Photoaffinity labeling of peripheral-type benzodiazepine receptors in rat kidney mitochondria with [3H]PK 14105

The peripheral-type benzodiazepine receptor in rat kidney has been identified by photoaffinity labeling. PK 14105, a derivative of the selective peripheral-type ligand PK 11195, was used to covalently label peripheral-type benzodiazepine receptors. In the absence of UV light PK 14105 demonstrated reversible, high affinity (KD = 4.8 nM) binding to rat kidney mitochondrial membranes. Inhibition of the reversible binding of [3H]PK 14105 by various benzodiazepine and other ligands demonstrated that this ligand bound with all the characteristics expected of a ligand interacting specifically with peripheral-type benzodiazepine receptors. A similar order of relative potencies was obtained for inhibition of photolabeling, indicating that reversible binding and photolabeling occurred at the same class of binding sites. Examination of photolabeled binding sites from kidney, heart, brain and adrenal membranes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the probe is photoincorporated into a single peptide of Mr = 18,500. The results indicate that [3H]PK 14105 identifies the ligand binding domain of the peripheral-type benzodiazepine receptor, which is a peptide with Mr = 18,500, that is of similar size in kidney, heart, brain and adrenals.     

Benzodiazepine receptors: labeling in intact animals with [3H] flunitrazepam.

[3H]Flumitrazepam appears to label specific benzodiazepine receptors in vitro after i.v. injection in mice. Benzodiazepine potencies in reducing [3H]flunitrazepam binding in vivo correspond to pharmacological potencies and parallel relative affinites for [3H]flunitrazepam binding sites in isolated brain membranes. However, 50% occupation of [3H]-flunitrazepam sites by benzodiazepines in vivo requires brain concentrations of the drugs about 1000 times higher than their Ki values for the binding sites in vitro. In pharmacologically active doses sodium pentobarbital, strychnine, picrotoxin and bicuculline fail to influence [3H] flunitrazepam binding in vivo.     

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.     

Comparative aspects of cerebellar [3H]flunitrazepam and [3H]GABA binding

[3H]Flu and [3H]GABA binding has been studied in mice and Rabbit cerebellum (Ce). The Bmax of [3H]Flu binding in Ce membranes is similar in all mice strains examined and Rabbit. However, in Ce homogenate there are significant differences in both kd and Bmax. Subcellular distribution shows higher [3H]Flu binding in the nuclear than in the membranous fraction. However, the [3H]GABA binding is lower in the nuclear than the membranous fraction.     

Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido[3H]GBR-12935

A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido[3H]GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido[3H]GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of [3H]GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido[3H]GBR-12935 binding to rat striatal membranes (r = 0.95, p less than 0.01). These data suggest that 3-azido[3H]GBR-12935, like other diphenylpiperazines such as [3H]GBR-12935 and [3H]GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido[3H]GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido[3H]GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine (but not other biogenic amines) and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido[3H]GBR-12935 is glycosylated.     

Isolation and purification of rat liver morphine UDP-glucuronosyltransferase

A UDP-glucuronosyltransferase (UDPGT) isoenzyme capable of morphine glucuronidation has been purified to apparent homogeneity and partially characterized from hepatic microsomes of female Wistar rats which have low 3 alpha-hydroxysteroid UDPGT. A rapid and sensitive assay was developed to quantify morphine glucuronide formation using 14C-UDP-glucuronic acid and reverse phase C-18 minicolumns whereby radioactive glucuronides were differentially eluted from 14C-UDP-glucuronic acid. Trisacryl-DEAE and chromatofocusing chromatographic procedures were employed to separate and purify morphine UDPGT in the presence of exogenous phosphatidylcholine. The addition of phospholipid was necessary to stabilize UDPGT activities throughout the purification procedures. Morphine UDPGT was isolated to apparent homogeneity and displayed a pl of 7.9 upon chromatofocusing. A monomeric molecular weight of 56,000 was obtained. The purified enzyme reacted with morphine but not with 4-hydroxybiphenyl, p-nitrophenol, testosterone, androsterone, estrone, bilirubin, 4-aminobiphenyl, or alpha-naphthylamine. The MgCl2 requirement for maximal expression of morphine glucuronidation was higher for the purified enzyme than for solubilized and intact microsomes. Codeine competitively inhibits morphine glucuronidation with an apparent Ki of 1.1 mM with the purified morphine UDPGT. 4-Hydroxybiphenyl UDPGT was separated from morphine UDPGT using a chromatofocusing procedure for Emulgen 911-solubilized microsomes. An apparent pl value of 5.5 was obtained for this protein. Based on this work we conclude that morphine and 4-hydroxybiphenyl can react with separate UDPGT isoforms.     

Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions

1. 2,6-Dinitrotoluene (2,6-DNT) metabolism by human liver and male Fischer F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubation conditions was examined. Under aerobic conditions the major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); under anaerobic conditions 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite.

2. Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132pmol/min per?mg protein, respectively. Rates of 2Am6NT formation by human and rat liver microsomes under anaerobic conditions were 292 and 285pmol/min per?mg protein, respectively. Anaerobic reduction of 2,6-DNT to 2Am6NT by rat and human liver microsomes was inhibited by carbon monoxide and metyrapone, which indicates that microsomal metabolism of 2,6-DNT to 2Am6NT is mediated by cytochrome P-450.

3. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. Formation of 2Am6NT by human and rat liver cytosols was supported by hypoxanthine, NADPH and NADH. Allopurinol inhibited the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by rat, but not human, liver cytosol. Dicumarol inhibited the NADPH-supported anaerobic metabolism of 2,6-DNT by human, but not rat, liver cytosol. These results indicate that xanthine oxidase contributes to the hypoxanthline-supported anaerobic metabolism of 2,6-DNT by human liver cytosol.     

Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP

The muscarinic receptors of rat submaxillary gland, rat heart and rat cortex were directly labeled using the ligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide [( 3H]4DAMP). In the rat submaxillary gland, [3H]4DAMP predominantly bound with high affinity (Kd = 0.2 nM) to a population of binding sites that displayed the pharmacology of the M3 muscarinic receptor subtype. In rat heart, [3H]4DAMP labeled the M2 muscarinic receptor with low affinity (Kd = 4 nM). In rat cortex [3H]4DAMP predominantly bound to a population of sites with high affinity (Kd = 0.2 nM). The pharmacology of these sites was consistent with [3H]4DAMP labeling both M1 and M3 muscarinic receptors present in rat cortex with high affinity. These data indicate that [3H]4DAMP represents a useful ligand for selectively labeling the M1 and M3 muscarinic receptor subtypes.     

Novobiocin-inhibition and magnesium-interaction of rat liver microsomal bilirubin UDP-glucuronosyltransferase

Novobiocin inhibited bilirubin UDP-glucuronosyltransferase (EC 2.4.1.17) from rat liver both in vitro and in vivo, in a dose-dependent fashion. This inhibition was immediate, and was fully reversed when novobiocin was removed by dialysis or by ultracentrifugation through 0.6 M sucrose. The inhibition could not be explained by an alteration in the membrane conformation of this enzyme, since the same kinetic changes were observed in digitonin-activated and in non-activated microsomes. Novobiocin exerted a non-competitive inhibition of bilirubin UDP-glucuronosyltransferase with either bilirubin or UDP-glucuronic acid as the substrate. Kinetic studies demonstrated uncompetitive inhibition of novobiocin or bilirubin UDP-glucuronosyltransferase as a function of Mg²⁺ concentration, whether the assays were EDTA-free or not. Thus, similarities seem to exist between the known effect of novobiocin on membrane-bound enzymes of the bacterial wall and its inhibitory effect on bilirubin UDP-glucuronosyltransferase: both these enzymic systems require metal divalent cations for maximal activity. The uncompetitive inhibition pattern observed with novobiocin with regard to Mg²⁺ suggests that this antibiotic acts on bilirubin conjugation by affecting Mg²⁺-enzyme complexes.     

Differential modulation of [3H]flunitrazepam binding in female rat brain by sex steroid hormones

Quantitative autoradiographic analysis revealed changes in [3H]flunitrazepam (a benzodiazepine agonist) binding in the anterior hypothalamus nucleus, the medial preoptic area and the cortico-medial amygdala nucleus following in vivo estradiol. The administration of 4 mg of progesterone, but not 1 mg, increased the binding of [3H]flunitrazepam in the basolateral amygdaloid nucleus and in the oriens-pyramidalis CA1 layer of the hippocampus. Exposure of brain sections in vitro to the potent, naturally occurring progesterone metabolite, 3 alpha-hydroxy-5 alpha-dihydroprogesterone, induced GABA-dependent changes in flunitrazepam binding, similar to the changes induced by progesterone, thus suggesting that different steroid mechanisms are implicated in the control of flunitrazepam binding.     

Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions.

1. 2,6-Dinitrotoluene (2,6-DNT) metabolism by human liver and male Fischer F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubation conditions was examined. Under aerobic conditions the major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); under anaerobic conditions 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite. 2. Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132 pmol/min per mg protein, respectively. Rates of 2Am6NT formation by human and rat liver microsomes under anaerobic conditions were 292 and 285 pmol/min per mg protein, respectively. Anaerobic reduction of 2,6-DNT to 2Am6NT by rat and human liver microsomes was inhibited by carbon monoxide and metyrapone, which indicates that microsomal metabolism of 2,6-DNT to 2Am6NT is mediated by cytochrome P-450. 3. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. Formation of 2Am6NT by human and rat liver cytosols was supported by hypoxanthine, NADPH and NADH. Allopurinol inhibited the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by rat, but not human, liver cytosol. Dicumarol inhibited the NADPH-supported anaerobic metabolism of 2,6-DNT by human, but not rat, liver cytosol. These results indicate that xanthine oxidase contributes to the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by human liver cytosol.     

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activities by various inducing agents

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methylcholanthrene β-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT₁ substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT₂ substrates) and bilirubin, similar to phenobarbital. (3) Clofibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT₁ and GT₂ reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT₁ and GT₂ activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450_MCand GT₁, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced gt₁ reactions without affecting benzo[a]pyrene monooxygenase.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam binding in rat brain by the prolonged ventricular infusion of ginsenoside Rc and Rg1.

In the present study, we have investigated the effects of centrally administered ginsenoside Rc and Rg1 on the modulation of the NMDA receptor and GABA(A)receptor binding in rat brain. The NMDA receptor binding was analysed by quantitative autoradiography using [(3)H]MK-801 binding, and the GABA(A)receptor bindings were analysed by using [(3)H]muscimol and [(3)H]flunitrazepam binding in rat brain slices. Rats were infused with ginsenoside Rc or Rg1 (10 microg/10 microl h(-1), i.c.v.) for 7 days, through pre-implanted cannula using osmotic minipumps (Alzet, model 2ML). The levels of [(3)H]MK-801 binding were highly decreased in part of the parietal layers of the cortex and cingulated by ginsenoside Rc and Rg1. The levels of [(3)H]muscimol binding were strongly elevated in almost all regions of the frontal cortex by the treatment of ginsenoside Rc but decreased by ginsenoside Rg1. However, the [(3)H]flunitrazepam binding was not modulated by ginsenoside Rc or Rg1 infusion. These results suggest that prolonged infusion of ginsenosides could differentially modulate [(3)H]MK-801 and [(3)H]muscimol binding in a region-specific manner.     

450088-S,a ring-opened prodrug of a 1,4-benzodiazepine,inhibited [3H]flunitrazepam labeling of rat cerebral cortex in vivo

We report here that a peptidoaminobenzophenone (450088-S), a ring-opened prodrug of a 1,4-benzodiazepine, effectively inhibited the labeling of receptors for 1,4-benzodiazepines by [³H]flunitrazepam in vivo despite the lack of receptor affinity for 450088-S in vitro. Labeling of cerebral cortex after an intravenous injection of [³H]flunitrazepam was inhibited by about 80% after an intraperitoneal injection of 450088-S or diazepam. 450088-S was found to be four times more potent than diazepam. The inhibition occurred within 5 and 10 min of administration of diazepam and 450088-S, respectively. These findings demonstrate the occupancy of 1,4-benzodiazepine receptors in brain upon administration of the prodrug 450088-S in rats.     

Deposition of [3H]cocaine, [3H]nicotine, and [3H]flunitrazepam in mouse hair melanosomes after systemic administration.

Microautoradiography was employed to show that association of drugs from the serum directly with forming hair pigment is a primary pathway of deposition into the hair. After systemic administration of [3H]flunitrazepam, [3H]nicotine, and [3H]cocaine, association of all three drugs with melanin in the forming hair was observed within minutes of dosage. Sebum was determined to be an insignificant deposition route for all three drugs. Pigmented mice had significantly higher concentrations of all three drugs than did nonpigmented mice. The results provide a better basis for ultimately using hair for reliable analysis of drug and environmental toxin exposure.     

Postnatal handling reduces novelty-induced fear and increases [3H]flunitrazepam binding in rat brain

Postnatal handling of rat pups alters their response to novelty. We have now shown that, as adults, rats handled for the first three weeks of life showed a suppression of feeding in a novel environment that was less, and a [3H]flunitrazepam binding in whole brain that was greater than in non-handled animals. These data suggest that variations in benzodiazepam binding capacity could be related to individual differences in the fear response to novelty.     

Anion-dependent modulation of [3H]muscimol binding and of GABA-stimulated [3H]flunitrazepam binding by picrotoxin and related CNS convulsants

Picrotoxin, isopropylbicyclophosphate (IPTBO) and related CNS-convulsants have allosteric effects on the binding of ligands to the GABA/benzodiazepine receptor complex. When binding experiments were performed at 23 degrees C and at 35 degrees C, these drugs inhibited [3H]muscimol binding and muscimol- or GABA-stimulated [3H]flunitrazepam binding, respectively. Both effects required the presence of C1-, Br-, I- but not of F- or SO4(2-). Picrotoxin and IPTBO could only partially inhibit [3H]muscimol binding. In contrast other GABA antagonists and convulsants like bicuculline, 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135), strychnine and d-tubocurarine interferred completely with [3H]muscimol binding, also in the absence of those ions mentioned above which were essential for the effects of picrotoxin. Our results support the notion that drugs like picrotoxin and IPTBO which interfere with the GABA receptor effector system, may lead to an allosteric perturbation of GABA-recognition sites.     

Kinetics of [3H]flunitrazepam binding to membrane-bound benzodiazepine receptors

The kinetics of [3H]flunitrazepam binding to synaptosomal (P2) membranes from rat cerebral cortex was studied. The pseudo-first order association at 0 degrees was curvilinear and the association rate was increased with increasing concentrations of ligand. The association curve was resolved best as two linear exponentials, representing a fast and a slow component. The percentage of each association component varied with the ligand concentration. Association was slightly decreased in the presence of gamma-aminobutyric acid. Dissociation at 0 degrees, whether equilibrium binding had occurred at 0 degrees or 37 degrees, was also biphasic. The dissociation of the complexes was more rapid at higher concentrations of ligand. Dissociation was slower in the presence of gamma-aminobutyric acid, and this effect was more pronounced at higher concentrations of ligand. Prolonged preincubation at 37 degrees either in the presence or in the absence of [3H]flunitrazepam also decreased the dissociation rate. Dissociation was faster before equilibrium than after equilibrium had been reached. These results are discussed in terms of one homogeneous type of benzodiazepine receptor with two interconvertible conformations.     

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.