Pharmacological Profile of U‐37883A,a Channel Blocker of Smooth Muscle‐Type ATP‐Sensitive K+ Channels

U‐37883A (PNU‐37883A, guanidine; 4‐morpholinecarboximidine‐N‐1‐adamantyl‐N′‐cyclohexyl hydrochloride) was originally developed as a potential diuretic with specific binding in kidney and vascular smooth muscle rather than in brain or pancreatic β cells. U‐37883A inhibits ATP‐sensitive K⁺ channels (K_ATP channels) in vascular smooth muscle at submicromolar concentrations whilst even at high concentrations (≥10 μM) it has no inhibitory effect at pancreatic, cardiac or skeletal K_ATP channels. Thus, it is generally thought that U‐37883A is a selective inhibitor of vascular smooth muscle K_ATP channels. Approximately one decade ago, K_ATP channels were cloned and found to consist of at least two subunits: an inwardly‐rectifying K⁺ channel six family (K_ir6.x; K_ir6.1 and K_ir6.2) which forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR.x; SUR1, SUR2A, and SUR2B) that accounts for several pharmacological properties. It is generally believed that different combinations of K_ir6.x and SUR.x determine the molecular properties of K_ATP channels. Thus, K_ir6.2/SUR1 channel represents the pancreatic β‐cell K_ATP channel, K_ir6.2/SUR2A channel is thought to represent the cardiac K_ATP channel, whereas K_ir6.1/SUR2B channel is likely to represent the vascular smooth muscle K_ATP channel. Recent molecular studies have shown that U‐37883A selectively suppresses the activity of recombinant K_ATP channels which contain K_ir6.1 subunits in the channel pore unit. It was thus thought that U‐37883A was a selective pharmacological tool which could be used to investigate the activity of vascular smooth muscle K_ATP channels. However, due to its multiple pharmacological actions on several ion channels and poor tissue selectivity, U‐37883A should not be viewed as a selective blocker of smooth muscle K_ATP channels.     

Selectivity of repaglinide and glibenclamide for the pancreatic over the cardiovascular KATP channels

Aims/hypothesis Sulfonylureas and glinides close beta cell ATP-sensitive K⁺ (K_ATP) channels to increase insulin release; the concomitant closure of cardiovascular K_ATP channels, however, leads to complications in patients with cardiac ischaemia. The insulinotrope repaglinide is successful in therapy, but has been reported to inhibit the recombinant K_ATP channels of beta cells, cardiocytes and non-vascular smooth muscle cells with similar potencies, suggesting that the (patho-)physiological role of the cardiovascular K_ATP channels may be overstated. We therefore re-examined repaglinide’s potency at and affinity for the recombinant pancreatic, myocardial and vascular K_ATP channels in comparison with glibenclamide.Methods K_ATP channel subunits (i.e. inwardly rectifying K⁺ channels [Kir6.x] and sulfonylurea receptors [SURx]) were expressed in intact human embryonic kidney cells and assayed in whole-cell patch-clamp and [³H]glibenclamide binding experiments at 37°C.Results Repaglinide and glibenclamide, respectively, were ≥30 and ≥1,000 times more potent in closing the pancreatic than the cardiovascular channels and they did not lead to complete inhibition of the myocardial channel. Binding assays showed that the selectivity of glibenclamide was essentially based on high affinity for the pancreatic SUR, whereas binding of repaglinide to the SUR subtypes was rather non-selective. After coexpression with Kir6.x to form the assembled channels, however, the affinity of the pancreatic channel for repaglinide was increased 130-fold, an effect much larger than with the cardiovascular channels. This selective effect of coexpression depended on the piperidino substituent in repaglinide.Conclusions/interpretation Repaglinide and glibenclamide show higher potency and efficacy in inhibiting the pancreatic than the cardiovascular K_ATP channels, thus supporting their clinical use.The first two authors listed contributed equally to this work.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

Facilitation of β-cell KATP channel sulfonylurea sensitivity by a cAMP analog selective for the cAMP-regulated guanine nucleotide exchange factor Epac

Clinical studies demonstrate that combined administration of sulfonylureas with exenatide can induce hypoglycemia in type 2 diabetic subjects. Whereas sulfonylureas inhibit β-cell K_ATP channels by binding to the sulfonylurea receptor-1 (SUR1), exenatide binds to the GLP-1 receptor, stimulates β-cell cAMP production, and activates both PKA and Epac. In this study, we hypothesized that the adverse in vivo interaction of sulfonylureas and exenatide to produce hypoglycemia might be explained by Epac-mediated facilitation of K_ATP channel sulfonylurea sensitivity. We now report that the inhibitory action of a sulfonylurea (tolbutamide) at K_ATP channels was facilitated by 2’-O-Me-cAMP, a selective activator of Epac. Thus, under conditions of excised patch recording, the dose-response relationship describing the inhibitory action of tolbutamide at human beta cell or rat INS-1 cell K_ATP channels was left-shifted in the presence of 2’-O-Me-cAMP, and this effect was abolished in INS-1 cells expressing a dominant-negative Epac2. Using an acetoxymethyl ester prodrug of an Epac-selective cAMP analog (8-pCPT-2'-O-Me-cAMP-AM), the synergistic interaction of an Epac activator and tolbutamide to depolarize INS-1 cells and to raise [Ca²⁺]_i was also measured. This effect of 8-pCPT-2'-O-Me-cAMP-AM correlated with its ability to stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis that might contribute to the changes in K^_ATP channel sulfonylurea-sensitivity reported here. On the basis of such findings, we propose that the adverse interaction of sulfonylureas and exenatide to induce hypoglycemia involves at least in part, a functional interaction of these two compounds to close K_ATP channels, to depolarize β-cells, and to promote insulin secretion.     

Ca2+-activated K+ channels in human smooth muscle cells of coronary atherosclerotic plaques and coronary media segments

The behavior of Ca²⁺-activated K⁺ channels of large conductance (BK_Ca) in smooth muscle cells, which were obtained from atherosclerotic plaque material (SMCP) and from media segments (SMCM) of human coronary arteries, were compared using the patch-clamp technique. Voltage-clamp protocols in cell-attached patches revealed the characteristic voltage-dependent activation of BK_Ca in both cell groups. Single-channel conduction was 216.4±16.7pS (n=6) in SMCP and 199.9±6.7pS (n=6) in SMCM in symmetrical 140 mMK⁺ solutions. Using outside-out patches, external perfusion with 500 M tetraethylammonium ions caused a typical flickery block of the unitary current. The selective BK_Ca channel inhibitor iberiotoxin (50 nM) effectively blocked BK_Ca channel activity. Comparing BK_Ca open-state probabilities (P₀) at +80 mV in cell-attached patches, a highly significant difference between SMCP (P₀=0.1438±0.1301; n=15) and SMCM (P₀=0.0093±0.0044; n=15; Kruskal-Wallis test, p<0.001) was found. In contrast to this finding, there was no significant difference in the open-state probability of BK_Ca between SMCP (P₀=0.0542±0.0237; n=9) and SMCM (P₀=0.0472±0.0218; n=10; p=n.s.) using inside-out patches. The results show an interesting difference in the behavior of large conductance Ca²⁺-activated K⁺ channel in SMCP compared to SMCM with a significantly higher channel activity in human smooth muscle cells obtained from coronary atherosclerotic plaque material. This finding may indicate an important functional role of BK_Ca channels in the development of atherosclerosis.     

Potent and selective activation of the pancreatic beta-cell type KATP channel by two novel diazoxide analogues

Aims/hypothesis We investigated the pharmacological properties of two novel ATP sensitive potassium (K_ATP) channel openers, 6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) and 6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414), on the cloned cardiac (Kir6.2/SUR2A), smooth muscle (Kir6.2/SUR2B) and pancreatic beta cell (Kir6.2/SUR1) types of K_ATP channel.Methods We studied the effects of these compounds on whole-cell currents through cloned K_ATP channels expressed in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes.Results In HEK 293 cells, NNC 55-0118 and NN414 activated Kir6.2/SUR1 currents with EC₅₀ values of 0.33 µmol/l and 0.45 µmol/l, respectively, compared with that of 31 µmol/l for diazoxide. Neither compound activated Kir6.2/SUR2A or Kir6.2/SUR2B channels expressed in oocytes, nor did they activate Kir6.2 expressed in the absence of SUR. Current activation was dependent on the presence of intracellular MgATP, but was not supported by MgADP.Conclusion/interpretation Both NNC 55-0118 and NN414 selectively stimulate the pancreatic beta-cell type of K_ATP channel with a higher potency than diazoxide, by interaction with the SUR1 subunit. The high selectivity and efficacy of the compounds could prove useful for treatment of disease states where inhibition of insulin secretion is beneficial.Abbreviations K_ATP channel ATP-sensitive potassium channel - SUR sulphonylurea receptor - KCO K⁺ channel opener - Kir inwardly rectifying K⁺ channel - TEVC two electrode voltage clamp - HEK293 cell Human Embryonic Kidney 293 cell     

Functional Communication Between Cardiac ATPSensitive K+ Channel and Na/K ATPase

K_ATP Channel and Na/K ATPase. Introduction: Functional interaction between K_ATP channel and Na/K ATPase was studied in single guinea pig ventricular myocytes because both membrane molecules are known to he involved in ischemic episodes. Methods and Results: K_ATP channel currents were recorded at 36°C by using whole cell, cell attached, inside-out, and open cell-attached modes of patch clamp techniques on enzymatically isolated ventricular myocytes. In the whole cell mode, ouabain (1 μM) reversibly inhibited the K_ATP currents induced by metabolic stress (ATP-free pipette solution and 1 mM NaCN), but not those activated by cromakalim (100 μM), a K_ATP channel opener. In the cell-attached mode, ouabain concentration dependently inhibited K_ATP, channel opening induced by metabolic suppression (5.5 μM 2-deoxyglucose and 1 mM CN). Half-inhibition concentration for ouabain was 21.0 ± 5.5 nM and the Hill coefficient was 0.8 ± 0.1 (n = 26). However, ouabain did not have an effect on the channel activity induced by cromakalim (100 μM). In the inside-out mode, ouabain applied to the internal side of membrane did not affect the channel. In the open cell-attached mode made by preincubation with streptolysin-0 (0.08 U/mL), the K_ATP channels were not activated by the metabolic inhibitors but were by reducing extracellular ATP concentrations, because subsarcolenimal ATP concentration could he controlled through tiny membrane holes. The channels thus activated were not suppressed by ouabain. Conclusion: The inhibition of Na/K ATPase by ouahain appeared to block the K_ATP channels by accumulating subsarcolemmal ATP caused by a decrease of the transition from ATP to ADP. In the presence of ischemic episodes, the administration of digitalis compounds may affect the opening of K_ATP channels, which is primarily protective against the development of irreversible myocardial damage.     

ATP-regulated potassium channels and voltage-gated calcium channels in pancreatic alpha and beta cells: similar functions but reciprocal effects on secretion

Closure of ATP-regulated K⁺ channels (K_ATP channels) plays a central role in glucose-stimulated insulin secretion in beta cells. K_ATP channels are also highly expressed in glucagon-producing alpha cells, where their function remains unresolved. Under hypoglycaemic conditions, K_ATP channels are open in alpha cells but their activity is low and only ~1% of that in beta cells. Like beta cells, alpha cells respond to hyperglycaemia with K_ATP channel closure, membrane depolarisation and stimulation of action potential firing. Yet, hyperglycaemia reciprocally regulates glucagon (inhibition) and insulin secretion (stimulation). Here we discuss how this conundrum can be resolved and how reduced K_ATP channel activity, via membrane depolarisation, paradoxically reduces alpha cell Ca²⁺ entry and glucagon exocytosis. Finally, we consider whether the glucagon secretory defects associated with diabetes can be attributed to impaired K_ATP channel regulation and discuss the potential for remedial pharmacological intervention using sulfonylureas.     

Suppressed Ca2+/CaM/CaMKII-dependent KATP channel activity in primary afferent neurons mediates hyperalgesia after axotomy

Painful axotomy decreases K_ATP channel current (IK_ATP) in primary afferent neurons. Because cytosolic Ca²⁺ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca²⁺–calmodulin (CaM)–Ca²⁺/CaM-dependent kinase II (CaMKII) regulates IK_ATP in large DRG neurons. Immunohistochemistry identified the presence of K_ATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200–positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK_ATP by ionomycin, a Ca²⁺ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K_ATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca²⁺-evoked K_ATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK_ATP by glybenclamide, a selective K_ATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K_ATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK_ATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via K_ATP channel inhibition. Unitary current conductance and sensitivity of K_ATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.     

Role of K+ATP Channels in Ischemic Preconditioning and Cardioprotection

Summary. Since the phenomenon of ischemic preconditioning was first described some 15 years ago, interest in strategies aimed at reducing infarct size has increased. During the past 10 years, investigations into the mechanism of ischemic preconditioning have clearly demonstrated the cardioprotective effect of K⁺ _ATP channel opening. Thus, K⁺ _ATP channel activation has been shown to be involved in this protection by a variety of stimuli, including a brief period of complete ischemia (classic ischemic preconditioning) or a partial coronary artery occlusion. In addition, ischemia in remote organs and nonischemic stimuli in the heart such as ventricular pacing, stretch, and heat stress also confer protection via K⁺ _ATP channel activation. Pharmacological agents that open K⁺ _ATP channels reduce infarct size, but K⁺ _ATP channel opening must occur prior to or early during the sustained infarct-producing coronary artery occlusion, while the degree and memory of cardioprotection are less than those produced by classic ischemic preconditioning. Although the exact mechanism by which K⁺ _ATP channel activation protects is still incompletely understood, recent studies indicate a role for the mitochondrial K⁺ _ATP channels. Before K⁺ _ATP channel opening can be employed in patients at increased risk of developing myocardial infarction (e.g., unstable angina), it is mandatory to determine whether tolerance (tachyphylaxia) occurs with repeated administration of K⁺ _ATP channel openers in a fashion similar to what occurs with ischemic preconditioning.     

Pancreatic islets from hypothalamic obese rats maintain K+ATP channel-dependent but not -independent pathways on glucose-induced insulin release process

One of the main features of obesity is hyperinsulinemia, which is related to insulin oversecretion. Glucose is by far the major physiological stimulator of insulin secretion. Glucose promotes an increase in the ATP/ADP ratio, which inactivates ATP-sensitive K⁺ channels (K⁺ _ATP) and induces beta cell depolarization with consequent calcium influx. Increased intracellular calcium concentration triggers insulin exocytosis. K⁺ _ATP channel function is important for K⁺ _ATP channel-dependent pathways involved in glucose-stimulated insulin secretion (GSIS). However, K⁺ _ATP channel-independent pathway has been identified and it has been found that this pathway sustains GSIS. Both pathways are critical to better GSIS control. GSIS was studied in pancreatic islets from hyperinsulinemic adult obese rats obtained by monosodium l-glutamate (MSG) neonatal treatment. Islets from MSG-obese rats were more glucose responsive than control ones. Diazoxide, a drug which maintains the K⁺ _ATP channels open without interfering with cell metabolism, blocked GSIS in islets from both groups. High extracellular potassium concentration plus diaz-oxide was used to study an alternative to the K⁺ _ATP channel pathway; in these conditions islets from MSG-obese rats did not respond, while islets from control animals showed enhanced GSIS. Results indicate that MSG-obese rats oversecreted insulin, even though the K⁺ _ATP channel-independent pathway is impaired in their beta cells.     

Disopyramide and its metabolite enhance insulin release from clonal pancreatic beta-cells by blocking K(ATP) channels

In an insulin-secreting pancreatic -cell line (MIN6), insulin release was caused by disopyramide, an antiarrhythmic drug with Na-channel blocking action, and its main metabolite mono-isopropyl disopyramide (MIP). Insulin secretion, measured as immunoreactive insulin (IRI), was accelerated to 265.7% of the control by disopyramide and to 184.4% by MIP, with half-effective concentrations (EC₅₀) of 30.9 ± 1.5 M and 92.4 ± 2.2 M. We tested the possibility that these drugs induce insulin release by inhibiting ATP-sensitive K⁺ (K_ATP) channels of MIN6 cells. In the cell-attached or ATP-free inside-out mode with patch membranes on MIN6 cells, K-selective channels were recorded with unitary conductance of 70.5 ± 3.5 pS (150 mM external K⁺ ions at room temperature). The channels were concluded to be MIN6-K_ATP channels because they were closed by extracellular high glucose (11.0 mM) or glibenclamide (200 nM) and were reversibly activated by diazoxide (50 M). In the inside-out patch mode, they were inhibited by micromolar ATP. In both cell-attached and insideout mode, disopyramide and MIP inhibited single MIN6-K_ATP channels. In the inside-out mode, they produced a dose-dependent inhibition of channel activity: the half-blocking concentrations (IC⁵⁰) were 4.8 ± 0.2 M for disopyramide and 40.4 ± 3.1 M for MIP. It was therefore concluded that both agents exert insulinotrphic effect through the inhibition of membrane K_ATP channels in MIN6 cells.     

HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels

Murine ventricular and atrial ATP-sensitive potassium (K_ATP) channels contain different sulfonylurea receptors (ventricular K_ATP channels are Kir6.2/SUR2A complexes, while atrial K_ATP channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K_ATP channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K_ATP channels over atrial K_ATP channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as ⁸⁶Rb⁺ efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K_ATP currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K_ATP currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits ⁸⁶Rb⁺ effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K_ATP channels.     

Sulphonylurea action revisited: the post-cloning era

Hypoglycaemic agents such as sulphonylureas and the newer group of "glinides" stimulate insulin secretion by closing ATP-sensitive potassium (K_ATP) channels in pancreatic beta cells, but have varying cross-reactivity with related channels in extrapancreatic tissues such as heart, vascular smooth and skeletal muscle. Experiments on the structure-function relationships of recombinant K_ATP channels and the phenotypes of mice deficient in different K_ATP channel subunits have provided important insights into the mechanisms underlying sulphonylurea selectivity, and the potential consequences of K_ATP channel blockade outside the pancreatic beta cell. The different pharmacological properties of K_ATP channels from beta cells compared with those from cardiac, smooth and skeletal muscle, are accounted for by the expression of alternative types of sulphonylurea receptor, with non-identical drug binding sites. The sulphonylureas and glinides are found to fall into two groups: one exhibiting selectivity for beta cell sulphonylurea receptors (SUR1), and the other blocking cardiovascular and skeletal muscle sulphonylurea receptors (SUR2) with potencies similar to their action on SUR1. In seeking potential side effects of K_ATP channel inhibitors in humans, it is essential to take these drug differences into account, along with the probability (suggested by the studies on K_ATP channel knockout mice) that the effects of extrapancreatic K_ATP channel inhibition might be either subtle or rare. Further studies are still required before a final decision can be made on whether non-selective agents are appropriate for the therapy of Type 2 diabetes.Abbreviations K_ATP channel ATP sensitive potassium channel - Kir inwardly-rectifying potassium channel - SUR sulphonylurea receptor - TMD transmembrane domain - NBD nucleotide binding domain - CL cytoplasmic linker - CHI congenital hyperinsulinism     

Gliclazide produces high-affinity block of KATP channels in mouse isolated pancreatic beta cells but not rat heart or arterial smooth muscle cells

Aims/hypothesis: Sulphonylureas stimulate insulin secretion by closing ATP-sensitive potassium (K_ATP) channels in the pancreatic beta-cell membrane. K_ATP channels are also found in other tissues, including heart and smooth muscle, where they link cellular metabolism to electrical activity. The sulphonylurea gliclazide blocks recombinant beta-cell K_ATP channels (Kir6.2/SUR1) but not heart (Kir6.2/SUR2A) or smooth muscle (Kir6.2/SUR2B) K_ATP channels with high potency. In this study, we examined the specificity of gliclazide for the native (as opposed to recombinant) K_ATP channels in beta cells, heart and smooth muscle. Methods: The action of the drug was studied by whole-cell current recordings of native K_ATP channels in isolated pancreatic beta-cells and myocytes from heart and smooth muscle. Results: Gliclazide blocked whole-cell beta-cell K_ATP currents with an IC ₅₀ of 184 ± 30 nmol/l (n = 6–10) but was much less effective in cardiac and smooth muscle (IC ₅₀s of 19.5 ± 5.4 μmol/l (n = 6–12) and 37.9 ± 1.0 μmol/l (n = 5–10), respectively). In all three tissues, the action of the drug on whole-cell K_ATP currents was rapidly reversible. In inside-out patches on beta-cells, gliclazide (1 μmol/l) produced a maximum of 66 ± 13 % inhibition (n = 5), compared with more than 98 % block in the whole-cell configuration. Conclusion/interpretation: Gliclazide is a high-potency sulphonylurea which shows specificity for the pancreatic beta-cell K_ATP channel over heart and smooth muscle. In this respect, it differs from glibenclamide. The difference in the maximal block observed in the excised patch and whole-cell recordings from beta-cells, may be due to the absence of intracellular Mg-nucleotides in the excised patch experiments. [Diabetologia (2001) 44: 1019–1025] Received: 21 March 2001 and in revised form: 30 April 2001     

Reduced sensitivity of dihydroxyacetone on ATP-sensitive K+ channels of pancreatic beta cells in GK rats

Summary In the GK (Goto-Kakizaki) rat, a genetic model of non-insulin-dependent diabetes mellitus, glucose-induced insulin secretion is selectively impaired. In addition, it has been suggested by previous studies that impaired glucose metabolism in beta cells of the GK rat results in insufficient closure of ATP-sensitive K⁺ channels (K_ATP channels) and a consequent decrease in depolarization, leading to a decreased insulin release. We have recently reported that the site of disturbed glucose metabolism is probably located in the early stages of glycolysis or in the glycerol phosphate shuttle. In the present study, in order to identify the impaired metabolic step in diabetic beta cells, we have investigated insulin secretory capacity by stimulation with dihydroxyacetone (DHA), which is known to be directly converted to DHA-phosphate and to preferentially enter the glycerol phosphate shuttle. In addition, using the patch-clamp technique, we also have studied the sensitivity of DHA on the K_ATP channels of beta cells in GK rats. The insulin secretion in response to 5 mmol/l DHA with 2.8 mmol/l glucose was impaired, and DHA sensitivity of the K_ATP channels was reduced in beta cells of GK rats. From these results, we suggest that the intracellular site responsible for impaired glucose metabolism in pancreatic beta cells of GK rats is located in the glycerol phosphate shuttle.Abbreviations DHA Dihydroxyacetone - K_ATP channel ATP-sensitive K⁺ channel - GK rat Goto-Kakizaki rat - KRBB Krebs Ringer bicarbonate buffer - BSA bovine serum albumin - NIDDM non-insulin-dependent diabetes     

A soluble epoxide hydrolase inhibitor--8-HUDE increases pulmonary vasoconstriction through inhibition of K(ATP) channels

Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, are endogenously produced epoxides that act as substrates for the soluble epoxide hydrolase (sEH). Recent studies indicate that EETs increase the tension of rat pulmonary arteries (PAs), and inhibition of sEH augments hypoxic pulmonary vasoconstriction. However, the mechanisms underlying the proconstrictive effects of sEH inhibitors in pulmonary artery smooth muscle cells (PASMCs) are unclear. In the present study, we used a sEH inhibitor, 12-(3-hexylureido) dodec-8-enoic acid (8-HUDE), to examine the ionic mechanisms underlying the constriction of PAs. 8-HUDE increased the tension of rat PAs to 145% baseline in a manner which was effectively eliminated by 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ (K_ATP) channels. Whole cell currents of HEK cells transfected with Kir6.1 or SUR2B were activated by K_ATP channel opener pinacidil, inhibited by K_ATP channel inhibitor glibenclamide or inhibited by 8-HUDE in a concentration-dependent manner with an IC50 value of 40 uM. In addition, 8-HUDE inhibited the expression of Kir6.1 and SUR2B at both mRNA and protein level in rat PASMCs. These observations suggest that 8-HUDE exerts acute effects on K_ATP channel activity as well as subacute effects through decreased channel expression, and these effects are, at least in part, via the Kir6.1/SUR2B channel.     

Long-Chain CoA esters activate human pancreatic beta-cell K<Subscript>ATP</Subscript> channels: potential role in Type 2 diabetes

Aims/hypothesis The ATP-regulated potassium (K_ATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the K_ATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent K_ATP channel activators in rodents, as studied in inside-out patches.Methods In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique.Results Human K_ATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg²⁺ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC ₅₀ of 0.7±0.1 µmol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell K_ATP channel currents increased approximately six-fold following addition of 1 µmol/l LC-CoA ester. The presence of 1 µmol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5±0.2 nS to 2.5±1.3 nS.Conclusion/interpretation Taken together, these results show that LC-CoA esters are potent activators of the K_ATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate K_ATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.Abbreviations K_ATP ATP sensitive potassium channel - LC-CoA Long-chain Co-enzyme A ester     

A potent diazoxide analogue activating ATP-sensitive K+ channels and inhibiting insulin release

Aims/hypothesis. To characterise the effects of BPDZ 73 (7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), a newly synthesised diazoxide analogue, on insulin secretory cells.¶Methods. Measurements of ⁸⁶Rb, ⁴⁵Ca outflow, membrane potential, [Ca²⁺]_i, insulin release in secretory cells as well as measurements of smooth muscle contractile activity and glycaemia were carried out.¶Results. The analogue BPDZ 73 induced a dose-dependent decrease in insulin output. The IC₅₀ value averaged 0.73 ± 0.05 μmol/l. The drug increased the rate of ⁸⁶Rb (⁴²K substitute) outflow from perifused rat pancreatic islets. This effect was inhibited by glibenclamide, a K_ATP channel blocker. Measurements of DiBAC₄(3) fluorescence further indicated that BPDZ 73 hyperpolarised the insulin secreting cells. It also decreased ⁴⁵Ca outflow from pancreatic islets perifused throughout in the presence of 16.7 mmol/l glucose and extracellular Ca²⁺. By contrast, the drug did not affect the increase in ⁴⁵Ca outflow mediated by K⁺ depolarisation. In single beta cells, BPDZ 73 inhibited the glucose-induced but not the K⁺-induced rise in [Ca²⁺]_i. Moreover, in Wistar rats, i. p. injection of BPDZ 73 provoked a considerable increase in blood glucose concentration whereas diazoxide induced a modest rise in glycaemia. Lastly, the vasorelaxant properties of BPDZ 73 were slightly less pronounced than those of diazoxide.¶Conclusion/interpretation. The inhibitory effect of BPDZ 73 on the insulin-releasing process results from the activation of K_ATP channels with subsequent decrease in Ca²⁺ inflow and [Ca²⁺]_i. The drug seems to be a K_ATP channel opener, more potent and more selective than diazoxide for insulin secreting cells. [Diabetologia (2000) 43: 723-732]     

Glucose‐stimulated insulin secretion: A newer perspective

Existing concepts and models for glucose‐stimulated insulin secretion (GSIS) are overviewed and a newer perspective has been formulated toward the physiological understanding of GSIS. A conventional model has been created on the basis of in vitro data on application of a square wave high glucose in the absence of any other stimulatory inputs. Glucose elicits rapid insulin release through an adenosine triphosphate‐sensitive K⁺ channel (K_ATP channel)‐dependent mechanism, which is gradually augmented in a K_ATP channel‐independent manner. Biphasic GSIS thus occurs. In the body, the β‐cells are constantly exposed to stimulatory signals, such as glucagon‐like peptide 1 (GLP‐1), parasympathetic inputs, free fatty acid (FFA), amino acids and slightly suprathreshold levels of glucose, even at fasting. GLP‐1 increases cellular cyclic adenosine monophosphate, parasympathetic stimulation activates protein kinase C, and FFA, amino acids and glucose generate metabolic amplification factors. Plasma glucose concentration gradually rises postprandially under such tonic stimulation. We hypothesize that these stimulatory inputs together make the β‐cells responsive to glucose independently from its action on K_ATP channels. Robust GSIS in patients with a loss of function mutation of the sulfonylurea receptor, a subunit of K_ATP channels, is compatible with this hypothesis. Furthermore, pre‐exposure of the islets to an activator of protein kinase A and/or C makes β‐cells responsive to glucose in a K_ATP channel‐ and Ca²⁺‐independent manner. We hypothesize that GSIS occurs in islet β‐cells without glucose regulation of K_ATP channels in vivo, for which priming with cyclic adenosine monophosphate, protein kinase C and non‐glucose nutrients are required. To understand the physiology of GSIS, comprehensive integration of accumulated knowledge is required.