA-induced neurotoxicity assessed by behavioral and neurochemical parameters, the authors suggest that MB may be of value to attenuate neurological deficits of methylmalonic acidemic patients.     

Chemical integrity of [3H]GABA used in binding studies

A method which is claimed to be able to determine the proportion of true GABA within radiolabeled GABA used in binding studies was tested using [³H]GABA. The method was found to be unsuitable for³H-labeled GABA and, furthermore, both theoretical considerations and the present experimental data indicated that it could also produce misleading results with [¹⁴C]GABA.     

Interaction between GABA agonists and the cholinergic muscarinic system in rat corpus striatum

Chronic treatment with γ-acetylenic GABA(GAG), a GABA transaminase inhibitor, causes an increase in striatal dopamine receptor binding and function in rat brain suggesting that extrapyramidal side effects may accompany the use of these agents. In the present investigation it was found that chronic administration of THIP, a direct acting GABA receptor agonist, induced a similar increase in dopamine receptor binding. In addition, co-administration of atropine, a cholinergic muscarinic antagonist, was found to completely prevent the GABA-induced dopamine receptor increase. Furthermore, high affinity choline uptake, a measure of cholinergic activity, in striatal synaptosomes is enhanced after the acute administration of either GAG or THIP. Taken together these results support the notion of an interaction between dopaminergic, cholinergic and GABA-ergic neurons in the extrapyramidal system and indicate that co-administration of an anticholinergic agent may be of benefit in preventing the extrapyramidal side effects which may accompany the use of GABAergic agonists.     

An analysis of the ionic regulation of the specific binding of 3H-diazepam depending on the phenotype of the emotional stress reaction]

It was shown that low NaCl concentrations (less than 50 mM) had more pronounced stimulatory effect on [3H]-diazepam ([3H]-DZ) binding in brain membranes of Balb/c (C) mice than in C57B1/6 (B6) mice. These interstrain differences disappeared after emotional stress in "open field" (OF) test. Low doses of diazepam (0.75 mg/kg) and hydazepam (1 mg/kg) induced anxiolytic effect in C mice and restored their normal [3H]-DZ binding level in the presence of NaCl. On the opposite, effects of the same doses of the benzodiazepines were not revealed either on the behavior in OF test or on stimulating properties of NaCl in B6 mice. Both benzodiazepines (10 mg/kg) induced similar behavior (sedative) and receptor (decrease of NaCl stimulating ability) in B6 and C mice. We made a conclusion that the ability of NaCl to increase [3H]-DZ binding is a physiological index which reflects hereditary differences in emotional-stress reactions and behavioral effects of benzodiazepine tranquillizers.     

A comparison of GABA and beta-alanine transport and GABA membrane binding in the rat brain

It was found that rat brain nerve endings contain a high affinity and Na^- dependent transport system for [³H]β-alanine ([³H]β-ala). As determined from Michaelis-Menten plots, the [³H]β-ala K_m was 2.8 × 10^-5 M and the V_max was 0.29 nmol/mg protein/5 min. Under similar incubation conditions the [³H]GABA K_m was 3.8 x 10^-6M and the V_max was 6.3 nmol/mg protein/5 min. The [³H]β-ala and [³H]GABA transport systems were further characterized by determining the IC₅₀ values for a number of compounds. The compounds tested were GABA, β-ala, l -2,4-diaminobutyric acid. DL-3-hyd-roxy-GABA, β-guanidopropionic acid, strychnine, γ-guanidobutyric acid, imidazole-4-acetic acid, DL-proline, bicuculline, L-serine, glycine, l -α-ala and taurine. DABA, dl -3-hydroxy-GABA, β-guanidopro-pionic acid and γ-guanidobutyric acid were more potent inhibitors of [³H]GABA than [³H]β-ala transport. Strychnine, imidazole-4-acetic acid, proline and glycine were between 2 and 6 times more potent inhibitors of [³H]β-ala than [³H]GABA transport. β-Ala, bicuculline, serine, α-alanine and taurine were all markedly more potent (12–150 times) inhibitors of [³H]β-ala than [³H]GABA transport. IC₅₀ values were also determined for the above compounds for the sodium-dependent and the sodium-independent binding of [³H]GABA to both fresh and frozen brain membranes. In general, the potency of these compounds to inhibit either sodium-independent or sodium-dependent binding was greater in fresh tissue. It was also observed that the neurophysiologically‘glycine-like’amino acids were more potent inhibitors in the presence of NaCl. No significant correlations were found between [³H]GABA binding under any condition and [³H]GABA or [³H]β-ala transport into nerve endings.     

Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [3H]SCH23390 and [3H]forskolin binding in rat striatum

The binding of [3H]forskolin to a homogeneous population of binding sites in rat striatum was enhanced by NaF, guanine nucleotides and MgCl2. These effects of NaF and guanylylimidodiphosphate (Gpp(NH)p) were synergistic with MgCl2, but NaF and Gpp(NH)p together elicited no greater enhancement of [3H]forskolin binding. These data suggest that [3H]forskolin may label a site which is modulated by the guanine nucleotide regulatory subunit which mediates the stimulation of adenylate cyclase (NS). The D1 dopamine receptor is known to stimulate adenylate cyclase via NS. In rat striatum, the Bmax of [3H]forskolin binding sites in the presence of MgCl2 and NaF was approximately two fold greater than the Bmax of [3H]SCH23390-labeled D1 dopamine receptors. Incubation of striatal homogenates with the protein modifying reagent EEDQ elicited a concentration-dependent decrease in the binding of both [3H]SCH23390 and [3H]forskolin, although EEDQ was approximately 14 fold more potent at inactivating the D1 dopamine receptor. Following in vivo administration of EEDQ there was no significant effect on [3H]forskolin binding sites using a dose of EEDQ that irreversibly inactivated greater than 90% of D1 dopamine receptors. These data suggest that EEDQ is a suitable tool for investigating changes in the stoichiometry of receptors and their second messenger systems.