oligomycins and Insulinoma

1. The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP-sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI-G1) cell line using a combination of whole cell and single channel recording techniques. 2. Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI-G1 cells with a concomitant increase in K+ conductance. This is due to activation of K(ATP) channels as the sulphonylurea tolbutamide (100 microM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 microM) or oligomycin (2 microM) did not hyperpolarize CRI-G1 cells or increase K+ conductance. 3. In cell-attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in K(ATP) channel activity, an action readily reversible by tolbutamide (100 microM). Application of sodium azide (3 mM), in the presence of Mg-ATP, to the intracellular surface of excised inside-out patches also increased K(ATP) channel activity, in a reversible manner. 4. In contrast, rotenone (10 microM) or oligomycin (2 microM) did not increase K(ATP) channel activity in either cell-attached, in the absence of glucose, or inside-out membrane patch recordings. 5. Addition of sodium azide (3 mM) to the intracellular surface of inside-out membrane patches in the presence of Mg-free ATP or the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) inhibited, rather than increased, K(ATP) channel activity. 6. In conclusion, sodium azide, but not rotenone or oligomycin, directly activates K(ATP) channels in CRI-G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Electric Conductivity; Enzyme Inhibitors; Insulinoma; Membrane Potentials; Oligomycins; Patch-Clamp Techniques; Potassium Channels; Rats; Rotenone; Sodium Azide; Tumor Cells, Cultured; Uncoupling Agents

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Eosine I Bluish; Fluorescein; Fluoresceins; Glyburide; Heart Ventricles; Insulinoma; Membrane Potentials; Oligomycins; Potassium Channels; Rats; Rose Bengal; Rubidium; Tumor Cells, Cultured

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Adenosine Triphosphate; Animals; Arachidonic Acid; Cricetinae; Enzyme Activation; Fatty Acids, Nonesterified; Insulinoma; Oligomycins; Potassium Channels; Protein Kinase C; Tumor Cells, Cultured

The uptake of Ca2+ by microsomes is thought to participate in the control of cytosolic Ca2+ activity in the insulin-producing pancreatic B-cell. In order to study such a phenomenon methods were developed to isolate microsomes from rat parotid cells, pancreatic islets and tumoral islet cells of the RINm5F line. In the latter case, a subcellular microsomal fraction was prepared in which the ratio of microsomal/mitochondrial enzyme markers, as well as that of ruthenium red-resistant/sensitive 45Ca2+ uptake was 20 times higher than in the corresponding mitochondrial subcellular fraction. The ATP-dependent net uptake of 45Ca2+ by RINm5F cell microsomes was inhibited at low temperature and by either vanadate or a monoclonal antibody to dog heart sarcoplasmic reticulum. Although the uptake of Ca2+ by microsomes may account for only a minor fraction of ATP consumption, its synarchistic regulation by ATP and Ca2+, at close-to-physiological concentrations, appeared well suited to play a major regulatory role in the control of cytosolic Ca2+ activity in intact islet cells.

Topics: Animals; Antibodies, Monoclonal; Antimycin A; Biological Transport, Active; Calcium; Calcium-Transporting ATPases; Cell Line; Dogs; Insulinoma; Islets of Langerhans; Kinetics; Microsomes; Mitochondria; Myocardium; Oligomycins; Pancreatic Neoplasms; Parotid Gland; Rats; Sarcoplasmic Reticulum; Vanadates

Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.

Topics: Adenoma, Islet Cell; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antimycin A; Calcium; Cell Membrane Permeability; Insulinoma; Magnesium; Oligomycins; Pancreatic Neoplasms; Phosphates; Tumor Cells, Cultured

Somatostatin, an hyperglycemia-inducing hormone, was studied in rat insulinoma (RINm5F) cells using 86Rb+ efflux techniques. 86Rb+ efflux is stimulated by somatostatin in a dose-dependent manner. The half-maximum value of activation is 0.7 nM. Somatostatin-induced 86Rb+ efflux is abolished by the hypoglycemia-inducing sulfonylurea, glibenclamide, a known blocker of ATP-regulated K+ channels. Somatostatin activation is prevented by pretreatment of insulinoma cells with pertussis toxin. 86Rb+ efflux studies show that somatostatin activates an ATP-dependent K+ channel.

Topics: Adenosine Triphosphate; Animals; Deoxyglucose; Glyburide; GTP-Binding Proteins; Insulinoma; Oligomycins; Pancreatic Neoplasms; Pertussis Toxin; Potassium Channels; Rats; Rubidium Radioisotopes; Somatostatin; Tumor Cells, Cultured; Virulence Factors, Bordetella