tram-34 and Disease-Models--Animal

Amyotrophic lateral sclerosis (ALS) patients exhibit dysfunctional energy metabolism and weight loss, which is negatively correlated with survival, together with neuroinflammation. However, the possible contribution of neuroinflammation to deregulations of feeding behaviour in ALS has not been studied in detail. We here investigated if microglial K. hSOD1. We found that treatment of hSOD1. Using ALS mouse models, we describe defects in the hypothalamic melanocortin system that affect appetite control. These results reveal a new regulatory role for K

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Energy Metabolism; Feeding Behavior; Homeostasis; Melanocortins; Mice; Mice, Transgenic; Microglia; Potassium Channels, Calcium-Activated; Pyrazoles; Receptors, Cannabinoid; Spinal Cord; Superoxide Dismutase-1; Weight Gain

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cornea; Corneal Diseases; Disease Models, Animal; Fibroblasts; Fibrosis; Gene Expression; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice, Inbred C57BL; Mice, Knockout; Molecular Targeted Therapy; Myofibroblasts; Pyrazoles; Wound Healing

Recent studies described a critical role for microglia in amyotrophic lateral sclerosis (ALS), where these CNS-resident immune cells participate in the establishment of an inflammatory microenvironment that contributes to motor neuron degeneration. Understanding the mechanisms leading to microglia activation in ALS could help to identify specific molecular pathways which could be targeted to reduce or delay motor neuron degeneration and muscle paralysis in patients. The intermediate-conductance calcium-activated potassium channel KCa3.1 has been reported to modulate the "pro-inflammatory" phenotype of microglia in different pathological conditions. We here investigated the effects of blocking KCa3.1 activity in the hSOD1

Topics: Amyotrophic Lateral Sclerosis; Animals; Cell Death; Disease Models, Animal; Disease Progression; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Motor Neurons; Phenotype; Potassium Channels, Calcium-Activated; Pyrazoles; Spinal Cord; Superoxide Dismutase

KCa3.1, a Ca

Topics: Actins; Animals; Cell Proliferation; Cells, Cultured; Collagen Type I; Disease Models, Animal; Fibroblasts; Intermediate-Conductance Calcium-Activated Potassium Channels; Lung; Male; Paraquat; Potassium Channel Blockers; Pulmonary Fibrosis; Pyrazoles; Rats, Sprague-Dawley; Signal Transduction; Time Factors; Transforming Growth Factor beta1

Partial bladder outlet obstruction (PBOO) usually induces overactive bladder (OAB) associated with detrusor overactivity (DO) which is related to the increased contractility of detrusor smooth muscle (DSM). The pharmacological activation of small-conductance Ca. Two weeks after surgically inducing PBOO in female guinea pigs, cystometry indicated that DO was achieved. Using this animal model, we conducted quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and isometric tension recordings.. The qRT-PCR experiments indicated that PBOO DSM had reduced SK channel mRNA expression. Isometric tension recordings showed a decreased inhibitory effect of NS309 on spontaneous phasic and electrical field stimulation-induced contractions via the activation of SK channels in PBOO DSM.. This study presents the novel finding that PBOO is associated with attenuated DSM SK channel expression and function, which results in increased DSM contractility and contributes to DO. Therefore, SK channels could be a therapeutic target to control OAB.

Topics: Animals; Apamin; Disease Models, Animal; Female; Gene Expression; Guinea Pigs; Indoles; Isometric Contraction; Muscle, Smooth; Oximes; Pyrazoles; RNA, Messenger; Small-Conductance Calcium-Activated Potassium Channels; Urinary Bladder; Urinary Bladder Neck Obstruction; Urinary Bladder, Overactive; Urodynamics

Tubular cell apoptosis significantly contributes to cisplatin-induced acute kidney injury (AKI) pathogenesis. Although KCa3.1, a calcium-activated potassium channel, participates in apoptosis, its involvement in cisplatin-induced AKI is unknown. Here, we found that cisplatin treatment triggered an early induction of KCa3.1 expression associated with HK-2 cell apoptosis, the development of renal tubular damage, and apoptosis in mice. Treatment with the highly selective KCa3.1 blocker TRAM-34 suppressed cisplatin-induced HK-2 cell apoptosis. We further assessed whether KCa3.1 mediated cisplatin-induced AKI in genetic knockout and pharmacological blockade mouse models. KCa3.1 deficiency reduced renal function loss, renal tubular damage, and the induction of the apoptotic marker caspase-3 in the kidneys of cisplatin-treated KCa3.1 (-/-) mice. Pharmacological blockade of KCa3.1 by TRAM-34 similarly attenuated cisplatin-induced AKI in mice. Furthermore, we dissected the mechanisms underlying cisplatin-induced apoptosis reduction via KCa3.1 blockade. We found that KCa3.1 blockade attenuated cytochrome c release and the increase in the intrinsic apoptotic mediators Bax, Bak, and caspase-9 after cisplatin treatment. KCa3.1 blocking inhibited the cisplatin-induced activation of the endoplasmic reticulum (ER) stress mediator caspase-12, which is independent of calcium-dependent protease m-calpain activation. Taken together, KCa3.1 blockade protects against cisplatin-induced AKI through the attenuation of apoptosis by interference with intrinsic apoptotic and ER stress-related mediators, providing a potential target for the prevention of cisplatin-induced AKI.

Topics: Acute Kidney Injury; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line; Cisplatin; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum Stress; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Kidney Tubules, Proximal; Mice, Inbred C57BL; Mice, Knockout; Potassium Channel Blockers; Pyrazoles; Signal Transduction; Time Factors

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1/KCNN4 plays an important role in the modulation of Ca(2+) signaling through the control of the membrane potential in T lymphocytes. Here, we study the involvement of KCa3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in a mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5% dextran sulfate sodium for 7 days. Inflammation-induced changes in KCa3.1 activity and the expressions of KCa3.1 and its regulators in MLN CD4(+) T lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp, and flow cytometric analyses. Concomitant with an upregulation of KCa3.1a and nucleoside diphosphate kinase B (NDPK-B), a positive KCa3.1 regulator, an increase in KCa3.1 activity was observed in MLN CD4(+) T lymphocytes in the IBD model. Pharmacological blockade of KCa3.1 elicited the following results: 1) a significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of KCa3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4(+) T lymphocytes. These findings suggest that the increase in KCa3.1 activity induced by the upregulation of KCa3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4(+) T lymphocyte and, therefore, that the pharmacological blockade of KCa3.1 may decrease the risk of IBD.

Topics: Animals; CD4-Positive T-Lymphocytes; Dextran Sulfate; Disease Models, Animal; DNA-Binding Proteins; Inflammatory Bowel Diseases; Intermediate-Conductance Calcium-Activated Potassium Channels; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; NM23 Nucleoside Diphosphate Kinases; Nuclear Proteins; Pyrazoles; Ubiquitin-Protein Ligases; Up-Regulation

The precise pathogenesis of allergic rhinitis (AR) remains unclear and AR is less easily cured. Recent evidence has suggested that calcium-activated K+ channel-3.1(KCa3.1) is implicated in the immune response of allergic and inflammatory diseases and TRAM-34 is a selective KCa3.1 blocker. However, little is known about its role in AR. We aimed to investigate the effect of TRAM-34 in a mouse model of AR induced by ovalbumin (OVA). The BALB/c mice were divided into six groups: untreated AR group, 200 μg TRAM-34 treated AR group, 400 μg TRAM-34 treated AR group, 200 μg TRAM-34 treated normal group, 400 μg TRAM-34 treated normal group and untreated normal control group. Histopathological characteristics were assessed by HE staining. KCa3.1 protein expression was investigated by immunohistochemistry and western blotting method, and mRNA expression of KCa3.1, stromal interaction molecule1 (STIM1) and Orai1 in nasal tissues were assessed by real-time PCR. Furthermore, concentrations of OVA-specific IgE, ECP, IL-4, IL-5, IL-17 and IL-1β in nasal lavage fluid (NLF) were analyzed by enzyme-linked immunosorbent assay (ELISA). Results showed that TRAM-34 administration into the nostril attenuated sneezing, nasal rubbing, epithelial cell proliferation, eosinophil infiltration and inhibited nasal mucosa KCa3.1, STIM1 and Orai1 expression in TRAM-34 treated mice compared with untreated AR mice and suppressed inflammatory cytokines in the NLF of TRAM-34 treated groups compared with untreated AR mice. In conclusion, TRAM-34 could effectively alleviate murine allergic rhinitis by suppressing KCa3.1 and leads to reduction of K+ efflux and Ca2 + influx, leading to inflammation reduction and allergic responses attenuation.

Topics: Administration, Intranasal; Animals; Blotting, Western; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Immunoglobulin E; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice, Inbred BALB C; Nasal Mucosa; Ovalbumin; Potassium Channel Blockers; Pyrazoles; Rhinitis, Allergic

Epidermal growth factor (EGF) is used to treat alkali-burned corneas. However, EGF-induced corneal angiogenesis, which is currently untreatable, is a side effect of this therapy. We therefore explored the role of the intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) in EGF-induced angiogenesis and tested whether KCa3.1 blockade can suppress EGF-induced corneal angiogenesis. The proliferation, migration and tube formation of HUVECs (human umbilical vein endothelial cells) in response to EGF, the MEK inhibitor PD98059 and the KCa3.1 inhibitor TRAM-34 were analyzed in vitro via MTT, cell counting, scratch and tube formation assays. The protein and mRNA levels of KCa3.1, phosphorylated-ERK (P-ERK), total-ERK (T-ERK), cyclin-dependent kinase 4 (CDK4), vimentin and MMP-2 were assessed via western blotting and RT-PCR. KCa3.1 and vimentin expression were also detected through immunofluorescence staining. Flow cytometry was performed to examine the cell cycle. Further, an in vivo murine alkali-burned cornea model was developed and treated with EGF and TRAM-34 eye drops to analyze the effect of these treatments on corneal healing and angiogenesis. The corneas were also analyzed by histological staining. The in vitro results showed that EGF induces the upregulation of KCa3.1 and P-ERK in HUVECs and that this upregulation is suppressed by PD98059. EGF stimulates proliferation, migration and tube formation in HUVECs, and this effect can be suppressed by TRAM-34. TRAM-34 also arrests HUVECs in the G1 phase of the cell cycle and downregulates CDK4, vimentin and MMP-2 in these cells. The in vivo results indicated that TRAM-34 suppresses EGF-induced corneal angiogenesis without affecting EGF-induced corneal wound healing. In summary, the upregulation of KCa3.1 may be crucial for EGF-induced angiogenesis through the MAPK/ERK signaling pathway. Thus, KCa3.1 may be a potential target for the treatment of EGF-induced corneal angiogenesis.

Topics: Animals; Blotting, Western; Burns, Chemical; Cell Movement; Cell Proliferation; Cornea; Corneal Neovascularization; Cyclin-Dependent Kinase 4; Disease Models, Animal; Epidermal Growth Factor; Eye Burns; Flavonoids; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Hydroxide; Up-Regulation; Vimentin; Wound Healing

The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of K(Ca)3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice.. The expression patterns of K(Ca)3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized, but non-challenged (Ox+/-) and Ox-sensitized and -challenged (Ox+/+)] using real-time polymerase chain reaction, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of K(Ca)3.1 blockade were examined by the administration of selective K(Ca)3.1 blockers.. Significant up-regulation of K(Ca)3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, K(Ca)3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor (REST) was significantly down-regulated. Pharmacological blockade of K(Ca)3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0/G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the K(Ca)3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+.. The up-regulation of K(Ca)3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice; and K(Ca)3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; Cell Cycle; Disease Models, Animal; Ear Auricle; Hypersensitivity, Delayed; Intermediate-Conductance Calcium-Activated Potassium Channels; Lymph Nodes; Male; Mice, Inbred BALB C; Oxazolone; Potassium Channel Blockers; Pyrazoles; Repressor Proteins

Key features of asthma include bronchial hyperresponsiveness (BHR), eosinophilic airway inflammation, and bronchial remodeling, characterized by subepithelial collagen deposition, airway fibrosis, and increased bronchial smooth muscle (BSM) mass. The calcium-activated K(+) channel K(Ca)3.1 is expressed by many cells implicated in the pathogenesis of asthma, and is involved in both inflammatory and remodeling responses in a number of tissues. The specific K(Ca)3.1 blocker 5-[(2-chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34) attenuates BSM cell proliferation, and both mast cell and fibrocyte recruitment in vitro. We aimed to examine the effects of K(Ca)3.1 blockade on BSM remodeling, airway inflammation, and BHR in a murine model of chronic asthma. BALB/c mice were sensitized with intraperitoneal ovalbumin (OVA) on Days 0 and 14, and then challenged with intranasal OVA during Days 14-75. OVA-sensitized/challenged mice received TRAM-34 (120 mg/kg/day, subcutaneous) from Days -7 to 75 (combined treatment), Days -7 to 20 (preventive treatment), or Days 21 to 75 (curative treatment). Untreated mice received daily injections of vehicle (n = 8 per group). Bronchial remodeling was assessed by histological and immunohistochemical analyses. Inflammation was evaluated using bronchoalveolar lavage and flow cytometry. We also determined BHR in both conscious and anesthetized mice via plethysmography. We demonstrated that curative treatment with TRAM-34 abolishes BSM remodeling and subbasement collagen deposition, and attenuates airway eosinophilia. Although curative treatment alone did not significantly reduce BHR, the combined treatment attenuated nonspecific BHR to methacholine. This study indicates that K(Ca)3.1 blockade could provide a new therapeutic strategy in asthma.

Topics: Airway Remodeling; Animals; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eosinophilia; Female; Flow Cytometry; Immunohistochemistry; Mice; Mice, Inbred BALB C; Potassium Channels, Calcium-Activated; Pyrazoles

The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD.. Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28.. Subepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes.. Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.

Topics: Animals; Bronchiolitis Obliterans; Cell Proliferation; Disease Models, Animal; Enzyme-Linked Immunospot Assay; Genetic Therapy; Intermediate-Conductance Calcium-Activated Potassium Channels; Isoantibodies; Lymphocyte Activation; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Potassium Channel Blockers; Pyrazoles; Respiratory Mucosa; Th1 Cells; Th2 Cells; Time Factors; Trachea

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K⁺ channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca²⁺ signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.

Topics: Analysis of Variance; Animals; Animals, Newborn; Calcium; Calcium Signaling; CD11b Antigen; Cells, Cultured; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Intermediate-Conductance Calcium-Activated Potassium Channels; Macrophages; Mice; Mice, Inbred C57BL; Microglia; Motor Activity; Nerve Tissue Proteins; Nitric Oxide Synthase Type II; Potassium Channel Blockers; Pyrazoles; RNA, Messenger; Spinal Cord Injuries; Thapsigargin; Time Factors; Up-Regulation; Uridine Triphosphate

New structural classes of K(V)1.3 and IK-1 ion channel blockers have been identified based on a virtual high throughput screening approach using a homology model of KcsA. These compounds display inhibitory effects on T-cell and/or keratinocyte proliferation and immunosuppressant activity within a DTH animal model.

Topics: Animals; Calcium Signaling; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Humans; Hypersensitivity, Delayed; Immunosuppressive Agents; Intermediate-Conductance Calcium-Activated Potassium Channels; Keratinocytes; Kv1.3 Potassium Channel; Patch-Clamp Techniques; Potassium Channel Blockers

The role of SK(Ca) and IK(Ca) channels in myogenic tone and endothelium-derived hyperpolarizing factor (EDHF) responsiveness was investigated under control conditions and after ischemia and reperfusion in parenchymal arterioles (PA) versus middle cerebral arteries (MCA).. MCA and PA were dissected from male Wistar rats that were ischemic for 1 hour with 24 hours of reperfusion (n=12) or sham controls (n=12). Basal tone and reactivity to apamin (300 nmol/L), TRAM-34 (1.0 micromol/L), and nitro-L-arginine (0.1 mmol/L) were compared in PA and MCA pressurized to 40 mm Hg and 75 mm Hg, respectively. SK(Ca) and IK(Ca) channel mRNA expression was measured using real-time PCR.. PA developed greater basal tone than MCA (42+/-4% versus 19+/-3%; P<0.01). Addition of apamin and TRAM-34 increased tone of PA by 25+/-3% and 16+/-2%, respectively, whereas MCA had no response to either inhibitor. After ischemia and reperfusion, the response to nitric oxide synthase inhibition (NOS) was diminished in PA, whereas EDHF responsiveness was preserved. In addition, stimulated EDHF dilation was partially reversed by apamin and completely reversed by TRAM-34 in both control and ischemic PA. SK(Ca) and IK(Ca) channel mRNA expression was similar in PA and MCA and not altered by ischemia and reperfusion. However, IK(Ca) channel mRNA expression was 4- to 5-fold greater than SK(Ca) channels.. It appears that SK(Ca) and IK(Ca) channel activity diminishes basal tone of PA, but not MCA. The preservation of EDHF responsiveness of PA after ischemia and reperfusion suggests an important role for this vasodilator under conditions when NOS is inhibited.

Topics: Animals; Apamin; Arterioles; Biological Factors; Brain Ischemia; Calcimycin; Disease Models, Animal; Enzyme Inhibitors; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionophores; Male; Middle Cerebral Artery; Nitric Oxide; Nitroarginine; Pyrazoles; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Small-Conductance Calcium-Activated Potassium Channels; Vasodilation

We previously demonstrated that upregulation of intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K(Ca)3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis.. Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K(Ca)3.1 blocker TRAM-34. Expression of K(Ca)3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K(Ca)3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K(Ca)3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level.. Blockade of K(Ca)3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.

Topics: Angioplasty, Balloon, Coronary; Animals; Cell Differentiation; Coronary Stenosis; Coronary Vessels; Disease Models, Animal; Intermediate-Conductance Calcium-Activated Potassium Channels; Male; Muscle, Smooth, Vascular; Phenotype; Potassium Channel Blockers; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Pyrazoles; Swine; Transcription Factors

Topics: Angioplasty, Balloon, Coronary; Animals; Cell Differentiation; Coronary Stenosis; Disease Models, Animal; Intermediate-Conductance Calcium-Activated Potassium Channels; Muscle, Smooth, Vascular; Phenotype; Pyrazoles; Swine

We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca(2+)-dependent K(+) channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (I(h)) antagonist Cs(+). It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

Topics: Action Potentials; Animals; Disease Models, Animal; Guinea Pigs; Ileitis; Ileum; Intermediate-Conductance Calcium-Activated Potassium Channels; Myenteric Plexus; Neurons; Phenotype; Potassium Channel Blockers; Pyrazoles; Synaptic Transmission; Tetrodotoxin; Time Factors; Trinitrobenzenesulfonic Acid

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4(+) T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype ("chronically activated") with large numbers of Kv1.3 voltage-gated channels (approximately 1500 per cell) and small numbers of IKCa1 Ca(2+)-activated K(+) channels (approximately 50-120 per cell). In contrast, resting T cells displayed 0-10 Kv1.3 and 10-20 IKCa1 channels per cell ("quiescent" phenotype), whereas T cells stimulated once or twice expressed approximately 200 Kv1.3 and approximately 350 IKCa1 channels per cell ("acutely activated" phenotype). Consistent with their channel phenotype, [(3)H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap(22)) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap(22) and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K(+) channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap(22) plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap(22), but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap(22) plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.

Topics: Animals; Calcium Channel Blockers; CD4-Positive T-Lymphocytes; Cells, Cultured; Cnidarian Venoms; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Guinea Pigs; Intermediate-Conductance Calcium-Activated Potassium Channels; Isotope Labeling; Kv1.3 Potassium Channel; Multiple Sclerosis; Phenotype; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Pyrazoles; Rats; Rats, Inbred Lew; Thymidine; Tritium