ABSTRACT
Vascular smooth muscle KATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP) over Kir6.2/SUR1 (pancreatic KATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270.
Subject(s)
KATP Channels , Animals , Mice , Glyburide , KATP Channels/antagonists & inhibitors , Muscle, Smooth, Vascular/metabolism , Sulfonylurea Receptors/antagonists & inhibitorsABSTRACT
ATP-sensitive potassium (KATP) channels are participants of mechanisms of pathological myocardial remodeling containment. The aim of our work was to find the association of changes in the expression of Kir6.1, Kir6.2, SUR1, and SUR2 subunits of KATP channels with changes in heart function and structure during aging under conditions of the constant increase of vascular pressure. The experiments were carried out on young and old spontaneously hypertensive rats (SHR) and Wistar rats. The expression levels of KATP channels subunits were determined using reverse transcription and quantitative PCR. It is shown that the mRNA expression level of Kir6.1 in young SHR rats is significantly lower (6.3-fold, p = 0.035) than that of young Wistar rats that may be one of the causes of arterial hypertension in SHR. At the same time, mRNA expression of both Kir6.1 and Kir6.2 in old SHR rats was significantly higher (6.8-fold, p = 0.003, and 5.9-fold, p = 0.006, respectively) than in young hypertensive animals. In both groups of old animals, SUR2 expression was significantly reduced compared to young animals, in Wistar rats at 3.87-fold (p = 0.028) and in SHR rats at 48.2-fold (p = 0.033). Changes in SUR1 expression were not significant. Thus, significant changes in the cardiovascular system, including impaired function and structure of the heart in old SHR rats, were associated with a significant decrease in SUR2 expression that may be one of the mechanisms of heart failure decompensation. Therefore, it can be assumed that increased expression of SUR2 may be one of the protective mechanisms against pathological myocardial remodeling.
Subject(s)
Heart Diseases/pathology , Hypertension/complications , Myocardium/pathology , Sulfonylurea Receptors/antagonists & inhibitors , Age Factors , Animals , Disease Models, Animal , Heart Diseases/etiology , Heart Diseases/metabolism , Male , Myocardium/metabolism , Rats , Rats, Inbred SHR , Rats, WistarABSTRACT
The sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) receptor is selectively expressed after intracerebral hemorrhage (ICH). This upregulation contributes to increases in intracellular sodium. Water follows sodium through aquaporin channels, leading to cytotoxic edema. Even after edema is thought to have resolved, ionic dyshomeostasis persists, as does blood-brain barrier (BBB) damage. Glibenclamide, a hypoglycemic agent that inhibits Sur1-Trpm4, has been shown to reduce BBB damage and edema following infusion of autologous blood into the brain (ICH) as well as after other brain injuries. In order to further assess efficacy, we used the collagenase ICH model in rats to test whether glibenclamide reduces edema, attenuates ion dyshomeostasis, improves BBB damage, and reduces lesion volume. We tested a widely-used glibenclamide dose shown effective in other studies (10 µg/kg loading dose followed by 200 ng/hr for up to 7 days). Early initiation of glibenclamide did not significantly impact edema (72 hours), BBB permeability (72 hours), or lesion volume after ICH (28 days). Recovery from neurological impairments was also not improved by glibenclamide. These results suggest that glibenclamide will not improve outcome in ICH. However, the treatment appeared to be safe as there was no effect on bleeding or other physiological variables.
Subject(s)
Cerebral Hemorrhage/drug therapy , Collagenases/metabolism , Glyburide/therapeutic use , Hypoglycemic Agents/therapeutic use , Animals , Behavior, Animal/drug effects , Blood Glucose/analysis , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Brain/drug effects , Brain/metabolism , Brain/pathology , Brain Edema/pathology , Cerebral Hemorrhage/etiology , Cerebral Hemorrhage/pathology , Collagenases/toxicity , Dose-Response Relationship, Drug , Drug Administration Schedule , Glyburide/pharmacology , Hypoglycemic Agents/pharmacology , Male , Rats , Rats, Sprague-Dawley , Sodium/metabolism , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/metabolism , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/metabolism , TemperatureABSTRACT
BACKGROUND AND PURPOSE: Sulfonylureas (SUs) have been suggested to have an insulin-independent blood glucose-decreasing activity due to an extrapancreatic effect. However, a lack of adequate in vivo evidence makes this statement controversial. Here, we aimed to evaluate whether glimepiride has extrapancreatic blood glucose-lowering activity in vivo. EXPERIMENTAL APPROACH: Sulfonylurea receptor 1 deficient (SUR1-/- ) rats were created by means of transcription activator-like effector nucleases (TALEN)-mediated gene targeting technology. Type 2 diabetic models were established by feeding a high-fat diet and administering a low-dose of streptozotocin. These rats were then randomly divided into four groups: glimepiride, gliclazide, metformin and saline. All rats were treated for 2 weeks. KEY RESULTS: Glimepiride decreased blood glucose levels and increased insulin sensitivity without elevating insulin levels. Gliclazide showed similar effects as glimepiride. Both agents were weaker than metformin. Further mechanistic investigations revealed that glimepiride increased hepatic glycogen synthesis and decreased gluconeogenesis, which were accompanied by the activation of Akt in the liver. Moreover, glimepiride increased both total and membrane glucose transporter 4 (GLUT4) levels in muscle and fat, which might be attributed to insulin receptor-independent IRS1/Akt activation. CONCLUSION AND IMPLICATIONS: Glimepiride possesses an extrapancreatic blood glucose-lowering effect in vivo, which might be attributed to its direct effect on insulin-sensitive tissues. Therefore, the combination of glimepiride with multiple insulin injections should not be excluded per se.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Hypoglycemic Agents/pharmacology , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/deficiency , Animals , Blood Glucose/drug effects , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Hypoglycemic Agents/administration & dosage , Rats , Sulfonylurea Compounds/administration & dosage , Sulfonylurea Receptors/metabolismABSTRACT
INTRODUCTION: Edema is a significant cause of neuromorbidity in children and adults with brain tumors. Agents used to control this effect, such as corticosteroids, have their own associated morbidities. Sulfonylurea receptor 1 (SUR1) is a transmembrane protein that regulates the activity of ion channels in neurons, glia, and endothelial cells. SUR1 expression is upregulated in neuroinflammatory conditions. Inhibition of SUR1 with glyburide decreases edema and neuroinflammation by countering cytotoxic edema and apoptosis in rodent models of subarachnoid hemorrhage, stroke, trauma, and cerebral metastases. However, the expression of SUR1 in human brain tumors has not been elucidated. The purpose of this study was to determine SUR1 expression and cellular colocalization in a variety of human brain tumor specimens. MATERIALS AND METHODS: Six glioblastoma, 12 cerebral metastases, 11 medulloblastoma, 9 supratentorial ependymoma, and 8 posterior fossa ependymoma specimens were analyzed using immunofluorescence. SUR1 expression and colocalization with blood vessels, neurons, and glial cells was analyzed and compared using ANOVA. RESULTS: SUR1 expression was found in all specimens examined as a percentage of the total tissue area (mean ± SD): glioblastoma 3.9 ± 4, cerebral metastases 4.1 ± 3.1, medulloblastoma 8.2 ± 7.2, supratentorial ependymoma 9.1 ± 7, and posterior fossa ependymoma 8.1 ± 5.9. SUR1 expression was greater in supratentorial ependymoma compared to glioblastoma and metastases (p < 0.05) and greater in medulloblastoma compared to glioblastoma (p < 0.05). SUR1 colocalized most reliably with the neuronal marker, NeuN, in glioblastoma, metastases, and posterior fossa ependymoma samples (p < 0.05). SUR1 colocalized most reliably with the endothelial cell marker, CD31, in medulloblastoma samples (p < 0.05). CONCLUSION: SUR1 is a putative therapeutic target to reduce neuroinflammation in adult and pediatric brain tumors. Inhibition of SUR1 may result in neuronal stabilization in glioblastoma, cerebral metastases, and posterior fossa ependymoma and reduced edema in medulloblastoma.â©.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Sulfonylurea Receptors/biosynthesis , Sulfonylurea Receptors/genetics , Adult , Brain Edema/etiology , Brain Edema/pathology , Brain Neoplasms/complications , Child , Endothelial Cells/metabolism , Humans , Inflammation/pathology , Neoplasm Metastasis , Neuroglia/metabolism , Neurons/metabolism , Sulfonylurea Receptors/antagonists & inhibitorsABSTRACT
Glyburide (also known as glibenclamide) is a second-generation sulfonylurea drug that inhibits sulfonylurea receptor 1 (Sur1) at nanomolar concentrations. Long used to target KATP (Sur1-Kir6.2) channels for the treatment of diabetes mellitus type 2, glyburide was recently repurposed to target Sur1-transient receptor potential melastatin 4 (Trpm4) channels in acute central nervous system injury. Discovered nearly two decades ago, SUR1-TRPM4 has emerged as a critical target in stroke, specifically in large hemispheric infarction, which is characterized by edema formation and life-threatening brain swelling. Following ischemia, SUR1-TRPM4 channels are transcriptionally upregulated in all cells of the neurovascular unit, including neurons, astrocytes, microglia, oligodendrocytes and microvascular endothelial cells. Work by several independent laboratories has linked SUR1-TRPM4 to edema formation, with blockade by glyburide reducing brain swelling and death in preclinical models. Recent work showed that, following ischemia, SUR1-TRPM4 co-assembles with aquaporin-4 to mediate cellular swelling of astrocytes, which contributes to brain swelling. Additionally, recent work linked SUR1-TRPM4 to secretion of matrix metalloproteinase-9 (MMP-9) induced by recombinant tissue plasminogen activator in activated brain endothelial cells, with blockade of SUR1-TRPM4 by glyburide reducing MMP-9 and hemorrhagic transformation in preclinical models with recombinant tissue plasminogen activator. The recently completed GAMES (Glyburide Advantage in Malignant Edema and Stroke) clinical trials on patients with large hemispheric infarctions treated with intravenous glyburide (RP-1127) revealed promising findings with regard to brain swelling (midline shift), MMP-9, functional outcomes and mortality. Here, we review key elements of the basic science, preclinical experiments and clinical studies, both retrospective and prospective, on glyburide in focal cerebral ischemia and stroke.
Subject(s)
Brain Edema/prevention & control , Brain/drug effects , Cerebral Infarction/drug therapy , Glyburide/administration & dosage , Neuroprotective Agents/administration & dosage , Administration, Intravenous , Animals , Aquaporin 4/metabolism , Brain/metabolism , Brain/pathology , Brain Edema/etiology , Brain Edema/metabolism , Brain Edema/pathology , Cerebral Infarction/complications , Cerebral Infarction/metabolism , Cerebral Infarction/pathology , Evidence-Based Medicine , Glyburide/adverse effects , Humans , Matrix Metalloproteinase 9/metabolism , Neuroprotective Agents/adverse effects , Signal Transduction/drug effects , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/metabolism , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/metabolism , Treatment OutcomeABSTRACT
Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic ß cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.
Subject(s)
Cryoelectron Microscopy/methods , KATP Channels/chemistry , Sulfonylurea Receptors/chemistry , Adenosine Triphosphate/metabolism , Animals , Humans , Ion Channel Gating , KATP Channels/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors/agonists , Sulfonylurea Receptors/antagonists & inhibitorsABSTRACT
Gliclazide, a sulfonylurea that is widely used to treat type II-diabetes, specifically blocks KATP channels and recombinant smooth muscle (SUR2B/Kir6.1) KATP channels with high potency. Furthermore, it exerts antioxidant properties and inhibits tumor cell proliferation. In this study, we investigated the inhibitory effect of gliclazide on vascular smooth muscle cell (VSMC) proliferation and tried to identify the underlying signaling pathway. We first investigated the effect of gliclazide-induced AMP-activated protein kinase (AMPK) activation on the proliferation of VSMCs. Gliclazide induced phosphorylation of AMPK in a dose- and time-dependent manner and inhibited VSMC proliferation following stimulation by platelet-derived growth factor (PDGF). However, KATP channel openers and Kir6.1 siRNA prevented gliclazide-mediated inhibition of VSMC proliferation. Gliclazide also increased the levels of Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß), an upstream kinase of AMPK. These findings suggested that the effects of KATP channels on AMPK activity were mediated by the regulation of intracellular Ca2+ levels. Oral administration of 2mg/kg gliclazide resulted in the activation of CaMKKß and AMPK in vivo, suggesting that gliclazide suppressed VSMC proliferation via the CaMKKß-AMPK signaling pathway. Taken together, our observations indicated that gliclazide-induced AMPK activation may act to prevent diabetes-associated atherosclerosis.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Cell Proliferation/drug effects , Gliclazide/pharmacology , KATP Channels/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Potassium Channel Blockers/pharmacology , Sulfonylurea Receptors/antagonists & inhibitors , Animals , Calcium/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Activation , KATP Channels/genetics , KATP Channels/metabolism , Male , Mice, Inbred C57BL , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Phosphorylation , RNA Interference , Rats, Sprague-Dawley , Signal Transduction/drug effects , Sulfonylurea Receptors/genetics , Sulfonylurea Receptors/metabolism , Time Factors , TransfectionABSTRACT
BACKGROUND: Stroke is a leading cause of long-term disability. All neuroprotectants targeting excitotoxicity have failed to become stroke medications. In order to explore and identify new therapeutic targets for stroke, we here reviewed present studies of ionic transporters and channels that are involved in ischemic brain damage. METHOD: We surveyed recent literature from animal experiments and clinical reports in the databases of PubMed and Elsevier ScienceDirect to analyze ionic mechanisms underlying ischemic cell damage and suggest promising ideas for stroke therapy. RESULTS: Dysfunction of ionic transporters and disrupted ionic homeostasis are most early changes that underlie ischemic brain injury, thus receiving sustained attention in translational stroke research. The Na+/K+-ATPase, Na+/Ca2+ Exchanger, ionotropic glutamate receptor, acid-sensing ion channels (ASICs), sulfonylurea receptor isoform 1 (SUR1)-regulated NCCa-ATP channels, and transient receptor potential (TRP) channels are critically involved in ischemia-induced cellular degenerating processes such as cytotoxic edema, excitotoxicity, necrosis, apoptosis, and autophagic cell death. Some ionic transporters/channels also act as signalosomes to regulate cell death signaling. For acute stroke treatment, glutamate-mediated excitotoxicity must be interfered within 2 hours after stroke. The SUR1-regulated NCCa-ATP channels, Na+/K+-ATPase, ASICs, and TRP channels have a much longer therapeutic window, providing new therapeutic targets for developing feasible pharmacological treatments toward acute ischemic stroke. CONCLUSION: The next generation of stroke therapy can apply a polypharmacology strategy for which drugs are designed to target multiple ion transporters/channels or their interaction with neurotoxic signaling pathways. But a successful translation of neuroprotectants relies on in-depth analyses of cell death mechanisms and suitable animal models resembling human stroke.
Subject(s)
Brain/drug effects , Ion Transport/drug effects , Membrane Transport Modulators/therapeutic use , Neuroprotective Agents/therapeutic use , Stroke/drug therapy , Water-Electrolyte Balance/drug effects , Acid Sensing Ion Channels/drug effects , Acid Sensing Ion Channels/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Animals , Apoptosis/drug effects , Autophagy/drug effects , Brain/metabolism , Brain/pathology , Brain/physiopathology , Humans , Membrane Transport Modulators/adverse effects , Necrosis , Neuroprotective Agents/adverse effects , Receptors, Ionotropic Glutamate/antagonists & inhibitors , Receptors, Ionotropic Glutamate/metabolism , Signal Transduction/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/metabolism , Stroke/metabolism , Stroke/pathology , Stroke/physiopathology , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/metabolism , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/metabolismABSTRACT
PURPOSE: The present study investigated the putative mechanisms underlying effects of KATP channel on high glucose (HG)-induced mesangial cell proliferation and tissue inhibitors of metalloproteinases (TIMP)-2 and Collagen IV production. METHODS: Rat mesangial cells were subjected to whole cell patch clamp to record the KATP channel currents under high glucose (HG, 30 mM) condition. Cell proliferation was measured using a CCK-8 assay. The production of TIMP-2 and Collagen IV and AMP-activated protein kinase (AMPK)-signaling pathway activity was assessed by ELISA and Western blotting, respectively. AMPK agonist (AICAR) was used to analyze the role of this kinase. The expression of KATP subunit (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B) was examined using quantitative real-time PCR (RT-PCR). RESULTS: We found that HG was significant decreases in the expression of Kir6.1, SUB2A and SUB2B, three subunits of KATP, TIMP-2 production, KATP channel activity and AMPK activity, while it promoted the cell proliferation and Collagen IV production in rat mesangial cells. Pretreatment with KATP selective opener (diazoxide, DZX) significantly inhibited HG-induced mesangial cell proliferation, Collagen IV production and decrease in KATP channel activity in rat mesangial cells, which were reversed by pretreatment of 5-hydroxydecanoate, a selective inhibitor of KATP. Moreover, AICAR pretreatment inhibited HG-induced decrease in KATP channel activity. CONCLUSIONS: Taken together, activating AMPK-KATP signaling may protect against HG-induced mesangial cell proliferation and Collagen IV production, and, thereby, provides new insights into the molecular mechanisms underlying early diabetic nephropathy (DN).
Subject(s)
AMP-Activated Protein Kinases/metabolism , Cell Proliferation/drug effects , Collagen Type IV/biosynthesis , Glucose/pharmacology , KATP Channels/metabolism , Mesangial Cells/metabolism , Sulfonylurea Receptors/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Cells, Cultured , Decanoic Acids/pharmacology , Diabetic Nephropathies/metabolism , Diazoxide/pharmacology , Glucose/administration & dosage , Hydroxy Acids/pharmacology , Hypoglycemic Agents/pharmacology , KATP Channels/antagonists & inhibitors , Male , Mesangial Cells/drug effects , Potassium Channels, Inwardly Rectifying/metabolism , Rats , Rats, Sprague-Dawley , Ribonucleotides/pharmacology , Signal Transduction/drug effects , Sulfonylurea Receptors/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-2/metabolismABSTRACT
Insulin secretagogues are used for treatment of type 2 diabetes. We attempted to discover novel small molecules to stimulate insulin secretion by using in silico similarity search using sulfonylureas as query, followed by measurement of insulin secretion. Among 38 compounds selected by in silico similarity search, we found three diphenylsemicarbazides and one quinolone that stimulate insulin secretion. We focused on compound 8 (C8), which had the strongest insulin-secreting effect. Based on the structure-activity relationship of C8-derivatives, we identified diphenylthiosemicarbazide (DSC) 108 as the most potent secretagogue. DSC108 increased the intracellular Ca2+ level in MIN6-K8 cells. Competitive inhibition experiment and electrophysiological analysis revealed sulfonylurea receptor 1 (SUR1) to be the target of DSC108 and that this diphenylthiosemicarbazide directly inhibits ATP-sensitive K+ (KATP) channels. Pharmacokinetic analysis showed that DSC108 has a short half-life in vivo. Oral administration of DSC108 significantly suppressed the rises in blood glucose levels after glucose load in wild-type mice and improved glucose tolerance in the Goto-Kakizaki (GK) rat, a model of type 2 diabetes with impaired insulin secretion. Our data indicate that DSC108 is a novel insulin secretagogue, and is a lead compound for development of a new anti-diabetic agent.
Subject(s)
Amino Acids, Cyclic/pharmacology , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Membrane Potentials/drug effects , Semicarbazides/pharmacology , Thiosemicarbazones/pharmacology , Administration, Oral , Amino Acids, Cyclic/chemistry , Amino Acids, Cyclic/metabolism , Amino Acids, Cyclic/therapeutic use , Animals , Blood Glucose/analysis , Calcium/metabolism , Cell Line , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/pathology , Glucose Tolerance Test , Glyburide/pharmacology , Glyburide/therapeutic use , Half-Life , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/metabolism , Hypoglycemic Agents/therapeutic use , Insulin Secretion , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , KATP Channels/antagonists & inhibitors , KATP Channels/metabolism , Male , Mice , Mice, Inbred C57BL , Protein Binding , Rats , Semicarbazides/blood , Semicarbazides/chemistry , Semicarbazides/metabolism , Structure-Activity Relationship , Sulfonylurea Compounds/chemistry , Sulfonylurea Compounds/metabolism , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/metabolism , Thiosemicarbazones/chemistry , Thiosemicarbazones/metabolism , Thiosemicarbazones/therapeutic useABSTRACT
Ischemia-reperfusion injury after central nervous system (CNS) injury presents a major health care challenge with few promising treatments. Recently, it has become possible to reduce edema after CNS injury by antagonizing a sulfonylurea receptor 1 (SUR1) regulated ion channel expressed after injury. SUR1 upregulation after injury is a necessary precondition for the formation of this channel, and has been implicated in white matter injury after clinical spinal cord trauma. Glibenclamide, an SUR1 antagonist, appears to have neuroprotective effect against cerebral stroke in an open-label small clinical trial and great effectiveness in reducing damage after varied experimental CNS injury models. Despite its importance in CNS injuries, SUR1 upregulation appears to play no part in rodent anterior ischemic optic neuropathy (rAION) injury as tested by real-time PCR and immunohistochemical staining of rAION-injured rat optic nerve (ON). Furthermore, the SUR1 antagonist glibenclamide administered immediately after rAION injury provided no protection to proximal ON microvasculature 1 day post-injury but may reduce optic nerve head edema in a manner unrelated to ON SUR1 expression. Our results suggest that there may be fundamental differences between rAION optic nerve ischemia and other CNS white matter injuries where SUR1 appears to play a role.
Subject(s)
Central Nervous System/physiopathology , Optic Neuropathy, Ischemic/physiopathology , Spinal Cord Injuries/physiopathology , Sulfonylurea Receptors/metabolism , Animals , Central Nervous System/drug effects , Central Nervous System/injuries , Glyburide/administration & dosage , Glyburide/pharmacology , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/pharmacology , Immunohistochemistry , Mice, Inbred C57BL , Mice, Knockout , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/pharmacology , Optic Neuropathy, Ischemic/genetics , Optic Neuropathy, Ischemic/metabolism , Rats , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stroke/physiopathology , Stroke/prevention & control , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/genetics , Time Factors , Tomography, Optical Coherence , Up-Regulation/drug effectsABSTRACT
BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), deletion of transient receptor potential melastatin 4 (Trpm4) and administration of glibenclamide were found to ameliorate disease progression, prompting speculation that glibenclamide acts by directly inhibiting Trpm4. We hypothesized that in EAE, Trpm4 upregulation is accompanied by upregulation of sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which are highly sensitive to glibenclamide, and that Sur1-Trpm4 channels are required for EAE progression. METHODS: EAE was induced in wild-type (WT) and Abcc8-/- mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). Lumbar spinal cords were examined by immunohistochemistry, immuno-Förster resonance energy transfer (immunoFRET), and co-immunoprecipitation for Sur1-Trpm4. WT/EAE mice were administered with the Sur1 inhibitor, glibenclamide, beginning on post-induction day 10. Mice were evaluated for clinical function, inflammatory cells and cytokines, axonal preservation, and white matter damage. RESULTS: Sur1-Trpm4 channels were upregulated in EAE, predominantly in astrocytes. The clinical course and severity of EAE were significantly ameliorated in glibenclamide-treated WT/EAE and in Abcc8-/-/EAE mice. At 30 days, the lumbar spinal cords of glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice showed significantly fewer invading immune cells, including leukocytes (CD45), T cells (CD3), B cells (CD20) and macrophages/microglia (CD11b), and fewer cells expressing pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17). In both glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice, the reduced inflammatory burden correlated with better preservation of myelin, better preservation of axons, and more numerous mature and precursor oligodendrocytes. CONCLUSIONS: Sur-Trpm4 channels are newly upregulated in EAE and may represent a novel target for disease-modifying therapy in multiple sclerosis.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Sulfonylurea Receptors/antagonists & inhibitors , TRPM Cation Channels/antagonists & inhibitors , Animals , Axons/pathology , Female , Gene Silencing , Glyburide/therapeutic use , Hypoglycemic Agents/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Sheath/drug effects , Myelin-Oligodendrocyte Glycoprotein , Neuroprotective Agents/therapeutic use , Peptide Fragments , Spinal Cord/pathology , Sulfonylurea Receptors/geneticsABSTRACT
The ABCC9 gene and its polypeptide product, SUR2, are increasingly implicated in human neurologic disease, including prevalent diseases of the aged brain. SUR2 proteins are a component of the ATP-sensitive potassium ("KATP") channel, a metabolic sensor for stress and/or hypoxia that has been shown to change in aging. The KATP channel also helps regulate the neurovascular unit. Most brain cell types express SUR2, including neurons, astrocytes, oligodendrocytes, microglia, vascular smooth muscle, pericytes, and endothelial cells. Thus it is not surprising that ABCC9 gene variants are associated with risk for human brain diseases. For example, Cantu syndrome is a result of ABCC9 mutations; we discuss neurologic manifestations of this genetic syndrome. More common brain disorders linked to ABCC9 gene variants include hippocampal sclerosis of aging (HS-Aging), sleep disorders, and depression. HS-Aging is a prevalent neurological disease with pathologic features of both neurodegenerative (aberrant TDP-43) and cerebrovascular (arteriolosclerosis) disease. As to potential therapeutic intervention, the human pharmacopeia features both SUR2 agonists and antagonists, so ABCC9/SUR2 may provide a "druggable target", relevant perhaps to both HS-Aging and Alzheimer's disease. We conclude that more work is required to better understand the roles of ABCC9/SUR2 in the human brain during health and disease conditions.
Subject(s)
Aging/physiology , Brain Diseases , Cardiomegaly , Hippocampus , Hypertrichosis , Osteochondrodysplasias , Sulfonylurea Receptors , Animals , Brain Diseases/drug therapy , Brain Diseases/metabolism , Brain Diseases/pathology , Cardiomegaly/genetics , Cardiomegaly/metabolism , Cardiomegaly/pathology , Hippocampus/metabolism , Hippocampus/pathology , Humans , Hypertrichosis/genetics , Hypertrichosis/metabolism , Hypertrichosis/pathology , Mutation , Osteochondrodysplasias/genetics , Osteochondrodysplasias/metabolism , Osteochondrodysplasias/pathology , Sulfonylurea Receptors/agonists , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/genetics , Therapies, InvestigationalABSTRACT
Ischemic and hemorrhagic strokes are associated with severe functional disability and high mortality. Except for recombinant tissue plasminogen activator, therapies targeting the underlying pathophysiology of central nervous system (CNS) ischemia and hemorrhage are strikingly lacking. Sur1-regulated channels play essential roles in necrotic cell death and cerebral edema following ischemic insults, and in neuroinflammation after hemorrhagic injuries. Inhibiting endothelial, neuronal, astrocytic and oligodendroglial sulfonylurea receptor 1-transient receptor potential melastatin 4 (Sur1-Trpm4) channels and, in some cases, microglial KATP (Sur1-Kir6.2) channels, with glibenclamide is protective in a variety of contexts. Robust preclinical studies have shown that glibenclamide and other sulfonylurea agents reduce infarct volumes, edema and hemorrhagic conversion, and improve outcomes in rodent models of ischemic stroke. Retrospective studies suggest that diabetic patients on sulfonylurea drugs at stroke presentation fare better if they continue on drug. Additional laboratory investigations have implicated Sur1 in the pathophysiology of hemorrhagic CNS insults. In clinically relevant models of subarachnoid hemorrhage, glibenclamide reduces adverse neuroinflammatory and behavioral outcomes. Here, we provide an overview of the preclinical studies of glibenclamide therapy for CNS ischemia and hemorrhage, discuss the available data from clinical investigations, and conclude with promising preclinical results that suggest glibenclamide may be an effective therapeutic option for ischemic and hemorrhagic stroke.
Subject(s)
Glyburide/therapeutic use , Stroke/drug therapy , Animals , Blood-Brain Barrier/metabolism , Humans , Hypoglycemic Agents/therapeutic use , KATP Channels/chemistry , KATP Channels/metabolism , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/metabolismABSTRACT
The ATP-sensitive K(+) (KATP) channel is part of a class of inward rectifier K(+) channels that can link local O2 availability to vasomotor tone across exercise-induced metabolic transients. The present investigation tested the hypothesis that if KATP channels are crucial to exercise hyperemia, then inhibition via glibenclamide (GLI) would lower hindlimb skeletal muscle blood flow (BF) and vascular conductance during treadmill exercise. In 27 adult male Sprague-Dawley rats, mean arterial pressure, blood lactate concentration, and hindlimb muscle BF (radiolabeled microspheres) were determined at rest (n = 6) and during exercise (n = 6-8, 20, 40, and 60 m/min, 5% incline, i.e., ~60-100% maximal O2 uptake) under control and GLI conditions (5 mg/kg intra-arterial). At rest and during exercise, mean arterial pressure was higher (rest: 17 ± 3%, 20 m/min: 5 ± 1%, 40 m/min: 5 ± 2%, and 60 m/min: 5 ± 1%, P < 0.05) with GLI. Hindlimb muscle BF (20 m/min: 16 ± 7%, 40 m/min: 30 ± 9%, and 60 m/min: 20 ± 8%) and vascular conductance (20 m/min: 20 ± 7%, 40 m/min: 33 ± 8%, and 60 m/min: 24 ± 8%) were lower with GLI during exercise at 20, 40, and 60 m/min, respectively (P < 0.05 for all) but not at rest. Within locomotory muscles, there was a greater fractional reduction present in muscles comprised predominantly of type I and type IIa fibers at all exercise speeds (P < 0.05). Additionally, blood lactate concentration was 106 ± 29% and 44 ± 15% higher during exercise with GLI at 20 and 40 m/min, respectively (P < 0.05). That KATP channel inhibition reduces hindlimb muscle BF during exercise in rats supports the obligatory contribution of KATP channels in large muscle mass exercise-induced hyperemia.
Subject(s)
Glyburide/pharmacology , Hyperemia/metabolism , Muscle, Skeletal/blood supply , Physical Exertion , Regional Blood Flow , Sulfonylurea Receptors/antagonists & inhibitors , Animals , Arterial Pressure , Hyperemia/physiopathology , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Sulfonylureas, which stimulate insulin secretion from pancreatic ß-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on ß-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on ß-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.
Subject(s)
Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Gliclazide/pharmacology , Ion Channel Gating , Potassium Channels, Inwardly Rectifying/metabolism , Sulfonylurea Receptors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Humans , Insulin-Secreting Cells/metabolism , Molecular Sequence Data , Potassium Channels, Inwardly Rectifying/chemistry , Protein Binding , Rats , Sulfonylurea Receptors/agonists , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/chemistry , XenopusABSTRACT
BACKGROUND AND PURPOSE: ATP-sensitive K(+)(K(ATP)) channels, which are composed of K(IR)6.x associated with sulphonylurea receptor (SUR) subunits, have been detected in native smooth muscle cells, but it is currently not known which of these is expressed in mouse vas deferens myocytes. EXPERIMENTAL APPROACH: Pharmacological and electrophysiological properties of K(ATP) channels in mouse vas deferens myocytes were investigated using patch clamp techniques. Molecular biological analyses were performed to examine the properties of these K(ATP) channels. KEY RESULTS: During conventional whole-cell recording, pinacidil elicited an inward current that was suppressed by glibenclamide, a sulfonylurea agent, and by U-37883A, a selective K(IR)6.1 blocker. When 0.3 mM ATP was added to the pipette solution, the peak amplitude of the pinacidil-induced current was much smaller than that recorded in its absence. When 3 mM UDP, GDP or ADP was included in the pipette solution, an inward current was elicited after establishment of the conventional whole-cell configuration, with potency order being UDP > GDP > ADP. These nucleoside diphosphate-induced inward currents were suppressed by glibenclamide. MCC-134, a SUR modulator, induced glibenclamide-sensitive K(ATP) currents that were similar to those induced by 100 µM pinacidil. In the cell-attached configuration, pinacidil activated channels with a conductance similar to that of K(IR)6.1. Reverse transcription PCR analysis revealed the expression of K(IR)6.1 and SUR2B transcripts and immunohistochemical studies indicated the presence of K(IR)6.1 and SUR2B proteins in the myocytes. CONCLUSIONS AND IMPLICATIONS: Our results indicate that native K(ATP) channels in mouse vas deferens myocytes are a heterocomplex of K(IR)6.1 channels and SUR2B subunits.
Subject(s)
Adenosine Triphosphate/metabolism , KATP Channels/metabolism , Myocytes, Smooth Muscle/metabolism , Sulfonylurea Receptors/metabolism , Vas Deferens/metabolism , Adenosine Diphosphate/metabolism , Animals , Dose-Response Relationship, Drug , Guanosine Diphosphate/metabolism , HEK293 Cells , Humans , KATP Channels/antagonists & inhibitors , KATP Channels/genetics , Male , Membrane Potentials , Mice , Mice, Inbred BALB C , Myocytes, Smooth Muscle/drug effects , Potassium Channel Blockers/pharmacology , Sulfonylurea Receptors/antagonists & inhibitors , Sulfonylurea Receptors/genetics , Transfection , Uridine Diphosphate/metabolism , Vas Deferens/cytology , Vas Deferens/drug effectsABSTRACT
BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) can leave patients with memory impairments that may not recover fully. Molecular mechanisms are poorly understood, and no treatment is available. The sulfonylurea receptor 1-transient receptor potential melastatin 4 (Sur1-Trpm4) channel plays an important role in acute central nervous system injury. We evaluated upregulation of Sur1-Trpm4 in humans with SAH and, in rat models of SAH, we examined Sur1-Trpm4 upregulation, its role in barrier dysfunction and neuroinflammation, and its consequences on spatial learning. METHODS: We used Förster resonance energy transfer to detect coassociated Sur1 and Trpm4 in human autopsy brains with SAH. We studied rat models of SAH involving filament puncture of the internal carotid artery or injection of blood into the subarachnoid space of the entorhinal cortex. In rats, we used Förster resonance energy transfer and coimmunoprecipitation to detect coassociated Sur1 and Trpm4, we measured immunoglobulin G extravasation and tumor necrosis α overexpression as measures of barrier dysfunction and neuroinflammation, and we assessed spatial learning and memory on days 7 to 19. RESULTS: Sur1-Trpm4 channels were upregulated in humans and rats with SAH. In rats, inhibiting Sur1 using antisense or the selective Sur1 inhibitor glibenclamide reduced SAH-induced immunoglobulin G extravasation and tumor necrosis α overexpression. In models with entorhinal SAH, rats treated with glibenclamide for 7 days after SAH exhibited better platform search strategies and better performance on incremental and rapid spatial learning than vehicle-treated controls. CONCLUSIONS: Sur1-Trpm4 channels are upregulated in humans and rats with SAH. Channel inhibition with glibenclamide may reduce neuroinflammation and the severity of cognitive deficits after SAH.
Subject(s)
Cognition Disorders/metabolism , Encephalitis/metabolism , Subarachnoid Hemorrhage/metabolism , Sulfonylurea Receptors/antagonists & inhibitors , TRPM Cation Channels/antagonists & inhibitors , Animals , Brain/drug effects , Brain/metabolism , Cognition Disorders/genetics , Cognition Disorders/physiopathology , Encephalitis/genetics , Encephalitis/physiopathology , Glyburide/pharmacology , Humans , Maze Learning/drug effects , Rats , Subarachnoid Hemorrhage/genetics , Subarachnoid Hemorrhage/physiopathology , Up-Regulation/drug effectsABSTRACT
BACKGROUND AND PURPOSE: The influence of sulfonylurea receptor 1 (SUR1) and its inhibitor glibenclamide on progressive secondary hemorrhage (PSH), progressive hemorrhagic necrosis (PHN), and brain edema has been studied in rat models of traumatic brain injury (TBI) and ischemia. These studies indicate that blocking SUR1 may exert protective effects in terms of outcome. METHODS: We discuss the effects of glibenclamide on outcome in patients with type 2 diabetes mellitus and TBI. We collected demographic, clinical, and imaging data from the clinical records of TBI patients with type 2 diabetes who were admitted to the neurosurgery department at Shanghai 6th People's Hospital between 2001 and 2012. Data from patients who met the inclusion criteria were analyzed. Patients were divided into glibenclamide group and insulin group. RESULTS: Of 70 patients fit criteria for inclusion, no significant difference was observed except for age and fasting plasma glucose between the two groups. Outcome indicators, including GCS discharge, GOS discharge, length of study in hospital (LOS-H), and the presence of PSH showed no significant difference too (p>0.05), except for length of stay in neuro-intensive care unit (LOS-NICU) (p<0.05). Age, hours between the initial CT scan and the injury (HCT1) and GCS at admission were observed as factors associated with PSH after logistic regression. CONCLUSIONS: In general, the use of glibenclamide to control plasma glucose after TBI had no significant effect on patient outcome at discharge but it could reduce the LOS-NICU (p<0.05). Glibenclamide also had no apparent effect on the presence of PSH in TBI patients with type 2 diabetes mellitus.