ns-309 and Disease-Models--Animal

Obesity usually induces overactive bladder (OAB) associated with detrusor overactivity, which is related to increased contractility of the detrusor smooth muscle (DSM). Small-conductance Ca. Female Sprague-Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD) and weighed after 12 weeks. Urodynamic studies, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and isometric tension recording were performed.. Increased average body weights and urodynamically demonstrated OAB were observed in HFD rats. qRT-PCR experiments revealed a decrease in the mRNA expression level of SK channel in DSM tissue of the HFD rats. Isometric tension recordings indicated an attenuated relaxation effect of NS309 on the spontaneous phasic and electrical field stimulation-induced contractions that occurred via SK channel activation in HFD DSM strips.. Reduced expression and activity of SK channels in the DSM contribute to obesity-related OAB, indicating that SK channels are a potential therapeutic target for OAB.

Topics: Anal Canal; Animals; Apamin; Diet, High-Fat; Disease Models, Animal; Female; Indoles; Muscle Contraction; Muscle, Smooth; Neuromuscular Agents; Obesity; Oximes; Rats; Rats, Sprague-Dawley; Small-Conductance Calcium-Activated Potassium Channels; Urinary Bladder, Overactive; Urodynamics

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

Plasmamembrane small conductance Ca2+-activated K+ (SK) channels were implicated in ventricular arrhythmias in infarcted and failing hearts. Recently, SK channels were detected in the inner mitochondria membrane (IMM) (mSK), and their activation protected from acute ischaemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK play an important role in regulating mitochondrial function in chronic cardiac diseases. We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using rat model of cardiac hypertrophy induced by banding of the ascending aorta thoracic aortic banding (TAB).. Dual Ca2+ and membrane potential optical mapping of whole hearts derived from TAB rats revealed that membrane-permeable SK enhancer NS309 (2 μM) improved aberrant Ca2+ homeostasis and abolished VT/VF induced by β-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging of cardiomyocytes derived from TAB hearts (TCMs) we found that membrane-permeable SK enhancers NS309 and CyPPA (10 μM) attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations. Furthermore, mSK inhibition enhanced (UCL-1684, 1 μM); while activation reduced mitochondrial ROS production in TCMs measured with MitoSOX. Protein oxidation assays demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by SK enhancers. Experiments in permeabilized TCMs showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function.. These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia and suggest that the protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.

Topics: Animals; Arrhythmias, Cardiac; Calcium Signaling; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Indoles; Kinetics; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocytes, Cardiac; Oxidation-Reduction; Oximes; Pyrazoles; Pyrimidines; Rats; Reactive Oxygen Species; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Small-Conductance Calcium-Activated Potassium Channels

N-methyl-D-aspartate receptor (NMDAR) antagonists have been shown to reduce mechanical hypersensitivity in animal models of inflammatory pain. However, their clinical use is associated with significant dose-limiting side effects. Small-conductance Ca-activated K channels (SK) have been shown to modulate NMDAR activity in the brain. We demonstrate that in vivo activation of SK channels in the spinal cord can alleviate mechanical hypersensitivity in a rat model of inflammatory pain. Intrathecal (i.t.) administration of the SK channel activator, 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), attenuates complete Freund adjuvant (CFA)-induced mechanical hypersensitivity in a dose-dependent manner. Postsynaptic expression of the SK channel subunit, SK3, and apamin-sensitive SK channel-mediated currents recorded from superficial laminae are significantly reduced in the dorsal horn (DH) after CFA. Complete Freund adjuvant-induced decrease in SK-mediated currents can be reversed in vitro by bath application of NS309. In addition, immunostaining for the SK3 subunit indicates that SK3-containing channels within DH neurons can have both somatic and dendritic localization. Double immunostaining shows coexpression of SK3 and NMDAR subunit, NR1, compatible with functional interaction. Moreover, we demonstrate that i.t. coadministration of NS309 with an NMDAR antagonist reduces the dose of NMDAR antagonist, DL-2-amino-5-phosphonopentanoic acid (DL-AP5), required to produce antinociceptive effects in the CFA model. This reduction could attenuate the unwanted side effects associated with NMDAR antagonists, giving this combination potential clinical implications.

Topics: Animals; Disease Models, Animal; Freund's Adjuvant; Indoles; Inflammation; Injections, Spinal; Male; Oximes; Pain; Pain Threshold; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Small-Conductance Calcium-Activated Potassium Channels; Spinal Cord; Treatment Outcome

Small conductance calcium (Ca(2+)) activated SK channels are critical regulators of neuronal excitability in hippocampus. Accordingly, these channels are thought to play a key role in controlling neuronal activity in acute models of epilepsy. In this study, we investigate the expression and function of SK channels in the pilocarpine model of mesial temporal lobe epilepsy. For this purpose, protein expression was assessed using western blotting assays and gene expression was analyzed using TaqMan-based probes and the quantitative real-time polymerase chain reaction (qPCR) comparative method delta-delta cycle threshold ( big up tri, open big up tri, openCT) in samples extracted from control and epileptic rats. In addition, the effect of SK channel antagonist UCL1684 and agonist NS309 on CA1 evoked population spikes was studied in hippocampal slices. Western blotting analysis showed a significant reduction in the expression of SK1 and SK2 channels at 10days following status epilepticus (SE), but levels recovered at 1month and at more than 2months after SE. In contrast, a significant down-regulation of SK3 channels was detected after 10days of SE. Analysis of gene expression by qPCR revealed a significant reduction of transcripts for SK2 (Kcnn1) and SK3 (Kcnn3) channels as early as 10days following pilocarpine-induced SE and during the chronic phase of the pilocarpine model. Moreover, bath application of UCL1684 (100nM for 15min) induced a significant increase of the population spike amplitude and number of spikes in the hippocampal CA1 area of slices obtained control and chronic epileptic rats. This effect was obliterated by co-administration of UCL1684 with SK channel agonist NS309 (1microM). Application of NS309 failed to modify population spikes in the CA1 area of slices taken from control and epileptic rats. These data indicate an abnormal expression of SK channels and a possible dysfunction of these channels in experimental MTLE.

Topics: Age Factors; Alkanes; Analysis of Variance; Animals; Disease Models, Animal; Drug Interactions; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Indoles; Male; Membrane Potentials; Muscarinic Agonists; Neurons; Oximes; Pilocarpine; Quinolinium Compounds; Rats; Rats, Sprague-Dawley; Small-Conductance Calcium-Activated Potassium Channels; Status Epilepticus; Time Factors