Fuel-induced amplification of insulin secretion in mouse pancreatic islets exposed to a high sulfonylurea concentration: role of the NADPH/NADP+ ratio.

AIMS/HYPOTHESIS: The aim of this study was to examine whether the cytosolic NADPH/NADP+ ratio of beta cells serves as an amplifying signal in fuel-induced insulin secretion and whether such a function is mediated by cytosolic alpha-ketoglutarate. METHODS: Pancreatic islets and islet cells were isolated from albino mice by collagenase digestion. Insulin secretion of incubated or perifused islets was measured by ELISA. The NADPH and NADP+ content of incubated islets was determined by enzymatic cycling. The cytosolic Ca2+ concentration ([Ca2+]c) in islets was measured by microfluorimetry and the activity of ATP-sensitive K+ channels in islet cells by patch-clamping. RESULTS: Both 30 mmol/l glucose and 10 mmol/l alpha-ketoisocaproate stimulated insulin secretion and elevated the NADPH/NADP+ ratio of islets preincubated in the absence of fuel. The increase in the NADPH/NADP+ ratio was abolished in the presence of 2.7 micromol/l glipizide (closing all ATP-sensitive K+ channels). However, alpha-ketoisocaproate, but not glucose, still stimulated insulin secretion. That glipizide did not inhibit alpha-ketoisocaproate-induced insulin secretion was not the result of elevated [Ca2+]c, as glucose caused a more marked [Ca2+]c increase. Insulin release triggered by glipizide alone was moderately amplified by dimethyl alpha-ketoglutarate (which is cleaved to produce cytosolic alpha-ketoglutarate), but there was no indication of a signal function of cytosolic alpha-ketoglutarate. CONCLUSIONS/INTERPRETATION: The results strongly suggest that the NADPH/NADP+ ratio in the beta cell cytosol does not serve as an amplifying signal in fuel-induced insulin release. The study supports the view that amplification results from the intramitochondrial production of citrate by citrate synthase and from the associated export of citrate into the cytosol.

Selective loss of glucose-induced amplification of insulin secretion in mouse pancreatic islets pretreated with sulfonylurea in the absence of fuels.

AIMS/HYPOTHESIS: The beta cell metabolism of glucose, and some other fuels, initiates insulin secretion by closure of ATP-sensitive K+ channels and amplifies the secretory response via unknown metabolic intermediates. The aim of this study was to further characterise the mechanism responsible for the metabolic amplification of insulin secretion. MATERIALS AND METHODS: Pancreatic islets were isolated from albino mice by collagenase digestion. Insulin secretion in perifused islets was determined by ELISA. Bioluminometry was used to determine the ATP and ADP content of the incubated islets. RESULTS: After perifusing islets for 60 min with 2.7 micromol/l glipizide (closing all ATP-sensitive K+ channels) in the absence of any fuel, perifusion with a test medium containing 2.7 micromol/l glipizide plus 30 mmol/l glucose did not enhance insulin secretion. However, test media supplemented with 2.7 micromol/l glipizide plus either 10 mmol/l alpha-ketoisocaproate or 10 mmol/l 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid amplified the glipizide-induced insulin secretion. In pancreatic islets preincubated for 60 min with 2.7 micromol/l glipizide in the absence of any fuel, 40 min incubations in the presence of 2.7 micromol/l glipizide plus 30 mmol/l glucose or plus 10 mmol/l alpha-ketoisocaproate produced an increase in the ATP content, no change in the ADP content and a rather small increase in the ATP:ADP ratio. The corresponding effects of glucose and alpha-ketoisocaproate were similar. CONCLUSIONS/INTERPRETATION: These results suggest that metabolic amplification of fuel-induced insulin secretion is not mediated by changes in the beta cell content of ATP and ADP, but might be due to export of citrate cycle intermediates to the beta cell cytosol.

Structural requirements of sulphonylureas and analogues for interaction with sulphonylurea receptor subtypes.

1. The structure-activity relationship for hypoglycaemic sulphonylureas and analogues was examined. Binding affinities were compared using membranes from HIT-T15 cells (beta-cell line) and from COS-7 cells transiently expressing sulphonylurea receptor subtypes (SUR1, SUR2A and SUR2B). Inhibition of adenosine-triphosphate-sensitive K+ channels (KATP-channels) was measured in mouse pancreatic beta-cells. 2. The tested compounds displayed similar binding affinities for SUR2A and SUR2B. 3. Meglitinide (benzoic acid derivative) bound to SUR1 and the SUR2 isoforms with similar affinities. Replacement of the carboxyl group of meglitinide by a methyl group significantly decreased the binding affinities for SUR1 and the SUR2 isoforms (>4 fold) and the potency to inhibit KATP-channel activity of beta-cells (24 fold). Replacement of the carboxyl group of meglitinide by a sulphonylurea group significantly increased the affinities for SUR1 (5 fold) and the SUR2 isoforms (13 - 16 fold). 4. Glibenclamide bound to the SUR2 isoforms with 300 - 500 fold lower affinity than to SUR1. Exchanging the cyclohexyl ring of glibenclamide by a methyl group or removal of the lipophilic side chain of glibenclamide (5-chloro-2-methoxy-benzamidoethyl chain) markedly reduced but did not abolish the selectivity for SUR1. 5. In conclusion, interaction of sulphonylureas and acidic analogues with SUR1, SUR2A and SUR2B is favoured by the anionic group of these drugs. Hypoglycaemic sulphonylureas (e.g. glibenclamide) owe selectivity for SUR1 to lipophilic substitution on their urea group. Sulphonylureas without lipophilic substitution on the urea group could represent lead compounds for the development of SUR2-selective drugs.

Interaction of N-benzoyl-D-phenylalanine and related compounds with the sulphonylurea receptor of beta-cells.

1. The structure activity relationships for the insulin secretagogues N-benzoyl-D-phenylalanine (NBDP) and related compounds were examined at the sulphonylurea receptor level by use of cultured HIT-T15 and mouse pancreatic beta-cells. The affinities of these compounds for the sulphonylurea receptor were compared with their potencies for K(ATP)-channel inhibition. In addition, the effects of cytosolic nucleotides on K(ATP)-channel inhibition by NBDP were investigated. 2. NBDP displayed a dissociation constant for binding to the sulphonylurea receptor (K(D) value) of 11 microM and half-maximally effective concentrations of K(ATP)-channel inhibition (EC50 values) between 2 and 4 microM (in the absence of cytosolic nucleotides or presence of 0.1 mM GDP or 1 mM ADP). 3. In the absence of cytosolic nucleotides or presence of GDP (0.1 mM) maximally effective concentrations of NBDP (0.1-1 mM) reduced K(ATP)-channel activity to 47% and 44% of control, respectively. In the presence of ADP (1 mM), K(ATP)-channel activity was completely suppressed by 0.1 mM NBDP. 4. The L-isomer of N-benzoyl-phenylalanine displayed a 20 fold lower affinity and an 80 fold lower potency than the D-isomer. 5. Introduction of a p-nitro substituent in the D-phenylalanine moiety of NBDP did not decrease lipophilicity but lowered affinity and potency by more than 30 fold. 6. Introduction of a p-amino substituent in the D-phenylalanine moiety of NBDP (N-benzoyl-p-amino-D-phenylalanine, NBADP) reduced lipophilicity and lowered affinity and potency by about 10 fold. This loss of affinity and potency was compensated for by formation of the phenylpropionic acid derivative of NBADP. A similar difference in affinity was observed for the sulphonylurea carbutamide and its phenylpropionic acid derivative. 7. Replacing the benzene ring in the D-phenylalanine moiety of NBDP by a cyclohexyl ring increased lipophilicity, and the K(D) and EC50 values were slightly lower than for NBDP. Exchange of both benzene rings in NBDP by cyclohexyl rings further increased lipophilicity without altering affinity and potency. 8. This study shows that N-acylphenylalanines interact with the sulphonylurea receptor of pancreatic beta-cells in a stereospecific manner. Their potency depends on lipophilic but not aromatic properties of their benzene rings. As observed for sulphonylureas, interaction of N-acylphenylalanines with the sulphonylurea receptor does not induce complete inhibition of K(ATP)-channel activity in the absence of inhibitory cytosolic nucleotides.

Association and stoichiometry of K(ATP) channel subunits.

ATP-sensitive potassium channels (K(ATP) channels) are heteromultimers of sulfonylurea receptors (SUR) and inwardly rectifying potassium channel subunits (K(IR)6.x) with a (SUR-K(IR)6.x)4 stoichiometry. Association is specific for K(IR)6.x and affects receptor glycosylation and cophotolabeling of K(IR)6.x by 125I-azidoglibenclamide. Association produces digitonin stable complexes with an estimated mass of 950 kDa. These complexes can be purified by lectin chromatography or by using Ni2(+)-agarose and a his-tagged SUR1. Expression of SUR1 approximately (K(IR)6.2)i fusion constructs shows that a 1:1 SUR1:K(IR)6.2 stoichiometry is both necessary and sufficient for assembly of active K(ATP) channels. Coexpression of a mixture of strongly and weakly rectifying triple fusion proteins, rescued by SUR1, produced the three channel types expected of a tetrameric pore.

Sulfonylurea receptors and mechanism of sulfonylurea action.

Binding of hypoglycemic sulfonylureas and their analogues to the sulfonylurea receptor in the beta-cell plasma membrane mediates closure of the ATP-sensitive K+-channel (KATP-channel) and thereby stimulation of insulin release. The sulfonylurea receptor is a member of the traffic ATPase family with two intracellular nucleotide binding folds. The receptor binding site for hypoglycemic drugs is located at the cytoplasmic face of the plasma membrane. Mutations in the sulfonylurea receptor gene have been detected which cause familial hyper-insulinism. Non-beta-cell sulfonylurea receptors do not contribute to the therapeutic benefit of sulfonylureas, but might be involved in presumed adverse effects of sulfonylureas in the cardiovascular and the central nervous system.

Coincidence of early glucose-induced depolarization with lowering of cytoplasmic Ca2+ in mouse pancreatic beta-cells.

1. The temporal relationship between the early glucose-induced changes of membrane potential and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in insulin-releasing pancreatic beta-cells. 2. The mean resting membrane potential and [Ca2+]i were about -70 mV and 60 nM, respectively, in 3 mM glucose. 3. Elevating the glucose concentration to 8-23 mM typically elicited a slow depolarization, which was paralleled by a lowering of [Ca2+]i. When the slow depolarization had reached a threshold of -55 to -40 mV, there was rapid further depolarization to a plateau with superimposed action potentials, and [Ca2+]i increased dramatically. 4. Imposing hyperpolarizations and depolarizations of 10 mV from a holding potential of -70 mV had no detectable effect on [Ca2+]i. Furthermore, glucose elevation elicited a decrease in [Ca2+]i even at a holding potential of -70 mV. 5. Step depolarizations induced [Ca2+]i transients, which decayed with time courses well fitted by double exponentials. The slower component became faster by a factor of about 4 upon elevation of glucose, suggesting involvement of ATP-dependent Ca2+ sequestration or extrusion of [Ca2+]i. 6. Glucose stimulation increased the size and accelerated the recovery of carbachol-triggered [Ca2+]i transients, and thapsigargin, an intracellular Ca(2+)-ATPase inhibitor, counteracted the glucose-induced lowering of [Ca2+]i, indicating that calcium transport into intracellular stores is involved in glucose-induced lowering of [Ca2+]i. 7. The results support the notion that in beta-cells, nutrient-induced elevation of ATP leads initially to ATP-dependent removal of Ca2+ from the cytoplasm, paralleled by a slow depolarization due to inhibition of ATP-sensitive K+ channels. Only after depolarization has reached a threshold do action potentials occur, inducing a sharp elevation in [Ca2+]i.

Location of the sulphonylurea receptor at the cytoplasmic face of the beta-cell membrane.

1. In insulin-secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexy moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH 7.4 compound IV is only present in charged form. 2. Lipid solubility declined in the order tolbutamide > compound III > compound IV. 3. The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT-cells (pancreatic beta-cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4. In mouse pancreatic beta-cells, drug concentrations inhibiting adenosine 5'-triphosphate-sensitive K+ channels (KATP-channels) half-maximally (EC50) were determined by use of the patch-clamp technique. When the drugs were applied to the extracellular side of outside-out or the intracellular side of inside-out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide. 5. In mouse pancreatic beta-cells, measurement of KATP-channel activity in cell-attached patches and recording of insulin release displayed much higher EC50 values for compound IV than inside-out patch experiments. A corresponding, but less pronounced difference in EC50 values was observed for compound III, whereas the EC50 values for tolbutamide did not differ significantly. 6. It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the beta-cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.

Photoaffinity labeling of the cerebral sulfonylurea receptor using a novel radioiodinated azidoglibenclamide analogue.

In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145-150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [3H]glibenclamide or a 125I-labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4-azido-5-iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high-affinity binding of this novel probe to the particulate (KD = 0.13 nM) and solubilized (KD = 0.56 nM) sulfonylurea receptor from cerebral cortex. The novel probe showed > 100-fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175-kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a KD value of 0.38 nM. Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed KD values for these sulfonylureas of 0.06 nM, 1.6 nM, and 1.2 microM, respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175-kDa protein represents the cerebral sulfonylurea receptor.

Identification of an ATP-sensitive K+ channel in spiny neurons of rat caudate nucleus.

On the somata of GABAergic spiny neurons in the caudate nucleus of the rat an ATP-sensitive K+ channel (KATP-channel) was identified. The KATP-currents in cell-attached patches were activated both by energy-depleting conditions (200 microM cyanide) and by diazoxide (300 microM) and were reversibly blocked by tolbutamide (EC50 = 5 microM). In inside-out patch membranes both ATP (1 mM) and its non-hydrolyzable analog AMP-PNP (adenylylimidodiphosphate; EC50 = 27 microM) reversibly inhibited channel activity. These results demonstrate that the KATP-channel in spiny neurons displays properties characteristic of the KATP-channel in hippocampal, neocortical and nigral neurons and in pancreatic beta-cells.

Interaction of tolbutamide and cytosolic nucleotides in controlling the ATP-sensitive K+ channel in mouse beta-cells.

1. In mouse pancreatic beta-cells the role of cytosolic nucleotides in the regulation of the sulphonylurea sensitivity of the adenosine 5'-triphosphate-sensitive K+ channel (KATP-channel) was examined. Patch-clamp experiments with excised inside-out membrane patches were carried out using an experimental protocol favouring phosphorylation of membrane proteins. 2. In the absence of Mg2+, the KATP-channel-inhibiting potency of cytosolic nucleotides decreased in the order ATP = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > adenosine 5'-diphosphate (ADP) > adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) = adenylyl-imidodiphosphate (AMP-PNP) > 2'-deoxyadenosine 5'-triphosphate (dATP) > uridine 5'-triphosphate (UTP) > 2'-deoxyadenosine 5'-diphosphate (dADP) > guanosine 5'-triphosphate (GTP) > guanosine 5'-diphosphate (GDP) > uridine 5'-diphosphate (UDP). 3. In the presence of Mg2+, the inhibitory potency of cytosolic nucleotides decreased in the order ATP gamma S > ATP > AMP-PNP > ADP beta S > dATP > UTP. In the presence of Mg2+, the KATP-channels were activated by dADP, GTP, GDP and UDP. 4. Tolbutamide inhibited the KATP-channels not only in the presence but also in the prolonged absence of Mg2+. In nucleotide-free solutions, the potency of tolbutamide was very low. When about half of the KATP-channel activity was inhibited by ATP, AMP-PNP, ADP beta S or ADP (absence of Mg2+), the potency of tolbutamide was increased. 5. Tolbutamide (100 microM) slightly enhanced the channel-inhibiting potency of AMP-PNP and inhibited the channel-activating effect of MgGDP in a non-competitive manner. 6. Channel activation by MgGDP (0.5 mM) competitively antagonized the inhibitory responses to AMP-PNP (1 MicroM- 1 mM). This effect of GDP was neutralized by tolbutamide (100 MicroM).7. The stimulatory effect of 0.5 mM MgGDP was neutralized by 200 MicroM AMP-PNP. Under these conditions the potency of tolbutamide was much higher than in the presence of 0.5 mM MgGDP alone or in the absence of any nucleotides.8. dADP (0.3-1 mM) increased the potency of tolbutamide. Additional application of 200 MicroM AMPPNP caused a further increase in the potency of tolbutamide.9. In conclusion, in the simultaneous presence of inhibitory and stimulatory nucleotides, binding of sulphonylureas to their receptor causes direct inhibition of channel activity, non-competitive inhibition of the action of stimulatory nucleotides and interruption of the competitive interaction between stimulatory and inhibitory nucleotides. The latter effect increases the proportion of KATP- channels staying in the nucleotide-blocked state. In addition, this state potentiates the direct effect of sulphonylureas.

Tolbutamide- and diazoxide-sensitive K+ channel in neurons of substantia nigra pars reticulata.

Single-channel K+ currents were recorded in cell-attached patches from slices of rat substantia nigra. On the somata of neurons in the caudal half of the substantia nigra pars reticulata a K+ selective channel with a unitary conductance of 71 pS (154 mmol/l K+ in pipette filling solution) was identified. The channel was activated both by application of diazoxide (300 mumol/l) and by energy-depleting conditions (200 mumol/l cyanide) and was reversibly blocked by tolbutamide (0.1-1 mmol/l). It is concluded that neurons in the substantia nigra pars reticulata of the rat contain a typical ATP-sensitive K+ channel the activity of which can be modulated by diazoxide and sulfonylureas.

Pancreatic and extrapancreatic sulfonylurea receptors.

The hypoglycemic effect of sulfonylureas and their analogues results from their binding to a high affinity site in the B-cell plasma membrane. This site seems to be a structural component of the ATP-sensitive K(+)-channel and represents the pancreatic sulfonylurea receptor. Binding of sulfonylureas causes closure of the ATP-sensitive K(+)-channel and thereby initiates a chain of events eventually leading to the release of insulin. Diazoxide inhibits insulin secretion via opening of the ATP-sensitive K(+)-channel. Sulfonylurea receptors resembling the pancreatic receptor occur in nerve cells, cardiac muscle, skeletal muscle and smooth muscle. Neither these extrapancreatic receptors nor low affinity receptors for sulfonylureas in myocytes and adipocytes contribute to the therapeutic benefit of sulfonylureas.

The binding properties of the particulate and solubilized sulfonylurea receptor from cerebral cortex are modulated by the Mg2+ complex of ATP.

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.

Diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel in mouse pancreatic beta-cells.

1. In mouse pancreatic beta-cells the regulation of the diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel (K-ATP-channel) was examined by use of the patch-clamp technique. 2. In intact beta-cells incubated at 37 degrees C in the presence of 3 mM D-glucose, diazoxide did not affect the single channel conductance but stimulated channel-opening activity. Diazoxide produced half-maximal effects at 82 microM and 13 fold activation at maximally effective concentrations (300-400 microM). The response to diazoxide (300 microM) was not completely suppressed by saturating tolbutamide concentrations (1 or 5 mM). 3. Inside-out patch-clamp experiments were carried out using an experimental protocol favouring phosphorylation of membrane proteins. Under these conditions diazoxide was ineffective in the absence of any nucleotides, weakly effective in the presence of MgATP (26 or 87 microM) and strongly effective in the presence of the Mg complexes of adenosine 5'-diphosphate, 2'-deoxyadenosine 5'-diphosphate or guanosine 5'-diphosphate (MgADP, MgdADP or MgGDP). 4. In inside-out patches exposed to nucleotide-free solutions, saturating concentrations of tolbutamide did not cause complete block of K-ATP-channels. When the channels were activated by MgdADP (48 microM), tolbutamide was even less effective. Sensitization of MgdADP-induced channel activation by diazoxide further weakened the effects of tolbutamide. 5. Diazoxide (50 or 300 microM) prevented the complete channel block induced by saturating tolbutamide concentrations in the presence of Mg2+ and ADP (1 mM). 6. In the presence of Mg2", the K-ATP-channel-blocking potency of cytosolic ATP decreased in the order inside-out> outside-out> whole-cell configuration of the patch-clamp technique.7. It is concluded that the K-ATP-channel is controlled via four separate binding sites for inhibitory nucleotides (e.g. free ATP and ADP), stimulatory nucleotides (MgADP, MgdADP, MgGDP), sulphonylureas and diazoxide. Strong inhibition of the channel openings by sulphonylureas results from occupation of both sites for nucleotides. Diazoxide is only effective when the site for stimulatory nucleotides is occupied.

The binding properties of the solubilized sulfonylurea receptor from a pancreatic B-cell line are modulated by the Mg(++)-complex of ATP.

Previous studies demonstrated that the Mg complex of ATP decreases glyburide- and increases diazoxide-binding to membranes from pancreatic islets. To examine further the mechanism of these effects, the sulfonylurea receptors in microsomes of the hamster B-cell line HIT-T15 were solubilized with detergents. Maximum recovery of receptors (40%) was obtained with Triton X-100. Specific binding of [3H]glyburide to the solubilized receptors (Kd = 0.35 nM, maximum number of binding sites = 170 fmol/mg of protein) corresponded well to specific binding to microsomes. In Triton X-100 extracts, MgATP (300 microM) reduced the number of high-affinity sites for [3H]glyburide by 50% and increased the dissociation constant for [3H]glyburide by 4-fold; MgATP was half-maximally effective at 20 microM. Development of MgATP-induced inhibition of [3H]glyburide binding to solubilized binding sites was not slower than dissociation of [3H]glyburide binding. Alkaline phosphatase accelerated the reversal of MgATP-induced inhibition of [3H]glyburide binding. In the presence of Mg++, not only ATP but also ADP, GTP and GDP inhibited [3H]glyburide binding to the solubilized receptor. However, MgADP did not inhibit [3H]glyburide binding when the MgATP concentration was kept low by the hexokinase reaction. MgATP significantly enhanced diazoxide-induced displacement of [3H]glyburide from the solubilized receptor. The MgATP-induced inhibition of binding was weakened by millimolar concentrations of free ATP. It is concluded that the binding sites for MgATP, glyburide and diazoxide are located at a single protein or at closely associated proteins which may include a protein kinase.

Effect of MgATP on pinacidil-induced displacement of glibenclamide from the sulphonylurea receptor in a pancreatic beta-cell line and rat cerebral cortex.

1. The effects of blockers and openers of K+ channels on binding of [3H]-glibenclamide to microsomes obtained from a pancreatic beta-cell line (HIT-T15) or rat cerebral cortex were examined. 2. The blockers quinine, chlorpromazine and thiopentone and the openers cromakalim [(+/- ) 6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1- pyrrolidyl)-2H-benzo[b]pyran-3-ol] and minoxidil sulphate did not significantly interact with the sulphonylurea receptor of HIT-cells both at phosphorylating (presence of MgATP) and dephosphorylating (absence of MgATP) conditions. 3. In the absence of MgATP, pinacidil (200-500 microM) did not significantly displace [3H]-glibenclamide binding to microsomes from HIT-cells. The displacement of [3H]-glibenclamide binding was strongly enhanced by MgATP and was due to a decrease in the number of high affinity binding sites for glibenclamide. 4. MgATP enhanced pinacidil-induced inhibition of [3H]-glibenclamide binding to microsomes from rat cerebral cortex. 5. The effect of MgATP on pinacidil-induced inhibition of [3H]-glibenclamide binding was maintained after solubilization of the membranes from HIT-cells or rat cerebral cortex. 6. It is concluded that the sulphonylurea receptor is regulated not only by sulphonylureas but also by the K+ channel openers, diazoxide and pinacidil, and by protein phosphorylation. The binding sites for sulphonylureas and these K+ channel openers are not identical, but appear to be located at a single protein or at tightly associated proteins.

Cytosolic nucleotides enhance the tolbutamide sensitivity of the ATP-dependent K+ channel in mouse pancreatic B cells by their combined actions at inhibitory and stimulatory receptors.

In the plasma membrane of pancreatic B cells, a K+ channel (K-ATP channel) has been identified that is regulated by cytoplasmic nucleotides. This channel is inhibited by sulfonylureas. We have previously shown that the potency of tolbutamide is much lower in excised membrane patches than in intact cells, unless the internal side of the membrane is exposed to the Mg2+ complex of ADP (MgADP). In the present study, the mechanism of this interactive control by sulfonylureas and nucleotides was examined using the inside-out configuration of the patch-clamp technique. When test solutions containing Mg2+ ions were applied, the opening activity of the K-ATP-channels was strongly stimulated by 2'-deoxyadenosine-5'-diphosphate (dADP) or GDP, slightly stimulated by ADP, and inhibited by adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) or adenylyl-imidodiphosphate (AMP-PNP). In the presence of Mg2+, not only ADP but also its analogues dADP (1 mM) and ADP beta S (0.1 mM) enhanced the potency of tolbutamide for channel inhibition; dADP at a low concentration (0.2 mM), GDP (0.2-1 mM), and AMP-PNP (0.2 mM) did not alter the potency of tolbutamide. The particular feature of the test solutions that enhanced the potency of tolbutamide was the presence of Mg(2+)-bound and free nucleotides at channel-stimulating and channel-inhibiting concentrations, respectively. In the presence of Mg2+ and 0.2 mM dADP or 0.2-1 mM GDP, 0.2 mM AMP-PNP intensified the response to tolbutamide by serving as channel-inhibiting component. MgAMP-PNP did not stimulate the opening activity of the K-ATP channel. The sensitivity to tolbutamide that was enhanced by a submaximally effective ADP concentration was further increased by AMP-PNP or ATP but not by GDP. The sensitivity to the sulfonylurea analogue meglitinide was also enhanced by ADP. It is concluded that nucleotides inhibit and activate the K-ATP channel by interaction with two separate receptor sites at the cytoplasmic face of the B cell membrane. Effective inhibition of the channel openings by sulfonylureas results from the simultaneous occupation of both sites by appropriate nucleotides.

Adenine nucleotide-induced inhibition of binding of sulphonylureas to their receptor in pancreatic islets.

1. The effects of the Mg complex of adenosine 5'-triphosphate (MgATP) on binding of sulphonylureas to microsomes obtained from mouse pancreatic islets were examined. 2. MgATP inhibited the binding of both glibenclamide and tolbutamide to microsomes. 3. Binding of [3H]-glibenclamide inhibited by MgATP was not further diminished by Mg(2+)-bound adenosine 5'-(beta, gamma-imidotriphosphate) (AMP-PNP) or free adenosine 5'-diphosphate (ADP). Higher concentrations of MgAMP-PNP induced a partial reversal of the inhibitory effect of MgATP on [3H]-glibenclamide binding. 4. The apparent dissociation constant (K'D) for binding of [3H]- glibenclamide remained constant when 5. Extracellular ADP did not markedly stimulate insulin release from mouse pancreatic islets. 6. It is concluded that sulphonylureas and cytosolic nucleotides exert their inhibitory effects on the K-ATP-channels of beta-cells by binding to different sites. The binding properties of the sulphonylurea receptor seem to be modulated by protein phosphorylation.