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.     

Reduced diazepam binding following chronic benzodiazepine treatment.

Specific ³H-diazepam binding was measured in rat cortex after 7–10 days of twice daily injection of a large dose of flurazepam. Compared to cortex taken from saline treated controls, there was a statistically significant decrease of about 15% in the maximum binding capacity (B_max). There was also a change in the dissociation constant (K_D) from 5.27 to 8.80 nM. Repeated washing of the tissue sample before the binding assay showed that the B_max was truly decreased in the treated animals, but the change in K_D was probably an artifact due to residual flurazepam remaining in the tissue that interfered with the binding assay. It is concluded that chronic benzodiazepine treatment caused an apparent decrease in the number of specific binding sites.     

Decreased benzodiazepine receptor binding in amygdala-kindled rat brains

3H-Flunitrazepam (3H-FLU) binding was measured in multiple brain regions of amygdala-kindled rats two weeks following the sixth Stage 5 convulsion. As compared to 'yoked' controls, the kindled animals displayed significant reductions in 3H-FLU binding in the ipsilateral cortex (20%) and in the hypothalamus (20%). Scatchard plots revealed that these reductions were due to changes in the maximal number of available binding sites (Bmax) rather than to alterations in receptor affinity (KD). No significant changes were found in the contralateral cortex, or in either the contralateral or ipsilateral amygdala, hippocampus or striatum. These data suggest that kindling is associated with long-lasting changes in the benzodiazepine receptor system and possibly with related changes in GABA-mediated neural inhibition.     

Analysis of the specific 3H-diazepam binding in the brain of inbred mice with differing emotionality

The influence of sodium chloride and gamma-aminobutyric acid (GABA) on H3-diazepam binding in CNS membrane fraction were investigated in C57Bl/6, BALB/c mice and their F1 hybrids. The addition of GABA (1, 10 and 100 microM) elevated the level of radioligand binding with CNS membranes in all the inbred mice in similar manner. The higher stimulating effect of sodium chloride (50, 100 and 150 mM) on the H3-diazepam reception was found in BALB/c mice and F1 hybrids, as compared to C57Bl/6 mice. It has been suggested that membrane-dependent conformational reconstructions of supramolecular receptor complex are the cause of genetic differences in the regulation of H3-diazepam reception by Cl(-)-ionophore.     

3H-diazepam binding to brain synaptic membranes during the development of generalized epileptic activity

The development of bemegride-induced generalized epileptic activity in rats was shown to reduce the constant (CB). of specific 3H-diazepam binding with synaptic membranes from 0.23 nM-1 to 0.15 nM-1 and to increase the maximum number of membrane binding sites (Bmax) from 410 fmol/mg protein to 550 fmol/mg protein. It is assumed that the changes of benzodiazepine receptor properties are due to alteration in physico-chemical characteristics of synaptic membrane lipids resulting from the activation of lipid peroxidation.     

Decreased benzodiazepine receptor binding in epileptic El mice: A quantitative autoradiographic study

Benzodiazepine receptors and subtypes were examined in El mice and normal ddY mice with a quantitative autoradiographic technique. Specific [³H]flunitrazepam binding in stimulated El mice, which had experienced repeated convulsions, was significantly lower in the cortex and hippocampus than in ddY mice and unstimulated El mice. In the amygdala, specific [³H]flunitrazepam binding in stimulated El mice was lower than in ddY mice. There was a tendency for the [³H]flunitrazepam binding in these regions in unstimulated El mice to be intermediate between that in stimulated El mice and that in ddY mice, but there was no significant difference between unstimulated El mice and ddY mice. [³H]Flunitrazepam binding displaced by CL218,872 was significantly lower in the cortex of stimulated El mice than in that of the other two groups, and in the hippocampus of stimulated than of unstimulated El mice. These data suggest that the decrease in [³H]flunitrazepam binding in stimulated El mice may be due mainly to that of type 1 receptor and may be the result of repeated convulsions.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

Amygdala kindled seizure stage is related to altered benzodiazepine binding site density

Benzodiazepine receptor binding was examined in rats at 3 stages of amygdaloid kindling (i.e., initial afterdischarge, Stage 3 and Stage 5) immediately or 24 hr after seizure. 3H-diazepam binding site density (Bmax) was significantly increased 24 hr after Stage 3 and Stage 5 kindled seizures in the hippocampus but not in the amygdala. There were no significant differences in the dissociation constants (KD) between kindled and control rats at any time point examined for either brain region. These results demonstrate that changes in benzodiazepine binding are observed with partial kindled seizures (i.e., Stage 3), indicating that generalized seizures are not prerequisite to increased benzodiazepine receptor site density.     

Biochemical identification of the site of action of benzodiazepines in human brain by 3H-diazepam binding.

Benzodiazepines, a group of minor tranquillizers, bind in a selective, stereo-specific fashion to a receptor site in human brain. Diazepam, their main representative, is bound with an apparent dissociation constant of 7 nM. The potency of various benzodiazepines in displacing diazepam parallels closely their therapeutic and pharmacological potencies. The density of the receptor site varies 24 fold in human brain, with the highest level in cerebral cortex and cerebellum. The apparent affinity of the receptor site, however, is remarkably similar in twenty different brain regions. The characteristics of the benzodiazepine receptor site suggest that it represents the site of therapeutic action of the benzodiazepines in human brain.     

Decreased cardiac response to isoproterenol infusion in acute and chronic hypoxia

The hypothesis of a blunted chronotropic response of cardiac beta-adrenergic receptors in altitude hypoxia was tested in nine subjects at sea level (SL) by infusion of isoproterenol. Observations were made at SL, in acute hypoxia (2 days at 4,350 m, condition H1), in more prolonged hypoxia [13 days between 850 and 4,800 m, condition H2] and in chronic hypoxia [21 days at 4,800 m, condition H3]. Resting heart rate was higher in all hypoxic conditions. Resting norepinephrine concentrations were found to be significantly higher in conditions H2 (1.64 +/- 0.59) and H3 (1.74 +/- 0.76) than at SL (0.77 +/- 0.18 ng/ml). Isoproterenol, diluted in saline, was infused at increasing doses of 0.0, 0.02, 0.04, and 0.06 micrograms.kg-1.min-1. For the highest dose, there was a significantly smaller increase in heart rate in conditions H1 (35 +/- 9), H2 (33 +/- 11), and H3 (31 +/- 11) than at SL (45 +/- 8 min-1). The increase in pulse (systolic/diastolic) pressure, considered as the vascular response to isoproterenol infusion, was smaller in condition H3 (29 +/- 16) than at SL (51 +/- 24 mmHg). There was a significant increase in the dose of isoproterenol required to increase heart rate by 25 min-1 and decrease in slope of heart rate increase vs. log(dose) relationship in conditions H2 and H3. Thus an hypoxia-related attenuated response of beta-adrenergic receptors to exogenous stimulation was found in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibition of GABA-stimulated benzodiazepine binding by a convulsant benzodiazepine

The binding of ³H-diazepam to benzodiazepine receptors of brain was studied in washed and pre-frozen preparations. The GABA enhancement of ³H-diazepam binding was found to be inhibited by a convulsant benzodiazepine, Ro 5-3663.     

Decreased antistereotypic effect of neuroliptics after additional treatment with a benzodiazepine, a GABA agonist or an anticholinergic compound.

Neuroleptics are very potent antagonists against stereotypies induced by DA-stimulants including methylphenidate. This effect of neuroleptics is usually related to the antipsychotic effect of these compounds. In contrast we found that GABA agonists potentiate stereotyped gnawing induced by methylphenidate. The GABA agonist muscimol in combination with neuroleptics will attenuate the antagonistic effect of these compounds on stereotyped gnawing induced by methylphenidate. However a differentiation between the neuroleptic drugs was found: Haloperidol, spiroperidol and pimozide were profoundly antagonized by muscimol whereas cis(Z) - flupenthixol and fluphenazine were less antagonized. Baclofen shows no significant effect. Diazepam and scopolamine also strongly antagonized the antistereotypic effect of the butyrophenone-like compounds whereas only scopolamine could antagonize fluphenazine and cis(Z) - flupenthixol. Therefore we conclude that if the antistereotypic effect of neuroleptics correlates to the antipsychotic effect, a GABA agonist would probably not potentiate the antipsychotic effect of neuroleptics but rather antagonize it.     

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 single histidine in GABAA receptors is essential for benzodiazepine agonist binding.

Benzodiazepines (BZ) modulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABAA) receptor, the major inhibitory ion channel in the mammalian brain. This receptor is composed of structurally distinct subunits whose numerous molecular variants underlie the observed diversity in the properties of the BZ site. Pharmacologically distinct BZ sites can be recreated by the recombinant coexpression of any one of six alpha subunits, a beta subunit variant, and the gamma 2 subunit. In these receptors the alpha variant determines the affinity for ligand binding of the BZ site. Notably, the alpha 1 and alpha 6 variants impart on alpha chi beta 2 gamma 2 receptors high and negligible affinity, respectively, to BZ ligands with sedative as well as anxiolytic activities. By exchanging domains between the alpha 1 and alpha 6 variants, we show that a portion of the large extracellular domain determines sensitivity toward these ligands. Furthermore, we identify a single histidine residue in the alpha 1 variant, replaced by an arginine in alpha 6, as a major determinant for high affinity binding of BZ agonists. This residue also plays a role in determining high affinity binding for BZ antagonists. Hence, this histidine present in the alpha 1, alpha 2, alpha 3, and alpha 5 subunits appears to be a key residue for the action of clinically used BZ ligands.     

Effects of GABA antagonist-induced seizures on 3H-muscimol and 3H-diazepam binding in the rat striatum]

The binding of 3H-muscimol and 3H-diazepam to rat striatum membranes after picrotoxin- and bicuculline-induced seizures was characterized. No alteration in the maximal binding capacity (Bmax) of 3H-muscimol was observed. However, bicuculline produced a 27% decrease in Kd. Both picrotoxin and bicuculline increased the binding capacity of 3H-diazepam. Bicuculline produced a 86% increase in Kd. These results suggest that the GABA antagonists-induced seizures may modulate 3H-muscimol and 3H-diazepam binding in rat striatum.     

Initiation of DNA synthesis in mouse 3T3 fibroblasts in response to a specific factor.

Antibodies specific for both the F 1 and F 2a-1 calf thymus histone fractions were prepared by use of highly purified histone fractions. With these antibodies, immunofluorescent studies were performed in cultured cells from a Syrian hamster, from human cancer and from rat embryonal cells. Specific staining of nuclei by both of the antibodies was seen in all the cell lines used. In the staining pattern of the cell nucleus, there was a distinct difference between the results obtained from anti-F 1 antibody and from anti-F2a-1 antibody. In the case of the anti-F 1, the nuclei were stained to be coarse-grained or clumped in appearance. However, the result from anti-F 2a-1 showed strong fluorescence in the peripheral part of the nucleus and a faint shaggy appearance in the central part of the nucleus. These differences in the nuclear fluorescent pattern between the results obtained from anti-F 1 antibody and from anti-F 2a-1 antibody were seen in all the cell lines used.     

Some properties of 3H-diazepam displacing activity from human urine.

A potent ³H-diazepam displacing factor (IC₅₀ < 0.2 μg/ml) is isolated from human urine by adsorption to Chromosorb, elution with 50% ethanol, heating at pH 1, extractions into ethyl acetate and diethyl ether, and separation on a Sephadex LH20 column. The lipophilic compound has a molecular weight below 500 daltons and is inactivated by chymotrypsin or by exposure to pH > 12. The ³H-diazepam displacing material is probably present in urine as a conjugate or in a proform.