Zoledronic Acid Blocks Overactive Kir6.1/SUR2-Dependent KATP Channels in Skeletal Muscle and Osteoblasts in a Murine Model of Cantú Syndrome.

Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1wt/V65M. Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1wt/V65MCS mice, with IC50 for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC50 of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2wt/A478V mice were less sensitive to ZOL inhibition, showing an IC50 of ~500 nM and a slope of ~0.3. In homozygous SUR2A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 µM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1wt/VMCS mice in both skeletal muscle and bone cells but was not effective in the SUR2[A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.

Resveratrol reduces cerebral edema through inhibition of de novo SUR1 expression induced after focal ischemia.

Cerebral edema is a clinical problem that frequently follows ischemic infarcts. Sulfonylurea receptor 1 (SUR1) is an inducible protein that can form a heteromultimeric complex with aquaporin 4 (AQP4) that mediate the ion/water transport involved in brain tissue swelling. Transcription of the Abcc8 gene coding for SUR1 depends on the activity of transcriptional factor SP1, which is modulated by the cellular redox environment. Since oxidative stress is implicated in the induced neuronal damage in ischemia and edema formation, the present study aimed to evaluate if the antioxidant resveratrol (RSV) prevents the damage by reducing the de novo expression of SUR1 in the ischemic brain. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by different times of reperfusion. RSV (1.9 mg/kg; i.v.) was administered at the onset of reperfusion. Brain damage and edema formation were recognized by neurological evaluation, time of survival, TTC (2,3,5-Triphenyltetrazolium chloride) staining, Evans blue extravasation, and water content. RSV mechanism of action was studied by SP1 binding activity measured through the Electrophoretic Mobility Shift Assay, and Abcc8 and Aqp4 gene expression evaluated by qPCR, immunofluorescence, and Western blot. We found that RSV reduced the infarct area and cerebral edema, prevented blood-brain barrier damage, improved neurological performance, and increased survival. Additionally, our findings suggest that the antioxidant activity of RSV targeted SP transcription factors and inhibited SUR1 and AQP4 expression. Thus, RSV by decreasing SUR1 expression could contribute to reducing edema formation, constituting a therapeutic alternative for edema reduction in stroke.

Glimepiride and glibenclamide have comparable efficacy in treating acute ischemic stroke in mice.

Glibenclamide protects against ischemic injury in both preclinical and clinical studies, presumably by blocking the de novo assembled sulfonylurea receptor 1-transient receptor potential M4 (Sur1-Trpm4) channel induced by ischemia. However, glibenclamide may cause unexpected serious hypoglycemia. Here, we tested whether glimepiride, another sulfonylurea with better safety, has comparable efficacy with glibenclamide and whether gene deletion of Trpm4 (Trpm4-/-) exerts similar effect. Wild-type (WT) mice subjected to temporary middle cerebral artery occlusion (tMCAO) were randomized to receive glibenclamide (an initial dose of 10 µg/kg and additional doses of 1.2 µg every 8 h), three different doses of glimepiride (10 µg/kg, 100 µg/kg and 1 mg/kg) or vehicle after ischemia, while tMCAO-treated Trpm4-/- mice were randomized to receive vehicle or glimepiride. Neurological function, infarct volume, edema formation, the integrity of blood-brain barrier and inflammatory reaction were evaluated at 24 h after ischemia. In tMCAO-treated WT mice, 10 µg/kg and 100 µg/kg glimepiride had comparable efficacy with glibenclamide in improving longa score and grip test score, reducing infarct volume, mitigating brain edema, lessening extravasation of Evans blue dye and IgG, restoring tight junction protein expression as well as suppressing inflammatory cytokines. Compared with WT mice, Trpm4-/- mice showed less neurological deficit, smaller cerebral infarction, lighter brain edema and more integrity of blood-brain barrier. As expected, glimepiride did not provide additional neuroprotection compared with vehicle in the tMCAO-treated Trpm4-/- mice. Glimepiride shows comparable efficacy with glibenclamide in alleviating brain injury after ischemic stroke in mice, possibly via targeting the Sur1-Trpm4 channel.

A Variation in the ABCC8 Gene Is Associated with Type 2 Diabetes Mellitus and Repaglinide Efficacy in Chinese Type 2 Diabetes Mellitus Patients.

Objective Previous studies have suggested that variations in the ABCC8 gene may be closely associated with T2DM susceptibility and repaglinide response. However, these results have not been entirely consistent, and there are no related studies in a Chinese population, suggesting the need for further exploration. The current study investigated the associations of the ABCC8 rs1801261 polymorphism with type 2 diabetes mellitus (T2DM) susceptibility and repaglinide therapeutic efficacy in Chinese Han T2DM patients. Methods A total of 234 T2DM patients and 105 healthy subjects were genotyped for ABCC8 rs1801261 polymorphism by a polymerase chain reaction-restriction fragment length polymorphism assay. A total of 70 patients with the same genotypes of CYP2C8*3 139Arg and OATP1B1 521TT were randomized to orally take 3 mg repaglinide per day (1 mg each time before meals) for 8 consecutive weeks. The pharmacodynamic parameters of repaglinide and biochemical indicators were then determined before and after repaglinide treatment. Results The frequency of ABCC8 rs1801261 allele was higher in T2DM patients than in the control subjects (22.6% vs.11.0%, p<0.01). After repaglinide treatment, T2DM patients carrying genotype CT showed a significantly attenuated efficacy on FPG (p<0.01) and HbA1c (p<0.01) compared with those with genotype CC. Conclusion These results suggested that the ABCC8 rs1801261 polymorphism might influence T2DM susceptibility and the therapeutic effect of repaglinide in Chinese Han T2DM patients. This study was registered in the Chinese Clinical Trial Register on May 14, 2013 (No. ChiCTR-CCC13003536).

Therapies and outcomes of congenital hyperinsulinism-induced hypoglycaemia.

Congenital hyperinsulinism is a rare disease, but is the most frequent cause of persistent and severe hypoglycaemia in early childhood. Hypoglycaemia caused by excessive and dysregulated insulin secretion (hyperinsulinism) from disordered pancreatic ß cells can often lead to irreversible brain damage with lifelong neurodisability. Although congenital hyperinsulinism has a genetic cause in a significant proportion (40%) of children, often being the result of mutations in the genes encoding the KATP channel (ABCC8 and KCNJ11), not all children have severe and persistent forms of the disease. In approximately half of those without a genetic mutation, hyperinsulinism may resolve, although timescales are unpredictable. From a histopathology perspective, congenital hyperinsulinism is broadly grouped into diffuse and focal forms, with surgical lesionectomy being the preferred choice of treatment in the latter. In contrast, in diffuse congenital hyperinsulinism, medical treatment is the best option if conservative management is safe and effective. In such cases, children receiving treatment with drugs, such as diazoxide and octreotide, should be monitored for side effects and for signs of reduction in disease severity. If hypoglycaemia is not safely managed by medical therapy, subtotal pancreatectomy may be required; however, persistent hypoglycaemia may continue after surgery and diabetes is an inevitable consequence in later life. It is important to recognize the negative cognitive impact of early-life hypoglycaemia which affects half of all children with congenital hyperinsulinism. Treatment options should be individualized to the child/young person with congenital hyperinsulinism, with full discussion regarding efficacy, side effects, outcomes and later life impact.

Mithramycin A Improves Functional Recovery by Inhibiting BSCB Disruption and Hemorrhage after Spinal Cord Injury.

After spinal cord injury (SCI), blood-spinal cord barrier (BSCB) disruption and progressive hemorrhage lead to secondary injury, subsequent apoptosis and/or necrosis of neurons and glia, causing permanent neurological deficits. Growing evidence indicates that mithramycin A (MA), an anti-cancer drug, has neuroprotective effects in ischemic brain injury and Huntington's disease (HD). However, the precise mechanism underlying its protective effects is largely unknown. Here, we examined the effect of MA on BSCB breakdown and hemorrhage as well as subsequent inflammation after SCI. After moderate spinal cord contusion injury at T9, MA (150 µg/kg) was immediately injected intraperitoneally (i.p.) and further injected once a day for 5 days. Our data show that MA attenuated BSCB disruption and hemorrhage, and inhibited the infiltration of neutrophils and macrophages after SCI. Consistent with these findings, the expression of inflammatory mediators was significantly alleviated by MA. MA also inhibited the expression and activation of matrix metalloprotease-9 (MMP-9) after injury, which is known to disrupt BSCB and the degradation of tight junction (TJ) proteins. In addition, the expression of sulfonylurea receptor 1 (SUR1) and transient receptor potential melastatin 4 (TRPM4), which are known to mediate hemorrhage at an early stage after SCI, was significantly blocked by MA treatment. Finally, MA inhibited apoptotic cell death and improved functional recovery after injury. Thus, our results demonstrated that MA improves functional recovery by attenuating BSCB disruption and hemorrhage through the downregulation of SUR1/TRPM4 and MMP-9 after SCI.

Effect of sulfur dioxide inhalation on the expression of KATP and L-Ca(2+) channels in rat hearts.

Epidemiological studies have revealed an association between sulfur dioxide (SO2) exposure and cardiovascular diseases. This study is designed to investigate the SO2 effect on the expression of ATP-sensitive K(+) (KATP) channel and L-type calcium (L-Ca(2+)) channel in rat hearts. The results show that the mRNA and protein levels of the KATP channel subunits Kir6.2 and SUR2A of rat hearts in SO2 groups were higher than those in control group. SO2 at 14mg/m(3) significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. This suggests that SO2 can activate the KATP channels by up-regulating the expression of Kir6.2 and SUR2A, while it inhibits the L-Ca(2+) channels by down-regulating the expression of Cav1.2 and Cav1.3 in rat hearts. The molecular mechanism of SO2-induced negative inotropic effect might be linked to the expression changes of these subunits, which may contribute to the pathogenesis of SO2-associated cardiovascular diseases.

17ß-estradiol inhibits MMP-9 and SUR1/TrpM4 expression and activation and thereby attenuates BSCB disruption/hemorrhage after spinal cord injury in male rats.

Blood-spinal cord barrier (BSCB) disruption and progressive hemorrhage after spinal cord injury (SCI) lead to secondary injury and the subsequent apoptosis and/or necrosis of neuron and glia, causing permanent neurological deficits. In this study, we examined the effect of 17ß-estradiol (E2) on BSCB breakdown and hemorrhage as well as subsequent inflammation after SCI. After a moderate contusion injury at the 9th thoracic segment of spinal cord, E2 (300 µg/kg) was administered by iv injection immediately after SCI, and the same dose of E2 was then administered 6 and 24 hours after injury. Our data show that E2 attenuated BSCB permeability and hemorrhage and reduced the infiltration of neutrophils and macorphages after SCI. Consistent with this finding, the expression of inflammatory mediators was significantly reduced by E2. Furthermore, E2 treatment significantly inhibited the expression of sulfonylurea receptor 1 and transient receptor potential melastatin 4 after injury, which are known to mediate hemorrhage at an early stage after SCI. Moreover, the expression and activation of matrix metalloprotease-9 after injury, which is known to disrupt BSCB, and the degradation of tight junction proteins, such as zona occludens-1 and occludin, were significantly inhibited by E2 treatment. Furthermore, the protective effects of E2 on BSCB disruption and functional improvement were abolished by an estrogen receptor antagonist, ICI 182780 (3 mg/kg). Thus, our study provides evidence that the neuroprotective effect of E2 after SCI is, in part, mediated by inhibiting BSCB disruption and hemorrhage through the down-regulation of sulfonylurea receptor 1/transient receptor potential melastatin 4 and matrix metalloprotease-9, which is dependent on estrogen receptor.

Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel complex.

Hydrogen sulfide (H2S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H2S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K(+) (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H2S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H2S in inflammation.

Combination of ACE inhibitor with nicorandil provides further protection in chronic kidney disease.

An inhibition in the renin-angiotensin system (RAS) is one of the most widely used therapies to treat chronic kidney disease. However, its effect is occasionally not sufficient and additional treatments may be required. Recently, we reported that nicorandil exhibited renoprotective effects in a mouse model of diabetic nephropathy. Here we examined if nicorandil can provide an additive protection on enalapril in chronic kidney disease. Single treatment with either enalapril or nicorandil significantly ameliorated glomerular and tubulointerstitial injury in the rat remnant kidney while the combination of these two compounds provided additive effects. In addition, an increase in oxidative stress in remnant kidney was also blocked by either enalapril or nicorandil while the combination of the drugs was more potent. A mechanism was likely due for nicorandil to preventing manganase superoxide dismutase (MnSOD) and sirtuin (Sirt)3 from being reduced in injured kidneys. A study with cultured podocytes indicated that the antioxidative effect could be mediated through sulfonylurea receptor (SUR) in the mitochondrial KATP channel since blocking SUR with glibenclamide reduced MnSOD and Sirt3 expression in podocytes. In conclusion, nicorandil may synergize with enalapril to provide superior protection in chronic kidney disease.

Molecular mechanism of sulphonylurea block of K(ATP) channels carrying mutations that impair ATP inhibition and cause neonatal diabetes.

Sulphonylurea drugs are the therapy of choice for treating neonatal diabetes (ND) caused by mutations in the ATP-sensitive K(+) channel (KATP channel). We investigated the interactions between MgATP, MgADP, and the sulphonylurea gliclazide with KATP channels expressed in Xenopus oocytes. In the absence of MgATP, gliclazide block was similar for wild-type channels and those carrying the Kir6.2 ND mutations R210C, G334D, I296L, and V59M. Gliclazide abolished the stimulatory effect of MgATP on all channels. Conversely, high MgATP concentrations reduced the gliclazide concentration, producing a half-maximal block of G334D and R201C channels and suggesting a mutual antagonism between nucleotide and gliclazide binding. The maximal extent of high-affinity gliclazide block of wild-type channels was increased by MgATP, but this effect was smaller for ND channels; channels that were least sensitive to ATP inhibition showed the smallest increase in sulphonylurea block. Consequently, G334D and I296L channels were not fully blocked, even at physiological MgATP concentrations (1 mmol/L). Glibenclamide block was also reduced in ß-cells expressing Kir6.2-V59M channels. These data help to explain why patients with some mutations (e.g., G334D, I296L) are insensitive to sulphonylurea therapy, why higher drug concentrations are needed to treat ND than type 2 diabetes, and why patients with severe ND mutations are less prone to drug-induced hypoglycemia.