Benzodiazepine Receptor Ligands Research Articles

Mitochondria protective and anti-apoptotic effects of peripheral benzodiazepine receptor and its ligands on the treatment of asthma in vitro and vivo.

Asthma is a prevalent respiratory inflammatory disease. Abnormal apoptosis of bronchial epithelial cells is one of the major factors in the progression of asthma. Peripheral benzodiazepine receptors are highly expressed in bronchial epithelial cells, which act as a component of the mitochondrial permeability transition pore to regulate its opening and closing and apoptosis of bronchial epithelial cells. We aimed to investigate the mechanisms by which peripheral benzodiazepine receptor and its ligands, agonist 4'-Chlorodiazepam (Ro5-4864) and antagonist 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11,195), modulate the mitochondrial function and cell apoptosis in the treatment of asthma. In vitro study, Ro5-4864 and PK 11,195 were utilized to pretreat cells prior to the inflammatory injury induced by Lipopolysaccharide. The reactive oxygen species, the apoptosis of cell, the mitochondrial membrane potentials, the ultrastructures of the mitochondria and the expression levels of peripheral benzodiazepine receptors and apoptosis-related proteins and genes were detected. In vivo study, mice were administrated intraperitoneally with Ro5-4864 and PK 11,195 before sensitized and challenged by ovalbumin. Serum IgE and bronchoalveolar lavage fluid cytokines were detected, and lung tissues were underwent the histopathological examination. The ligands of peripheral benzodiazepine receptor counteracted the effects of the increase of reactive oxygen species, the elevated extent of apoptosis, the decrease of mitochondrial membrane potentials and the disruption of mitochondrial ultrastructures induced by Lipopolysaccharide. The ligands also promoted the expression of anti-apoptosis-related proteins and genes and inhibited the expression of pro-apoptosis-related proteins and genes. Besides, the ligands reduced the levels of serum IgE and bronchoalveolar lavage fluid cytokines in asthmatic mice and attenuated the histopathological damage of lungs. Peripheral benzodiazepine receptor serves as a potential therapeutic target for the treatment of asthma, with its ligands exerting mitochondrial protective and anti-apoptotic effects on bronchial epithelial cells.

Benzodiazepine diazepam regulates cell surface β1-adrenergic receptor density in human monocytes

Downregulation of cell surface β-adrenergic receptors (β-AR) is an important adaptive response that prevents deleterious effects of receptor overstimulation. Various factors including reactive oxygen species cause β-AR downregulation. In this study, we evaluated the effects of ligands of the peripheral benzodiazepine receptor (PBR), a key protein in regulating oxidative stress, on surface density of endogenous β1-and β2-ARs in highly differentiated cells such as human monocytes, which express both β-AR subtypes. β-AR expression in human monocytes was evaluated by flow cytometry, qPCR and western blotting. Monocyte treatment with β-AR agonist isoproterenol did not change surface β1-AR density while downregulating surface β2-AR density. This effect was antagonized by the β-blocker propranolol. An opposite response was observed with benzodiazepine diazepam that led to a time-dependent reduction in β1-AR density. In particular, while no significant downregulation was observed after 3 h of treatment, only 63% of β1-ARs were still present on the cell surface after 48 h of treatment with diazepam at 1 μM. Treatment with the PBR antagonist PK11195, but not with propranolol, antagonized the effects of diazepam. No change in β1-AR-mRNA or protein levels was observed at any time after diazepam treatment. We also found that diazepam did not affect Gs-protein or β-arrestin-2 recruitment for both β-ARs in engineered fibroblasts, further suggesting that diazepam activity on β1-AR density is mediated by PBR. Finally, no sex-related differences were found. Collectively, these results indicate that monocyte β1-ARs are resistant to catecholamine-mediated downregulation and suggest that PBR plays an important role in regulating β1-AR density.

Synthesis of 4,6-diphenylpyrimidin-2-ol derivatives as new benzodiazepine receptor ligands

Benzodiazepines (BZDs) have been widely used in neurological disorders such as insomnia, anxiety, and epilepsy. The use of classical BZDs, e.g., diazepam, has been limited due to adverse effects such as interaction with alcohol, ataxia, amnesia, psychological and physical dependence, and tolerance. In the quest for new benzodiazepine agonists with more selectivity and low adverse effects, novel derivatives of 4,6-diphenylpyrimidin-2-ol were designed, synthesized, and evaluated. In this series, compound 2, 4-(2-(benzyloxy)phenyl)-6-(4-fluorophenyl)pyrimidin-2-ol, was the most potent analogue in radioligand binding assay with an IC50 value of 19 nM compared to zolpidem (IC50 = 48 nM), a nonbenzodiazepine central BZD receptor (CBR) agonist. Some compounds with a variety of affinities in radioligand receptor binding assay were selected for in vivo evaluations. Compound 3 (IC50 = 25 nM), which possessed chlorine instead of fluorine in position 4 of the phenyl ring, exhibited an excellent ED50 value in most in vivo tests. Proper sedative-hypnotic effects, potent anticonvulsant activity, appropriate antianxiety effect, and no memory impairment probably served compound 3, a desirable candidate as a benzodiazepine agonist. The pharmacological effects of compound 3 were antagonized by flumazenil, a selective BZD receptor antagonist, confirming the BZD receptors’ involvement in the biological effects of the novel ligand.

Novel Derivatives of diphenyl-1,3,4-oxadiazol as Ligands of Benzodiazepine Receptors; Synthesize, Binding Assay and Pharmacological Evaluation.

Benzodiazepines (BZD) are among the main classes of tranquilizing drugs, bearing much less toxicity compared to other drugs acting on the CNS. Considering the pharmacophore model of BZD binding to GABA-A receptor, novel diphenyl 1,3,4-oxadiazole compounds as BZD ligands were designed. The compounds were synthesized and structurally confirmed using LCMS, IR and NMR techniques. We investigated the affinity of the compounds to BZD receptors using radioligand [3H]-flumazenil by in-vitro studies. In addition, sedative-hypnotic, anxiety, anticonvulsant, muscle relaxant, memory impairment, and motor coordination activities of the synthesized compounds were evaluated using in-vivo studies. Based on in-vitro studies, compounds 7i and 7j were the most potent with IC50 values of 1.54 and 1.66 nM respectively. In-vivo studies showed that compound 7i has the highest impact on increased sedation, muscle relaxation, and decreased anxiety and these observations were antagonized by flumazenil. Compounds 7e and 7i were the most potent anticonvulsant agents among synthesized compounds in both MES and PTZ induced seizure tests. All synthesized compounds significantly decreased latency to fall in the Rotarod test but none of them had a significant impact on the memory impairment test.

Design, synthesis and biological evaluation of 7-substituted 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f] [1,4]diazepines as safe anxiolytic agents

A series of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate esters were synthesized and tested as central benzodiazepine receptor (CBR) ligands by the ability to displace [3H]flumazenil from rat cortical membranes. All the compounds showed high affinity with IC50 values ranging from 5.19 to 16.22 nM. In particular, compounds 12b (IC50 = 8.66 nM) and 12d (IC50 = 5.19 nM) appeared as the most effective ligands being their affinity values significantly lower than that of diazepam (IC50 = 18.52 nM). Compounds 12a-f were examined in vivo for their pharmacological effects in mice and five potential benzodiazepine (BDZ) actions were thus taken into consideration: anxiolytic, anticonvulsant, anti-amnesic, hypnotic, and locomotor activities. All the new synthesized compounds were able to induce a significant antianxiety effect and, among them, compound 12f protected pentylenetetrazole (PTZ)-induced convulsions in a dose-dependent manner reaching a 40% effect at 30 mg/kg. In addition, all the compounds were able to significantly prevent the memory impairment evoked by scopolamine, while none of them was able to interfere with pentobarbital-evoked sleep and influence motor coordination. Moreover, title compounds did not affect locomotor and exploratory activity at the same time and doses at which the anti-anxiety effect was observed. Finally, molecular docking simulations were carried out in order to assess the binding mode for compounds 12a-f. The obtained results demonstrated that these compounds bind the BDZ binding site in a similar fashion to flumazenil.

Effects of the Astrocyte Endozepine Octadecaneuropeptide (ODN) on Hypoglycemia Patterns of Ventromedial Hypothalamic Nucleus (VMN) Metabolic‐Sensory Neuron Transmitter Signaling and Energy Sensor Activation in Male and Female Rats

The astrocyte gliotransmitter octadecaneuropeptide (ODN) is a cleavage product of diazepam‐binding inhibitor (DBI) and an endogenous central benzodiazepine receptor ligand. ODN regulation of feeding responses to excess or diminished glucose availability in the brain infers ability to modulate neural metabolic‐sensory function [Lanfray et al., 2013]. The ventromedial hypothalamic nucleus (VMN), a principal component of the neural glucostatic network, integrates nutrient and other cues to shape glucose counter‐regulation. Our studies show that VMN gluco‐stimulatory nitric oxide (NO) and gluco‐inhibitory g‐aminobutyric acid (GABA) neurons express the ultra‐sensitive energy sensor 5′‐AMP‐activated protein kinase (AMPK). This research examined the premise that ODN exerts sex‐specific effects on hypoglycemic patterns of VMN nitrergic and/or GABAergic nerve cell sensor activation and neurotransmitter signaling. Testes‐intact male and ovariectomized, estradiol‐replaced female rats were pretreated by intracerebroventricular administration of the ODN octapeptide (OP) fragment or vehicle prior to insulin injection. VMN NO and GABA cells were identified by immunocytochemistry prior to combinatory laser‐catapult dissection/high‐sensitivity Western blotting. Results identified nitrergic cells exhibiting OP‐reversible effects of hypoglycemia on expression profiles of the NO marker protein neuronal nitric oxide synthase (nNOS), AMPK, and phospho (activated)‐AMPK in distinctive regions of the male and female VMN. In each sex, OP effects on GABA neuron AMPK protein content and activation were also observed at specific levels of the VMN. Outcomes provide novel evidence that ODN may critically impact VMN substrate fuel screening. Further studies are needed to characterize neuroanatomical connectivity of OP‐sensitive nitrergic and GABAergic neurons within central metabolic pathways, and to examine whether metabolic cues that regulate VMN astrocyte ODN release, astrocyte reactivity to ODN‐controlling signals, and metabolic‐sensory nerve cell receptivity to ODN are sex‐dimorphic.Support or Funding InformationNIH DK 109382

Neuroprotection with the Endozepine Octadecaneuropeptide, ODN

The term endozepines designates a family of astroglia-secreted proteins including the diazepambinding inhibitor (DBI) and its processing products, which have been originally isolated and characterized as endogenous ligands of benzodiazepine receptors. It is now clearly established that the octadecaneuropeptide ODN (DBI33-50), acting through the central-type benzodiazepine receptor or a metabotropic receptor, exerts important functions such as proconflict behavior, induction of anxiety, inhibition of pentobarbital-provoked sleep, decrease of water consumption and reduction of food intake. To mediate its effects, ODN regulates both glial cell and neuronal activities by acting on neurosteroid biosynthesis and/or neuropeptide expression. In addition, ODN stimulates astrocyte proliferation and protects both neurons and astrocytes from oxidative stress-induced cell death. The antiapoptotic effect of ODN on neural cells is mediated through activation of the ODN metabotropic receptor positively coupled to PKA, PKC and MAPK/ERK transduction pathways, which ultimately reduces the pro-apoptotic gene Bax and stimulates Bcl-2 expressions, and inhibits intracellular reactive oxygen species accumulation. The imbalance in favor of Bcl2 promotes mitochondria functions and blocks in turn caspases activation while at the same time, ODN also activates the endogenous antioxidant system i.e. glutathione biosynthesis, and expression and activities of antioxidant enzymes. In cultured astrocytes, DBI expression is up-regulated during moderate oxidative stress, and authentic ODN production is increased, suggesting that ODN may act as a paracrine factor protecting neighboring neurons. Taken together, the remarkable effect of ODN on the apoptotic cascade suggests that innovative ODN derivatives could potentially be useful for treatment of cerebral injuries involving oxidative stress and neurodegeneration.

Discovery of Imidazoquinazolinone Derivatives as TSPO Ligands Modulating Neurosteroidogenesis and Cellular Bioenergetics in Neuroblastoma Cells Expressing Amyloid Precursor Protein

AbstractStarting from the central benzodiazepine receptor (CBR) ligand CGS‐13767, we designed a series of imidazoquinazolinone derivatives acting as 18 kDa translocator protein (TSPO) ligands, and deprived of affinity for CBR. These compounds, as well as ligands of reference (Ro5‐4864, XBD173, SSR180575), were assessed for their ability to restore neurosteroidogenesis and energy production in SH‐SY5Y neuroblastoma cell lines expressing amyloid protein precursor (APP). Our results show the potential neuroprotective effect of TSPO ligands, but highlight a strong discrepancy in the active concentrations, nanomolar for ATP production while pregnenolone production is restored in the micromolar range.

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

The constitutive androstane receptor (CAR) plays an important role in xenobiotic metabolism, energy homeostasis, and cell proliferation. Antagonism of the CAR represents a key strategy for studying its function and may have potential clinical applications. However, specific human CAR (hCAR) antagonists are limited and conflicting data on the activity of these compounds have been reported. 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a typical peripheral benzodiazepine receptor ligand, has been established as a potent hCAR deactivator in immortalized cells; whether it inhibits hCAR activity under physiologically relevant conditions remains unclear. Here, we investigated the effects of PK11195 on hCAR in metabolically competent human primary hepatocytes (HPH) and HepaRG cells. We show that although PK11195 antagonizes hCAR in HepG2 cells, it induces the expression of CYP2B6 and CYP3A4, targets of hCAR and the pregnane X receptor (PXR), in HPH, HepaRG, and PXR-knockout HepaRG cells. Utilizing a HPH-HepG2 coculture model, we demonstrate that inclusion of HPH converts PK11195 from an antagonist to an agonist of hCAR, and such conversion was attenuated by potent CYP3A4 inhibitor ketoconazole. Metabolically, we show that the N-desmethyl metabolite is responsible for PK11195-mediated hCAR activation by facilitating hCAR interaction with coactivators and enhancing hCAR nuclear translocation in HPHs. Structure-activity analysis revealed that N-demethylation alters the interaction of PK11195 with the binding pocket of hCAR to favor activation. Together, these results indicate that removal of a methyl group switches PK11195 from a potent antagonist of hCAR to an agonist in HPH and highlights the importance of physiologically relevant metabolism when attempting to define the biologic action of small molecules.

Increased Hypothalamic Levels of Endozepines, Endogenous Ligands of Benzodiazepine Receptors, in a Rat Model of Sepsis.

The mechanisms involved in septic anorexia are mainly related to the secretion of inflammatory cytokines. The term endozepines designates a family of neuropeptides, including the octadecaneuropeptide (ODN), originally isolated as endogenous ligands of benzodiazepine receptors. Previous data showed that ODN, produced and released by astrocytes, is a potent anorexigenic peptide. We have studied the effect of sepsis by means of a model of cecal ligation and puncture (CLP) on the hypothalamic expression of endozepines (DBI mRNA and protein levels), as well as on the level of neuropeptides controlling energy homeostasis mRNAs: pro-opiomelanocortin, neuropeptide Y, and corticotropin-releasing hormone. In addition, we have investigated the effects of two inflammatory cytokines, TNF-α and IL-1β, on DBI mRNA levels in cultured rat astrocytes. Studies were performed on Sprague-Dawley male rats and on cultures of rat cortical astrocytes. Sepsis was induced using the CLP method. Sham-operated control animals underwent the same procedure, but the cecum was neither ligated nor incised. Sepsis caused by CLP evoked an increase of DBI mRNA levels in ependymal cells bordering the third ventricle and in tanycytes of the median eminence. CLP-induced sepsis was also associated with stimulated ODN-like immunoreactivity (ODN-LI) in the hypothalamus. In addition, TNF-α, but not IL-1β, induced a dose-dependent increase in DBI mRNA in cultured rat astrocytes. An increase in the mRNA encoding the precursor of the anorexigenic peptide α-melanocyte stimulating hormone, the pro-opiomelanocortin, and the corticotropin-releasing hormone was observed in the hypothalamus. These results suggest that during sepsis, hypothalamic mRNA encoding endozepines, anorexigenic peptide as well as stress hormone could play a role in the anorexia/cachexia associated with inflammation due to sepsis and we suggest that this hypothalamic mRNA expression could involve TNF-α.

Hypothermic activity of acetaminophen; involvement of GABAA receptor, theoretical and experimental studies.

The mechanism of hypothermia action of acetaminophen (APAP) remains unclear even 125 years after its synthesis. Acetaminophen produces hypothermia. The mechanism of this reduction in core body temperature is not clear but evidence shows that it is not dependent on opioid and cannabinoid receptors. Because of strong documents about the roles of GABA and benzodiazepine receptors in hypothemic activity of some drugs such as diazepam, we determined if these receptors also contributes to the hypothermic effect of APAP. Diazepam (5 mg/kg, IP) was used for induction of hypothermia. Flumazenil (10 mg/kg, IP) or picrotoxin (2 mg/kg, IP) used for reversal of this effect. Rats injected with APAP (100, 200 or 300 mg/kg, IP). Baseline temperature measurements were taken with a digital thermometer via rectum. To evaluate the structural correlation between APAP and benzodiazepine receptor ligands, numerous models are selected and studied at HF/6-31G* level of theory. Relative energies, enthalpies and Gibbs free energies were calculated for all selected drugs. Diazepam induced hypothermia was reversed by flumazenil or picrotoxin. Rats injected with APAP displayed dose- and time-related hypothermia. For combined administration, the hypothermic effect of APAP (200 mg/kg) was strongly reduced by pretreatment with picrotoxin or flumazenil P<0.0001and P<0.01, respectively. Selective structural data, bond length, dihedral angles, and related distance in pharmacophore of APAP and BZDR models were the same. Some significant structural analogues were obtained between these drugs. Results suggest hypothermic action of acetaminophen may be mediate by its effect at GABAA benzodiazepine receptor.

Design, Synthesis, and Biological Evaluation of Imidazo[1,5-a]quinoline as Highly Potent Ligands of Central Benzodiazepine Receptors.

A series of imidazo[1,5-a]quinoline derivatives was designed and synthesized as central benzodiazepine receptor (CBR) ligands. Most of the compounds showed high CBR affinity with Ki values within the submicromolar and subnanomolar ranges with interesting modulations in their structure-affinity relationships. In particular, fluoroderivative 7w (Ki = 0.44 nM) resulted in the most potent ligand among the imidazo[1,5-a]quinoline derivatives described so far. Overall, these observations confirmed the assumption concerning the presence of a large though apparently saturable lipophilic pocket in the CBR binding site region interacting with positions 4 and 5 of the imidazo[1,5-a]quinoline nucleus. The in vivo biological characterization revealed that compounds 7a,c,d,l,m,q,r,w show anxiolytic and antiamnestic activities without the unpleasant myorelaxant side effects of the classical 1,4-BDZ. Furthermore, the effect of 7l,q,r, and 8i in lowering lactate dehydrogenase (LDH) release induced by ischemia-like conditions in rat brain slices suggested neuroprotective properties for these imidazo[1,5-a]quinoline derivatives.

PET imaging studies show enhanced expression of mGluR5 and inflammatory response during progressive degeneration in ALS mouse model expressing SOD1-G93A gene.

BackgroundAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motor neuron disorder. Genetic studies have linked mutation of the gene SOD1 to ALS pathology as well as several other pathological processes including modulation of glutamatergic function and inflammatory processes. Since therapeutic approaches for ALS are focused on glutamatergic function, we investigated modulation of glutamate transport based on its receptor function as well as excitotoxicity-induced inflammatory response.MethodsIn vivo positron emission tomography (PET) imaging studies of metabotropic glutamate receptor subtype 5 (mGluR5) using [18F]FPEB ([18F]3-fluoro-5-(2-pyridylethynyl)benzonitrile) and inflammatory response using [11C]PBR28 (peripheral benzodiazepine receptor ligand 28) were done in an early and a late phase of neurodegeneration in four ALS mice expressing SOD1-G93A gene and four control base mice (C57/BL6). Accumulation of [18F]FPEB and [11C]PBR28 were quantitated in several brain areas and spinal cord to determine degeneration-induced modulation. The studies were completed with immunohistochemical analyses of mGluR5 and inflammatory response.ResultsThese studies showed enhanced binding potential of [18F]FPEB in several brain areas including striatum, hippocampus, and frontal cortex. In the whole brain, the binding potential increased 49 ± 9 % from base mice to ALS-type mice and further enhanced 23 ± 4 % during disease progression. Also, in the spinal cord 6–22 %, enhanced accumulation of [18F]FPEB was observed during progression of the disease. The accumulation of [11C]PBR28 increased by 110 ± 33 % in the whole brain during progression of the disease indicating significant inflammatory process. [11C]PBR28 accumulation enhanced 89–264 % in the spinal cord and 204 % in the lungs. The end point immunohistochemical analyses verified the enhanced mGluR5 expression and inflammation.ConclusionsThese results confirm the role of glutamate and inflammation in ALS-type pathology. These data also support the hypothesis that excessive glutamate may contribute to inflammation in the chronic neurodegenerative processes in ALS.

Standing & walking posture in children with bilateral spastic cerebral palsy: Are they related?

Regional depolarization of the mitochondrial network can alter cellular electrical excitability and increase the propensity for reentry, in part, through the opening of sarcolemmal KATP channels. Mitochondrial inner membrane potential (ΔΨm) instability or oscillation can be induced in myocytes by exposure to reactive oxygen species (ROS), laser excitation, or glutathione depletion, and is thought to be a major factor in arrhythmogenesis during ischemia–reperfusion. Nevertheless, the correlation between ΔΨm recovery kinetics and reperfusion-induced arrhythmias has been difficult to demonstrate experimentally. Here, we investigate the relationship between subcellular changes in ΔΨm, cellular glutathione redox potential, electrical excitability, and wave propagation during coverslip-induced ischemia–reperfusion (IR) in neonatal rat ventricular myocyte (NRVM) monolayers. Ischemia led to decreased action potential amplitude and duration followed by electrical inexcitability after ~ 15 min of ischemia. ΔΨm depolarization occurred in two phases during ischemia: in phase 1 (< 30 min ischemia), mitochondrial clusters within individual NRVMs depolarized, while phase 2 ΔΨm depolarization (30–60 min) was characterized by global functional collapse of the mitochondrial network across the whole ischemic region of the monolayer, typically involving a propagating metabolic wave. Oxidation of the glutathione (GSSG:GSH) redox potential occurred during ischemia, followed by recovery upon reperfusion (i.e., lifting the coverslip). ΔΨm recovered in the mitochondria of individual myocytes quite rapidly upon reperfusion (< 5 min), but was highly unstable, characterized by subcellular oscillations or flickering of clusters of mitochondria in NRVMs across the reperfused region. Electrical excitability also recovered in a heterogeneous manner, providing an arrhythmogenic substrate which led to formation of sustained reentry. Treatment with 4′-chlorodiazepam, a peripheral benzodiazepine receptor ligand, prevented ΔΨm oscillation, improved GSH recovery rate, and prevented reentry during reperfusion, indicating that stabilization of mitochondrial network dynamics is important for preventing post-ischemic arrhythmias. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Increased plasma levels of endozepines, endogenous ligands of benzodiazepine receptors, during systemic inflammation: a prospective observational study.

IntroductionRecent work has shown that benzodiazepines interact with the immune system and exhibit anti-inflammatory effects. By using in vitro models, researchers in several studies have shown that the peptidergic endogenous ligands of benzodiazepine receptors, named endozepines, are involved in the immune response. All endozepines identified so far derive from diazepam-binding inhibitor (DBI), which generates several biologically active fragments. The aim of the present study was to measure plasma levels of DBI-like immunoreactivity (DBI-LI) in a rat model of sepsis and in patients with systemic inflammation from septic or non-septic origin.MethodsCecal ligation and puncture (CLP) or sham surgery was performed in rats. Blood samples were taken from animals, patients hospitalized for digestive surgery with inflammatory diseases, and healthy volunteers. Measurements of plasma DBI-related peptides were carried out by radioimmunoassay in animal and human samples.ResultsIn the rats, CLP provoked an increase of plasma DBI-LI (+37%) 6 hours postsurgery. In humans, DBI-LI levels were significantly higher in the systemic inflammation group than in the healthy volunteer group (48.6 (32.7 to 77.7) pg/ml versus 11.1 (5.9 to 35.3) pg/ml, P < 0.001). We found a positive correlation between endozepine levels and Acute Physiology and Chronic Health Evaluation II score (rs = 0.33 (0.026 to 0.58), P < 0.05) and tumor necrosis factor α levels (rs = 0.43 (0.14 to 0.65), P < 0.01). The area under the receiver operating characteristic curve for endozepines was 0.842 (95% CI (0.717 to 0.966), P < 0.0001) for discriminating patients with inflammation from healthy volunteers.ConclusionsEndozepines might be involved in the inflammatory response in patients with systemic inflammation.

Mitochondrial instability during regional ischemia–reperfusion underlies arrhythmias in monolayers of cardiomyocytes

Regional depolarization of the mitochondrial network can alter cellular electrical excitability and increase the propensity for reentry, in part, through the opening of sarcolemmal KATP channels. Mitochondrial inner membrane potential (ΔΨm) instability or oscillation can be induced in myocytes by exposure to reactive oxygen species (ROS), laser excitation, or glutathione depletion, and is thought to be a major factor in arrhythmogenesis during ischemia–reperfusion. Nevertheless, the correlation between ΔΨm recovery kinetics and reperfusion-induced arrhythmias has been difficult to demonstrate experimentally. Here, we investigate the relationship between subcellular changes in ΔΨm, cellular glutathione redox potential, electrical excitability, and wave propagation during coverslip-induced ischemia–reperfusion (IR) in neonatal rat ventricular myocyte (NRVM) monolayers. Ischemia led to decreased action potential amplitude and duration followed by electrical inexcitability after ~15min of ischemia. ΔΨm depolarization occurred in two phases during ischemia: in phase 1 (<30min ischemia), mitochondrial clusters within individual NRVMs depolarized, while phase 2 ΔΨm depolarization (30–60min) was characterized by global functional collapse of the mitochondrial network across the whole ischemic region of the monolayer, typically involving a propagating metabolic wave. Oxidation of the glutathione (GSSG:GSH) redox potential occurred during ischemia, followed by recovery upon reperfusion (i.e., lifting the coverslip). ΔΨm recovered in the mitochondria of individual myocytes quite rapidly upon reperfusion (<5min), but was highly unstable, characterized by subcellular oscillations or flickering of clusters of mitochondria in NRVMs across the reperfused region. Electrical excitability also recovered in a heterogeneous manner, providing an arrhythmogenic substrate which led to formation of sustained reentry. Treatment with 4′-chlorodiazepam, a peripheral benzodiazepine receptor ligand, prevented ΔΨm oscillation, improved GSH recovery rate, and prevented reentry during reperfusion, indicating that stabilization of mitochondrial network dynamics is important for preventing post-ischemic arrhythmias. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

18F]DPA-C5yne, a novel fluorine-18-labelled analogue of DPA-714: radiosynthesis and preliminary evaluation as a radiotracer for imaging neuroinflammation with PET.

DPA-C5yne, the lead compound of a novel series of DPA-714 derivatives in which the fluoroethoxy chain linked to the phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn-1-yl moiety, is a high affinity (Ki : 0.35 nM) and selective ligand targeting the translocator protein 18 kDa. In the present work, DPA-C5yne was labelled with no-carrier-added [(18)F]fluoride based on a one-step tosyloxy-for-fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3-5.2 GBq of [(18)F]DPA-C5yne, ready-to-use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to 110 GBq/µmol within 50-60 min, starting from a 30 GBq [(18)F]fluoride batch (14-17%). LogP and LogD of [(18)F]DPA-C5yne were measured using the shake-flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies performed on slices of ((R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA)-lesioned rat brains showed a high target-to-background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated using DPA-C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore, DPA-C5yne proved to be stable in plasma at 37°C for at least 90 min.

GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABAAR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABAARs consistently deactivating ∼10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABAARs was due to longer burst durations at low GABA concentrations, corresponding to a ∼4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABAARs containing γ1 and γ2 use different mechanisms for synaptic clustering.

Optimum conditions of radioligand receptor binding assay of ligands of benzodiazepine receptors.

To obtain drugs which are more selective at benzodiazepine (BZD) receptors, design and synthesis of functionally selective ligands for BZD receptors is the current strategy of our pharmaceutical chemistry department. The affinity of newly synthesized ligands is assessed by radioligand receptor binding assays. Based on our previous studies, 2-phenyl-5-oxo-7-methyl-1,3, 4-oxadiazolo[a,2,3]-pyrimidine (compound A) was chosen for design and synthesis of new triazole derivatives as GABAA BZD receptor agonist. The cortical membrane of male Sprague-Dawley rats was prepared as the source of the BZD receptors. Different concentrations of membrane protein and [3H]-flumazenil were incubated at room temperature at different time periods to reach the steady-state. To saturate the receptors, increased amounts of radioligand were incubated with membrane protein. The bound and un-bound ligands were separated by centrifugation. The affinity of compound A was measured in competition studies at optimum conditions by displacement of [3H]-Flumazenil from rat cortical membrane. Based on results, the optimum conditions of radioligand receptor binding assay of benzodiazepines were 35 min incubation of ligands with 100 μg cortical membrane protein and 8.6 × 10-5 nmole 3H-flumazenil in a final volume of 0.5 mL Tris-HCl buffer (50 mM, pH 7.4) at 30 °C. The binding parameters of [3H]-flumazenil, Bmax and Kd were determined through saturation studies as 0.638 ± 0.099 pmol/mg and 1.35 ± 0.316 nM respectively. The affinity of compound A was 1.9 nM comparable with diazepam (1.53nM). This finding makes the compound an interesting lead for further optimization. Starting from this compound, new ligands were synthesized and screened in-vitro by competitive binding assays.

Radioiodination of Zaleplon and Its in-vivo Biologic Behavior in Mice: An Imaging Probe for Brain

The hypnotic zaleplon displays novel receptor selectivity different from other benzodiazepine receptor ligands in clinical use. Zaleplon was successfully labeled with iodine-125 via electrophilic substitution reaction producing 125I-Zaleplon tracer. This reaction proceeds well in acidic pH of value equal to 4, due to the solubility of the Zaleplon in this acidic pH value. Chloramine-T (CAT) was used as oxidizing agent, at low amount of (CAT) (25–50 ug) the radiochemical yield of the labeled compound was low (80%) while at 200 ug, an optimum yield (97%) was obtained.Heating the reaction mixture to 40 oC for 15 min was recommended to get a yield more than 97%, but heating for longer time causes a decomposition of the labeled Zaleplon. The in-vitro stability of 125I-Zaleplon was determined along 24 hours; the data confirms that 125I-Zaleplon tracer was stable along eight hours without the detection of any by-products in the reaction mixture. The biodistribution data of the labeled Zaleplon shows rapid blood clearance passes through the blood brain barrier (BBB) and the activity detected in the brain exceeds 4.4% at 1 h post injection.