Synthesis and evaluation of a glibenclamide glucose-conjugate: a potential new lead compound for substituted glibenclamide derivatives as islet imaging agents.

The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.

In vitro and in vivo evaluation of novel glibenclamide derivatives as imaging agents for the non-invasive assessment of the pancreatic islet cell mass in animals and humans.

Pancreatic islet cell mass (PICM) is a major determinant of the insulin secretory capacity in humans. Currently, the only method for accurate assessment of the PICM is an autopsy study. Thus, development of a technique allowing the non-invasive quantification of PICM is of great interest. The aim of this study was to develop such a non-invasive technique featuring novel fluorine- and (99m)Tc-labelled glibenclamide derivatives. Despite the structural modifications necessary to introduce fluorine into the glibenclamide molecule, all derivatives retained insulin stimulating capacity as well as high affinity binding to human SUR1 when compared to the original glibenclamide. Contrastingly, the lipophilicity of the fluorine-labelled derivatives was altered depending on the particular modification. In the human PET-study a constant but weak radioactive signal could be detected in the pancreas using a fluorine-labelled glibenclamide derivative. However, a reliable assessment and visualisation of the PICM could not be obtained. It can be assumed that the high uptake of the fluorine-labelled tracer e.g. into the the liver and the high plasma protein binding leads to a relatively low signal-to-noise ratio. In case of the presented fluorine-labelled glibenclamide based compounds this could be the result of their invariably high lipophilicity. The development of a (99 m)Tc-labelled glibenclamide derivative with a lower lipophilicity and differing in vivo behaviour, glibenclamide based compounds for non-invasive imaging of the pancreatic islet cell mass may be possible.

Synthesis and evaluation of (S)-2-(2-[18F]fluoroethoxy)-4-([3-methyl-1-(2-piperidin-1-yl-phenyl)-butyl-carbamoyl]-methyl)-benzoic acid ([18F]repaglinide): a promising radioligand for quantification of pancreatic beta-cell mass with positron emission tomography (PET).

18F-labeled non-sulfonylurea hypoglycemic agent (S)-2-(2-[(18)F]fluoroethoxy)-4-((3-methyl-1-(2-piperidin-1-yl-phenyl)-butylcarbamoyl)-methyl)-benzoic acid ([(18)F]repaglinide), a derivative of the sulfonylurea-receptor (SUR) ligand repaglinide, was synthesized as a potential tracer for the non-invasive investigation of the sulfonylurea 1 receptor status of pancreatic beta-cells by positron emission tomography (PET) in the context of type 1 and type 2 diabetes. [(18)F]Repaglinide could be obtained in an overall radiochemical yield (RCY) of 20% after 135 min with a radiochemical purity higher than 98% applying the secondary labeling precursor 2-[(18)F]fluoroethyltosylate. Specific activity was in the range of 50-60 GBq/micromol. Labeling was conducted by exchanging the ethoxy-moiety into a 2-[(18)F]fluoroethoxy group. To characterize the properties of fluorinated repaglinide, the affinity of the analogous non-radioactive (19)F-compound for binding to the human SUR1 isoform was assessed. [(19)F]Repaglinide induced a complete monophasic inhibition curve with a Hill coefficient close to 1 (1.03) yielding a dissociation constant (K(D)) of 134 nM. Biological activity was proven via insulin secretion experiments on isolated rat islets and was comparable to that of repaglinide. Finally, biodistribution of [(18)F]repaglinide was investigated in rats by measuring the concentration of the compound in different organs after i.v. injection. Pancreatic tissue displayed a stable accumulation of approximately 0.12% of the injected dose from 10 min to 30 min p.i. 50% of the radioactive tracer could be displaced by additional injection of unlabeled repaglinide, indicating that [(18)F]repaglinide might be suitable for in vivo investigation with PET.

ATP4- mediates closure of pancreatic beta-cell ATP-sensitive potassium channels by interaction with 1 of 4 identical sites.

In pancreatic beta-cells, cytosolic [ATP(4-)] critically controls insulin secretion via inhibition of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel subunit (Kir6.2) plus a regulatory sulfonylurea receptor. To elucidate stoichiometry of ATP(4-) action, we analyzed ATP(4-) sensitivity of channels coassembled from wild-type Kir6.2 and a loss of ATP(4-) sensitivity mutant (G334D). Concentration-inhibition curves for cDNA ratios of 1:1 or 1:10 resembled those for channel block resulting from interaction with 1 of 4 sites, whereas models for inhibition requiring occupation of 2, 3, or 4 sites were incongruous. Random assembly of wild-type Kir6.2 with the G334D mutant was confirmed by controls, which assessed the effect of an additional mutation that induced strong rectification (N160D). We conclude 4 identical noncooperative ATP(4-) sites to be grouped within 1 KATP channel complex, with occupation of 1 site being sufficient to induce channel closure. This architecture might facilitate coupling of [ATP(4-)] to insulin secretion and may protect against diabetic dysregulation resulting from heterozygous mutations in Kir6.2.

Stoichiometry of potassium channel opener action.

Potassium channel openers (KCOs; e.g., P1075, pinacidil) exert their effects on excitable cells by opening ATP-sensitive potassium channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K(+) channel subunit plus a regulatory subunit comprising the receptor sites for hypoglycemic sulfonylureas and KCOs (a sulfonylurea receptor). To elucidate stoichiometry of KCO action, we analyzed P1075 sensitivity of channels coassembled from sulfonylurea receptor isoforms with high or low P1075 affinity. Concentration activation curves for cDNA ratios of 1:1 or 1:10 resembled those for channel opening resulting from interaction with a single site, whereas models for activation requiring occupation of two, three, or four sites were incongruous. We conclude KCO-induced channel activation to be mediated by interaction with a single binding site per tetradimeric complex.

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.

Identification of the potassium channel opener site on sulfonylurea receptors.

Diversity of sulfonylurea receptor (SUR) subunits underlies tissue specific pharmacology of K(ATP) channels, which represent critical regulators of electrical activity in numerous cells. Notably, the neuronal/pancreatic beta-cell receptor, SUR1, imparts high sensitivity to hypoglycemic sulfonylureas (SUs; e.g. glibenclamide) and low to potassium channel openers (KCOs; e.g. P1075), whereas the opposite drug sensitivities are conferred by cardiovascular receptors, SUR2A and SUR2B. By exchanging domains between SUR1 and SUR2B, we identify two regions (KCO I: Thr(1059)-Leu(1087) and KCO II: Arg(1218)-Asn(1320); rat SUR2 numbering) within the second set of transmembrane domains (TMDII) as critical for KCO binding. Swapping both regions reconstitutes KCO affinities and sensitivities of the donor SUR isoform. High glibenclamide affinity of SUR1 is not reduced by transfer of KCO I plus II from SUR2B, demonstrating that high SU and KCO affinity can coexist in the same SUR molecule. Consistently, high SU affinity was imparted on SUR2B by substituting the region separating KCO I and II (Ile(1088)-Val(1217)) with the corresponding domain of SUR1. We infer the receptor sites for KCOs and SUs to be closely associated within a regulatory domain (Thr(1059)-Asn(1320)) in TMDII of SURs.

Stoichiometry of sulfonylurea-induced ATP-sensitive potassium channel closure.

Hypoglycemic sulfonylureas (e.g., glibenclamide, glipizide, and tolbutamide) exert their stimulatory effect on excitatory cells by closure of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel (KIR) subunit 6.x plus a sulfonylurea receptor (SUR). SUR1/KIR6.2 reconstitutes the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular smooth muscle-type KATP channels, respectively. SUR2A and SUR2B are splice variants of a single gene differing only in their C-terminal 42 amino acids. Affinities of sulfonylureas for rat SUR2A, rat or human SUR2B, and a SUR2 chimera containing the C-terminal 42 amino acids of SUR1 did not differ significantly, implying that the C terminus does not form part of the binding pocket. Consistent with these findings, reconstituted SUR2A/KIR6.2 and SUR2B/KIR6.2 channels revealed similar sensitivities for glibenclamide and tolbutamide. Dissociation constants of sulfonylureas for SUR2A and SUR2B were 10- to 400-fold higher than for SUR1, however, amazingly the benzoic acid derivative meglitinide did not show lower affinity for SUR2 isoforms. Potencies of glibenclamide, glipizide, tolbutamide, and meglitinide to inhibit activity of SUR1/KIR6.2 and SUR2B/KIR6.2 channels were 3- to 6-fold higher than binding affinities of these drugs with concentration-inhibition relations being significantly steeper (Hill coefficients 1.23-1.32) than binding curves (Hill coefficients 0.93-1.06). The data establish that the C terminus of SURs does not affect sulfonylurea affinity and sensitivity. We conclude that occupation of one of the four SUR sites per channel complex is sufficient to induce KATP channel closure.

Potassium channel openers require ATP to bind to and act through sulfonylurea receptors.

KATP channels are composed of a small inwardly rectifying K+ channel subunit, either KIR6.1 or KIR6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP-binding cassette superfamily. SUR1/KIR6.2 reconstitute the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular-smooth-muscle-type KATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUR2A and SUR2B requires ATP. Non-hydrolysable ATP-analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide-binding folds (NBFs) abolish or weaken [3H]P1075 binding to SUR2B, rendering reconstituted SUR2B/KIR6.2 channels insensitive towards KCOs. The C-terminus of SUR affects KCO affinity with SUR2B approximately SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [3H]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5-8-fold higher than their potencies for activation of SUR2B/KIR6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.

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.

KATP-channel on the somata of spiny neurones in rat caudate nucleus: regulation by drugs and nucleotides.

1. The aim of the present study was to characterize the pharmacological properties of the adenosine 5'-triphosphate(ATP)-sensitive K+ channel (KATP-channel) on the somata of spiny neurones in rat caudate nucleus and to compare them with those of beta-cells. For that purpose we tested the effects of several KATP-channel-inhibiting and -activating drugs on the opening activity of the KATP-channel in caudate nucleus by use of the patch-clamp technique. In addition, the modulation of drug responses by cytosolic nucleotides was examined. 2. When KATP-channels in caudate nucleus were activated in cell-attached patches by inhibition of mitochondrial energy production, meglitinide (a benzoic acid derivative), Hoe36320 (a sulphonylurea of low lipophilicity) and glipizide reduced KATP-channel activity half-maximally at 0.4 microM, 0.4 microM and about 0.5 nM, respectively. 3. In inside-out patches (presence of 0.7 mM free Mg2+ at the cytoplasmic membrane side), tolbutamide (0.1 mM) caused only partial inhibition of KATP-channels in the absence of cytosolic nucleotides but complete inhibition in the simultaneous presence of the channel-activating nucleotide guanosine 5'-diphosphate (GDP; 1 mM) and the channel-inhibiting nucleotide adenylyl-imidodiphosphate (AMP-PNP; 0.2 mM). 4. Diazoxide (0.3 mM) strongly increased channel activity in the presence of ATP (0.1 mM) or GDP (0.03 mM), but was ineffective in the presence of AMP-PNP (0.1 mM). In the absence of cytosolic nucleotides diazoxide even decreased channel activity. 5. In the presence of 0.1 mM ATP, diazoxide activated KATP-channels half-maximally at 38 microM. 6. When KATP-channel activity was inhibited by 0.1 mM ATP, (-)-pinacidil (0.5 mM) elicited a slight activation of KATP-channels in caudate nucleus, whereas (+)-pinacidil (0.5 mM) and lemakalim (0.3 mM) were ineffective. 7. Since our data indicate similar control by drugs and nucleotides of KATP-channels in the somata of spiny neurones and pancreatic beta-cells, we conclude that the high affinity sulphonylurea receptors of these tissues are probably closely related.

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.

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.

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.

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.

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.