calcimycin and iberiotoxin

Cytoprotective properties of potassium channel openers (KCOs) have been demonstrated in several models of cell injury, mainly in ischemia-reperfusion-induced damage of cardiac muscle. The mechanism responsible for the observed cytoprotection and the relative contribution of plasma membrane or inner mitochondrial membrane potassium channels regarding the beneficial effects exerted by KCOs remain unclear. Our work demonstrates the cytoprotective properties of NS1619, an opener of large-conductance calcium-activated potassium channels (BKCa channels), using C2C12 myoblasts injured by calcium ionophore A23187 treatment. Application of two BKCa channel inhibitors, paxilline and iberiotoxin, abolished this cytoprotective effect. At concentrations of 10-100 μM, NS1619 increased the respiration rate and decreased mitochondrial membrane potential (Δψ) in C2C12 cells in a dose-dependent manner. At a concentration of 0.2 μM, paxilline, which effectively abolished the protective effect of NS1619, failed to counteract the opener-induced mitochondrial depolarization and increase in cellular respiration. This result indicates that the NS1619-mediated increase in the survival rate of A23187-treated C2C12 cells occurs in a manner distinct from its effect on mitochondrial functioning and suggests that activation of BKCa channels in the plasma membrane is the mechanism responsible for cytoprotection by NS1619.

Topics: Animals; Benzimidazoles; Calcimycin; Calcium; Cell Line; Cell Membrane; Cell Respiration; Cell Survival; Cytoprotection; Dose-Response Relationship, Drug; Homeostasis; Indoles; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potential, Mitochondrial; Mice; Myoblasts; Peptides

We have previously identified a human vascular smooth muscle clone that can reversibly convert between proliferative and contractile phenotypes. Here we compared receptor-channel coupling in these cells using fura-2 to monitor [Ca(2+)](i) and patch-clamp to record currents. Histamine elevated [Ca(2+)](i) in all cells and caused contraction of cells exhibiting the contractile phenotype. The rise of [Ca(2+)](i) persisted in Ca(2+)-free solution and was abolished by thapsigargin, indicating involvement of stores. Whole cell electrophysiological recording revealed that histamine evoked transient outward K(+) current, indicating functional receptor-channel coupling. The time-course and amplitude of the histamine-activated current were similar in cells of the proliferative and contractile phenotypes. Moreover, a large conductance K(+) channel was recorded in cell-attached patches and was activated by histamine as well as the Ca(2+) ionophore A-23187, identifying it as the large conductance Ca(2+)-dependent K(+) channel. This K(+) channel showed similar characteristics and activation in both proliferative and contractile phenotypes, indicating that expression was independent of phenotype. In contrast, histamine also elicited an inward Cl(-) current in some contractile cells, suggesting differential regulation of this current depending on phenotype. These studies demonstrate the usefulness of this human vascular cell clone for studying functional plasticity of smooth muscle, while avoiding complications arising from extended times in culture.

Topics: Calcimycin; Calcium; Cell Division; Cells, Cultured; Chlorides; Enzyme Inhibitors; Histamine; Humans; Ionophores; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Muscle Contraction; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Peptides; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Tetraethylammonium; Thapsigargin; Vasoconstriction

Voltage-dependent delayed rectifier K+ (KV) channels and Ca(2+)-activated K+ (KCa) channels both play important roles in the regulation of the membrane potential and contractility of vascular smooth muscle cells (SMCs). The expression and function of these K+ channels in cultured vascular SMCs have been extensively studied. The long-term in vitro cell culture would change the properties of K+ channels in SMCs. However, whether the short-term culture could differentially affect the expression and function of KV and KCa channels was not clear. In the present study, both KV and KCa channel currents were identified in freshly dissociated SMCs and in 1-day-cultured SMCs from rat tail arteries. KCa currents were inhibited by iberiotoxin or tetraethylammonium (TEA), and amplified by the calcium ionophore A-23187. Kv currents were inhibited by 4-aminopyridine or beta-dendrotoxin. By using different pharmacological agents and manipulating the calcium concentrations in the recording solutions, it was revealed that in freshly dissociated SMCs the predominant component of total outward K+ currents is KCa current, and KV current a minor component. In contrast, KV current was found to be the predominant component of total outward K+ currents in SMCs primarily cultured with 10% fetal bovine serum at 37 degrees C for 24 h. Differential expression of KV and KCa channels in 1-day-cultured SMCs was thus demonstrated under our experimental conditions. Our results are important for interpreting the electrophysiological properties of vascular SMCs under different cell culture conditions and for understanding the relative contributions of KV and KCa channels to different cellular functions.

Topics: 4-Aminopyridine; Animals; Arteries; Calcimycin; Cells, Cultured; Chelating Agents; Egtazic Acid; Electric Conductivity; Ionophores; Male; Membrane Potentials; Muscle, Smooth, Vascular; Peptides; Potassium Channels; Rats; Rats, Sprague-Dawley; Tail; Tetraethylammonium

We investigated the effects of the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on endothelin-1 (ET-1)-induced constriction of rat small pulmonary arteries (diameter 100-400 microm) in vitro, following endothelium removal. ET-1 (30 nM) induced a sustained constriction of rat pulmonary arteries in physiological salt solution. Arteries preconstricted with ET-1 were relaxed by niflumic acid (IC50: 35.8 microM) and NPPB (IC50: 21.1 microM) in a reversible and concentration-dependent manner. However, at concentrations known to block Ca++-activated Cl- channels, DIDS (

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Angiotensin II; Animals; Calcimycin; Calcium Channels; Chloride Channels; Endothelin-1; Glyburide; In Vitro Techniques; Male; Niflumic Acid; Nitrobenzoates; Peptides; Pulmonary Artery; Rats; Rats, Wistar; Uridine Triphosphate; Vasoconstriction; Vasodilation

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth mu

Topics: Acetylcholine; Animals; Apamin; Calcimycin; Carotid Arteries; Cell Membrane; Cyclic GMP; Glyburide; In Vitro Techniques; Membrane Potentials; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Peptides; Potassium Channel Blockers; Rabbits

Spontaneous transient outward potassium currents (STOCs) induce myogenic relaxation in small cerebral vessels. We found STOCs in human coronary artery vascular smooth muscle cells (VSMCs) and studied their regulation.. K+ currents were recorded in human coronary VSMCs by current- and voltage-clamp techniques. STOCs were recorded in the presence of 200 mumol/L Cd2+ and 10 mumol/L verapamil, which block voltage-dependent Ca2+ channels. STOCs were inhibited by iberiotoxin (100 nmol/L), a selective blocker of Ca(2+)-activated potassium channels (BKCa), and disappeared in a Ca(2+)-free bath. Iberiotoxin depolarized the VSMCs within 20 minutes from -44 +/- 7 to -18 +/- 5 mV (n = 17). The Ca2+ ionophore A23187 increased intracellular Ca2+ and stimulated whole-cell BKCa current. Depletion of Ca2+ from the sarcoplasmic reticulum with caffeine (4 mmol/L) abolished STOCs for several minutes. Ryanodine (50 mumol/L) transiently stimulated STOCs but then completely inhibited STOCs within 10 minutes. The firing frequency of STOCs was directly correlated with intracellular Na+ concentrations from 0 to 24 mmol/L. Lowering intracellular Na+ to zero abolished STOCs. We next gave monensin (30 mumol/L) to increase intracellular Na+. This maneuver resulted in an increase in whole-cell current fluctuations and STOCs. Monensin-induced STOCs were abolished by either lowering extracellular Ca2+ to zero or chelating Ca2+ intracellularly with BAPTA-AM (30 mumol/L).. STOCs resulted from BKCa activity and were dependent on extracellular Ca2+ but not significantly on voltage-dependent Ca2+ channels. STOCs were dependent on intracellular Na+ and intracellular calcium store refilling state. We suggest that Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchange determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.

Topics: Arteries; Caffeine; Calcimycin; Calcium; Coronary Vessels; Electric Conductivity; Electrophysiology; Extracellular Space; Humans; Intracellular Membranes; Ionophores; Muscle, Smooth, Vascular; Peptides; Potassium; Ryanodine; Sodium

Vascular endothelial cells have several types of Ca2+-dependent K+ current (I(K-Ca)). Here, we describe apamin-sensitive I(K-Ca) which is activated by treatment with histamine (His) in human umbilical vein endothelial cells (HUVECs). In 65 % of HUVECs examined, 100 nM apamin potently inhibited I(K-Ca) and hyperpolarization induced by His (19 and 7 % of control, respectively). In contrast, application of 5 mM tetraethylammonium, a non-selective K channel blocker, or 100 nM iberiotoxin, a selective K channel blocker for a large conductance Ca2+-dependent K+ channel, had small (78 % of control) or no effects (102 % of control) on I(K-Ca), respectively. These findings suggest that apamin-sensitive Ca2+-dependent K+ channels are expressed in HUVECs and activated by receptor stimulation.

Topics: Apamin; Calcimycin; Calcium; Cells, Cultured; Endothelium, Vascular; Histamine; Humans; Ion Channel Gating; Ionophores; Membrane Potentials; Peptides; Potassium Channels; Tetraethylammonium; Tetraethylammonium Compounds

Our goal was to study the activation current in physiologically competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa or aged in vitro, and, in particular, to determine whether a soluble sperm factor triggers a fertilization current comparable to that observed with intact spermatozoa and to characterize the current involved.. The whole-cell voltage-clamp technique was used on spare metaphase II oocytes, obtained with patient consent from IVF programs. In this configuration a soluble fraction from human spermatozoa was microinjected, and the current recorded.. Metaphase II human oocytes generate bell-shaped outward currents of 400-1000 pA (X = 706 +/- 322; n = 10), following injection of a cytosolic extract from human spermatozoa. The amount of sperm extract injected was less than 10% of the total oocyte volume and was equivalent to 1-10 spermatozoa. A similar current was generated following exposure to 20 microM of the calcium ionophore A23187 (n = 10). The steady-state conductance of the oocyte increased from 10 to 19.8 nS (n = 10) following injection of the sperm factor and from 5.3 to 27.7 nS following ionophore exposure. Both sperm factor- and ionophore-induced currents were reduced in amplitude when the unfertilized oocyte was preexposed to 25-75 microM iberiotoxin (n = 8) and eliminated at a concentration of 100 microM iberiotoxin.. The data support the hypothesis of a soluble sperm factor involved in the activation of human oocytes and shows that the initial activation response in the human oocyte is the gating of Ca(2+)-activated K+ channels.

Topics: Calcimycin; Calcium; Cell Extracts; Female; Humans; Ion Channel Gating; Ionophores; Male; Oocytes; Patch-Clamp Techniques; Peptides; Potassium Channels; Solubility; Sperm-Ovum Interactions; Spermatozoa

This study provides evidence for the presence of large conductance Ca(2+)-dependent K-channels in guinea pig and human urinary bladder smooth muscle. A23187, a Ca(2+)-ionophore, increased charybdotoxin and iberiatoxin sensitive 42K efflux in human urinary bladder smooth muscle cells, suggesting that large conductance Ca(2+)-dependent K-channels are present in these cells. NS004, a large conductance Ca(2+)-dependent K-channel opener, relaxed guinea pig bladder strips precontracted with 15 mM KCl which is inhibited by iberiatoxin. In addition, NS004 also evoked an iberiatoxin sensitive increase in 86Rb/42K efflux in guinea pig and human urinary bladder smooth muscle cells, demonstrating that NS004 activates large conductance Ca(2+)-dependent K-channels to achieve its relaxation effect in the bladder.

Topics: Animals; Benzimidazoles; Calcimycin; Calcium; Charybdotoxin; Chlorophenols; Electric Conductivity; Guinea Pigs; Humans; Muscle Relaxation; Muscle, Smooth; Peptides; Potassium Channels; Potassium Chloride; Rubidium Radioisotopes; Scorpion Venoms; Urinary Bladder