Phospholipids and benzodiazepine recognition sites of brain synaptic membranes

Benzodiazepine binding to cerebral synaptic membranes and its modulation by membraneous phospholipids were studied. [³H]diazepam and [³H]flunitrazepam binding to the cerebral synaptic membrane were saturable and consisted of one component with a high affinity. Both [³H]diazepam and [³H]flunitrazepam binding was significantly decreased by pretreatment of the membrane with trypsin and pronase. In contrast, treatment of the membrane with phospholipase C and phospholipase A₂, induced a significant increase in binding. Kinetic studies have indicated that the treatment with phospholipase C induces an increase in density of the receptors, whereas treatment with phospholipase A₂ increases the affinity of the receptor. The addition of phospholipids, such as phosphatidyl serine and phosphatidic acid, abolished the increase in [³H]diazepam binding induced by phospholipase C and phospholipase A₂ treatment. The GABA-induced increase in [³H]diazepam binding was significantly attenuated following phospholipase A₂ treatment, but not in the control or in phospholipase C-treated membranes. In addition, the binding of [³H]flunitrazepam to the solubilized benzodiazepine receptor from the synaptic membrane was not affected by phospholipase C and phospholipase A₂ treatments.

The present results indicate that the benzodiazepine receptor binding in synaptic membranes is modulated, at least in part, by membraneous phospholipids susceptible to treatment with phospholipase C and phospholipase A₂.     

Interaction between Erythromycin and the Benzodiazepines Diazepam and Flunitrazepam

Abstract: The effect of erythromycin on the pharmacokinetics and pharmacodynamics of diazepam and flunitrazepam was investigated in two randomized, double-blind, cross-over studies. Healthy volunteers ingested erythromycin for one week 500 mg t.i.d. On the 4th day they ingested a single 5 mg dose of diazepam (6 subjects, Study 1) or 1 mg dose of flunitrazepam (5 subjects, Study 2), respectively. Plasma drug concentrations and psychomotor effects were measured during 42 hr after the ingestion of diazepam or flunitrazepam. In Study 1 erythromycin increased the area under the diazepam plasma concentration-time curve [AUC (0-42 hr)] by 15% (P<0.05) and the concentration of diazepam in plasma at 42 hr by 63% (P<0.05). The median peak concentration (C_max) and the half-life (t_1/2) of diazepam were increased but they did not change significantly (P=0.17 and 0.12, respectively). Plasma N-desmethyldiazepam concentrations were slightly reduced during erythromycin treatment up to 8 hr (P<0.05). In Study 2 the AUC (0-42 hr) of flunitrazepam was increased by 25% (P<0.05) during the erythromycin treatment. The t_1/2 of flunitrazepam increased significantly (P<0.05), but the C_max remained unchanged. The psychomotor effects of diazepam or flunitrazepam were not changed significantly by erythromycin. These pharmacokinetic interactions can be explained by the reduced metabolic elimination of diazepam and flunitrazepam. The interactions of erythromycin with diazepam and flunitrazepam seem to be slight and of limited clinical significance only.     

Regional GABA/benzodiazepine receptor/chloride channel coupling after acute and chronic benzodiazepine treatment

GABA/benzodiazepine coupling was evaluated in 8 regions of rat brain by the ability of GABA to stimulate 0.5 nM [3H]flunitrazepam binding. Rats were treated acutely with diazepam (p.o) or chronically with flurazepam, offered in the drinking water for 4 weeks, and compared to a pair-handled vehicle-treated control group. Regional variations in GABA/benzodiazepine coupling were found in control membranes. GABA increased benzodiazepine binding maximally (40%) in cerebellum and medulla, and least (25%) in olfactory bulb. A significant decrease in the effect of GABA was found in cortex of chronically treated rats immediately after, but not 2 days following treatment. The Emax for GABA stimulation of [3H]flunitrazepam binding was significantly increased in medulla after acute treatment but was not altered after acute or chronic treatment in other brain areas evaluated. Treatment had no effect on the ability of bicuculline to inhibit [3H]flunitrazepam binding in cortex. Benzodiazepine/Cl- coupling in cortex or hippocampus of acutely and chronically treated rats, evaluated by the ability of Cl- to stimulate specific [3H]flunitrazepam binding, was not changed. The results support the hypothesis that a functional uncoupling of the benzodiazepine recognition site from the GABA receptor in cortex, but not from the anion recognition site, may play a role in tolerance development.     

Interaction of the hypnotic lormetazepam with central benzodiazepine receptor subtypes omega 1, omega 2 and omega 3]

To clarify the action of lormetazepam, 3-hydroxybenzodiazepine, on the benzodiazepine (BZ) receptor subtypes, effects of lormetazepam on motor performance in the traction test and hexobarbital-induced loss of righting reflex in mice and the binding to BZ receptor subtypes were investigated in comparison with those of other BZ hypnotics. Lormetazepam prolonged the duration of hexobarbital-induced loss of righting reflex. The minimal effective dose (1 mg/kg, p.o.) was higher than that of flunitrazepam, lower than those of diazepam and zopiclone, and the same as those of triazolam and brotizolam. Lormetazepam showed the ataxic effect at 10 mg/kg, p.o., but the separation between its effective doses for the hypnotic and ataxic effects was the largest among the hypnotics tested. In the displacement study on [3H]flumazenil binding to cerebellar and spinal cord membranes, lormetazepam bound with a higher affinity to omega 1 receptor (Ki = 10 nM) than to omega 2 receptor (Ki = 29 nM). The GABA-ratios of lormetazepam to omega 1 and omega 2 receptors were 3.9 and 4.0, respectively; and they were higher and lower than those of flunitrazepam to omega 1 and omega 2 receptors, respectively. In the displacement study on [3H] Ro5-4864 binding to kidney membranes, lormetazepam bound with a lower affinity to the omega 3 receptor (Ki = 213 nM) than flunitrazepam. Thus, lormetazepam was suggested to be a potent hypnotic with weaker ataxic effects than other BZ hypnotics, which may be due to its selective and potent agonistic action on central omega 1 receptors.     

Locomotor responses to benzodiazepines,barbiturates and ethanol in diazepam-sensitive (DS) and -resistant (DR) mice

Diazepam-sensitive (DS) and -resistant (DR) mice were selectively bred for increased and reduced sensitivity to the ataxic effects of diazepam (40 mg/kg). Other response differences between DS and DR mice may reflect pleiotropic effects of the genes fixed during their selection. These mice were tested for their sensitivity to the locomtor stimulant effects of several doses of diazepam, flunitrazepam, pentobarbital, phenobarbital, and ethanol. DR mice were more sensitive than DS mice to the locomotor stimulant effects of all drugs except phenobarbital. These results largely support the hypothesis that a common biological mechanism mediates sensitivity to the stimulant effects of sedative-hypnotic drugs. Receptor mediation of the benzodiazepine effects was examined by administering the benzodiazepine receptor antagonist, RO15-1788. Locomotor depression produced by diazepam and flunitrazepam in DS mice was blocked by RO15-1788. However, while the locomotor stimulation produced by diazepam in DR mice was antagonized, the stimulant effect of flunitrazepam was not. This suggests that binding of flunitrazepam to the GABA_A-benzodiazepine receptor is not necessary for production of locomotor stimulation.     

The ontogeny of benzodiazepine receptors in selected regions of the rat brain: effect of perinatal exposure to diazepam

The postnatal development of the binding of [3H]flunitrazepam to benzodiazepine receptors has been studied in the frontal cortex, cerebellum, striatum, hypothalamus and hippocampus of the rat after prenatal, perinatal and postnatal exposure to diazepam. The dams were injected subcutaneously with single daily doses of 1 mg/kg of diazepam from day 7-20 of gestation or from day 15 of gestation day 6 after birth. Offspring of untreated dams were injected in the same way from postnatal day 7-20. The developmental profiles of the binding of [3H]flunitrazepam obtained in both control and diazepam-treated groups of animals were very similar. This supported the idea of a sequential development of benzodiazepine receptors in relation to the different rates of maturation of certain structures of the brain. Prenatal administration of diazepam resulted in an increase of the binding of [3H]flunitrazepam in the frontal cortex by 20% at postnatal day 90 and in a decrease of binding in the hippocampus by 14% at postnatal day 60 and an increase of binding by 18% at postnatal day 90. Perinatal exposure to diazepam did not affect the binding of [3H]flunitrazepam in the hippocampus in young adult offspring. Postnatal application of diazepam resulted in a transiently decreased binding of [3H]flunitrazepam in the cerebellum by 20% at postnatal day 28. The results obtained point to the necessity for a prolonged evaluation of events after exposure to diazepam in early stages of development of the brain.     

Properties of [3H]flunitrazepam binding to different benzodiazepine binding proteins

Membranes from cerebellum or hippocampus were incubated with various concentrations of [³H]flunitrazepam in the absence or presence of diazepam, Cl 218 872 or ethyl-β-carboline-3-carboxylate (β-CCE). After binding equilibrium of [³H]flunitrazepam had been established, the membranes were either filtered for determination of reversible binding or were irradiated with UV light for determination of irreversible binding. Irradiated membranes were then subjected to SDS-polyacrylamide gel electrophoresis and fluorography. Individual photolabeled proteins were identified, appropriate sections cut out of the gel, and the radioactivity in the gel pieces measured. The results indicate that [³H]flunitrazepam binding to individual benzodiazepine binding proteins and its inhibition by various drugs can be measured by the present technique and support previous evidence for the independent existence of various proteins irreversibly labeled by [³H]flunitrazepam and their possible association with different benzodiazepine receptors.     

Modulation of [3H]flunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine

The influence of phosphatidylserine on ligand binding to the benzodiazepine/GABA receptor complex was assessed in rat cerebellar synaptic membranes and in a detergent-solubilized membrane preparation. Intact synaptic membranes or membranes solubilized with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate) were incubated with a range of concentrations of phosphatidylserine for 2 h at 4 degrees C, prior to use in radioligand binding assays. Phosphatidylserine, an endogenous membrane phospholipid, facilitated the site-specific binding of [3H]flunitrazepam to synaptic membranes and CHAPS-solubilized preparations. In addition, phosphatidylserine inhibited the facilitation of [3H]flunitrazepam binding induced by either cartazolate or gamma-aminobutyric acid (GABA). Although the maximum effect (38% facilitation of [3H]flunitrazepam binding; greater than 90% inhibition of the cartazolate action) was produced using 130 microM phosphatidylserine, a significant enhancement of [3H]flunitrazepam binding could be observed upon preincubation of synaptic membranes with concentrations of phosphatidylserine as low as 5 microM. These results suggest that endogenous phosphatidylserine may play a role in the regulation of benzodiazepine/GABA receptor function, possibly through modulation of the mechanisms which functionally link the various components of this complex receptor system.     

Chronic RO 15-1788 treatment increases the number of benzodiazepine receptors in rat cerebral cortex and hippocampus

Administration of 4 mg/kg per day of diazepam in the drinking water for 14 days produced a small, but not significant decrease of benzodiazepine binding sites for [3H]flunitrazepam and [3H]ethyl-beta-carboline-3-carboxylate. Similar treatment with the benzodiazepine antagonist RO 15-1788 resulted in a marked increase in the density of sites for both ligands in the synaptosomal membranes of cerebral cortex and hippocampal formation.     

6-Chloro-3'-nitroflavone is a potent ligand for the benzodiazepine binding site of the GABA(A) receptor devoid of intrinsic activity

6-Chloro-3'-nitroflavone integrates a list of nearly 70 flavone derivatives synthesized in our laboratories. The effects of 6-chloro-3'-nitroflavone on the benzodiazepine binding sites (BDZ-BSs) of the GABA(A) receptor were examined in vitro and in vivo. 6-Chloro-3'-nitroflavone inhibited the [3H]flunitrazepam ([3H]FNZ) binding to rat cerebral cortex membranes with a Ki of 6.68 nM and the addition of GABA to extensively washed membranes did not modify its affinity for the BDZ-BSs (GABA-shift = 1.16+/-0.12). The binding assays performed in rat striatal and cerebellar brain membranes showed that this compound has similar affinity to different populations of BDZ-BSs. Electrophysiological experiments revealed that 6-chloro-3'-nitroflavone did not affect GABA(A)-receptors (GABA(A)-Rs) responses recorded in Xenopus oocytes expressing alpha1beta2gamma2s subunits, but blocked the potentiation exerted by diazepam (DZ) on GABA-activated chloride currents. In vivo experiments showed that 6-chloro-3'-nitroflavone did not possess anxiolytic, anticonvulsant, sedative, myorelaxant actions in mice or amnestic effects in rats; however, 6-chloro-3'-nitroflavone antagonized diazepam-induced antianxiety action, anticonvulsion, short-term, and long-term amnesia and motor incoordination. These biochemical, electrophysiological, and pharmacological results suggest that 6-chloro-3'-nitroflavone behaves as an antagonist of the BDZ-BSs.     

Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.     

Acute handling stress downregulates benzodiazepine receptors: Reversal by diazepam

In rats naive to handling, the effects of an acute handling stress led to lower [³H]flunitrazepam binding in the frontal cortex, compared with animals previously habituated to handling for 2 or 21 days. This decreased binding was due to reductions in the number of receptors, not to a change in affinity. Pre-incubation with diazepam (0.3, 3.0 or 30 μM) of frontal cortex homogenates taken from naive rats exposed to acute handling stress (followed by extensive washing to remove residual diazepam and endogenous modulators) led to a concentration-dependent increase in the number of benzodiazepine receptors, without any change in K_D. Acute in vivo administration of diazepam (4 mg/kg) prior to the handling stress was without significant effect in handling-habituated animals, but increases [³H]flunitrazepam binding in handling naive rats. Thus, handling habituation and diazepam treatment have similar actions on benzodiazepine binding and represent two ways of adapting to the stress of handling by increasing the number of benzodiazepine receptors.     

Inhibition of benzodiazepine and GABA receptor binding by amino-gamma-carbolines and other amino acid pyrolysate mutagens

The effect of several pyrolysate mutagens on the benzodiazepine and GABA receptors was investigated. Of amino-gamma-carbolines, Trp-P-1 antagonized the suppressive effect of diazepam on the pentylenetetrazol-induced convulsions and death, whereas Trp-P-2 by itself precipitated seizures and death in male mice. Both Trp-P-1 and Trp-P-2 inhibited the specific binding of [3H]diazepam and [3H]muscimol in rat brain membranes mainly by increasing Kd, indicating that these gamma-carbolines bind on benzodiazepine and GABA receptors. IC50S of Trp-P-1 and Trp-P-2 on specific [3H]flunitrazepam binding were not changed by addition of GABA. The Hill coefficient of Trp-P-1 for displacing [3H]diazepam binding was about unity whereas that of Trp-P-2 was less than unity. These results suggest that Trp-P-1 and Trp-P-2 act as active antagonists or inverse agonists at benzodiazepine receptors. The convulsant effect of the gamma-carbolines may be mediated by an action on the central benzodiazepine receptors; however, the role of the effect on GABA receptors is not clear.     

Group-selective reagent modification of the benzodiazepine-gamma-aminobutyric acid receptor-ionophore complex reveals that low-affinity gamma-aminobutyric acid receptors stimulate benzodiazepine binding

The modification of membrane proteins with diethylpyrocarbonate (DEP) and diazotized sulfanilate was investigated on the binding of three benzodiazepine radioligands in three brain regions. Both of these reagents produced a dose-dependent inactivation of [3H] diazepam, [3H]flunitrazepam, and [3H]propyl beta-carboline-3-carboxylate binding to cortex, cerebellum, and hippocampus. Both DEP and diazotized sulfanilate decrease the Bmax of the benzodiazepine binding sites without altering the KD. The ability of muscimol and pentobarbital to enhance [3H]diazepam binding was not altered by DEP pretreatment in any of the three regions. Scatchard analysis indicated that, following the inactivation of 40-50% of [3H]diazepam binding by 1 mM DEP, pentobarbital and muscimol were still able to increase the affinity of [3H]diazepam binding in cortex, cerebellum, and hippocampus. In contrast, diazotized sulfanilate pretreatment abolishes the ability of muscimol and pentobarbital to enhance [3H]diazepam binding in these three regions. The effects of these reagents on [3H] gamma-aminobutyric acid (GABA) binding revealed that sulfanilate but not DEP eliminates the low-affinity GABA receptor sites in cortex and cerebellum. Thus, while both DEP and sulfanilate inactivate benzodiazepine binding sites, only sulfanilate abolishes the low-affinity GABA binding sites and the ability of the GABA agonists to enhance [3H]diazepam binding. These results suggest that the stimulation of benzodiazepine binding appears to be mediated by the low-affinity GABA receptors.     

Long-term diazepam treatment produces changes in cholecystokinin receptor binding in rat brain

This study examined the effect of chronic diazepam administration on central benzodiazepine and CCK-8 receptor binding in rat brain. After a two-week treatment with diazepam (5 mg/kg per day) tolerance developed towards the sedative but not towards the anxiolytic action of this drug as determined using elevated plus-maze and open field tests. The % entries the rats made onto open arms and % time the rats spent in open arms were markedly decreased 24 h after the last dose of diazepam, probably indicating withdrawal anxiety. There were no changes in [3H]flunitrazepam binding either 30 min or 24 h after the last diazepam dose. However, 30 min after the last diazepam administration the apparent number of sulphated [3H]CCK-8 binding sites was significantly increased in the primary olfactory cortex. Acute diazepam treatment (5 mg/kg) had no influence on [3H]flunitrazepam or sulphated [3H]CCK-8 binding in any brain region studied. Cessation of chronic diazepam treatment was followed after 24 h by an increase in the number of CCK-8 receptors in frontal cortex and hippocampus as compared to the vehicle group. These results demonstrate that certain alterations in CCK-8 receptor characteristics may be important in the anti-anxiety effect, tolerance, and withdrawal reaction reaction after benzodiazepine administration.     

Ovarian steroids modify the behavioral and neurochemical responses of the central benzodiazepine receptor

The effect of ovarian steroids on the benzodiazepine receptor was assessed in the elevated plus-maze and, after restraint stress, in benzodiazepine receptor binding assays. Vehicle-treated proestrous rats displayed anxiolytic behavior, relative to diestrous or estrous rats. Anxiolytic behavior was observed after 1 or 2 mg/kg diazepam in diestrus and estrus. However, whereas 4 mg/kg increased open arm exploration in diestrus, a decrease in the same measure was found at estrus. At proestrus, a decrease in anxiolytic behavior was observed after 2 and 4 mg/kg. In ovariectomized vehicletreated rats, restraint stress increased NaCl-induced potentiation of³H-flunitrazepam binding in cortical and cerebellar, but not in hippocampal membranes. Estradiol benzoate (2 µg) prevented the potentiation of flunitrazepam binding by NaCl in nonstressed and stressed animals, whereas progesterone (0.5 mg) increased the NaCl-induced potentiation of flunitrazepam binding in both nonstressed and stressed animals. Combined estradiol benzoate and progesterone treatment produced effects that were intermediate to those seen after injection of either steroid alone. The potentiation of flunitrazepam binding by NaCl observed in vehicle-treated stressed or progesterone-treated nonstressed animals was mimicked in vitro by addition to reaction test tubes of the neuroactive metabolite of progesterone, 3-hydroxy-5-pregnan-20-one (allopregnanolone). These results point to a significant role of ovarian hormones in modifying the stress response of the benzodiazepine receptor.     

In vivo determination of efficacy of pyrazoloquinolinones at the benzodiazepine receptor

The pyrazoloquinolinones CGS 9896, CGS 9895 and CGS 8216 potently displace the benzodiazepines from their CNS binding site in vitro but have been reported to display agonist, partial agonist and antagonist activity respectively in a number of in vivo tests in rats. We found CGS 9896 to have only weak antipentylenetetrazole activity in mice at doses which produced near maximal displacement of [3H]flunitrazepam in vivo and were not able to detect any antipentylenetetrazole activity of CGS 9895. However, CGS 9895 blocked the anticonvulsant effect of diazepam at doses closely related to those which inhibited the in vivo binding of [3H]flunitrazepam and CGS 9896 also showed some reversal of the antipentylenetetrazole action of diazepam. This is consistent with the notion that CGS 9896 is a partial agonist and CGS 9895 an antagonist at the benzodiazepine receptor.     

Interaction of purines and related compounds with photoaffinity-labelled benzodiazepine receptors in rat brain membranes

The interaction of purine-receptor agonists and antagonists with [3H]Ro15-1788 binding sites in rat brain membranes was examined before and after UV-photoaffinity labelling of a proportion of the sites with flunitrazepam. Whereas photoaffinity labelling of the receptors reduced benzodiazepine agonist affinity but not benzodiazepine antagonist affinity, the IC50S of adenosine-receptor agonists, partial agonists and antagonists were unaltered by the conformational changes in the benzodiazepine receptors which are thought to be induced by the photolabelling process. The affinity of dipyridamole, a potent adenosine uptake blocker and potent displacer of [3H]diazepam binding, was drastically reduced by photolabelling.     

Enhancement of benzodiazepine and GABA binding by the novel anxiolytic, tracazolate

Tracazolate (ICI 136,753) 4-butylamine-1-ethyl-6-methyl-1H-pyrazolo[3,4]pyridine-5-carboxylic acid ethyl ester is a non-benzodiazepine with anxiolytic-like activity in animal models. In contrast to the benzodiazepines, it enhances [3H]flunitrazepam binding in rat synaptic membrane fragments. The enhancement is potential by chloride ion and is due to an increase in affinity of the receptor. The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) is additive with that of tracazolate; however, the GABA antagonist bicuculline blocks the enhancement by both compounds. Tracazolate enhances [3H]GABA binding to frozen and thawed Triton X-100-treated membrane fragments. The enhancement is due to an increase in the number of sites and potentiated by chloride. Benzodiazepines also enhanced GABA binding but the effect was due to an apparent change in affinity and not potentiated by chloride. The rank order to chlorodiazepoxide, diazepam and flunitrazepam for enhancement of GABA binding and displacement of [3H]flunitrazepam binding were the same. The enhancement of [3H]GABA binding by flunitrazepam and tracazolate were additive. Possible interactions between these various receptors are discussed.