sq-23377 and iberiotoxin

Mammalian K(+) homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K(+) intake. Aldosterone is known to enhance both renal and colonic K(+) secretion. In mouse distal colon K(+) secretion occurs exclusively via luminal K(Ca)1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K(+) secretion via BK channels. Luminal Ba(2+) and iberiotoxin (IBTX)-sensitive electrogenic K(+) secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K(+) diet. High K(+) diet led to a 2-fold increase of luminal Ba(2+) and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK alpha-subunit-deficient (BK(-/-)) mice. The resting and diet-induced K(+) secretion was stimulated by luminal ionomycin. In BK(-/-) mice luminal ionomycin did not stimulate K(+) secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K(+) secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK alpha- and beta(2)-subunits in high K(+) diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K(+)-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K(+) secretion occurs via increased expression of luminal BK channels.

Topics: Aldosterone; Animals; Barium; Colon; Female; Gene Deletion; Gene Expression Regulation; Immunohistochemistry; Ionomycin; Ionophores; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Male; Mice; Mineralocorticoid Receptor Antagonists; Peptides; Polymerase Chain Reaction; Potassium; Spironolactone; Up-Regulation

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).

Topics: Acetylcholine; Animals; Calcium; Cell Membrane Permeability; Cyclic GMP; Endothelium, Vascular; Ionomycin; Male; Middle Cerebral Artery; Nitric Oxide; Nitric Oxide Donors; Peptides; Potassium Channels; Rats; Rats, Sprague-Dawley; Serotonin; Type C Phospholipases; Vasoconstriction; Vasodilation

Effects of a switched, time-varying 1.7 T magnetic field on Rb(+)(K+) uptake by HeLa S3 cells incubated in an isosmotic high K(+) medium were examined. The magnetic flux density was varied intermittently from 0.07-1.7 T at an interval of 3 s. K(+) uptake was activated by replacement of normal medium by high K(+) medium. A membrane-permeable Ca(2+) chelating agent (BAPTA-AM) and Ca(2+)-dependent K(+) channel inhibitors (quinine, charibdotoxin, and iberiotoxin) were found to reduce the Rb(+)(K+) uptake by about 30-40%. Uptake of K(+) that is sensitive to these drugs is possibly mediated by Ca(2+)-dependent K(+) channels. The intermittent magnetic field partly suppress ed the drug-sensitive K(+) uptake by about 30-40% (P < 0.05). To test the mechanism of inhibition by the magnetic fields, intracellular Ca(2+) concentration ([Ca(2+)]c) was measured using Fura 2-AM. When cells were placed in the high K(+) medium, [Ca(2+)]c increased to about 1.4 times the original level, but exposure to the magnetic fields completely suppressed the increase (P < 0.01). Addition of a Ca(2+) ionophore (ionomycin) to the high K(+) medium increased [Ca(2+)]c to the level of control cells, regardless of exposure to the magnetic field. But the inhibition of K(+) uptake by the magnetic fields was not restored by addition of ionomycin. Based on our previous results on magnetic field-induced changes in properties of the cell membrane, these results indicate that exposure to the magnetic fields partly suppresses K(+) influx, which may be mediated by Ca(2+)-dependent K(+) channels. The suppress ion of K(+) fluxes could relate to a change in electric properties of cell surface and an inhibition of Ca(2+) influx mediated by Ca(2+) channels of either the cell plasma membrane or the inner vesicular membrane of intracellular Ca(2+) stores.

Topics: Calcium; Cell Membrane; Charybdotoxin; Chelating Agents; Culture Media; Egtazic Acid; Electromagnetic Fields; Electrophysiology; Environmental Exposure; Fluorescent Dyes; Fura-2; HeLa Cells; Humans; Intracellular Membranes; Ionomycin; Ionophores; Magnetics; Peptides; Potassium; Potassium Channel Blockers; Potassium Channels; Quinine; Rubidium

1. The effects of adrenergic agonists on K+ currents were studied in cultured rabbit pigmented ciliary epithelial (PCE) cells. 2. Outward K+ current (IK) was reduced by tetraethylammonium chloride, the Ca2+-activated K+ (K(Ca)) channel blocker iberiotoxin (IbTX), or Ca2+-free external Ringer solution. The calcium ionophore ionomycin increased an IbTX-sensitive IK in PCE cells. 3. The adrenergic agonists adrenaline and phenylephrine increased IK in PCE cells. The induced current was blocked by IbTX and the alpha1-antagonist prazosin, suggesting that adrenergic agonists activate IK(Ca) via alpha1-adrenoreceptors. 4. Internal dialysis of D-myo-inositol 1,4, 5-trisphosphate (IP3) increased IK, whilst pre-incubation of PCE cells with thapsigargin or the phospholipase C (PLC) inhibitor U-73122 reduced phenylephrine-induced increases in IK(Ca). Adrenergic increases in IK(Ca) were mediated by a pertussis toxin-insensitive G protein. 5. These results demonstrate that IK(Ca) channels in rabbit PCE cells are coupled to alpha1-adrenergic receptors and a PLC/IP3 signalling pathway. Activation of these channels may modulate fluid secretion by the ciliary epithelium.

Topics: Adrenergic alpha-Agonists; Animals; Calcium-Transporting ATPases; Cells, Cultured; Ciliary Body; Enzyme Inhibitors; Epinephrine; Epithelial Cells; Estrenes; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Ionomycin; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Peptides; Phenylephrine; Potassium Channels; Potassium Channels, Calcium-Activated; Prazosin; Pyrrolidinones; Rabbits; Receptors, Adrenergic, alpha-1; Scorpion Venoms; Signal Transduction; Tetraethylammonium; Thapsigargin; Type C Phospholipases

1. Previous electrophysiological studies have suggested the presence of KCa and Kv channels in human platelets. However, the pharmacology of these channels has not been defined. 2. We have studied potassium channels in human platelets by measuring the efflux of 86Rb+ (a marker for K+) from 86Rb(+)-loaded cells, and have defined their responses to stimulation by the platelet agonist thrombin and the calcium ionophore ionomycin. 3. Thrombin (0.1-0.6 i.u./ml) stimulated an increase in 86Rb+ efflux from the platelets in a concentration-dependent manner. This efflux was significantly inhibited by apamin (100 nmol/l), charybdotoxin (300 nmol/l) and alpha-dendrotoxin (100-200 nmol/l), blockers of SKCa channels, KCh channels and Kv channels respectively. Iberiotoxin (300 nmol/l), a specific inhibitor of BKCa channels, had no effect on the thrombin-stimulated 86Rb+ efflux. Although glibenclamide, an inhibitor of KATP channels, inhibited the thrombin-stimulated efflux, it did so only in a high concentration (20 mumol/l). 4. Ionomycin (1-5 mumol/l) stimulated an increase in 86Rb+ efflux from the platelets in a concentration-dependent manner. This efflux was significantly inhibited by apamin (100 nmol/l) and charybdotoxin (300 nmol/l). However, iberiotoxin (300 nmol/l) had no effect on the ionomycin-stimulated 86Rb+ efflux. 5. These findings suggest that 86Rb+ efflux from platelets stimulated by thrombin and ionomycin occurs via two types of KCa channel: SKCa and KCh channels. Thrombin also stimulated efflux via Kv channels.

Topics: Apamin; Blood Platelets; Calcium; Charybdotoxin; Dose-Response Relationship, Drug; Elapid Venoms; Glyburide; Humans; Hypoglycemic Agents; Ion Channel Gating; Ionomycin; Ionophores; Peptides; Potassium Channel Blockers; Potassium Channels; Rubidium Radioisotopes; Stimulation, Chemical; Thrombin

Calcium-activated potassium current was studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell and single channel patch-clamp recording techniques. When K+ was the principal cation in the electrode, depolarizing voltage steps from a holding potential of -60 mV activated outwardly rectifying current. Outward K+ current was increased by the Ca2+ ionophore ionomycin and reduced when the extracellular Ca2+ concentration was decreased from 2.5 mM to 100 nM in the presence of ionomycin. Outward K+ current recorded in the presence of ionomycin was blocked by iberiotoxin and by charybdotoxin. Single channel recording from cell-attached and excised membrane patches revealed a large conductance Ca2+-activated K+ (K(Ca)) channel. Identification of K(Ca) channels was based on: 1) the voltage-dependence of channel opening; 2) the large unitary conductance (> 200 pS with symmetrical 130 mM K+); 3) the dependence of the reversal potential on the K+ gradient; and 4) increased channel opening after exposure of the cytosolic surface of excised membrane patches to elevated Ca2+. These results demonstrate that Ca2+-activated K+ channels are present in rabbit RPE cells and may play an essential role in the regulation of membrane potential and ion transport.

Topics: Animals; Calcium; Cells, Cultured; Charybdotoxin; Electric Conductivity; Ionomycin; Ionophores; Patch-Clamp Techniques; Peptides; Pigment Epithelium of Eye; Potassium; Potassium Channels; Rabbits; Toxins, Biological