No changes in rat benzodiazepine receptors after withdrawal from continuous treatment with lorazepam and diazepam.

Five and 11 days after withdrawal from 8 weeks of treatment with 90 mg/kg/day of diazepam p.o. or 60 mg/kg/day of lorazepam p.o. there were no consistent changes in the number of benzodiazepine receptors or apparent affinity $\text{in vitro}$ for ³H-diazepam at 0°C in rat forebrain membranes. Daily exposure of rats from 10 days before birth until 7 days after birth was also without gross effects on the benzodiazepine receptor. Abstinence and tolerance to benzodiazepines were thus not attributable to changes in brain benzodiazepine receptors.     

Accelerated skeletal muscle recovery after in vivo polyphenol administration

Acute skeletal muscle damage results in fiber disruption, oxidative stress and inflammation. We investigated cell-specific contributions to the regeneration process after contusion-induced damage (rat gastrocnemius muscle) with or without chronic grape seed-derived proanthocyanidolic oligomer (PCO) administration. In this placebo-controlled study, male Wistar rats were subjected to PCO administration for 2 weeks, after which they were subjected to a standardised contusion injury. Supplementation was continued after injury. Immune and satellite cell responses were assessed, as well as oxygen radical absorption capacity and muscle regeneration. PCO administration resulted in a rapid satellite cell response with an earlier peak in activation (Pax7(+), CD56(+), at 4 h post-contusion) vs. placebo groups (PLA) (P<.001: CD56(+) on Day 5 and Pax7(+) on Day 7). Specific immune-cell responses in PLA followed expected time courses (neutrophil elevation on Day 1; sustained macrophage elevation from Days 3 to 5). PCO dramatically decreased neutrophil elevation to nonsignificant, while macrophage responses were normal in extent, but significantly earlier (peak between Days 1 and 3) and completely resolved by Day 5. Anti-inflammatory cytokine, IL-10, increased significantly only in PCO (Day 3). Muscle fiber regeneration (MHC(f) content and central nuclei) started earlier and was complete by Day 14 in PCO, but not in PLA. Thus, responses by three crucial cell types involved in muscle recovery were affected by in vivo administration of a specific purified polyphenol in magnitude (neutrophil), time course (macrophages), or time course and activation state (satellite cell), explaining faster effective regeneration in the presence of proanthocyanidolic oligomers.     

Putative endogenous ligands for the benzodiazepine receptor.

The recent discovery of pharmacologically relevant, high affinity, stereospecific binding sites for the benzodiazepines in the central nervous system (CNS) has rekindled investigations concerning the mechanism of action of these drugs. It has become increasingly clear that elucidation of benzodiazepine action will provide new and important insights into the neurochemical substances of seizure activity, centrally mediated muscle relaxation and anxiety, three major actions of this class of drugs.The existence of a functional receptor for the benzodiazepines, compounds not present $\text{in vivo}$, suggests that endogenous substances exist that serve as natural substrates for this receptor. Furthermore, the characterization of endogenous benzodiazepine receptor ligands affords an opportunity to determine the neurochemical mechanisms underlying the pharmacologic and behavioral effects manifested by the benzodiazepines.Using receptor binding methodology to assay tissue extracts for [³H] diazepam binding inhibitory activity, putative endogenous ligands for the benzodiazepine receptor have been isolated and identified as the purine nucleosides. Compounds such as inosine and hypoxanthine exhibit competitive inhibition of [³H] diazepam binding. The low affinity purinergic inhibition of diazepam binding is consistent with their $\text{in vivo}$ concentrations. Distinct structure-activity relationships exist for the purines with subtle structural alterations having marked effects on diazepam binding inhibitory potency. The methylxanthine stimulants, caffeine, theophylline, and theobromine, also competitively inhibit diazepam binding, suggesting that some of their actions may be mediated by the benzodiazepine receptor.The purines also have “benzodiazepine-like” pharmacologic properties, since they have been shown to antagonize pentylenetetrazol induced seizures in mice in a dose dependent manner. Neurophysiologic studies have also shown that iontophoresis of inosine on cultured mouse primary neurons produce neurotransmitter like effects. Furthermore, these effects are similar to those observed with flurazepam, a finding that provides additional evidence for the “benzodiazepine-like” properties of the purines.The preliminary studies outlined below indicate that the purines are good candidates as putative endogenous ligands for the benzodiazepine receptor and provide a foundation for future studies that concern the homeostatic mediation of seizure activity and anxiety.     

Tissue specific regulation of "peripheral-type" benzodiazepine receptor density after chemical sympathectomy

The characteristics of [3H]Ro 5-4864 binding to "peripheral" benzodiazepine receptors (PBR) in the central nervous system and peripheral tissues were examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). One week after the intracisternal administration of 6-OHDA, the number of [3H]Ro 5-4864 binding sites (Bmax) in the hypothalamus and striatum increased 41 and 50%, respectively, concurrent with significant reductions in catecholamine content. An increase (34%) in the Bmax of [3H]Ro 5-4864 to cardiac ventricle was observed one week after parenteral 6-OHDA administration. In contrast, the Bmax of [3H]Ro 5-4864 to pineal gland decreased 48% after 6-OHDA induced reduction in norepinephrine content. The Bmax values for [3H]Ro 5-4864 binding to other tissues (including lung, kidney, spleen, cerebral cortex, cerebellum, hippocampus and olfactory bulbs) were unaffected by 6-OHDA administration. The density of pineal, but not cardiac PBR was also reduced after reserpine treatment, an effect reversed by isoproterenol administration. These findings demonstrate that alterations in sympathetic input may regulate the density of PBR in both the central nervous system and periphery in a tissue specific fashion.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues.

Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.     

Accelerated marrow recovery following total-body irradiation after treatment with vincristine, lithium or combined vincristine-lithium

Accelerated post-irradiation recovery of hematopoietic marrow has been reported following treatment with lithium (Li) or vincristine (VcR). Because these two agents appear to exert their effects on different, albeit overlapping, hematopoietic populations, it was felt that combining them might lead to a wider spectrum of enhanced post-irradiation marrow regeneration. Results demonstrated that an accelerated recovery, which appeared to be additive in nature, was observed in the marrow following combined VcR-Li/4.5 Gy total-body irradiation. The combined schedule significantly enhanced post-irradiation recovery of white blood cells, 12-day spleen colony-forming units, erythroid burst-forming units, and fibroblastic colony-forming units over radiation alone; and recovery of marrow cellularity, multipotential colony-forming units (CFU-gemm) and granulocytic/monocytic colony-forming units (CFU-gm) over both radiation alone and either drug given singly with the 4.5 Gy. In addition, while data on the ability of regenerating stroma to support CFU-gm and CFU-gemm did not suggest that VcR was acting to enhance post-irradiation marrow recovery by increasing stromal production of hematopoietic growth factors, Li did appear to increase production of one or more of these factors, and this may be part of its mechanism of action.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Effect of picrotoxinin on benzodiazepine receptor binding

The effect of picrotoxinin on [³H]flunitrazepam binding to benzodiazepine receptors was investigated. In mouse forebrain membranes, picrotoxinin inhibited basal, GABA- and pentobarbital-stimulated [³H]flunitrazepam binding; this inhibitory activity was temperature- and chloride ion-dependent. Scatchard analysis of the data indicates that picrotoxinin decreases the number of binding sites without alterating binding affinity. In cerebellar membranes, picrotoxinin did not alter [³H]flunitrazepam receptor binding.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Olfactory memory in rats, cholinergic agents and benzodiazepine receptor ligands.

Drugs and their effects on olfactory learning processes in rats were tested using a modified version of the runway apparatus developed by Ades. Rats were first exposed to a conspecific urine sample and 24 h later were exposed to the same stimulus in the runway. Observations recorded the time spent investigating the urine and the number of sniffs at the site, these being considered to be indices of memory. Diazepam-treated rats (4 or 6 mg/kg) and scopolamine-treated rats (0.5 or 1 mg/kg) showed increases for both parameters. When both drugs were administered simultaneously, the impairing effect was potentiated. However, no changes in learning responses were observed in rats treated with physostigmine (0.125, 0.25, 0.5 mg/kg) or methyl beta-carboline-3-carboxylate (0.3, 0.5, 1 mg/kg), although the administration of physostigmine or methyl beta-carboline-3-carboxylate was shown to antagonize the impairing effect of diazepam or scopolamine respectively. These observations support the hypothesis of interactions existing between cholinergic agents and benzodiazepine receptor ligands and of such interactions affecting olfactory acquisition processes. The runway apparatus appears to be a valid candidate model to be used for the assessment of pharmacological influences on olfactory learning in rats.     

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.