Characterization of peripheral benzodiazepine receptors in purified large mammal pancreatic islets
ABSTRACT 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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- "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. "
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.British Journal of Cancer 12/2001; 85(11):1771-80. DOI:10.1054/bjoc.2001.2181 · 4.82 Impact Factor
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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.Biochemical Pharmacology 01/1998; 55(2):209-14. DOI:10.1016/S0006-2952(97)00433-4 · 4.65 Impact Factor
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ABSTRACT: Pancreatic islet desensitization by high glucose concentrations is a temporary and reversible state of beta-cell refractoriness to glucose (and possibly other secretagogues), due to repeated or prolonged pre-exposure to increased glucose concentrations. We evaluated whether the oral antidiabetic agent metformin affects this phenomenon in isolated, human pancreatic islets, and whether the possible effects of the biguanide are influenced by the presence of a sulphonylurea, glyburide. Islets prepared from five human pancreases were incubated for 24 h in M199 culture medium containing either 5.5 or 22.2 mmol/l glucose, with or without a therapeutic concentration (2.4 microg/ml) of metformin. Then, the islets were challenged with either 3.3 mmol/l glucose, 16.7 mmol/l glucose, or 3.3 mmol/l glucose + 10 mmol/l arginine, and insulin release was measured. After incubation in the absence of metformin, the human islets exposed to 22.2 mmol/l glucose showed no significant increase in insulin release when challenged with 16.7 mmol/l glucose (confirming that hyperglycemia desensitizes pancreatic beta-cells). In the presence of metformin, the islets fully maintained the ability to significantly increase their insulin release in response to glucose, even when previously exposed to 22.2 mmol/l glucose. No major effect on arginine-induced insulin release was observed, whatever the culture conditions. The protective action of metformin was observed also when glyburide was present in the incubation medium, whereas the sulphonylurea alone did not affect insulin release from the islets previously exposed to high glucose concentrations. These in vitro results suggest that metformin can prevent the desensitization of human pancreatic islets induced by prolonged exposure to increased glucose concentrations.European Journal of Pharmacology 02/1999; 364(2-3):205-9. DOI:10.1016/S0014-2999(98)00807-3 · 2.68 Impact Factor