thiourea and iberiotoxin

Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF.. Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BK. Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BK

Topics: Adult; Aged; Aged, 80 and over; Arrhythmia, Sinus; Atrial Fibrillation; Atrial Remodeling; Calcium; Cells, Cultured; Female; Gene Knockdown Techniques; Heart Atria; Humans; Indoles; Ion Channels; Ion Transport; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Male; Middle Aged; Myofibroblasts; Peptides; Signal Transduction; Tetrazoles; Thiourea; Transfection

Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca2+-activated K+ (BK) channels in offspring mesenteric arteries following prenatal hypoxia.. Pregnant rats were exposed to normoxic control (21% O2, Con) or hypoxic (10.5% O2, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR.. Prenatal hypoxia increased pressor responses and vasoconstrictions to phenylephrine in the offspring. Whole-cell currents density of BK channels and amplitude of spontaneous transient outward currents (STOCs), not the frequency, were significantly reduced in Hy vascular myocytes. The sensitivity of BK channels to voltage, Ca2+, and tamoxifen were reduced in Hy myocytes, whereas the number of channels per patch and the single-channel conductance were unchanged. Prenatal hypoxia impaired NS1102- and tamoxifen-mediated relaxation in mesenteric arteries precontracted with phenylephrine in the presence of Nω-nitro-L-arginine methyl ester. The mRNA and protein expression of BK channel β1, not the α-subunit, was decreased in Hy mesenteric arteries.. Impaired BK channel β1-subunits in vascular smooth muscle cells contributed to vascular dysfunction in the offspring exposed to prenatal hypoxia.

Topics: Animals; Blood Pressure; Down-Regulation; Female; Fetal Hypoxia; Gestational Age; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Mesenteric Arteries; Myocytes, Smooth Muscle; Patch-Clamp Techniques; Peptides; Phenylephrine; Pregnancy; Protein Subunits; Rats; Rats, Sprague-Dawley; Tamoxifen; Tetrazoles; Thiourea; Vasoconstriction

Large-conductance Ca(2+) -activated K(+) channels (BKC a ), located on the vascular smooth muscle, play an important role in regulation of vascular tone. In penile corpus cavernosum tissue, opening of BKC a channels leads to relaxation of corporal smooth muscle, which is essential during erection; however, there is little information on the role of BKC a channels located in penile vascular smooth muscle. This study was designed to investigate the involvement of BKC a channels in endothelium-dependent and endothelium-independent relaxation of human intracavernous penile arteries. In human intracavernous arteries obtained in connection with transsexual operations, change in isometric force was recorded in microvascular myographs, and endothelium-dependent [nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type] and endothelium-independent (NO-donor) relaxations were measured in contracted arteries. In penile small arteries contracted with phenylephrine, acetylcholine evoked NO- and EDH-type relaxations, which were sensitive to iberiotoxin (IbTX), a selective blocker of BKC a channels. Iberiotoxin also inhibited relaxations induced by a NO-donor, sodium nitroprusside. NS11021, a selective opener of BKC a channels, evoked pronounced relaxations that were inhibited in the presence of IbTX. NS13558, a BKC a -inactive analogue of NS11021, failed to relax human penile small arteries. Our results show that BKC a channels are involved in both NO- and EDH-type relaxation of intracavernous penile arteries obtained from healthy men. The effect of a selective opener of BKC a channels also suggests that direct activation of the channel may be an advantageous approach for treatment of impaired endothelium-dependent relaxation often associated with erectile dysfunction.

Topics: Acetylcholine; Adolescent; Adult; Arteries; Endothelium; Humans; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Penis; Peptides; Phenylephrine; Tetrazoles; Thiourea; Young Adult

We examined the electrophysiological properties of reverse-mode Na(+) /Ca(2+) exchange (NCX) in mouse airway smooth muscle (ASM), assessing its contributions to regulation of [Ca(2+) ], and its expression in acute and chronic airway hyperresponsiveness (AHR).. Membrane currents were studied in single murine ASM cells under voltage clamp at -60 mV using ramp depolarizing commands to +80 mV. Confocal fluorimetric and RT-PCR techniques were used to monitor changes in cytosolic [Ca(2+) ] and NCX expression, respectively.. With standard KCl-containing electrode, 30 μm KB-R7943 (an inhibitor of reverse-mode NCX activity) exhibited variable effects on membrane current, indicating modulation of more than one conductance. KB-R7943 activated outwardly rectifying current that was inhibited by 100 μm iberiotoxin (blocker of large-conductance Ca(2+) -dependent K(+) channels), indicating a direct enhancing effect of KB-R7943 on those K(+) channels. After obviating K(+) currents, we found that a current sensitive to 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid (blocker of Ca(2+) -dependent Cl- channels) was markedly increased by elevating [Na(+) ] in the electrode solution to 13, 15.5 and 18 mm and suppressed by KB-R7943, indicating Ca(2+) influx via reverse-mode NCX activity. With conditions preventing Ca(2+) influx through voltage-dependent Ca(2+) channels but promoting that through NCX, we found that introduction of Ca(2+) led to marked but transient KB-R7943-sensitive elevation of [Ca(2+) ]. Additionally, KB-R7943 suppressed cholinergically evoked Ca(2+) waves. Finally, NCX1 expression was not significantly changed in allergen-induced AHR acute model but increased approx. 2.5-fold in a chronic model.. Reverse-mode NCX activity leads to a physiologically relevant increase in [Ca(2+) ] even under control conditions, and this may be exaggerated in allergen-induced AHR and asthma.

Topics: Allergens; Animals; Antigens, Dermatophagoides; Bronchi; Bronchial Hyperreactivity; Calcium; Female; Membrane Potentials; Mice; Muscle Contraction; Muscle, Smooth; Peptides; Potassium Channels, Calcium-Activated; Sodium-Calcium Exchanger; Thiourea; Trachea

Large-conductance Ca(2+)-activated potassium (BK) channels play an important role in regulating the function and activity of urinary bladder smooth muscle (UBSM), and the loss of BK channel function has been shown to increase UBSM excitability and contractility. However, it is not known whether activation of BK channels has the converse effect of reducing UBSM excitability and contractility. Here, we have sought to investigate this possibility by using the novel BK channel opener NS11021. NS11021 (3 microM) caused an approximately threefold increase in both single BK channel open probability (P(o)) and whole cell BK channel currents. The frequency of spontaneous action potentials in UBSM strips was reduced by NS11021 from a control value of 20.9 + or - 5.9 to 10.9 + or - 3.7 per minute. NS11021 also reduced the force of UBSM spontaneous phasic contractions by approximately 50%, and this force reduction was blocked by pretreatment with the BK channel blocker iberiotoxin. NS11021 (3 microM) had no effect on contractions evoked by nerve stimulation. These findings indicate that activating BK channels reduces the force of UBSM spontaneous phasic contractions, principally through decreasing the frequency of spontaneous action potentials.

Topics: Action Potentials; Animals; Electric Stimulation; Electrophysiology; Guinea Pigs; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Muscle Contraction; Muscle, Smooth; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Tetrazoles; Thiourea; Urinary Bladder

Large conductance calcium-activated potassium (BK(Ca)) channels are membrane proteins contributing to electrical propagation through neurons. Calcitonin gene-related peptide (CGRP) is a neuropeptide found in the trigeminovascular system (TGVS). Both BK(Ca) channels and CGRP are involved in migraine pathophysiology. Here we study the expression and localization of BK(Ca) channels and CGRP in the rat trigeminal ganglion (TG) and the trigeminal nucleus caudalis (TNC) as these structures are involved in migraine pain. Also the effect of the BK(Ca) channel blocker iberiotoxin and the BK(Ca) channel opener NS11021 on CGRP release from isolated TG and TNC was investigated. By RT-PCR, BK(Ca) channel mRNA was detected in the TG and the TNC. A significant difference in BK(Ca) channel mRNA transcript levels were found using qPCR between the TNC as compared to the TG. The BK(Ca) channel protein was more expressed in the TNC as compared to the TG shown by western blotting. Immunohistochemistry identified BK(Ca) channels in the nerve cell bodies of the TG and the TNC. The beta2- and beta4-subunit proteins were found in the TG and the TNC. They were both more expressed in the TNC as compared to TG shown by western blotting. In isolated TNC, the BK(Ca) channel blocker iberiotoxin induced a concentration-dependent release of CGRP that was attenuated by the BK(Ca) channel opener NS11021. No effect on basal CGRP release was found by NS11021 in isolated TG or TNC or by iberiotoxin in TG. In conclusion, we found both BK(Ca) channel mRNA and protein expression in the TG and the TNC. The BK(Ca) channel protein and the modulatory beta2- and beta4-subunt proteins were more expressed in the TNC than in the TG. Iberiotoxin induced an increase in CGRP release from the TNC that was attenuated by NS11021. Thus, BK(Ca) channels might have a role in trigeminovascular pain transmission.

Topics: Animals; Blotting, Western; Calcitonin Gene-Related Peptide; Immunohistochemistry; In Vitro Techniques; Ion Channel Gating; Large-Conductance Calcium-Activated Potassium Channel beta Subunits; Large-Conductance Calcium-Activated Potassium Channels; Male; Neurons; Peptides; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Tetrazoles; Thiourea; Trigeminal Ganglion; Trigeminal Nuclei

Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blunted outward K(+) currents in carotid body (CB) glomus cells from chronic heart failure (CHF) rabbits. In the present study, we measured the effects of the neuronal nitric oxide synthase (nNOS) transgene on the K(+) currents in CB glomus cells from pacing-induced CHF rabbits. Using single-cell real-time RT-PCR and immunofluorescent techniques, we found that nNOS mRNA and protein are expressed in the rabbit CB glomus cells and CHF decreased the expression of nNOS mRNA and protein in CB glomus cells. After 3 days of an adenoviral nNOS (Ad.nNOS) gene transfection, the expression of nNOS protein was increased to the level found in sham CB glomus cells. In whole cell patch-clamp experiments, Ad.nNOS markedly reversed the attenuated K(+) currents in CB glomus cells from CHF rabbits. The specific nNOS inhibitor (S-methyl-l-thiocitrulline [SMTC]) and large-conductance Ca(2+)-activated K(+) (BK) channel blocker (iberiotoxin) fully abolished the effect of Ad.nNOS on the K(+) currents in the CB glomus cells from CHF rabbits. However, neither CHF nor Ad.nNOS altered the protein expression of BK channel alpha-subunit. These results suggest that a decrease of NO induced by an attenuated nNOS activity lowers the activation of the BK channels but not the protein expression of the BK channel alpha-subunit in the CB glomus cells during CHF.

Topics: Analysis of Variance; Animals; Body Weight; Calcium; Carotid Body; Chemoreceptor Cells; Citrulline; Disease Models, Animal; Enzyme Inhibitors; Genetic Vectors; Green Fluorescent Proteins; Heart Failure; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Nitric Oxide Synthase Type I; Pacemaker, Artificial; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Rabbits; RNA, Messenger; Thiourea; Transduction, Genetic; Tyrosine 3-Monooxygenase