benzofurans and Insulinoma

Pancreatic β cells are very sensitive to oxidative stress and this might play an important role in β cell death with diabetes. The protective effect of dieckol, one of the phlorotannin polyphenol compounds purified from Ecklonia cava (E. cava), against high glucose-induced oxidative stress was investigated by using rat insulinoma cells. A high-glucose (30 mM) treatment induced the death of rat insulinoma cells, but dieckol, at a concentration 17.5 or 70 µM, significantly inhibited the high-glucose induced glucotoxicity. Treatment with dieckol also dose-dependently reduced thiobarbituric acid reactive substances (TBARS), the generation of intracellular reactive oxygen species (ROS), and the nitric oxide level increased by a high glucose concentration. In addition, the dieckol treatment increased the activities of antioxidative enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) in high glucose-pretreated rat insulinoma cells. Dieckol protected rat insulinoma cells damage under high glucose conditions. These effects were mediated by suppressing apoptosis and were associated with increased anti-apoptotic Bcl-2 expression, and reduced pro-apoptotic cleaved caspase-3 expression. These findings indicate that dieckol might be useful as a potential pharmaceutical agent to protect against the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.

Topics: Animals; Antioxidants; Apoptosis; Benzofurans; Caspase 3; Catalase; Dose-Response Relationship, Drug; Gene Expression; Glucose; Glutathione Peroxidase; Insulinoma; Nitric Oxide; Oxidative Stress; Phaeophyceae; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Tumor Cells, Cultured

A novel ATP-sensitive potassium channel (K(ATP)) channel agonist, BPDZ 154 (6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), was synthesized, and its effects on insulin-secreting cells were evaluated using electrophysiology, (86)Rb(+) and (45)Ca(2+) efflux, and RIA determinations of insulin secretion. BPDZ 154, an analog of diazoxide, inhibited both glucose-induced insulin secretion from isolated perifused islets and the secretion of insulin induced by glucose and tolbutamide. These effects were mediated by the activation of ATP-sensitive potassium channels because BPDZ 154 induced a concentration-dependent increase in channel activity that was inhibited by the sulfonylurea tolbutamide and the imidazoline efaroxan. In beta-cells isolated from patients with either nontypical hyperinsulinism (preserved K(ATP) channel function) or from the control areas of the pancreas of patients with focal hyperinsulinism, BPDZ 154 activated K(ATP) channels and was found to be more effective and less readily reversible than diazoxide. By contrast, it was not possible to activate K(ATP) channels by either diazoxide or BPDZ 154 in beta-cells from patients with hyperinsulinism as a consequence of defects in K(ATP) channel function. In beta-cells isolated from a patient with pancreatic insulinoma, K(ATP) channels were readily recorded and modulated by BPDZ 154. These data suggest that BPDZ 154 or BPDZ 154-like compounds may have therapeutic potential in the treatment of certain forms of hyperinsulinism.

Topics: Adenosine Triphosphate; Adolescent; Adrenergic alpha-Antagonists; Animals; Benzofurans; Benzothiadiazines; Calcium Radioisotopes; Cell Line; Child, Preschool; Cyclic S-Oxides; Female; Glucose; Humans; Hyperinsulinism; Hypoglycemic Agents; Imidazoles; Infant; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Male; Pancreatic Neoplasms; Potassium Channels; Rats; Rats, Wistar; Rubidium Radioisotopes; Tolbutamide

The properties of ATP-sensitive K+ (K(ATP)) channels were explored in the electrofusion-derived, glucose-responsive, insulin-secreting cell line BRIN-BD11 using patch-clamp techniques. In intact cells, K(ATP) channels were inhibited by glucose, the sulfonylurea tolbutamide, and the imidazoline compounds efaroxan and phentolamine. Each of these agents initiated insulin secretion and potentiated the actions of glucose. K(ATP) channels were blocked by ATP in a concentration-dependent manner and activated by ADP in the presence of ATP. In both intact cells and excised inside-out patches, the K(ATP) channel agonists diazoxide and pinacidil activated channels, and both compounds inhibited insulin secretion evoked by glucose, tolbutamide, and imidazolines. The mechanisms of action of imidazolines were examined in more detail. Pre-exposure of BRIN-BD11 cells to either efaroxan or phentolamine selectively inhibited imidazoline-induced insulin secretion but not the secretory responses of cells to glucose, tolbutamide, or a depolarizing concentration of KCl. These conditions did not result in the loss of depolarization-dependent rises in intracellular Ca2+ ([Ca2+]i), K(ATP) channel operation, or the actions of either ATP or efaroxan on K(ATP) channels. Desensitization of the imidazoline receptor following exposure to high concentrations of efaroxan, however, was found to result in an increase in SUR1 protein expression and, as a consequence, an upregulation of K(ATP) channel density. Our data provide 1) the first characterization of K(ATP) channels in BRIN-BD11 cells, a novel insulin-secreting cell line produced by electrofusion techniques, and 2) a further analysis of the role of imidazolines in the control of insulin release.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adrenergic alpha-Antagonists; Animals; Benzofurans; Cell Fusion; Cell Line; Diazoxide; Glucose; Imidazoles; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Membrane Potentials; Pancreatic Neoplasms; Phentolamine; Pinacidil; Potassium Channels; Tolbutamide; Tumor Cells, Cultured

The effect on insulin release of efaroxan, an alpha 2-adrenoceptor antagonist and a highly potent drug at imidazoline I1 receptors, and the effects of seven other imidazoline compounds selective for the imidazoline I1 or I2 receptors, were studied in the rat insulinoma cell line RIN-5AH. The cells released insulin in response to glucose (0.3-10 mM), and efaroxan (100 microM) potentiated glucose-induced insulin release. (-)-Adrenaline completely displaced the binding of [125I]p-iodoclonidine to membranes of RIN-5AH cells, indicating that these cells do not express imidazoline I1 receptors. Cirazoline and idazoxan (100 microM), both highly potent drugs at imidazoline I2 receptors, and the guanidines guanoxan and amiloride (200 microM), also promoted insulin release from RIN-5AH cells. Irreversible blockade of imidazoline I2 receptors with 10 microM clorgyline did not prevent the stimulatory effects of cirazoline or idazoxan; however, these compounds completely reversed the inhibition by diazoxide (250 microM), an opener of ATP-dependent K+ channels (K+ATP channels), of glucose-induced insulin release. These data indicate that the imidazoline/guanidine compounds promote insulin release from RIN-5AH cells, by interacting with a novel binding site related to K+ATP channels that does not represent any of the known imidazoline I1 or I2 receptors.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Adrenergic beta-Agonists; Affinity Labels; Animals; Benzofurans; Binding, Competitive; Clonidine; Clorgyline; Diazoxide; Dioxanes; Guanidines; Idazoxan; Imidazoles; Imidazoline Receptors; Insulin; Insulinoma; Ligands; Pancreatic Neoplasms; Potassium Channels; Rats; Receptors, Drug; Regression Analysis; Software; Tumor Cells, Cultured

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.

Topics: Adenoma, Islet Cell; Animals; Benzofurans; Calcium; Carbachol; Cell Line; Cell Membrane; Cytosol; Ethers; Fluorescent Dyes; Fura-2; Insulinoma; Ionomycin; Kinetics; Membrane Potentials; Pancreatic Neoplasms; Potassium Chloride; Rats; Tetradecanoylphorbol Acetate