Antidiabetic Sulfonylureas Research Articles

ATP-sensitive potassium channel and hormone/neuropeptide

ATP-sensitive potassium channels (KATP) are the ion channels which are closely associated with cellular metabolism. A number of chemical compounds which block KATP facilitate the release of hormones or neuropeptides. For example, KATP-blocking agents such as antidiabetic sulfonylureas and imidazolines stimulate insulin secretion from pancreatic beta-cells by decreasing KATP activity. On the other hand, so-called potassium channel openers, KATP-activating drugs which constitute a chemically diverse group of compounds, inhibit growth hormone secretion from anterior pituitary cells and release of gamma-aminobutylic acid from substantia nigra. Several endogenous substances also modulate release of hormone or neuropeptide by affecting KATP activity. Acetylcholine and histamine stimulate the release of endothelium-derived hyperpolarizing factor, which activates KATP in the plasma membrane of vascular smooth muscle cells. Both galanin and somatostatin inhibit insulin release from pancreatic beta-cells by opening KATP through the activation of G-protein. Glucagon-like peptide-1[7-36], which stimulates insulin secretion by indirectly blocking KATP in beta-cells, shows antidiabetic effects in patients with non-insulin-dependent diabetes mellitus. Endosulphine, an endogenous inhibitor of KATP, stimulates insulin secretion from pancreatic beta-cells. Accumulating knowledge of the modulation and function of KATP would help our understanding of the regulation and physiological role of hormones and neuropeptides.

Therapeutic potential of potassium channel activators in coronary heart disease.

Potassium channel activators have the ability to open potassium channels in a variety of cells. Since most of their effects are antagonized by antidiabetic sulfonylureas, the ATP-sensitive potassium channel is their likely target. Opening of potassium channels leads to hyperpolarization of the surface membrane with consequent closure of voltage-dependent ion channels and reduction of free intracellular calcium ions. Currently available potassium channel activators, including aprikalim, bimakalim, cromakalim, emakalim, nicorandil, pinacidil etc., display a high affinity for potassium channels of vascular smooth muscle. Vasodilation and a reduction in systemic vascular resistance are their prominent pharmacological effects. Coronary and cerebral arteries are highly sensitive to potassium channel activator-induced dilation. Apart from treatment of hypertension, potassium channel activators appear to have therapeutic potential in coronary heart disease. They reduce cardiac afterload, increase native and collateral coronary blood flow and reduce the size of experimental myocardial infarcts. This last effort cannot be satisfactorily explained entirely by haemodynamic or coronary vascular actions of potassium channel activators and a cardioprotective effect is postulated. For these drugs ischaemia-induced and activator-induced opening of cardiac ATP-sensitive potassium channels appear to work in concert. Nicorandil combines the pharmacological properties of an organic nitrate with those of potassium channel activators. Experimental and clinical results characterize nicorandil as a unique and promising drug for the treatment of coronary heart disease.

Regional distribution of sulfonylurea receptors in the brain of rodent and primate

Glibenclamide, one of the most potent antidiabetic sulfonylureas, inhibits the activity of ATP-sensitive K + channels in the pancreas as well as in the brain through its binding to specific receptors. Quantitative autoradiography was used to localize such receptors in the brain of rat, mouse, guinea-pig and marmoset, using [ 3H]glibenclamide as radioligand. In all four species, specific glibenclamide binding sites were found to be heterogeneously distributed. The highest densities were in the cerebral cortex, the molecular layer of the cerebellar cortex, the thalamus and the caudate-putamen. The globus pallidus and the substantia nigra were highly labelled in rat and mouse but poorly labelled in guinea-pig and marmoset. The distribution of glibenclamide binding sites in the hippocampus was different between the rodents and marmoset; in rodents, most binding sites were distributed in the fascia dentata and the CA3-CA4 fields of Ammon's horn, contrasting with a very homogeneous distribution in all subfields of the marmoset hippocampus. In conclusion, we demonstrate that primate brain contains specific binding sites for [ 3H]glibenclamide with a distribution not exactly similar to that in rodent brain.

A class-independent drug screen in forensic toxicology using a photodiode array detector.

A class-independent drug screen, that is suitable for use in clinical and forensic toxicology and uses gradient-elution high-performance liquid chromatography and photodiode array detection, is presented. The assay is capable of detecting and identifying therapeutic and toxic amounts of barbiturates, anti-convulsants, diuretics, non-steroidal anti-inflammatory drugs, sulfonylurea anti-diabetic drugs, theophylline, and analgesic drugs. The assay has been successfully used to screen over 1000 postmortem blood specimens.

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.

Effectors of ATP-sensitive K + channels inhibit the regulatory effects of somatostatin and GH-releasing factor on growth hormone secretion

Somatostatin inhibition of growth hormone (GH) secretion from adenohypophysis cells in culture was antagonized by the antidiabetic sulfonylurea glipizide (K 0.5 = 10 ± 5 nM). Although all cells that hyperpolarize with somatostatin have ATP-sensitive K + channels, the antagonistic actions of the hormone and of the antidiabetic drug are due to effects on different types of K + channels. Diazoxide, an opener of ATP-sensitive K + channels, abolished the increase of intracellular Ca 2+ provoked by growth hormone releasing factor (GRF) and induced inhibition of GRF stimulated GH secretion (K 0.5 = 138 μM). This inhibition by diazoxide was largely suppressed by glipizide which blocked the ATP-sensitive K + channels opened by diazoxide. In summary, hormonal activation of GH secretion is inhibited by openers of ATP-sensitive K + channels, while hormonal inhibition of GH secretion is suppressed by blockers of ATP-sensitive K + channels.

Endosulfine, an endogenous peptidic ligand for the sulfonylurea receptor: purification and partial characterization from ovine brain.

Antidiabetic sulfonylureas act through receptors coupled to ATP-dependent potassium channels. Using the binding of [3H]glibenclamide, a highly potent sulfonylurea, to rat brain membranes to follow the purification procedure, we extracted from ovine brain, purified, and partially characterized two peptides that are endogenous ligands for the central nervous system sulfonylurea receptors. These peptides, referred to as alpha and beta endosulfine, differ by their isoelectric points, the beta form being more basic. Each form of endosulfine is recognized equally by the sulfonylurea receptors from the central nervous system and from insulin-secreting beta cells. In the same concentration range that is active on the receptors, beta endosulfine releases insulin from a beta-cell line. Endosulfine is a good candidate for being implicated in the physiology of beta cells and their disorders (e.g., type II diabetes) and in certain pathologies related to modifications of ion fluxes.

Specificity of photolabeling of beta-cell membrane proteins with an 125I-labeled glyburide analog.

The interaction between sulfonylureas and membrane proteins from a hamster insulin-secreting tumor (HIT) cell line has been examined. Four HIT cell membrane proteins were covalently linked to an 125I-labeled glyburide analog by photolabeling. Three photolabeled polypeptides of M(r) 65,000, 55,000, and 30,000 were identified as low affinity "glyburide receptors." These proteins appear to be of similar abundance, when quantitated by photolabeling, with half-maximal displacements (Ki values) by glyburide, glipizide, and tolbutamide in the low micromolar range. The glyburide analog is more tightly bound to a M(r) 140,000 protein with dissociation constants, determined by filtration binding assays and by photolabeling, of 7 and 9.0 nM, respectively. The labeled analog was displaced from the M(r) 140,000 protein by glyburide, glipizide and tolbutamide with Ki values of 3.3 nM, 103 nM, and 25 microM, respectively, as estimated by photolabeling. Optimal conditions established for visualizing the M(r) 140,000 band on autoradiograms prepared after UV cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis include irradiating the radioligand-receptor complex at 1.5 J/cm2 at 312 nm, followed by heating samples in pH 9.0 sodium dodecyl sulfate-gel sample buffer. With receptor sites partially occupied (5 nM radioligand), approximately 0.75% of the protein is photocoupled to the radioligand and visualized by autoradiography. Our results confirm that the M(r) 140,000 polypeptide contains the beta-cell high affinity glyburide binding site and show that the second generation sulfonylurea antidiabetic drugs have a selective increase in affinity for this receptor.

ATP/ADP binding sites are present in the sulfonylurea binding protein associated with brain ATP-sensitive K+ channels.

Covalent labeling of nucleotide binding sites of the purified sulfonylurea receptor has been carried out with alpha-32P-labeled oxidized ATP. The main part of 32P incorporation is in the 145-kDa glycoprotein that has been previously shown to be the sulfonylurea binding protein (Bernardi et al., 1988). ATP and ADP protect against this covalent labeling with K0.5 values of 100 microM and 500 microM, respectively. Non-hydrolyzable analogs of ATP also inhibit 32P incorporation. Interactions between nucleotide binding sites and sulfonylurea binding sites have then been observed. AMP-PNP, a nonhydrolyzable analog of ATP, produces a small inhibition of [3H]glibenclamide binding (20-25%) which was not influenced by Mg2+. Conversely, ADP, which also produced a small inhibition (20%) in the absence of Mg2+, produced a large inhibition (approximately 80%) in the presence of Mg2+. This inhibitory effect of the ADP-Mg2+ complex was observed with a K0.5 value of 100 +/- 40 microM. All the results taken together indicate that ATP and ADP-Mg2+ binding sites that control the activity of KATP channels are both present on the same subunit that bears the receptors for antidiabetic sulfonylureas.

ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids.

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.

Antidiabetic sulfonylureas relax isolated rabbit coronary arteries

Antidiabetic sulfonylureas completely relaxed isolated rabbit coronary arteries contracted by prostaglandin F 2α. The order of potency was glibenclamide (EC 50 = 4.75 μM) > glipizide = glibornuride = tolbutamide = chlorpropamide. The drugs also relaxed the contractions induced by 30 mM K + but much less potently. The effectiveness of the drugs as vascular smooth muscle relaxants did not correlate with their ability to antagonize the vasorelaxant action of cromakalim. Sulfonylurea-induced vasorelaxation probably involves mechanisms other than an interaction with ATP-regulated K + channels.

Two different types of channels are targets for potassium channel openers in Xenopus oocytes

K + channel openers elicit K + currents in follicle-enclosed Xenopus oocytes. The most potent activators are the pinacidil derivatives P1075 and P1060. The rank order of potency to activate K + currents in follicle-enclosed oocytes was: P1075 (K 0.5: 5 μM)>P1060 (K 0.5: 12 μM)> BRL38227 (lemakalim) (K 0.5:77 μM)>RP61419 (K 0.5:100 μM)>(−)pinacidil (K 0.5:300 μM). Minoxidil sulfate, nicorandil. RP49356 and diazoxide were ineffective. Activation by the K + channel openers could be abolished by the antidiabetic sulfonylurea glibenclamide. It was not affected by the blocker of the Ca 2+-activated K + channels charybdotoxin. The various K + channel openers failed to activate glibenclamide-sensitive K + channels in defolliculated oocytes, but BRL derivatives (K 0.5 for BRL38226 is 150 μM) and RP61419 inhibited a background current. The channel responsible for this background current is K + permeable but not fully selective for K +. It is resistant to glibenclamide. It is inhibited by Ba 2+, 4-aminopyridine. Co 2+,.Ni 2+ and La 3+.

Hormone-regulated K+ channels in follicle-enclosed oocytes are activated by vasorelaxing K+ channel openers and blocked by antidiabetic sulfonylureas.

Follicular oocytes from Xenopus laevis contain K+ channels activated by members of the recently recognized class of vasorelaxants that include cromakalim and pinacidil and blocked by antidiabetic sulfonylureas, such as glibenclamide. These channels are situated on the adherent follicular cells and are not present in denuded oocytes. Cromakalim-activated K+ channels are also activated by increases in intracellular cAMP, and cAMP-activated K+ channels are blocked by glibenclamide. Although cromakalim and cAMP effects are synergistic, cromakalim activation of K+ channels is drastically reduced or abolished by treatments that stimulate protein kinase C (e.g., muscarinic effectors, phorbol esters). Gonadotropins, known to play an essential role in ovarian physiology, also activate cromakalim and sulfonylurea-sensitive K+ channels. Follicular oocytes constitute an excellent system for studying regulation of cromakalim-sensitive K+ channels that are important in relation to a variety of disease processes, such as cardiovascular dysfunction and asthma, as well as brain function.

Brain ischemia alters the density of binding sites for glibenclamide, a specific blocker of ATP-sensitive K + channels

Transient ischemia in normoglycemic animals leads to delayed neuronal damage which is confined to selectively vulnerable regions. In at least one of these, the CA1 sector of the hippocampus, cell death is preceded by neuronal hyperactivity, presumed to be caused by loss of inhibitory control. Hyperglycemic subjects develop postischemic seizures, andshow enhanced damage. The ATP-sensitive K + channel, which may be important in inhibitory control, is the target of antidiabetic sulfonylureas. We determined densities of sulfonylurea binding sites in rat brain after forebrain ischemia. Normoglycemic animals showed a decrease of glibenclamide receptor binding in the CA3 field, hilus and dentate gyrus of the hippocampus after 1 day of recovery. After 4 days of recovery, levels of sulfonylurea binding sites decreased mainly in the CA1 field and in the hilus, as well as in the substantia nigra. After 1 day of recovery, hyperglycemic animals did not show any significant variations of densities of sites compared to control animals. It is proposed that reduction of inhibitory control by ATP-senstivve K + channels may be associated with delayed neuronal death.

Sulfonylurea binding sites associated with ATP-regulated K + channels in the central nervous system: autoradiographic analysis of their distribution and ontogenesis, and of their localization in mutant mice cerebellum

The localization of a putative ATP-regulated K + channel in normal rat and neurological mutant mice was studied by light microscopic quantitative autoradiography using a tritiated glibenclamide, an antidiabetic sulfonylurea. Glibenclamide binding sites presented a heterogeneous distribution in the rat central nervous system. Their density was particularly important in substantia nigra reticulata, septohippocampal nucleus, globus pallidus, neocortex, molecular layer of cerebellum, CA3 field and dentate gyrus of hippocampus. Conversely hypothalamic areas, medulla oblongata and spinal cord contained only low amounts of glibenclamide receptors. The ontogenesis of sulfonylurea binding sites was a postnatal phenomenon and seemed to correlate with the maturation of neuronal connectivity. In the cerebellum of neurological mutant mice, the autoradiographic patterns were different to that of wild-type cerebellum. In particular, in the molecular layer of weaver cerebellum, a decrease of 82% of binding site density suggested a presynaptic position of glibenclamide receptors in parallel fibers.

K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.

Glucose, Sulfonylureas, and Neurotransmitter Release: Role of ATP-Sensitive K + Channels

Sulfonylurea-sensitive adenosine triphosphate (ATP)-regulated potassium (KATP) channels are present in brain cells and play a role in neurosecretion at nerve terminals. KATP channels in substantia nigra, a brain region that shows high sulfonylurea binding, are inactivated by high glucose concentrations and by antidiabetic sulfonylureas and are activated by ATP depletion and anoxia. KATP channel inhibition leads to activation of gamma-aminobutyric acid (GABA) release, whereas KATP channel activation leads to inhibition of GABA release. These channels may be involved in the response of the brain to hyper- and hypoglycemia (in diabetes) and ischemia or anoxia.

Reduction of ischemic K+ loss and arrhythmias in rat hearts. Effect of glibenclamide, a sulfonylurea.

Glibenclamide, one of the antidiabetic sulfonylureas, is known to block ATP-dependent K+ channels. We used this drug to determine to what extent K+ loss from acutely ischemic myocardium is mediated via these channels. We also investigated whether glibenclamide would influence ischemic arrhythmias. Isolated rat hearts rendered globally ischemic showed no correlation between early lactate and K+ efflux rates. Cumulative K+ loss during 11 minutes of global ischemia (0.5 ml min-1 g-1) was reduced, from 3.2 +/- 0.3 to 2.5 +/- 0.1 mueq/g (p less than 0.025) by 1 microM glibenclamide and from 3.3 +/- 0.2 to 1.9 +/- 0.2 mueq/g (p less than 0.005) by 10 microM glibenclamide, while lactate efflux was unaltered by the drug. Glibenclamide also exhibited potent antifibrillatory activity, abolishing irreversible ventricular fibrillation during regional ischemia (0/6 vs. 5/6 controls; p less than 0.02) and during global ischemia (0/7 vs. 9/9 controls; p less than 0.01). Heart rate, coronary flow rate, peak systolic pressure, and myocardial oxygen consumption were unaltered by the drug (1 microM). Similarly, glibenclamide (1 microM) did not alter myocardial ATP, phosphocreatine or lactate content, or glucose utilization. Ventricular fibrillation threshold during normoxia was also unaltered by glibenclamide (1 microM). We conclude that K+ loss during acute myocardial ischemia is mediated partly by ATP-dependent K+ channels, and not by a tightly coupled co-efflux with anionic lactate.

Antidiabetic sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices

The distribution of antidiabetic sulfonylurea ([ 3H]glibenclamide) binding sites is heterogeneous in rat brain. Pyramidal and extrapyramidal motor system contain the highest densities of sites, particularly in the substantia nigra and in the globus pallidus. Only low levels are present in the hypothalamic nuclei and the main medulla oblongata regions. In hippocampal formation the stratum lucidum and the stratum lacunosum moleculare of CA3 show an important density of glibenclamide binding sites. Electrophysiological studies with hippocampal slices show that glibenclamide blocks hyperpolarization induced by anoxia, suggesting the involvement of adenosine triphosphate-sensitive K + channel in this early hyperpolarization event.

Antidiabetic sulfonylureas control action potential properties in heart cells via high affinity receptors that are linked to ATP-dependent K+ channels.

Both avian and mammalian heart cells have high affinity receptors for antidiabetic sulfonylureas. The biochemical identification of these receptors has been carried out with [3H]glibenclamide. The Kd values for the most potent sulfonylureas, such as glibenclamide itself, are in the nanomolar range. Comparative studies of structure-function relationships indicate high similarities of binding properties between the sulfonylurea receptors in cardiac cells and insulinoma cells, respectively. The duration of the action potential of guinea pig cardiac cells was drastically reduced by decreasing intracellular ATP concentrations by perfusion or by blockade of oxidative phosphorylation. Glibenclamide was found to restore normal or nearly normal action potential properties in [ATP]in-depleted cardiac cells. Single channel recording using the patch-clamp technique has shown that this effect is associated with high affinity blockade of ATP-sensitive K+ channels by sulfonylureas.