Angiotensin II type 1 receptor, but no type 2 receptor, interferes with the insulin-induced nitric oxide production in HUVECs

Objective

Two subtypes of angiotensin II (ATII) receptor have been defined on the basis of their differential pharmacological and biochemical properties: ATII-type1 receptors (AT₁-R) and ATII-type2 receptors (AT₂-R). It has been hypothesized that part of the protective effects on the cardiovascular system of AT₁-R blockers is mediated by an ATII-mediated overstimulation of AT₂-R.We hypothesized that the inhibition of AT₁-R has a stronger impact on insulin-induced nitric oxide (NO) production than ATII-mediated overstimulation of AT₂-R. Therefore we studied the effect of the inhibition of AT₁-R and AT₂-R on ATII-mediated actions in Human Umbilical Vein Endothelial Cells (HUVECs).

Methods

We analyzed the phosphorylation state of IRS1 at Ser⁶¹⁶ and Ser³¹² and on tyrosines after preincubation with PD123319, an inhibitor of AT₂-R, alone and in combination whit losartan, an inhibitor of AT₁-R. In addition we measured eNOS and Akt activation through the evaluation of their phosphorylation at Ser¹¹⁷⁷ and Ser⁴⁷³ sites respectively.

Results

ATII induces IRS-1 phosphorylation at Ser³¹² and Ser⁶¹⁶ through the activation of JNK and ERK 1/2, resulting in the inhibition of the insulin-induced phosphorylation of IRS1 tyrosines, Akt and eNOS. Treatment of HUVECs with AT₁-R inhibitor restored the insulin signaling leading to NO production, whereas AT₂-R inhibitor did not have effects on NO production in presence of ATII.

Conclusion

Our results demonstrate that in presence of AT₁-R antagonist, the AT₂-R blockage does not modify the effect obtained with the AT₁-R inhibition alone. Therefore, a possible positive role of an AT₂-R overstimulation in condition of AT₁-R antagonism seems to be irrelevant.     

H2 Mediates Cardioprotection Via Involvements of KATP Channels and Permeability Transition Pores of Mitochondria in Dogs

Purpose

Inhalation of hydrogen (H₂) gas has been shown to limit infarct size following ischemia-reperfusion injury in rat hearts. However, H₂ gas-induced cardioprotection has not been tested in large animals and the precise cellular mechanism of protection has not been elucidated. We investigated whether opening of mitochondrial ATP-sensitive K+ channels (mK_ATP) and subsequent inhibition of mitochondrial permeability transition pores (mPTP) mediates the infarct size-limiting effect of H₂ gas in canine hearts.

Methods

The left anterior descending coronary artery of beagle dogs was occluded for 90?min followed by reperfusion for 6?h. Either 1.3% H₂ or control gas was inhaled from 10?min prior to start of reperfusion until 1?h of reperfusion, in the presence or absence of either 5-hydroxydecanoate (5-HD; a selective mK_ATP blocker), or atractyloside (Atr; a mPTP opener).

Results

Systemic hemodynamic parameters did not differ among the groups. Nevertheless, H₂ gas inhalation reduced infarct size normalized by risk area (20.6?±?2.8% vs. control gas 44.0?±?2.0%; p?2 gas (42.0?±?2.2% with 5-HD and 45.1?±?2.7% with Atr; both p?2 group). Neither Atr nor 5-HD affected infarct size per se. Among all groups, NAD content and the number of apoptotic and 8-OHdG positive cells was not significantly different, indicating that the cardioprotection afforded by H₂ was not due to anti-oxidative actions or effects on the NADH dehydrogenase pathway.

Conclusions

Inhalation of H₂ gas reduces infarct size in canine hearts via opening of mitochondrial K_ATP channels followed by inhibition of mPTP. H₂ gas may provide an effective adjunct strategy in patients with acute myocardial infarction receiving reperfusion therapy.     

Mice expressing a human K(ATP) channel mutation have altered channel ATP sensitivity but no cardiac abnormalities

Aims/hypothesis  

Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K_ATP) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay—a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K_ATP channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K_ATP channel ATP sensitivity.     

Continuous hypothalamic KATP activation blunts glucose counter-regulation in vivo in rats and suppresses KATP conductance in vitro

Aims/hypothesis

Acute systemic delivery of the sulfonylurea receptor (SUR)-1-specific ATP-sensitive K⁺ channel (K_ATP) opener, NN414, has been reported to amplify glucose counter-regulatory responses (CRRs) in rats exposed to hypoglycaemia. Thus, we determined whether continuous NN414 could prevent hypoglycaemia-induced defective counter-regulation.

Methods

Chronically catheterised male Sprague–Dawley rats received a continuous infusion of NN414 into the third ventricle for 8 days after implantation of osmotic minipumps. Counter-regulation was examined by hyperinsulinaemic–hypoglycaemic clamp on day 8 after three episodes of insulin-induced hypoglycaemia (recurrent hypoglycaemia [RH]) on days 5, 6 and 7. In a subset of rats exposed to RH, NN414 infusion was terminated on day 7 to wash out NN414 before examination of counter-regulation on day 8. To determine whether continuous NN414 exposure altered K_ATP function, we used the hypothalamic glucose-sensing GT1-7 cell line, which expresses the SUR-1-containing K_ATP channel.

Results

Continuous exposure to NN414 in the setting of RH increased, rather than decreased, the glucose infusion rate (GIR), as exemplified by attenuated adrenaline (epinephrine) secretion. Termination of NN414 on day 7 with subsequent washout for 24 h partially diminished the GIR. The same duration of exposure of GT1-7 cells to NN414 substantially reduced K_ATP conductance, which was also reversed on washout of the agonist. The suppression of K_ATP current was not associated with reduced channel subunit mRNA or protein levels.

Conclusions/interpretation

These data indicate that continuous K_ATP activation results in suppressed CRRs to hypoglycaemia in vivo, which in vitro is associated with the reversible conversion of K_ATP into a stable inactive state.     

Coronary spasm and acute myocardial infarction due to a mutation (V734I) in the nucleotide binding domain 1 of ABCC9

Background

Alterations in coronary vasomotor tone may participate in the pathogenesis of acute myocardial infarction (AMI). Vascular ATP-sensitive K⁺ (K_ATP) channels, formed by Kir6.x/SUR2B, are key regulators of coronary tone and mutations in cardiac (Kir6.2/SUR2A) K_ATP channels result in heart disease. Here we explore the pathophysiological mechanism of a rare mutation (V734I) found in exon 17 of the ABCC9 gene, estimated to cause a 6.4-fold higher risk of AMI before the age of 60.

Methods and results

Eleven patients carrying the mutation were identified; they presented AMI of vasospastic origin associated with increased plasma levels of endothelin-1 and increased leukocyte ROCK activity. The effects of the mutation on the functional properties of the two splice variants of ABCC9 (SUR2A and SUR2B) were studied using patch-clamp electrophysiology. The mutation reduced the sensitivity to MgATP inhibition of Kir6.2/SUR2B channels but not of Kir6.2/SUR2A and Kir6.1/SUR2B channels. Furthermore, the stimulatory effects of MgNDP (MgADP, MgGDP and MgUDP) were unaltered in mutant Kir6.2/SUR2A and Kir6.1/SUR2B channels. In contrast, mutant channels composed of Kir6.2 and SUR2B were less sensitive to MgNDP activation, assessed in the presence of MgATP. The antianginal drug nicorandil activated Kir6.2/SUR2B–V734I channels, thus substituting for the loss of MgNDP stimulation, suggesting that this drug could be of therapeutic use in the treatment of AMI associated with V734I.

Conclusions

The 734I allele in ABCC9 may influence susceptibility to AMI by impairing the response of vascular, but not cardiac, K_ATP channels to intracellular nucleotides. This is the first human mutation in an ion channel gene to be implicated in AMI.     

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     

Effect of repaglinide on cloned beta cell, cardiac and smooth muscle types of ATP-sensitive potassium channels

Aims/hypothesis. The carbamoylbenzoic acid derivative repaglinide is a potent short-acting insulin secretagogue that acts by closing ATP-sensitive potassium (K_ATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated the specificity of repaglinide for three types of cloned (K_ATP) channel composed of the inwardly rectifying potassium channel Kir6.2 and either the sulphonylurea receptor SUR1, SUR2A or SUR2B, corresponding to the beta cell, cardiac and either smooth muscle types of K_ATP channel, respectively. Methods. The action of the drug was studied by whole-cell current recordings of K_ATP channels expressed either in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes for detailed analysis of repaglinide action. Results. The drug blocked all three types of K_ATP channel with similar potency, by interacting with a low-affinity site on the pore-forming subunit of the channel (Kir6.2: half-maximal inhibition 230 μmol/l) and with a high-affinity site on the regulatory subunit, the sulphonylurea receptor (SUR: half-maximal inhibition 2–8 nmol/l). There was no difference in potency between channels containing SUR1, SUR2A or SUR2B. MgADP potentiated the inhibitory effect of repaglinide on Kir6.2/SUR1 and (to a lesser extent) Kir6.2/SUR2B, but not on Kir6.2/SUR2A. Conclusion/interpretation. Repaglinide interacts with a site common to all three types of sulphonylurea receptor leading to inhibition of the K_ATP channel. The fact that MgADP potentiated this effect in the case of the beta cell, but not cardiac, type of channel could help explain why the drug shows no adverse cardiovascular side-effects in vivo. [Diabetologia (2001) 44: 747–756] Received: 13 December 2000 and in revised form: 14 February 2001     

Characterisation of new K<Subscript>ATP</Subscript>-channel mutations associated with congenital hyperinsulinism in the Finnish population

Aims/hypothesis ATP-sensitive potassium (K_ATP) channels are crucial for the regulation of insulin secretion from pancreatic beta cells and mutations in either the Kir6.2 or SUR1 subunit of this channel can cause congenital hyperinsulinism (CHI). The aim of this study was to analyse the functional consequences of four CHI mutations (A1457T, V1550D and L1551V in SUR1, and K67N in Kir6.2) recently identified in the Finnish population.Methods Wild type or mutant Kir6.2 and SUR1 subunits were coexpressed in Xenopus oocytes. The functional properties of the channels were examined by measuring currents in intact oocytes or giant inside-out membrane patches. Surface expression was measured by enzyme-linked immunosorbance assay, using HA-epitope-tagged subunits.Results Two mutations (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. The L1551V mutation reduced surface expression 40-fold, and caused loss of MgADP and diazoxide activation. Both these factors will contribute to the lack of K_ATP current activation observed in response to metabolic inhibition in intact oocytes. The L1551V mutation also increased the channel open probability, thereby producing a reduction in ATP-sensitivity (from 10 µmol/l to 120 µmol/l). The fourth mutation (K67N mutation in Kir6.2) did not affect surface expression nor alter the properties of K_ATP channels in excised patches, but resulted in a reduced K_ATP current amplitude in intact cells on metabolic inhibition, through an unidentified mechanism.Conclusion/interpretation The four CHI mutations disrupted K_ATP channel activity by different mechanisms. Our results are discussed in relation to the CHI phenotype observed in patients with these mutations.Abbreviations CHI Congenital hyperinsulinism - HA haemagluttinin - K_ATP channel ATP-sensitive potassium channel - Po open probability - PIP₂ phosphatidyl inositol bis-phosphate - RLU relative light units - SUR sulphonylurea receptor - UTR untranslated region An erratum to this article can be found at     

Cardioprotective-Mimetics Reduce Myocardial Infarct Size in Animals Resistant to Ischemic Preconditioning

Summary Background: Ischemic preconditioning (IPC) elicits two distinct windows of cardioprotection, an early phase that lasts for 1–2 h and a delayed phase that lasts for 24–72 h. However, there is conflicting data as to how long the heart is resistant to IPC-induced cardioprotection after the initial protection wanes, leading to the demonstration of IPC-resistance. This resistance to IPC appears to be dependent on the timing of the next IPC stimulus, the species of animals used and the model studied. Furthermore, the mechanisms responsible IPC-resistance are unknown. It is also important to demonstrate therapeutic interventions that will produce cardioprotection during this period of IPC-resistance. Methods and Results: To examine potential mechanisms responsible for acute IPC-induced resistance, the NHE-1 inhibitor EMD 85131 (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoylguanidine), which exerts its effects via mechanisms distinct from IPC, and the K_ATP channel opener bimakalim, which bypasses the signaling mechanisms of IPC to directly open K_ATP channels, were examined in a canine model of IPC-resistance. One 10 min. IPC stimulus followed by 10 min. of reperfusion produced a significant reduction in IS/AAR compared to Control (7.1 ± 2.6% versus 26.0 ± 6.2%; P < 0.05). However, IPC did not significantly protect the myocardium if a 2 h reperfusion period occurred between the initial IPC stimulus and the subsequent prolonged (60 min) ischemic challenge (IS/AAR: 22.5 ± 4.8%: P > 0.05). Furthermore, hearts treated with IPC followed by 2 h of reperfusion were resistant to an additional IPC stimulus administered just prior to the subsequent 60 min. occlusion period (IS/AAR: 22.9 ± 3.2%: P > 0.05). In contrast, administration of the NHE-1 inhibitor EMD 85131 (IS/AAR: 7.4 ± 2.5%: P < 0.05) or the K_ATP channel opener bimakalim (IS/AAR: 11.8 ± 2.4%: P < 0.05) both afforded significant cardioprotection when administered at 2 h of reperfusion in previously preconditioned canine hearts resistant to IPC. Conclusions: IPC resistance occurs in this canine model of ischemia-reperfusion injury. However, in spite of IPC resistance, hearts can still be pharmacologically protected by direct application of the K_ATP channel opener bimakalim or the NHE inhibitor EMD 85131.     

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.     

Vascular KATP channel structural dynamics reveal regulatory mechanism by Mg-nucleotides

Vascular tone is dependent on smooth muscle K_ATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantú syndrome. Unique among K_ATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined cryogenic electron microscopy structures of vascular K_ATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic K_ATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational “propeller” and “quatrefoil” geometries surrounding their Kir6.1 core. The glutamate/aspartate-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. Molecular dynamics simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B, and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated, and MgADP-bound activated conformations wherein the glutamate/aspartate-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward K_ATP channel activation.

Dynamic regulation of K⁺ channel gating is a primary point of control for processes governed by electrical excitability. ATP-sensitive potassium (K_ATP) channels, regulated by intracellular ATP to ADP ratios, transduce metabolic changes into electrical signals to govern many physiological processes (1). They are uniquely evolved hetero-octameric complexes comprising four pore-forming inwardly rectifying potassium channel subunits, Kir6.x, and four regulatory sulfonylurea receptors, SURx, nontransporting members of the ABCC subfamily of ABC transporters (2). Various Kir6.x/SURx combinations generate channel isoforms with distinct tissue distribution and function (3, 4). Kir6.2/SUR1 channels are expressed in pancreatic β cells and control glucose-stimulated insulin secretion. Kir6.2/SUR2A channels are the predominant isoform in myocardium, while Kir6.1/SUR2B channels are the major isoform found in vascular smooth muscle. SUR2A and 2B are two splice variants of ABCC9 that differ in their C-terminal 42 amino acids (aa). In vascular smooth muscle, K_ATP activation leads to membrane hyperpolarization and vasodilation (5), while inhibition or deletion causes membrane depolarization, vasoconstriction, and hypertension (5–8). Mutations in the vascular K_ATP channel genes (KCNJ8 and ABCC9) cause Cantú syndrome (9–11), a severe pleiotropic systemic hypotension disorder including hypertrichosis, osteochondrodysplasia, and cardiomegaly (12).K_ATP channel gating by intracellular ATP and ADP involves allosteric sites on both subunits. ATP binding to Kir6.x inhibits the channel. SURx, through induced dimerization of the paired nucleotide binding domains (NBDs), requiring MgADP bound to NBD2 and MgATP bound to noncatalytic NBD1, activates the channel (1, 4, 13). Like all Kir channels, opening further requires PIP₂ bound to Kir6.x (14–16). Despite these commonalities, vascular Kir6.1/SUR2B K_ATP channels have distinct biophysical properties, nucleotide sensitivities, and pharmacology that differentiate them from other isoforms (17–19). First, vascular K_ATP channel unitary conductance is half that of Kir6.2-containing channels. Second, vascular channels lack spontaneous activity, only opening in the presence of NBD-dimerizing Mg-dinucleotides/trinucleotides; in contrast, pancreatic or cardiac channels containing Kir6.2 open spontaneously in the absence of ATP. Third, once activated, vascular K_ATP channels are relatively insensitive to ATP inhibition, requiring mM concentrations to observe an effect, while their pancreatic or cardiac counterparts are blocked by ATP at μM concentrations. Lastly, the antidiabetic sulfonylurea drug glibenclamide (Glib), which inhibits SUR1-containing pancreatic channels with high affinity, is ∼10-fold less potent toward the vascular and cardiac channels containing SUR2. Glib has been shown to reverse defects from gain-of-function Cantú mutations in mice (20). However, clinical application in Cantú patients is hindered by hypoglycemia from inhibition of pancreatic channels (21). Structural mechanisms underlying unique biophysical, physiological, and pharmacological properties among K_ATP channels are unknown.Here, we report cryogenic electron microscopy (cryoEM) structures for the vascular K_ATP channel, Kir6.1/SUR2B, in the presence of ATP and Glib. The structures show conformations not previously seen in pancreatic K_ATP channels prepared under the same condition (22–24). First, unlike in Kir6.2, Kir6.1 cytoplasmic domains (CDs) were displaced from the membrane too far to interact with PIP₂ for channel opening. Second, unlike pancreatic channels, which have a predominant propeller-shaped conformation when bound to ATP and Glib (22, 24), vascular K_ATP channels held four distinct conformations, two resembling propellers and two quatrefoils, marked by varying degrees of rotation of SUR2B toward the core Kir6.1 tetramer. Importantly, a long segment of SUR not previously resolved in any K_ATP structures, linking NBD1 and transmembrane domain 2 (TMD2), was revealed within vascular K_ATP structures to mediate the cytosolic interface between SUR2B and Kir6.1. In particular, the linker’s unique 15 glutamate/aspartate residues termed the ED-domain (25) established a nexus of interactions engaging SUR2B-NBD2 with Kir6.1 C-terminal domain (CTD). Molecular dynamics (MD) simulations showed MgADP binding to NBD2 was accompanied by substantial reconfiguration at this nexus, revealing the ED-domain provides a mobile autoinhibitory interaction that guards the transition of SUR2B from MgADP-free inactivated state to MgADP-bound activated state. Together, our findings point to a structural pathway through which SUR regulates Kir6 channel gating.     

Role of PI3Kα and sarcolemmal ATP-sensitive potassium channels in epoxyeicosatrienoic acid mediated cardioprotection

AimsEpoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that have known cardioprotective properties. While the mechanism(s) remains unknown, evidence suggests that phosphoinositide 3-kinase (PI3K) and sarcolemmal ATP-sensitive potassium channels (pmK_ATP) are important. However the role of specific PI3K isoforms and corresponding intracellular mechanisms remains unknown.Methods and resultsTo study this, mice hearts were perfused in Langendorff mode for 40 min of baseline and subjected to 20 or 30 min of global no-flow ischemia followed by 40 min of reperfusion. C57BL6 mice perfused with 11,12-EET (1 μM) had improved postischemic recovery, whereas co-perfusion with PI3Kα inhibitor, PI-103 (0.1 μM), abolished the EET-mediated effect. In contrast, blocking of PI3Kβ or PI3Kγ isoforms failed to inhibit EET-mediated cardioprotection. In addition to the improved post-ischemic recovery, increased levels of p-Akt, decreased calcineurin activity and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET-treated hearts. Perfusion of 11,12-EET to Kir6.2 deficient mice (pmK_ATP) failed to improve postischemic recovery, decrease calcineurin activity and translocation of proapoptotic protein BAD, however increased levels of p-Akt were still observed. Patch-clamp experiments demonstrated that 11,12-EET could not activate pmK_ATP currents in myocytes pre-treated with PI-103. Mechanistic studies in H9c2 cells demonstrate that 11,12-EET limits anoxia–reoxygenation triggered Ca^2 + accumulation and maintains mitochondrial ΔΨm compared to controls. Both PI-103 and glibenclamide (10 μM, pmK_ATP inhibitor) abolished EET cytoprotection.ConclusionTogether our data suggest that EET-mediated cardioprotection involves activation of PI3Kα, upstream of pmK_ATP, which prevents Ca^2 + overload and maintains mitochondrial function.     

Hyperinsulinism in mice with heterozygous loss of KATP channels

Aims/hypothesis ATP-sensitive K⁺ (K_ATP) channels couple glucose metabolism to insulin secretion in pancreatic beta cells. In humans, loss-of-function mutations of beta cell K_ATP subunits (SUR1, encoded by the gene ABCC8, or Kir6.2, encoded by the gene KCNJ11) cause congenital hyperinsulinaemia. Mice with dominant-negative reduction of beta cell K_ATP (Kir6.2[AAA]) exhibit hyperinsulinism, whereas mice with zero K_ATP (Kir6.2^−/−) show transient hyperinsulinaemia as neonates, but are glucose-intolerant as adults. Thus, we propose that partial loss of beta cell K_ATP in vivo causes insulin hypersecretion, but complete absence may cause insulin secretory failure. Materials and methods Heterozygous Kir6.2^+/− and SUR1^+/− animals were generated by backcrossing from knockout animals. Glucose tolerance in intact animals was determined following i.p. loading. Glucose-stimulated insulin secretion (GSIS), islet K_ATP conductance and glucose dependence of intracellular Ca²⁺ were assessed in isolated islets. Results In both of the mechanistically distinct models of reduced K_ATP (Kir6.2^+/− and SUR1^+/−), K_ATP density is reduced by ∼60%. While both Kir6.2^−/− and SUR1^−/− mice are glucose-intolerant and have reduced glucose-stimulated insulin secretion, heterozygous Kir6.2^+/− and SUR1^+/− mice show enhanced glucose tolerance and increased GSIS, paralleled by a left-shift in glucose dependence of intracellular Ca²⁺ oscillations. Conclusions/interpretation The results confirm that incomplete loss of beta cell K_ATP in vivo underlies a hyperinsulinaemic phenotype, whereas complete loss of K_ATP underlies eventual secretory failure.     

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K+ channel activity

Aims/hypothesis

The TGF-β superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-β family signalling pathway is required for essential islet functions in the adult pancreas.

Methods

To identify distinct roles for the downstream components of the canonical TGF-β signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-β signals, in pancreatic beta cells (i.e. Smad2β knockout [KO] mice). The activity of ATP-sensitive K⁺ channels (K_ATP channels) was recorded in mutant beta cells using patch-clamp techniques.

Results

The Smad2βKO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in beta cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of K_ATP channels was decreased in mutant beta cells.

Conclusions/interpretation

These results suggest that in the adult pancreas, TGF-β signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of K_ATP channel activity.     

Exercise-induced expression of cardiac ATP-sensitive potassium channels promotes action potential shortening and energy conservation

Physical activity is one of the most important determinants of cardiac function. The ability of the heart to increase delivery of oxygen and metabolic fuels relies on an array of adaptive responses necessary to match bodily demand while avoiding exhaustion of cardiac resources. The ATP-sensitive potassium (K_ATP) channel has the unique ability to adjust cardiac membrane excitability in accordance with ATP and ADP levels, and up-regulation of its expression that occurs in response to exercise could represent a critical element of this adaption. However, the mechanism by which K_ATP channel expression changes result in a beneficial effect on cardiac excitability and function remains to be established. Here, we demonstrate that an exercise-induced rise in K_ATP channel expression enhanced the rate and magnitude of action potential shortening in response to heart rate acceleration. This adaptation in membrane excitability promoted significant reduction in cardiac energy consumption under escalating workloads. Genetic disruption of normal K_ATP channel pore function abolished the exercise-related changes in action potential duration adjustment and caused increased cardiac energy consumption. Thus, an expression-driven enhancement in the K_ATP channel-dependent membrane response to alterations in cardiac workload represents a previously unrecognized mechanism for adaptation to physical activity and a potential target for cardioprotection.     

Differential sensitivity of beta-cell and extrapancreatic KATP channels to gliclazide

Aims/hypothesis. To investigate the tissue specificity of gliclazide for cloned beta-cell, cardiac and smooth muscle ATP-sensitive K-channels (K_ATP channels). These channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). Methods. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes, and channel activity was measured by recording macroscopic currents in giant inside-out membrane patches. Gliclazide was added to the intracellular membrane surface. Results. We reported previously that Kir6.2-SUR1 currents are blocked at two sites by tolbutamide: a high-affinity site on SUR1 and a low-affinity site on Kir6.2. We now show that gliclazide also inhibits beta-cell K_ATP channels at two sites: a high-affinity site, which is half-maximally blocked (K _i) at 50 ± 7 nmol/l (n = 8) and a low-affinity site with a K _i of 3.0 ± 0.6 mmol/l (n = 4). The high-affinity site on SUR1 was thus about 40-fold more sensitive to gliclazide than to tolbutamide (K _i∼ 2 μmol/l). Cloned cardiac and smooth muscle K_ATP channels did not show high-affinity block by gliclazide. Kir6.2-SUR2A currents exhibited a single low-affinity site with a K _i of 0.8 ± 0.1 mmol/l (n = 5), which is likely to reside on the Kir6.2 subunit. Conclusion/interpretation. Our results show that gliclazide is a sulphonylurea with high affinity and strong selectivity for the beta-cell type of K_ATP channel. [Diabetologia (1999) 42: 845–848] Received: 20 January 1999 and in final revised form: 4 March 1999     

MgATP activates the β cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (K_ATP) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca²⁺ influx and exocytosis. Metabolic regulation of K_ATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell K_ATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate K_ATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg²⁺ caused an apparent reduction in the inhibitory action of ATP on wild-type K_ATP channels, and MgATP actually activated K_ATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates K_ATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg²⁺.     

Cognitive deficits and impaired hippocampal long-term potentiation in KATP-induced DEND syndrome

ATP-sensitive potassium (K_ATP) gain-of-function (GOF) mutations cause neonatal diabetes, with some individuals exhibiting developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. Mice expressing K_ATP-GOF mutations pan-neuronally (nK_ATP-GOF) demonstrated sensorimotor and cognitive deficits, whereas hippocampus-specific hK_ATP-GOF mice exhibited mostly learning and memory deficiencies. Both nK_ATP-GOF and hK_ATP-GOF mice showed altered neuronal excitability and reduced hippocampal long-term potentiation (LTP). Sulfonylurea therapy, which inhibits K_ATP, mildly improved sensorimotor but not cognitive deficits in K_ATP-GOF mice. Mice expressing K_ATP-GOF mutations in pancreatic β-cells developed severe diabetes but did not show learning and memory deficits, suggesting neuronal K_ATP-GOF as promoting these features. These findings suggest a possible origin of cognitive dysfunction in DEND and the need for novel drugs to treat neurological features induced by neuronal K_ATP-GOF.

ATP-sensitive potassium (K_ATP) channels are a unique link between cellular metabolism and membrane excitability. K_ATP gain-of-function (GOF) mutations have been identified as the most common cause of neonatal diabetes (1, 2), which, in many cases, manifests neurological features in a novel syndrome known as developmental delay, epilepsy, and neonatal diabetes (DEND) (3–6). Neurological symptoms of DEND include motor and developmental delays, severe epileptic phenotypes, and lifelong intellectual disabilities (7, 8). Diabetic features arise from suppression of insulin secretion by expression of K_ATP-GOF channels in pancreatic insulin-producing β-cells, and mice pan-neuronally expressing a DEND-associated K_ATP-GOF mutation showed sensorimotor deficits attributed to loss of excitability in cerebellar Purkinje neurons (9, 10). However, the involvement of K_ATP-GOF mutations in other neurological features as well as the treatability of these features remain unknown.K_ATP channels are hetero-octameric complexes comprising four pore-forming Kir6.x and four sulfonylurea receptor subunits, with Kir6.2 and SUR1 compositions predominating in neurons of the hippocampus and cerebellum (11, 12) as well as in pancreatic insulin-producing β-cells (13). SUR1 subunits provide pharmacological sensitivity to K_ATP channel openers (diazoxide) and blockers (e.g., sulfonylureas such as glibenclamide and tolbutamide). Kir6.2 and SUR1 subunits each contain RKR endoplasmic reticulum retention motifs, with the expression of both subunits required to form functional channels (14). Mice globally lacking K_ATP demonstrate spatial learning deficits (15, 16), intrahippocampal application of the K_ATP channel opener diazoxide impairs spatial learning and memory (17), and intraseptal application of glibenclamide improved spatial memory defects induced by galanin or morphine in rats (18, 19). K_ATP currents regulate spike rates and spontaneous bursting activity in hippocampal CA1/CA3 neurons (20) and gate epileptic seizures (21), suggesting that neurological features may arise from alterations to excitability in hippocampal neurons (10). In human neonatal diabetes, sulfonylureas are effective in normalizing blood glucose (22) and often successful in restoring muscular tone, but they are not nearly as effective in treating neurological, especially cognitive, features of DEND (4, 23–25). These findings raise questions about the pathophysiology of DEND, particularly the relative contributions of neuronal and pancreatic expression of K_ATP-GOF channels in the development of neurological features. Here, we explored the origin, underlying mechanisms, and treatability of the cognitive deficits of DEND in mouse models expressing K_ATP-GOF channels in central neurons (pan-neuronal or hippocampus specific) or in pancreatic β-cells.     

Angiotensin II Type 2 Receptor (AT2R) is Associated with Increased Tolerance of the Hyperthyroid Heart to Ischemia-Reperfusion

Background

Thyroid hormone induces cardiac hypertrophy and preconditions the myocardium against Ischemia/Reperfusion (I/R) injury. Type 2 Angiotensin II receptors (AT2R) are shown to be upregulated in cardiac hypertrophy observed in hyperthyroidism and this receptor has been reported to mediate cardioprotection against ischemic injury.

Methods

The aim of the present study was to evaluate the role of AT2R in the recovery of myocardium after I/R in isolated hearts from T3 treated rats. Male Wistar rats were treated with triiodothyronine (T3; 7 μg/100 g BW/day, i.p.) in the presence or not of a specific AT2R blocker (PD123,319; 10 mg/Kg) for 14 days, while normal rats served as control. After treatment, isolated hearts were perfused in Langendorff mode; after 30 min of stabilization, hearts were subjected to 20 min of zero-flow global ischemia followed by 25 min, 35 min and 45 min of reperfusion.

Results

T3 treatment induced cardiac hypertrophy, which was not changed by PD treatment. Post-ischemic recovery of cardiac function was increased in T3-treated hearts after 35 min and 45 min of reperfusion as compared to control and the ischemic contracture was accelerated and intensified. AT2R blockade was able to return the evaluated functional parameters of cardiac performance (LVDP, +dP/dt_máx and ?dP/dt_min) to the control condition. Furthermore, AT2R blockade prevented the increase in AMPK expression levels induced by T3, suggesting its possible involvement in this process.

Conclusion

AT2R plays a significant role in T3-induced cardioprotection.     

The L-Type Ca+ and KATP channels may contribute to pacing-induced protection against anoxia-reoxygenation in the embryonic heart model

Aims: The L‐type Ca²⁺ channel, the sarcolemmal (sarcK_ATP), and mitochondrial K_ATP (mitoK_ATP) channels are involved in myocardial preconditioning. We aimed at determining to what extent these channels can also participate in pacing‐induced cardioprotection. Methods: Hearts of 4‐day‐old chick embryos were paced in ovo during 12 hour using asynchronous intermittent ventricular stimulation at 110% of the intrinsic rate. Sham operated and paced hearts were then submitted in vitro to anoxia (30 minutes) and reoxygenation (60 minutes). These hearts were exposed to L‐type Ca²⁺ channel agonist Bay‐K‐8644 (BAY‐K) or blocker verapamil, nonselective K_ATP channel antagonist glibenclamide (GLIB), mitoK_ATP channel agonist diazoxide (DIAZO), or antagonist 5‐hydroxydecanoate. Electrocardiogram, electromechanical delay (EMD) reflecting excitation‐contraction (E‐C) coupling, and contractility were determined. Results: Under normoxia, heart rate, QT duration, conduction, EMD, and ventricular shortening were similar in sham and paced hearts. During reoxygenation, arrhythmias ceased earlier and ventricular EMD recovered faster in paced hearts than in sham hearts. In sham hearts, BAY‐K (but not verapamil), DIAZO (but not 5‐hydroxydecanoate) or GLIB accelerated recovery of ventricular EMD, reproducing the pacing‐induced protection. By contrast, none of these agents further ameliorated recovery of the paced hearts. Conclusion: The protective effect of chronic asynchronous pacing at near physiological rate on ventricular E‐C coupling appears to be associated with subtle activation of L‐type Ca²⁺ channel, inhibition of sarcK_ATP channel, and/or opening of mitoK_ATP channel.