Characterization of peripheral benzodiazepine receptors in purified large mammal pancreatic islets

Istituto Policattedra di Discipline Biologiche, Università di Pisa, Via Bonanno 6, 56126, Pisa, Italy
Biochemical Pharmacology (Impact Factor: 5.01). 06/1996; 51(11):1437-1442. DOI: 10.1016/0006-2952(96)00082-2


In this work, we evaluated the biochemical properties of peripheral benzodiazepine receptors (PBRs) in the porcine endocrine pancreas and their role in insulin release. Binding of [3H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3-isoquinolinecarboxamide ([3H]PK-11195), a specific ligand of PBRs, to islet membranes was saturable and Scatchard's analysis of saturation curve demonstrated the presence of a single population of binding sites, with a dissociation constant (Kd) value of 4.75 ± 0.70 nM and a maximum amount of specifically bound ligand (Bmax) of 4505 ± 502 fmol/mg of proteins. The pharmacological profile of PBRs was determined as the ability of PK-11195 and several benzodiazepine compounds to displace [3H]PK-11195 from these binding sites. The rank order of potency yielded the following affinity results: PK-11195 > 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2-on (Ro 5-4864) > diazepam ⩾ flunitrazepam ⪢ flumazenil. Secretion studies demonstrated that PK-11195 (1 and 10 μM) and Ro 5-4864 (10 and 50 μM) significantly potentiated insulin secretion from freshly isolated porcine islets at 3.3 mM glucose. This potentiating effect was not observed at 16.7 mM glucose concentration nor by the addition of clonazepam. These results show the presence of PBRs in purified porcine pancreatic islets and suggest an implication of PBRs in the mechanisms of insulin release.

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    • "In patients treated with the imidazopyridine compound alpidem, which binds benzodiazepine receptors [3], we reported an alteration of glucose tolerance after one week administration [4]. Studies in vitro have shown that some benzodiazepines could affect insulin secretion differently according to the experimental model [5,6]. "
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    ABSTRACT: The present study aimed at investigating in healthy volunteers the effects of diazepam and clonazepam on beta-cell function, insulin sensitivity and glucose effectiveness based on the frequently sampled intravenous (0.5 gkg-1) glucose tolerance test with minimal-model analysis. The study was designed as a double-blind, placebo-controlled, cross-over clinical trial. Diazepam (10 mg) and clonazepam (1 mg) were infused during 30 min to 15 male subjects with a mean age of 22 years (range: 20–29), after informed consent was given. Benzodiazepines were assayed by capillary gas chromatography with electron capture, insulin by radioimmunoassay and glucose by the enzymatic glucose oxidase method. Both benzodiazepines induced significant psychotropic effects. The acute insulin responses (AIR) were significantly and negatively correlated with the clonazepam plasma concentrations (r = -0.609, P < 0.05, n = 14). However, the mean AIR was not significantly different between the benzodiazepine-treated subjects and the controls. In addition, the parameters of glucose assimilation were significantly decreased as compared with placebo in the subgroup of 7 subjects with plasma clonazepam concentrations higher than 6.0 ng ml-1 (median and lower limit of effective therapeutic concentrations): 1.37 ± 0.3 versus 2.84 ± 0.60 × 10-2min-1 (P = 0.028) for the coefficient of glucose tolerance (Kg), 2.18 ± 0.29 versus 3.71 ± 0.89 × 10-4μUml-1min-1 (P = 0.018) for insulin sensitivity (Si) and 1.80 ± 0.39 versus 3.59 ± 0.71 × 10-2min-1 (P = 0.028) for glucose effectiveness at basal insulin (Sg). These parameters were not significantly modified when diazepam was administered; plasma levels of this drug however, were below the effective therapeutic concentrations (300 ng ml-1) from min 15 after the end of the perfusion. The present results suggest that a benzodiazepine, in particular clonazepam, may alter insulin secretion and insulin sensitivity after a single administration in healthy volunteers.
    Full-text · Article · Apr 2004 · BMC Clinical Pharmacology
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    • "to PBR such as regulation of steroid production (Papadopoulos, 1993), inflammatory response (Torres et al, 1999), insulin secretion (Marchetti et al, 1996b), mitochondrial respiration (Hirsch et al, 1989; Krueger, 1995), cell differentiation (Canat et al, 1993) and cell proliferation. Proliferation of various tumours including breast cancer (Beinlich et al, 1999; Carmel et al, 1999), melanoma (Landau et al, 1998), testis (Garnier et al, 1993) and astrocytoma (Neary et al, 1995) was shown to be inhibited by PBR ligands at micromolar concentrations. "
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    ABSTRACT: The peripheral benzodiazepine receptor (PBR) has been implicated in growth control of various tumour models. Although colorectal cancers were found to overexpress PBR, the functional role of PBR in colorectal cancer growth has not been addressed to date. Using primary cell cultures of human colorectal cancers and the human colorectal carcinoma cell lines HT29, LS174T, and Colo320 DM we studied the involvement of PBR in the growth control and apoptosis of colorectal cancers. Both mRNA and protein expression of PBR were detected by RT-PCR and flow cytometry. Using confocal laser scanning microscopy and immunohistochemistry the PBR was localized in the mitochondria. The specific PBR ligands FGIN-1-27, PK 11195, or Ro5-4864 inhibited cell proliferation dose-dependently. FGIN-1-27 decreased the mitochondrial membrane potential, which indicates an early event in apoptosis. Furthermore, FGIN-1-27, PK 11195 or Ro5-4864 increased caspase-3 activity. In addition to their apoptosis-inducing effects, PBR ligands induced cell cycle arrest in the G(1)/G(0)-phase. Thus, our data demonstrate a functional involvement of PBR in colorectal cancer growth and qualify the PBR as a possible target for innovative therapeutic approaches in colorectal cancer.
    Full-text · Article · Dec 2001 · British Journal of Cancer
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    ABSTRACT: Benzodiazepines have been shown to inhibit salivary secretion from the rat salivary gland. This action is mediated by specific benzodiazepine binding sites in the glands. The presence and characteristics of central- and peripheral-type benzodiazepine receptors in rat parotid and submandibular glands were examined employing [3H]Ro15-1788 and [3H]PK11195 as radioligands. [3H]Ro15-1788 and [3H]PK11195 bound with high affinity for both salivary glands ([3H]Ro15-1788: 24.5 and 37.4 mM, [3H]PK11195: 1.37 and 1.88 nM, for parotid and submandibular glands, respectively). [3H]Ro15-1788 binding sites occupied only 0.22 to 0.43% of the total binding for benzodiazepine receptors in the glands. The rank order of the competing potency of [3H]Ro15-1788 binding (Ro15-1788 = clonazepam > diazepam > flunitrazepam > PK11195 > Ro5-4864) and [3H]PK11195 binding (Ro5-4864 = PK11195 > diazepam = flunitrazepam > clonazepam) demonstrated that [3H]Ro15-1788 and [3H]PK11195 binding sites were characteristic of the central and peripheral type, respectively. These studies show that both central- and peripheral-type benzodiazepine receptors exist in rat parotid and submandibular glands.
    No preview · Article · Jan 1998 · Biochemical Pharmacology
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