Modulation Of Benzodiazepine Receptors Research Articles

Anti-seizure effects of walnut peptides in mouse models of induced seizure: The involvement of GABA and nitric oxide pathways

Epilepsy is one of the foremost medical disorders. Oxidative stress is a well-known mechanism in epileptogenesis, and many studies suggest that oxidative stress affects the onset and evolution of epilepsy. Here we evaluated the walnut peptide extracts’ anti-seizure property in three different mouse seizure models including pentylenetetrazole-induced clonic seizure, chemical kindling, and maximal electroshock. Walnut peptides (20 mg/Kg) were administered by intraperitoneal (IP) injection of mice 60 min before seizure induction in the three models. To delineate the mechanisms of walnut peptides anti-seizure activity, we evaluated the impact of diazepam, flumazenil, and a NOS inhibitor on this activity. Intraperitoneal administration of walnut peptides significantly increased the seizure threshold. Our results also demonstrated that walnut peptides exert their anti-seizure properties through the modulation of benzodiazepine receptors. Thus, walnut peptides may be considered as a new anti-convulsion agent, which can reduce seizure occurrence and slow down seizure progression.

Protective Effects of Artemisia persica Essential Oil Against Pentylenetetrazol-Induced Seizure in Male Mice with Emphasizing its Mechanism of Action

Background: A new approach in the treatment of epilepsy is to use new drugs with neuroprotective, antioxidant, and anti-inflammatory effects. Objectives: The study aimed to investigate the protective effects of Artemisia persica essential oil (EO) against pentylenetetrazol (PTZ)-induced seizure in mice. Methods: This experimental study was conducted at the Izeh Islamic Azad University, Iran. 70 male BALB/c mice were divided into seven groups of 10 using simple random allocation, including control (normal saline), PTZ (35 mg/kg i.p. with 48 hours intervals and then 60 mg/kg on the 10th day), interventions (PTZ plus daily i.p. injection of EO at doses of 50, 75, and 100 mg/kg), diazepam (PTZ plus EO at a dose of 100 mg/kg + diazepam), and flumazenil (PTZ plus EO at a dose of 100 mg/kg + flumazenil) groups. Results: The treatment of PTZ-kindled mice with 50 mg/kg of EO significantly reduced the seizure onset latency (P < 0.05). EO at a dose of 100 mg/kg significantly decreased tonic seizures, head tics, and repeated spinning and jumping (P < 0.05). Diazepam improved, and flumazenil weakened the anticonvulsant effects of the EO. The treatment of PTZ-kindled mice with EO (100 mg/kg) significantly decreased nitric oxide and malondialdehyde, and increased the total antioxidant capacity in both serum and brain (P < 0.05). EO at a dose of 100 mg/kg could significantly decrease IL-1β and TNF-α expression in the brain of epileptic mice (P < 0.05). Conclusions: A. persica EO shows anticonvulsant effects through benzodiazepine receptor binding activity and modulation of oxidative stress and the inflammatory process.

Nociception-specific blink reflex: pharmacology in healthy volunteers.

BackgroundThe physiology and pharmacology of activation or perception of activation of pain-coding trigeminovascular afferents in humans is fundamental to understanding the biology of headache and developing new treatments.MethodsThe blink reflex was elicited using a concentric electrode and recorded in four separate sessions, at baseline and two minutes after administration of ramped doses of diazepam (final dose 0.07 mg/kg), fentanyl (final dose 1.11 μg/kg), ketamine (final dose 0.084 mg/kg) and 0.9 % saline solution. The AUC (area under the curve, μV*ms) and the latency (ms) of the ipsi- and contralateral R2 component of the blink reflex were calculated by PC-based offline analysis. Immediately after each block of blink reflex recordings certain psychometric parameters were assessed.ResultsThere was an effect due to DRUG on the ipsilateral (F3,60 = 7.3, P < 0.001) AUC as well as on the contralateral (F3,60 = 6.02, P < 0.001) AUC across the study.A significant decrement in comparison to placebo was observed only for diazepam, affecting the ipsilateral AUC. The scores of alertness, calmness, contentedness, reaction time and precision were not affected by the DRUG across the sessions.ConclusionPrevious studies suggest central, rather than peripheral changes in nociceptive trigeminal transmission in migraine. This study demonstrates a robust effect of benzodiazepine receptor modulation of the nociception specific blink reflex (nBR) without any μ-opiate or glutamate NMDA receptor component. The nociception specific blink reflex offers a reproducible, quantifiable method of assessment of trigeminal nociceptive system in humans that can be used to dissect pharmacology relevant to primary headache disorders.

509 POSTER In vitro evaluation of glioma cell lines and primary glioma cell cultures chemosensitivities: the effect of pharmacological modulation of peripheral benzodiazepine receptors

Carboplatin is a platinum based drug used in the treatment of several malignancies. Due to poor cellular uptake, generally, a larger dose of drug is administered to achieve therapeutic levels, causing harmful side-effects such as hematologic toxicity. In order to enhance the cellular uptake of carboplatin, we have developed carboplatin loaded nanoparticles using the biodegradable polymer poly (ɛ-caprolactone) (PCL). Nanoparticles ranging from the size of 23.77 ± 1.37 to 96.73 ± 2.79 nm with positive zeta potential and moderate entrapment efficiency (54.21 ± 0.98%) were obtained. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed the spherical morphology and smooth surface of all nanoformulations. The concentrations of PCL and the stabilizer (DMAB) are found to play a role in determining the size and the entrapment efficiency of the nanoparticles. Drug release from nanoparticles followed a biphasic pattern with an initial burst release followed by a sustained release for 10 h. Results of in vitro cellular uptake and cytotoxicity studies revealed that carboplatin in the form of PCL-nanoparticles were efficiently up taken and displayed profound cytotoxicity to U-87 MG (human glioma) cells than the free drug. Importantly, unlike the free carboplatin, carboplatin in the form of PCL nanoparticles did not present any haemolytic activity in rat erythrocytes, a major side effect of this chemotherapeutic drug. This suggests that poly (ɛ-caprolactone) nanoencapsulation of carboplatin might be an efficient approach to treat cancer, while reducing carboplatin induced haemolysis.

CCK-8 modulates GABA-stimulated [3H]-flunitrazepam binding in vitro.

In membranes from rat cerebral cortex, cholecystokinin-8 (CCK-8) did not modulate basal [(3)H]-flunitrazepam binding at either 4 degrees C or 37 degrees C. At a concentration of 10(-6) M, CCK(-8) significantly decreased gamma-amino-butyric acid (GABA)-stimulated (10(-6) M) [(3)H]-flunitrazepam binding at 37 degrees C. Scatchard analyses suggest that the decreased GABA-stimulated binding might be due to a decrease in the affinity of benzodiazepine receptors rather than to a decrease of number of binding sites. The observed modulation of benzodiazepine receptors by CCK-8 in vitro might explain some of the functional interactions between CCK and benzodiazepine systems.

Anti-depressant action of melatonin in chronic forced swimming-induced behavioral despair in mice, role of peripheral benzodiazepine receptor modulation

The possible antidepressant effect of physiological and pharmacological doses of melatonin was investigated in the Porsolt forced swimming-induced behavioral despair test. The duration of immobility period of BALB/c and C57BL/6J mice during a 6-min swim test was measured at noon (11:00–12:00 h), early dark (20:00–21:00 h) and at midnight (1:00–2:00 h), respectively. The circadian time cycle did not alter the duration of immobility in either strains of mice. Similarly, exogenously administered melatonin (10–1000 μg/kg≅50 nM to 5 μM/mouse), a dose that could act on high affinity melatonin receptors, did not modify the duration of immobility period at any of the time intervals studied in either strains of the mice. This suggested that neither circadian variation influenced the duration of immobility period of BALB/c and C57BL/6J mice nor at physiological doses melatonin showed any anti-depressant action. Acute administration of higher doses of melatonin (2.5–10 mg/kg) failed to induce any anti-depressant activity in mice which were subjected to forced swimming test for the first time. However, daily administration of melatonin (2.5–10 mg/kg) prior to swimming test significantly reversed the increase in immobility period that was observed on chronic exposure to swimming test. This effect was comparable with the effect of GABA-benzodiazepine (BZ) receptor agonists. Similarly, like GABAergic drugs, acute administration of melatonin also showed anti-depressant activity in a mice which were exposed to chronic forced swimming test. The anti-depressant action of melatonin was sensitive to reversal by peripheral BZ receptor antagonist, PK11195. Whereas, flumazenil failed to reverse the anti-depressant action of melatonin, thereby suggesting that central BZ receptor were not involved in its action. In conclusion the study showed that at pharmacological doses melatonin has anti-depressant action in chronic forced swimming-induced despair behavior by an action involving peripheral BZ receptors.

89-8 - Modulation of lymphocyte peripheral benzodiazepine receptors in patients with hyperthyroidism

89-8 - Modulation of lymphocyte peripheral benzodiazepine receptors in patients with hyperthyroidism

The transcriptional and translational control of diazepam binding inhibitor expression in rat male germ-line cells.

The diazepam binding inhibitor [DBI, also known as acyl-CoA-binding protein, (ACBP), or endozepine] is a 10-kD protein that has been suggested to be involved in the regulation of several biological processes such as acyl-CoA metabolism, steroidogenesis, insulin secretion, and gamma-aminobutyric acid type A (GABA(A))/benzodiazepine receptor modulation. DBI has been cloned from vertebrates, insects, plants, and yeasts. In mammals, DBI is expressed in almost all the tissues studied. Nevertheless, DBI expression is restricted to specific cell types. Here we have studied DBI gene expression in the germ-line cells of rat testis. The DBI gene was intensively transcribed in postmeiotic round spermatids from stages VI to VIII of the seminiferous epithelial cycle. A prominent, spermatid-specific upstream transcription initiation site was identified in addition to the multiple common transcriptional initiation sites found in the somatic tissues. However, no DBI protein was detected in round spermatids, suggesting that the DBI transcripts were translationally arrested. The DBI protein was detected in the late spermatogenic stages starting from elongating spermatids from step 18 (stage VI) onward. The DBI protein was also detected in mature spermatozoa and in ejaculated human sperms. The majority of DBI was located at the middle piece of the spermatozoons tail enriched with mitochondria. On the basis of this observation and the well-established role of DBI in acyl-CoA metabolism, we propose that DBI expression in spermatozoa reflects the usage of fatty acids as a primary energy source by spermatozoa. The biological function of DBI in spermatozoa could thus be related to the motility function of sperm.

Participation of ACTH1-10 and ACTH4-10 on the melatonin modulation of benzodiazepine receptors in rat cerebral cortex.

In this study, we examined the effect of intracerebroventricular (i.c.v) injection of melatonin and/or ACTH1-10 and ACTH4-10 on [3H]flunitrazepam binding sites in the cerebral cortex of hypophysectomized rats. Hypophysectomy increased the Bmax (maximum number of binding sites) of benzodiazepine (BNZ) receptors for at least 7 days after surgery, without changing KD (dissociation constant). The i.c.v. injection of melatonin to hypophysectomized rats significantly increased Bmax, whereas the same doses of melatonin were ineffective in sham-operated animals. In both cases, KD values were unchanged. The i.c.v. injection of ACTH1-10 to hypophysectomized animals significantly increased Bmax, an effect that was enhanced by simultaneous i.c.v. injection of ACTH1-10 + melatonin, reaching higher values of Bmax than the i.c.v. injection of these hormones individually. No significant changes in KD values were found after ACTH1-10 and/or melatonin administration. However, the i.c.v. injection of ACTH4-10 to hypophysectomized rats did not change Bmax, although it significantly increased KD values, indicating a decrease in the BNZ binding affinity. Melatonin injection counteracted this effect of ACTH4-10, returning KD to the control value. Moreover, although the lower dose of i.c.v. melatonin used, 10 ng, was unable to modify Bmax of BNZ binding in the ACTH4-10-injected group, the higher dose, 20 ng, significantly increased Bmax. The results suggest that these ACTH-derived peptides can modulate the effect of melatonin on brain benzodiazepine receptors.

Modulation of peripheral benzodiazepine receptors in female rat genital organs by various gonadal steroids

Peripheral benzodiazepine receptors (PBR) in the ovary, oviduct, uterus, and kidney of immature rats were studied under short- and long-term treatment with testosterone (T), progesterone (P 4), and diethylstilbestrol (DES). A significant increase in PBR specific binding was observed after 4 days′ treatment with T in the ovary (1.6-fold), oviduct (2.0-fold), and uterus (1.4-fold) compared with intact rats. Four days′ treatment with P 4 increased PBR specific binding in the ovary (1.5-fold), but no changes were detected in the oviduct or uterus. In contrast, PBR specific binding was significantly reduced by 10 days′ treatment with T or P 4: 40 and 12%, respectively, in the ovary and 35 and 40%, respectively, in the oviduct. Ten days′ treatment with T reduced PBR specific binding in the uterus by 25%, but the same interval of treatment with P 4 did not alter specific binding in the uterus. Four or 10 days′ treatment wit DES signuificantly increased PBR specific binding in the ovary (1.5-fold), oviduct (2.4-fold), and uterus (1.9-fold). Scatchard analysis revealed that the changes in the PBR specific binding were due to a change in PBR density values rather than PBR affinity values. No change in PBR specific binding was found in the kidney following any of these treatments. Taken together, it is suggested that PBR density in the ovary is altered by exogenously administered steroids that usually are biosynthesized in the ovary. Additionally, the altered PBR density in the oviduct and uterus via the various steroids employed may imply that changes occurring in ovarian steroidogenesis should affect PBR density in these organs.

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain.

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [3H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [3H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.

Peripheral benzodiazepine receptor modulation with phagocyte differentiation

Peripheral benzodiazepine receptor (PBR) was found to be less expressed in the immature phagocytic HL-60 and U-937 cell lines than in the more mature monocytic THP-1 cell line. Cell differentiation by several agents induced a strong enhancement of PBR density on these three phagocytic cell lines but not on the lymphocytic CEM line line. Detailed analysis of phorbol 12-myristate 13-acetate-treated THP-1 cells showed an increased PBR expression and the rise came along with an increase of CD11a and CD11b antigens and a secretion of macrophagic cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-8. Quantitation of mRNA using polymerase chain reaction (PCR)-based technique showed that overexpression of PBR did not parallel mRNA expression, indicating a gene-independent regulation. These results suggest that PBR predominance on phagocytic cells could be related to maturation process.

Calcium Induced Modulation of Peripheral-Type Benzodiazepine Receptors in Rat Kidney Membranes

The effects of Ca2+ ions on 3H-RO 5-4864 binding to the peripheral benzodiazepine receptor were examined. Preincubation of rat kidney membranes with Ca2+ at 37 degrees C produced a dose-dependent inhibition of 3H-RO 5-4864 binding. No inhibition was observed in membranes preincubated at 0 degrees C. The effect of Ca2+ was competitive in nature and was fully reversed by the addition of EGTA. At 1 mM, the maximal effect was achieved with CaCl2, whereas CoCl2 and CdCl2 had lesser effects. No other divalent cation salts examined decreased 3H-RO 5-4864 binding to rat kidney membranes. Collectively, these data demonstrate that the affinity of 3H-RO 5-4864 binding to rat kidney membranes is regulated by Ca2+ and suggest the presence of cation recognition binding sites coupled to the peripheral benzodiazepine receptor.

In vivo and in vitro modulation of central type benzodiazepine receptors by phosphatidylserine

The in vivo and in vitro modulation of central benzodiazepine binding sites (BDZ-R) by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was studied. Five days i.p. administration of 15 mg/kg/day of BC-PS liposomes increased the maximal number of binding sites ( B max) for [ 3H]flunitrazepam in cerebral cortical membranes. In contrast, the density of hippocampal benzodiazepine recognition binding sites decreased. In cerebellar membranes, BC-PS treatment did not alter the characteristics of [ 3H]flunitrazepam binding. Similar experiments using phosphatidylcholine extracted from bovine brain (BC-PC) resulted in no changes in the [ 3H]flunitrazepam binding in the 3 neural structures studied. Confirming previous results, rats submitted to an acute swimming stress showed a decrease in the density of cerebral cortex BDZ-R. Animals treated with BC-PS liposomes before stress showed cortical [ 3H]flunitrazepam binding significantly below treated, unstressed animals but not below controls. The effects of BC-PS liposomes appeared to be selective for the central type of BDZ-R since no changes were observed in [ 3H]RO 5-4864 binding, a radioligand specific for the peripheral type BDZ-R. Preincubation of cerebral cortical and cerebellar synaptosomal membranes with BC-PS liposomes (1–300 μg per assay) significantly increased in a concentration-dependent manner (up to 100 μg) the [ 3H]flunitrazepam binding. Scatchard analysis revealed changes in the apparent affinity without alterations in the B max. Very similar results were obtained using a purified PS from spinal cord. BC-PC, phosphatidylinositol, phosphatidic acid and the lyso derivatives of PS and PC (lysoPS and lysoPC) were found to be ineffective. The facilitating effects of BC-PS and γ-aminobutyric acid (GABA) on [ 3H]flunitrazepam binding were not additive. As with BC-PS, the diglyceride cardiolipine added to cerebral cortical and cerebellar membranes induced a concentration dependent (up to 25 μg) increase in [ 3H]flunitrazepam binding. Higher concentrations of cardiolipine produced a decrease of [ 3H]flunitrazepam binding. These results indicate that the central type benzodiazepine receptors are under the modulatory action of PS and suggest that this endogenous phospholipid may play a regulatory role on the benzodiazepine/GABA receptor function.

Benzodiazepine receptor modulation of [ 35S]TBPS binding to the chloride channel. Noncompetitive inhibition of classical benzodiazepines and competitive inhibition of the partial agonist, CGS 9895, by CGS 8216

Benzodiazepine receptor ligands are known to modulate the binding of [ 35S]TBPS to the chloride channel via an allosteric action. In the cases of diazepam, zopiclone and CGS 9895, the enhanced binding of [ 35S]TBPS induced by these benzodiazepine agonists was antagonized by CGS 8216. This antagonism was characterized both by a shift to the right and a decrease in the maximal stimulation, for the doseresponse curves of diazepam and zopiclone. In the case of CGS 9895, the maximal response was not decreased. These data indicate that CGS 8216 is a noncompetitive antagonist of classical benzodiazepine receptor agonists but is a competitive inhibitor of the partial agonist, CGS 9895.

Increase of peripheral type benzodiazepine binding sites in kidney and olfactory bulb in acutely stressed rats

Fifteen minutes after the initiation of swimming stress in the rat we observed a 50% increase in the number of [ 3H]RO 5-4864 binding sites in kidney and a 37% increase in the olfactory bulb, without change in affinity. The binding in heart and cerebral cortex remained unchanged after the stress. These results are discussed in relation to previous work on both the action of an acute stress in central benzodiazepine receptors and the possible modulation of peripheral benzodiazepine receptors of the kidney by adrenocortical hormones.

Aging: effect on the interaction of ethanol and pentobarbital with the benzodiazepine-GABA receptor-ionophore complex.

Benzodiazepine receptor binding and modulation by pentobarbital and ethanol was studied using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate to solubilize the gamma-aminobutyric acid (GABA)-benzodiazepine receptor-ionophore complex from the brains of Fischer 344 rats of 3-4, 12-15 and more than 28 months of age. The affinity of the benzodiazepine binding site was significantly lower in the young rats compared to either the mature or senescent animals. However, no age-related changes in the maximum number of benzodiazepine binding sites or GABA concentrations occurred in the detergent extract. Pentobarbital produced practically identical dose-dependent enhancement of [3H]flunitrazepam specific binding in all 3 age groups. In contrast, ethanol between 0.1 and 200 microM failed to produce a dose-dependent effect on [3H]flunitrazepam specific binding in any age group. The effect of pentobarbital and ethanol on [35S]t-butyl-bicyclophosphorothionate [( 35S]TBPS) specific binding to the picrotoxinin binding site was examined in the above solubilized receptor/ionophore complex under the same binding conditions. Both sedative-hypnotics produced a dose-dependent decrease in [35S]TBPS specific binding. However, pentobarbital was over 10,000 times more potent. It appears that ethanol may not enhance [3H]flunitrazepam specific binding in this solubilized preparation because of its weak action at the picrotoxinin binding site.

Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation

In rats rendered hyperthyroid by chronic treatment with L-triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]beta-CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 microM, whereas in hyperthyroid rats, it was 83 microM, a 92-fold difference. In control rats, the Ki was 11 microM. These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.

The adenosine receptor activity of EMD 28422, a purine derivative with reported actions on benzodiazepine receptors.

The effects of a novel purine derivative, N6-[2-(4-chlorophenyl)-bicyclo-2.2.2.octyl-(3)]-adenosine (EMD 28422), that has been found to influence central benzodiazepine receptors, has been compared to those of other adenosine analogues such as L-phenylisopropyladenosine (L-PIA), cyclohexyladenosine (CHA) and adenosine-5'-N-ethyl-carboxamide (NECA). EMD 28422 was about 30 times less potent than CHA and 4 times less potent than NECA in displacing bound [3H]-L-PIA from specific binding sites in the rat brain, presumably reflecting adenosine A1-receptors. A similar relative potency was found using depression of field e.p.s.p. in the hippocampal slice in vitro. In isolated fat cells EMD 28422 was antilipolytic, but some 1000 times less potent than L-PIA. In rat isolated hippocampal slices, which have adenosine A2-receptors, EMD 28422 was more than 300 times less potent than NECA and in guinea-pig thymocytes, which similarly have A2-receptors, EMD 28422 was about 60 times less potent. The results are compatible with the opinion that EMD 28422 is a rather weak agonist at adenosine receptors, with limited selectivity for A1- or A2-receptors. The compound is highly lipophilic, which plays a role in determining its potency in a given biological system. The results are discussed in relation to reported adenosine modulation of benzodiazepine receptors.