Disulfiram and diethyldithiocarbamate are competitive inhibitors at the peripheral benzodiazepine receptor.

In the present study in vitro interactions of disulfiram (an agent used to induce ethanol intolerance in alcoholics), diethyldithiocarbamate (DDC), and metronidazole with central benzodiazepine receptors (CBR) and peripheral benzodiazepine (BZ) receptors (PBR) were investigated in rat tissues. Disulfiram displaced specific binding of [3H]PK 11195 from PBR in the cerebral cortex with an IC50 value of 5 x 10(-7) M. The binding of [3H]PK 11195 and [3H]Ro 5-4864 to PBR in the kidney was displaced by disulfiram with IC50 values of 7 x 10(-7) and 2 x 10(-7) M, respectively. DDC displaced [3H]PK 11195 binding to kidney membranes with an IC50 value of 5 x 10(-5) M. Binding of [3H] flunitrazepam to CBR in the cerebral cortex was not affected by either disulfiram or DDC. Metronidazole (up to 10(-4) M), a disulfiram congener, did not affect [3H]flunitrazepam (FNZ) and [3H] PK 11195 binding to CBR and PBR, respectively. Scatchard analysis of [3H]PK 11195 binding to kidney membranes, performed in the absence or presence of 7 x 10(-7) M disulfiram, decreased ligand affinity without influencing the maximal number of binding sites, suggesting a competitive inhibition. Beta-Mercaptoethanol (2 x 10(-2) M), which blocks the inhibitory activity of disulfiram and DDC at the acetaldehyde dehydrogenase, did not affect the inhibitory potency of disulfiram at the kidney PBR. Removal of disulfiram from kidney by repeated washing with Tris-HCl buffer resulted in the restoration of binding properties to control values, suggesting reversibility of disulfiram binding to PBR.     

Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes

The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.     

Involvement of peripheral-type benzodiazepine receptors in the proconvulsant actions of pyrethroid insecticides

It has been demonstrated previously that select Type II pyrethroids are potent proconvulsants in the rat and that the proconvulsant actions of deltamethrin are blocked by administration of PK 11195, an antagonist of the peripheral-type benzodiazepine receptor (PTBR). The present investigation has extended these findings to include various Type I pyrethroids as proconvulsants. Additionally, the proconvulsant activity of cismethrin was reversed by administration of PK 11195. Pyrethroid displacement of specific [3H]Ro5-4864 binding to rat brain membranes was investigated to further define the interaction of pyrethroids with the PTBR. Both Type I and Type II pyrethroids potently inhibited [3H]Ro5-4864 binding with affinities ranging from nanomolar to micromolar. The ED50 values for the proconvulsant effects of both Type I and Type II pyrethroids were significantly correlated with their respective IC50 values as inhibitors of [3H]Ro5-4864 binding. [3H]Ro5-4864 saturation isotherms performed in the presence of fixed concentrations of deltamethrin or cismethrin showed that these pyrethroids increased the observed Kd values for [3H]Ro5-4864 with no change in the maximum number of binding sites. However, Schild plot analysis of the effect of deltamethrin on [3H]Ro5-4864 affinity was nonlinear with the Kd shift approaching a limiting value. Considered together these results suggest an allosteric effect of pyrethroids on [3H]Ro5-4864 binding, and provide additional support for the involvement of the PTBR in the proconvulsant actions of pyrethroids.     

Effects of peripheral benzodiazepine receptor ligands on hypothalamic-pituitary-adrenal axis function in the rat

High concentrations of the "peripheral" benzodiazepine (pBZD) binding site ("receptor") have been described in the hypothalamus, the pituitary and the adrenal glands. This study was undertaken to examine the effects of ligands of this binding site on the hypothalamic-pituitary-adrenal axis (HPA). To accomplish this we administered graded doses of the pBZD receptor agonist 4-chloro-diazepam (Ro5-4864) i.v. to catheterized, freely moving adult male Sprague-Dawley rats. Serial blood samples for plasma adrenocorticotropin hormone (ACTH) and corticosterone determinations were drawn from the catheter before and after the injection of the drug. Ro5-4864 significantly stimulated ACTH and corticosterone secretion in a dose-dependent fashion. To examine whether this effect could be antagonized by the pBZD binding site antagonist PK 11195, we treated rats with PK 11195 at doses 10- and 50-times higher than Ro5-4864 before administration of a maximally effective dose of Ro5-4864. Neither dose of PK 11195 antagonized Ro5-4864-induced plasma ACTH or corticosterone elevations. However, this agent, given alone, stimulated ACTH and corticosterone release. Similarly, carbamazepine (CBZ), which binds to the pBZD binding site with low affinity, stimulated weakly the HPA in vivo, reaching statistical significance only at the highest dose tested. To examine the site(s) of action of these compounds on the HPA, we evaluated their effects on hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion in vitro. Ro5-4864 stimulated hypothalamic CRH, but not pituitary ACTH secretion. Neither PK 11195 nor CBZ had any agonist effect on hypothalamic CRH secretion in vitro, whereas both antagonized Ro5-4864-induced CRH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a peptide antagonist to the peripheral-type benzodiazepine receptor that inhibits hormone-stimulated leydig cell steroid formation

Peripheral-type benzodiazepine receptor (PBR) is an 18-kDa high-affinity cholesterol and drug ligand-binding protein involved in various cell functions, including cholesterol transport and steroid biosynthesis. To aid our investigation of the biological function of PBR, we have set out to identify functional antagonists. By screening phage display libraries, we have identified peptides that displace the high-affinity PBR benzodiazepine drug ligand, Ro5-4864 (4'-chlorodiazepam). Among these peptides, STPHSTP was the most potent (IC(50) = 10 microM). All of the isolated peptides showed a conserved motif STXXXXP. The role of these peptides in Leydig cell steroidogenesis was examined using a transducible peptide composed of the TAT domain of human immunodeficiency virus and the peptides under investigation. Synthesized peptides efficiently transduced into MA-10 Leydig cells, and the peptide TAT-STPHSTP inhibited Ro5-4864- and human chorionic gonadotropin-stimulated steroid production in a dose-dependent manner (ED(50) = 5 microM). TAT-STPHSTP behaved as a competitive PBR antagonist, which did not affect 22R-hydroxycholesterol-supported steroidogenesis. These results yield leads for the development of potent PBR antagonists and indicate that endogenous PBR agonist-receptor interaction is critical for hormone-induced steroidogenesis.     

QUANTITATIVE AUTORADIOGRAPHIC DETERMINATION OF BINDING SITES FOR A PERIPHERAL BENZODIAZEPINE LIGAND ([3H]PK 11195) IN HUMAN IRIS

Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 micron) labelled with [3H]PK 11195 (1 nM) at 25 degrees C. Nonspecific binding was determined with PK 11211 (5 microM) or Ro 5-4864 (5 microM). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris, and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively, were: 42.7 +/- 0.2, 30.1 +/- 0.5, and 37.4 +/- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.     

Local anesthetics differentiate dihydropyridine calcium antagonist binding sites in rat brain and cardiac membranes

Local anesthetics were used to probe differences in the binding of [3H]nitrendipine to dihydropyridine calcium antagonist binding sites on rat brain and cardiac membranes. Local anesthetics inhibited [3H]nitrendipine binding to brain and cardiac membranes with the rank order of potency, dibucaine = proadifen much greater than tetracaine greater than meproadifen greater than RAC-109 (S) greater than RAC-109 (R) greater than benzocaine. Lidocaine, procaine, piperocaine and bupivacaine produced either a small potentiation or inhibition of [3H]nitrendipine binding. Dibucaine inhibited [3H]nitrendipine binding to brain membranes (IC50, 4.9 +/- 0.5 microM) by increasing the Kd, whereas in cardiac membranes (IC50, 8.5 +/- 0.9 microM) it both increased the Kd and decreased the maximum binding site capacity of [3H]nitrendipine. The potency of dibucaine to inhibit [3H]nitrendipine binding was reduced in both tissues by monovalent (Li+ greater than Na+ = K+ = Rb+; EC50, 40-50 mM) and divalent (Ca++, Mg++ and Mn++; EC50, 10-50 microM) cations. These cations reduced the effect of dibucaine on the Kd of [3H]nitrendipine in brain and on the maximum binding site capacity of [3H]nitrendipine in cardiac membranes. Inhibition of [3H]nitrendipine binding by dibucaine was best described by high (2 microM) and low (50 microM) affinity sites. The apparent affinities of these sites, but not the fractional occupancies, were similar in brain and cardiac membranes. Na+ modulated the occupancies of these sites in brain, but not in cardiac membranes, whereas Ca++ inhibited occupancy of the high affinity site in both tissues. The effects of Li+ were similar to those of Ca++. These findings indicate that brain and cardiac dihydropyridine calcium antagonist binding sites are coupled to different allosteric effectors or exist in a different membrane environment.     

Solubilization and characterization of the liver peripheral-type benzodiazepine receptor

The rat liver membrane-bound and digitonin-solubilized peripheral-type benzodiazepine receptors (mPBZR and dsPBZR, respectively) were characterized. Forty percent of the receptors were solubilized from a liver homogenate with 0.25% digitonin. Scatchard analysis of saturation data for the mPBZR and the dsPBZR showed Kd = 1.5 nM and maximum number of binding sites = 3.12 pmol/mg of protein and Kd = 9.2 nM and maximum number of binding sites = 1.10 pmol/mg of protein, respectively. Estimates of Kd calculated from kinetic data agree with estimates from Scatchard analysis. The affinity of the PBZR for [3H]Ro5-4864 was not affected by guanosine 5'0-(3-thiotriphosphate) which suggests the receptor is not coupled to a G-protein. Competition for specific [3H]Ro5-4864 binding by various ligands demonstrated the same rank order potency of binding inhibition for the membrane bound and solubilized receptors (PK-11195 greater than or equal to Ro5-4864 greater than diazepam greater than clonazepam). Thus, the soluble receptor had ligand binding characteristics similar to those of the membrane PBZR. [3H]PK-14105 was used to photoaffinity label the PBZR in a rat liver homogenate. Labeling was specific for the PBZR and the molecular weight of the digitonin-solubilized photoaffinity-labeled receptor was estimated to be 170 kDa by gel filtration chromatography. Estimation of the molecular weight of the [3H]PK-14105 labeled receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein corresponding to 19 kDa.     

Microglia imaging

Imaging of microglia cells using the radiolabeled ligand toward peripheral benzodiazepine receptor(PBR) is applied to brain injury and neurodegenerative diseases. A novel PBR radioligand other than [11C] PK11195 might be desirable to detailedly delineate this complicated phenomenon and a novel idea other than in vitro PBR binding ability is desirable to expect feasibility of a new candidate. Current status, problems and future prospects associated with indirect imaging of microglial activity with [11C] PK11195 will be discussed.     

SPM analysis of parametric (R)-[11C]PK11195 binding images: plasma input versus reference tissue parametric methods

(R)-[11C]PK11195 has been used for quantifying cerebral microglial activation in vivo. In previous studies, both plasma input and reference tissue methods have been used, usually in combination with a region of interest (ROI) approach. Definition of ROIs, however, can be labourious and prone to interobserver variation. In addition, results are only obtained for predefined areas and (unexpected) signals in undefined areas may be missed. On the other hand, standard pharmacokinetic models are too sensitive to noise to calculate (R)-[11C]PK11195 binding on a voxel-by-voxel basis. Linearised versions of both plasma input and reference tissue models have been described, and these are more suitable for parametric imaging. The purpose of this study was to compare the performance of these plasma input and reference tissue parametric methods on the outcome of statistical parametric mapping (SPM) analysis of (R)-[11C]PK11195 binding. Dynamic (R)-[11C]PK11195 PET scans with arterial blood sampling were performed in 7 younger and 11 elderly healthy subjects. Parametric images of volume of distribution (Vd) and binding potential (BP) were generated using linearised versions of plasma input (Logan) and reference tissue (Reference Parametric Mapping) models. Images were compared at the group level using SPM with a two-sample t-test per voxel, both with and without proportional scaling. Parametric BP images without scaling provided the most sensitive framework for determining differences in (R)-[11C]PK11195 binding between younger and elderly subjects. Vd images could only demonstrate differences in (R)-[11C]PK11195 binding when analysed with proportional scaling due to intersubject variation in K1/k2 (blood-brain barrier transport and non-specific binding).     

Electrophysiological actions of Ro5-4864 on cerebellar Purkinje neurons: evidence for "peripheral" benzodiazepine receptor-mediated depression

The effects of Ro 5-4864 (4'-chlorodiazepam; the archetypic peripheral benzodiazepine receptor ligand) were examined on the electrophysiological responses of rat cerebellar Purkinje neurons maintained in vitro. Ro 5-4864 produced a biphasic response (consisting of an increase followed by a decrease in spontaneous firing) in approximately 50% of the neurons studied. The remaining neurons responded to Ro 5-4864 application with decreased spontaneous activity. The EC50 and IC50 values for the excitatory and depressant responses to Ro 5-4864 were 490 and 450 nM, respectively. Preincubation with PK 11195 [N-methyl-N-(methyl-1-propyl)chloro-2-phenyl-1-isoquinoline-3-carboxamid e; a peripheral benzodiazepine receptor antagonist] reduced the potency of Ro 5-4864 to inhibit cell firing in a competitive fashion, but did not alter Ro 5-4864-elicited excitations. A similar effect was observed with the monocarboxylic acid anion transport inhibitor UK 5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate]. In contrast, the increases in cell firing elicited by t-butylbicyclophosphorothionate (a GABA-gated chloride channel blocker) were enhanced 6-fold by coperfusion with both Ro 5-4864 and PK 11195. These findings suggest that the Ro 5-4864 induced depressions of Purkinje neuron spontaneous activity is mediated by peripheral benzodiazepine receptors, whereas the excitation may result from modulation of the gamma-aminobutyric acid-gated chloride ionophore.     

3H]PK11195 binding sites in human neutrophils: effect of fMLP stimulation and modulation in rheumatic diseases

OBJECTIVE: The objectives of this study were to evaluate the [3H]PK11195 binding parameters in a model of acute inflammation, the N-formylmethionine-leucine-phenylalanine (fMLP)-stimulated neutrophil cell membranes, and to analyze if alterations of peripheral-type benzodiazepine receptor (PBR) characteristics occurred in neutrophil cell membranes of patients affected by osteoarthritis (OA), rheumatoid arthritis (RA), and psoriasic arthritis (PA). DESIGN AND METHODS: Neutrophils were obtained from 15 patients with OA, 15 patients with RA, and 15 patients with PA. fMLP stimulation was performed to aliquots of neutrophils from six healthy individuals. Evaluation of kinetic parameters of PBR was performed using [3H]PK11195, as specific radioligand compared with 15 healthy volunteers. RESULTS: The results showed a significant decrease of Kd and Bmax in fMLP-stimulated neutrophil membranes. Moreover, an increase of PBR binding sites and affinity value was observed in neutrophils membranes from PA patients. CONCLUSIONS: Our data suggested a fMLP modulation on [3H]PK11195 binding in human neutrophils. Moreover, our results showed an up-regulation of PBR in neutrophils of PA patients.     

Peripheral type benzodiazepine receptors in human and guinea pig lung: characterization and autoradiographic mapping

Binding of [3H]Ro5-4864, a ligand selective for peripheral-type benzodiazepine receptors, to human and guinea pig lung membranes was characterized and the binding sites localized in lung sections by light microscopic autoradiography. Inhibition of [3H]Ro5-4864 binding by unlabeled Ro5-4864, PK11195, midazolam, diazepam, clonazepam and flumazenil indicated that [3H]Ro5-4864 bound to specific receptors and binding was reversible. Scatchard analysis indicated a single class of binding site, with a dissociation constant (Kd) of 12.8 +/- 2.1 nM with maximum binding capacity of 2.6 +/- 0.6 pmol/mg of protein for human lung and Kd of 8.4 +/- 1.5 nM and maximum binding capacity of 3.5 +/- 0.3 pmol/mg of protein for guinea pig lung. Autoradiograms showed specific labeling over discrete structures in both human and guinea pig lung. Labeling was particularly dense over submucosal glands in intrapulmonary bronchi of human. Airways were also labeled, with epithelium having a higher grain density than smooth muscle. Labeling over smooth muscle was denser in small compared to large airways. Vascular smooth muscle was not labeled in either human and guinea pig. The alveolar walls were uniformly labeled in both species. The functional significance of pulmonary peripheral-type benzodiazepine receptors remains to be determined.     

Stimulation of aldosterone secretion by benzodiazepines in bovine adrenocortical cells

Previous studies have indicated that peripheral benzodiazepine receptor (PBR) ligands inhibit aldosterone secretion in isolated adrenal zona glomerulosa cells although positive responses have been demonstrated in other steroidogenic tissues. In the present study, aldosterone secretion was measured in bovine cells after 6 days of primary culture. At this time, basal aldosterone secretion was very low and cells appeared less sensitive to the steroidogenic effects of extracellular [K+] (maximal response required K+ concentration > 32 mmol/L) but were sensitised to angiotensin II (maximal response achieved with 3 nM) when compared with previous studies with freshly isolated cells. Diazepam concentration in the range 0.1 nM to 1 microM increased basal aldosterone secretion, an effect which was not enhanced by pre-treatment with diazepam. The effects were small compared with those of angiotensin II or K+. Over the same concentration range, diazepam also potentiated the stimulatory effects of sub-maximally effective concentrations of angiotensin II. When cells were treated with high-density lipoprotein (HDL-3) as a source of cholesterol, diazepam and the PBR ligands Ro5-4864 and PK11195 also stimulated aldosterone secretion at nanomolar concentrations. In addition, the conversion of added 11-deoxycorticosterone (DOC) to aldosterone was increased by nanomolar concentrations of diazepam and Ro5-4864 but inhibited by high micromolar concentrations of these drugs (100 microM). We conclude that adrenocortical responses to PBR ligands are complex. At high concentrations, inhibitory effects involving competition for steroidogenic enzymes and calcium channel blockage predominate. At low concentrations, an enhancement of basal, angiotensin-II and cholesterol-dependent aldosterone synthesis is revealed which may involve a PBR-mediated mitochondrial uptake of cholesterol and DOC.     

Cardiac cellular actions of hydrochlorothiazide

In long term treatment, thiazide diuretics such as hydrochlorothiazide (HCTZ) lower blood pressure by decreasing peripheral resistance rather than by their diuretic effect. This action has been attributed to the opening of Ca2+-activated K+ channels in vascular smooth muscle cells. However, little is known about their cardiac cellular actions. Here we investigated the possible actions of HCTZ on action potential and contraction of rat ventricular muscle strips and on the ionic currents of isolated rat ventricular cardiomyocytes. HCTZ depressed ventricular contraction with an IC30 of 1.85 microM (60% decrease at 100 microM). Action potential duration at -60 mV and maximal rate of depolarization were, however, only slightly decreased by 12% and 22%, respectively, at 100 microM. At the single cell level, HCTZ (100 microM) depressed the fast Na+ current (INa) and the L-type Ca2+ current (ICaL) by 30% and 20%, respectively. The effects on ICaL were not voltage-or frequency-dependent. In cells intracellularly perfused with 50 microM cyclic adenosine, monophosphate HCTZ reduced ICaL by 33%. The transient (Ito), the delayed rectifier and the inward rectifier potassium currents were decreased by 20% at 100 microM HCTZ. The effects on Ito were voltage-dependent. In conclusion, HCTZ at high concentrations possesses a negative inotropic action that could be in part due to its blocking action on INa and ICaL. The actions of HCTZ on multiple cardiac ionic currents could explain its weak effect on action potential duration.     

Inhibitory action of peripheral-type benzodiazepines on dopamine release from PC12 pheochromocytoma cells.

Characteristics of the benzodiazepine inhibition of dopamine (DA) release in PC12 cells were investigated. Diazepam inhibited DA release evoked by high concentrations of extracellular K+ in a dose-dependent manner (IC50, 10 microM). Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine- 2-one], a peripheral-type benzodiazepine, also inhibited DA release effectively. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide], a benzodiazepine generally considered a peripheral-type benzodiazepine receptor antagonist, did not antagonize the inhibition induced by diazepam, but rather inhibited DA release itself. On the other hand, the central-type benzodiazepines, clonazepam and Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) did not affect the DA release. Diazepam, Ro 5-4864 and PK 11195 also inhibited a Ba(++)-current carried by voltage-gated Ca++ channels, and diazepam suppressed an increase in intracellular Ca++ evoked by 80 mM extracellular K+ as measured by the fura-2 method. These results suggest that the inhibitory action of diazepam and other benzodiazepines on DA release from PC12 cells may be mediated through one type of peripheral-type benzodiazepine receptors which are coupled to voltage-gated Ca++ channels and that these receptors may not necessarily be the same as those in other tissues.     

Cardiac arrhythmias in hypertensive outpatients on various diuretics. Correlation between incidence and serum potassium and magnesium levels

Twenty-four hypertensive out-patients (WHO I) underwent an ambulatory 24-hour electrocardiogram-monitoring both before and after 8 weeks treatment with either hydrochlorothiazide (20-50 mg/day, N = 6), indacrinone (50-100 mg/day, N = 6) or indacrinone plus amiloride (50-100 mg and 5-10 mg/day, N = 12) in a double-blind fashion in 3 parallel groups. A 4-weeks placebo run-in period preceded the active treatment. Serum potassium fell significantly in the hydrochlorothiazide group (p less than 0.001) and in the indacrinone group (p less than 0.005). Serum magnesium values remained unchanged. In 4 out of the 24 patients ventricular ectopic activity increased but there was no correlation with changes in serum potassium or magnesium values in these patients. Treatment with different diuretics had a similar influence on the occurrence of ventricular extrasystoles.     

Comparison of [11C]-(R)-PK 11195 and [11C]PBR28, two radioligands for translocator protein (18 kDa) in human and monkey: Implications for positron emission tomographic imaging of this inflammation biomarker

Ten percent of humans lack specific binding of [¹¹C]PBR28 to 18 kDa translocator protein (TSPO), a biomarker for inflammation. “Non-binders” have not been reported using another TSPO radioligand, [¹¹C]-(R)-PK 11195, despite its use for more than two decades. This study asked two questions: (1) What is the cause of non-binding to PBR28? and (2) Why has this phenomenon not been reported using [¹¹C]-(R)-PK 11195? Methods: Five binders and five non-binders received whole-body imaging with both [¹¹C]-(R)-PK 11195 and [¹¹C]PBR28. In vitro binding was performed using leukocyte membranes from binders and non-binders and the tritiated versions of the ligand. Rhesus monkeys were imaged with [¹¹C]-(R)-PK 11195 at baseline and after blockade of TSPOs. Results: Using [¹¹C]PBR28, uptake in all five organs with high densities of TSPO (lung, heart, brain, kidney, and spleen) was 50% to 75% lower in non-binders than in binders. In contrast, [¹¹C]-(R)-PK 11195 distinguished binders and non-binders in only heart and lung. For the in vitro assay, [³H]PBR28 had more than 10-fold lower affinity to TSPO in non-binders than in binders. The in vivo specific binding of [¹¹C]-(R)-PK 11195 in monkey brain was ～80-fold lower than that reported for [¹¹C]PBR28. Conclusions: Based on binding of [³H]PK 11195 to leukocyte membranes, both binders and non-binders express TSPO. Non-binding to PBR28 is caused by its low affinity for TSPO in non-binders. Non-binding may be differentially expressed in organs of the body. The relatively low in vivo specific binding of [¹¹C]-(R)-PK 11195 may have obscured its detection of non-binding in peripheral organs.     

Regional distribution of a Ro5 4864 binding site that is functionally coupled to the gamma-aminobutyric acid/benzodiazepine receptor complex in rat brain

The hypothesis that a novel drug binding site linked to a gamma-aminobutyric acid (GABA)-regulated chloride ionophore mediates the excitatory effects of the atypical benzodiazepine (BZ) Ro5 4864 is further evaluated in the present study. Dose-dependent inhibition of [3H]flunitrazepam to the central BZ receptor in rat cerebral cortex by the cage convulsant t-butylbicyclophosphorotionate (TBPS) is modulated by Ro5 4864 and the isoquinoline PK 11195 in a manner consistent with their reported pro/anticonvulsant effects. The ability of Ro5 4864 to enhance the binding of [35S]TBPS to a GABA-regulated chloride ionophore in rat cortex is unchanged after the irreversible labeling of the central BZ receptor by the photoaffinity label Ro15 4513. Together, these observations further suggest that 1) the effect of Ro5 4864 on [35S]TBPS is not mediated by the central BZ receptor and 2) the Ro5 4864 binding site is allosterically coupled to the GABA/BZ receptor-chloride ionophore complex in rat cerebral cortex. Anatomical localization of Ro5 4864-stimulated [35S]TBPS binding in rat brain by autoradiography reveals a distribution of chloride ionophore-coupled Ro5 4864 sites which is in many instances similar to that of the GABA/BZ receptor-chloride ionophore complex. These studies lend additional support to the postulate that this drug binding site represents an additional locus for the regulation of GABAergic neurotransmission in the central nervous system.