scholarly journals Cl− and K+ conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules

1997 ◽  
Vol 273 (5) ◽  
pp. F680-F697 ◽  
Author(s):  
I. Rubera ◽  
M. Tauc ◽  
C. Poujeol ◽  
M. T. Bohn ◽  
M. Bidet ◽  
...  
Keyword(s):  
Osmotic Shock ◽  
Ion Selectivity ◽  
Primary Cultures ◽  
Regulatory Volume Decrease ◽  
Ionic Currents ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl− currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl− currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I−= Br− > Cl− ≫ glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid and 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 μM LaCl3, 10 μM nifedipine, 100 μM verapamil, 5 μM tamoxifen, and 50 μM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl−currents. Osmotic shock also activated K+ currents in Cl−-free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl− and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.

Whole cell Cl- currents in human neutrophils induced by cell swelling

1993 ◽  
Vol 265 (1) ◽  
pp. C156-C165 ◽  
Author(s):  
J. S. Stoddard ◽  
J. H. Steinbach ◽  
L. Simchowitz
Keyword(s):  
Regulatory Volume Decrease ◽  
Reversal Potential ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Human Neutrophils ◽  
Patch Clamp Technique ◽  
Transport Pathway ◽  
Pipette Solution ◽  
Whole Cell ◽  
Cl Channels

The properties of the conductive Cl- transport pathway underlying regulatory volume decrease (RVD) in human neutrophils were investigated using the whole cell patch-clamp technique. Cell swelling was induced during whole cell recordings by making the patch pipette solution hyperosmotic (approximately 20%) relative to the bath by addition of sucrose. Immediately after establishment of the whole cell configuration, no measurable Cl- currents were evident. Over a period of several minutes the outwardly rectifying Cl- current that developed displayed no apparent voltage dependence of activation and did not inactivate with time during voltage steps over the range of -80 to +80 mV. Reduction of Cl- currents by application of suction to the interior of the pipette implied that the swelling-induced Cl- channels are activated by membrane stretch. Based on reversal potential measurements, the volume-induced Cl- conductance was found to discriminate poorly among Cl-, Br-, I-, and NO3-, to possess a finite permeability to glucuronate (Pglucuronate/PCl approximately 0.1) and to be impermeable to cations. Single-channel conductance was estimated to be 1.5 pS from analysis of the variance of membrane current fluctuations. The activated Cl- currents were blocked by 100 microM of the compound MK-447 analogue A (inhibitor constant Ki = 37 microM) and by 200 microM 3,5-diiodosalicylate, 500 microM 4-acetamido-4'-iodothiocyanostilbene-2,2'-disulfonic acid, and 200 microM UK-5099. These results suggest that the initial event triggering RVD in neutrophils may be activation of stretch sensitive Cl- channels in the plasma membrane.

Whole cell membrane currents in cultured pig endocardial endothelial cells

1995 ◽  
Vol 268 (5) ◽  
pp. H2036-H2047 ◽  
Author(s):  
P. F. Fransen ◽  
M. J. Demolder ◽  
D. L. Brutsaert
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelium ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
K Current ◽  
Endocardial Endothelial Cells ◽  
Inwardly Rectifying

The whole cell mode of the patch-clamp technique was applied to cultured endocardial endothelial cells from the porcine right ventricle to study their electrophysiological properties. With isotonic pipette and bathing solutions (300-310 mosmol/kgH2O), single endocardial endothelial cells had resting membrane potentials ranging from -20 to -90 mV (mean = -55 +/- 20 mV, n = 48). In voltage-clamp experiments, the main membrane current was an inwardly rectifying K+ current with all characteristics described for the inwardly rectifying K+ current in vascular endothelium. Outward currents at positive clamp potentials were small, but when cell swelling was induced by means of a hypertonic pipette or hypotonic bathing solution and ATP (5 mM) was present in the pipette solution, a large outwardly rectifying current developed. This volume-activated current was insensitive to extracellular K+ or Na+ concentration variations but sensitive to changes in extracellular Cl- concentrations. It was inhibited in the presence of 4,4'-diisothiocyanostilbene-2,2 disulfonic acid (100-300 microM) and flufenamic acid (50-100 microM). Volume-activated Cl- channels are different from the stretch-activated cationic channels described in vascular endothelium and might be involved in the regulation of cell volume or the response to mechanical stretch.

Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule

1996 ◽  
Vol 271 (4) ◽  
pp. F940-F950 ◽  
Author(s):  
M. Bidet ◽  
M. Tauc ◽  
I. Rubera ◽  
G. de Renzis ◽  
C. Poujeol ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Distal Convoluted Tubule ◽  
Disulfonic Acid ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Steady State Current ◽  
Cl Permeability

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal “bright” convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.

Transmembrane chloride currents in human atrial myocytes

1996 ◽  
Vol 270 (2) ◽  
pp. C500-C507 ◽  
Author(s):  
G. R. Li ◽  
J. Feng ◽  
Z. Wang ◽  
S. Nattel
Keyword(s):  
Membrane Conductance ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Whole Cell ◽  
Human Atrial Myocytes ◽  
Cyclic Monophosphate ◽  
Whole Cell Patch Clamp

The present study was designed to evaluate the presence of basal, swelling-induced, and cAMP-dependent Cl- currents in human atrial myocytes studied with the whole cell patch-clamp technique. Under basal conditions, a small outwardly rectifying background conductance was noted that reversed close to 0 mV and was not altered by Cl- replacement. Isoproterenol (1 microM), forskolin (3 microM), and 8-bromoadenosine 3',5'-cyclic monophosphate (50 microM) did not increase membrane conductance, even when responsiveness to isoproterenol was confirmed by an increase in Ca2+ current and when perforated-patch techniques (nystatin) were used. Exposure to hyposmotic solutions increased cell volume and induced a whole cell conductance that showed outward rectification, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM), and responded to changes in Cl- gradient in a fashion consistent with a Cl(-)-selective conductance, with estimated relative permeabilities of 1, 0.25, and 0.07 for Cl-, methanesulfonate, and aspartate, respectively. The results suggest that human atrial cells lack basal and adenosine 3',5'-cyclic monophosphate-dependent Cl- current but manifest a substantial Cl- conductance in the presence of cell swelling.

cAMP- and swelling-activated chloride conductance in rat hepatocytes

1996 ◽  
Vol 271 (1) ◽  
pp. C112-C120 ◽  
Author(s):  
X. J. Meng ◽  
S. A. Weinman
Keyword(s):  
Rat Hepatocytes ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Ionic Selectivity ◽  
Patch Clamp Technique ◽  
Isolated Rat Hepatocytes ◽  
Pipette Solution ◽  
Set Point ◽  
Cl Conductance

An outwardly rectifying Cl- conductance was identified in primary isolated rat hepatocytes, and the whole cell patch-clamp technique was used to characterize its properties and mechanisms of activation. With symmetrical Cl(-)-containing solutions on both sides and adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM) in the pipette solution, a large outwardly rectifying conductance (1,014 +/- 153 pS/pF, n = 20) developed in all cells within 3 min. This cAMP-activated conductance was highly anion selective and slowly inactivated at voltages > 80 mV. It was completely inhibited by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM, n = 6) and partially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM, n = 7). It displayed a halide selectivity of I- > Br- > Cl-. In the absence of cAMP, a functionally similar conductance was activated by cell swelling. Reduction of bath osmolality from 300 to 250 mosmol/kg increased membrane conductance from 64 +/- 16.4 to 487 +/- 23 pS/pF (n = 4). This swelling-activated conductance was also highly anion selective and had identical halide selectivity and blocker sensitivity as the cAMP-activated conductance. Although cell swelling was not necessary for cAMP activation, cell shrinkage with hyperosmotic bath (350 mosmol/kg), either before or after exposure to cAMP, inhibited the cAMP-activated conductance. By the determination of conductance as a function of bath osmolality in the presence and absence of cAMP, it was observed that cAMP shifted the osmotic set point for conductance activation without changing either the maximum or minimum conductance. In conclusion, both cAMP and cell swelling activate a large outwardly rectifying Cl- conductance in rat hepatocytes. Its ionic selectivity and sensitivity to channel blockers are identical to those seen for swelling-activated Cl- conductances in many cell types. The conductive properties are not those of cystic fibrosis transmembrane conductance regulator-mediated Cl- conductance. cAMP appears to activate this conductance by altering the volume set point of a swelling-activated channel.

scholarly journals Metabolic stress in isolated mouse ventricular myocytes leads to remodeling of t tubules

2011 ◽  
Vol 301 (5) ◽  
pp. H1984-H1995 ◽  
Author(s):  
Lu-Feng Cheng ◽  
Fuzhen Wang ◽  
Anatoli N. Lopatin
Keyword(s):  
Membrane Potential ◽  
Ventricular Myocytes ◽  
Ionic Currents ◽  
Inward Rectifier ◽  
Short Time Scale ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
T Tubules

Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents ( IK1,tail), and inward rectifier potassium current ( IK1) “inactivation”. At room temperatures and in the absence of Mg2+ ions in pipette solution, the amplitude of IK1,tail measured ∼10 min after the establishment of whole cell configuration was reduced by ∼18%, but declined nearly twofold in the presence of 1 mM cyanide. At ∼35°C IK1,tail was essentially preserved in intact cells, but its amplitude declined by ∼85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg2+ ions played protective role at all temperatures. Decline of IK1,tail was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of IK1 inactivation, a marker of depletion of t-tubular K+. The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced IK1,tail, as well as the inactivation of inward IK1. Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.

Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle

1996 ◽  
Vol 270 (6) ◽  
pp. G932-G938 ◽  
Author(s):  
J. Jury ◽  
K. R. Boev ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Circular Muscle ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Outward Currents

Single smooth muscle cells from the opossum body circular muscle were isolated and whole cell currents were characterized by the whole cell patch-clamp technique. When the cells were held at -50 mV and depolarized to 70 mV in 20-mV increments, initial small inactivating inward currents were evoked (-30 to 30 mV) followed by larger sustained outward currents. Depolarization from a holding potential of -90 mV evoked an initial fast inactivating outward current sensitive to 4-aminopyridine but not to high levels of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The outward currents reversed near K+ equilibrium potential and were abolished when KCl was replaced by CsCl in the pipette solution. The sustained outward current was inhibited by quinine and cesium. High EGTA in the pipette solution reduced but did not abolish the sustained outward currents, suggesting that both Ca(2+)-dependent and -independent currents were evoked. The nitric oxide (NO)-releasing agents Sin-1 and sodium nitroprusside increased outward K+ currents. High levels of EGTA in the pipette solution abolished the increase in outward current induced by Sin-1. The presence of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, blocked the effects of NO-releasing agents. We conclude that NO release activates K+ outward currents in opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.

Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell)

2004 ◽  
Vol 287 (5) ◽  
pp. C1426-C1435 ◽  
Author(s):  
Muriel Nobles ◽  
Christopher F. Higgins ◽  
Alessandro Sardini
Keyword(s):  
Carboxylic Acid ◽  
Ion Selectivity ◽  
Cell Types ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Biophysical Properties ◽  
Extracellular Acidification ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Rate Of Activation

A Cl− current activated by extracellular acidification, ICl(pHac), has been characterized in various mammalian cell types. Many of the properties of ICl(pHac) are similar to those of the cell swelling-activated Cl− current ICl(swell): ion selectivity (I− > Br− > Cl− > F−), pharmacology [ ICl(pHac) is inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 1,9-dideoxyforskolin (DDFSK), diphenylamine-2-carboxylic acid (DPC), and niflumic acid], lack of dependence on intra- or extracellular Ca2+, and presence in all cell types tested. ICl(pHac) differs from ICl(swell) in three aspects: 1) its rate of activation and inactivation is very much more rapid, currents reaching a maximum in seconds rather than minutes; 2) it exhibits a slow voltage-dependent activation in contrast to the fast voltage-dependent activation and time- and voltage-dependent inactivation observed for ICl(swell); and 3) it shows a more pronounced outward rectification. Despite these differences, study of the transition between the two currents strongly suggests that ICl(swell) and ICl(pHac) are related and that extracellular acidification reflects a novel stimulus for activating ICl(swell) that, additionally, alters the biophysical properties of the channel.

ATP is not required for anion current activated by cell swelling in multidrug-resistant lung cancer cells

1994 ◽  
Vol 267 (3) ◽  
pp. C688-C699 ◽  
Author(s):  
J. D. Jirsch ◽  
D. W. Loe ◽  
S. P. Cole ◽  
R. G. Deeley ◽  
D. Fedida
Keyword(s):  
Multidrug Resistant ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Open Probability ◽  
Pipette Solution ◽  
Current Cell ◽  
Absolute Requirement ◽  
Cell Recording ◽  
Volume Response ◽  
Cytoskeletal Elements

During whole cell recording with 4 mM ATP and 0.1 mM GTP in the pipette, outwardly rectifying Cl- currents (155 +/- 20.5 pA/pF) were repetitively activated on reduction of bath solution osmolarity from 290 mosM (control) to 210 mosM. These currents were sensitive to 0.1-1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Omission of ATP from the pipette solution reduced the current magnitude to 42.7 +/- 9.5 pA/pF and prevented repetitive activation. More hyposmotic solutions (160 mosM) usually elicited current repetitively despite an ATP-free pipette solution. In cells depleted of ATP (to < 5% of control) by preincubation with 2-deoxyglucose (10 mM) and rotenone (100 nM), hyposmotic solutions failed to activate significant current. Cell volume increased to 230 +/- 18% of control (19.1 +/- 1.2 microns) in 210 mosM bath (normal cells) but only to 114 +/- 13% of control in ATP-depleted cells exposed to 160 mosM solution. This failure of ATP-depleted cells to swell in hypotonic external solutions was reversed by overnight pretreatment with cytochalasin D (2 micrograms/ml; n = 6) but not by colchicine (250 microM; n = 8). In outside-out patches of membrane dialyzed with zero ATP and excised from swollen cells, we observed sustained activation of a 53-pS outwardly rectifying channel (chord conductance, +100 mV; open probability approximately 1.0). In cell-attached patches from normal and ATP-depleted cells, we activated similar channels by suction. ATP does not appear to be an absolute requirement for the activation of this Cl- channel in H69AR cells but may be essential for the normal volume response and channel activation mediated through cytoskeletal elements within cells.

Regulation of whole cell currents by cytosolic cAMP, Ca2+, and Cl- in rat fetal distal lung epithelium

1995 ◽  
Vol 269 (1) ◽  
pp. C156-C162 ◽  
Author(s):  
T. Nakahari ◽  
Y. Marunaka
Keyword(s):  
Lung Epithelium ◽  
Cellular Mechanisms ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Adrenergic Receptor Agonist ◽  
K Currents

The whole cell patch-clamp technique was used to study ionic conductances in fetal distal lung epithelial (FDLE) cells. In unstimulated FDLE cells, K+ conductances were detected in lowered intracellular Cl- concentration ([Cl-]i, < or = 50 mM). The whole cell currents of FDLE cells were increased by elevation of intracellular Ca2+ concentration ([Ca2+]i) or intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration ([cAMP]i). The elevation of [Ca2+]i activated the K+ currents. The amiloride-blockable whole cell currents were activated by [cAMP]i of 1 mM with [Cl-]i of 20 mM and were more frequently detected in the pipette solution without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) than with it (0.5 mM). When the [Cl-]i was fixed at 50 or 145 mM, however, the increase in these currents was not detected even with cAMP and without EGTA. The amiloride-blockable currents were detected in both the Na+ and K+ pipette solutions. Thus the increase in amiloride-blockable whole cell currents was due to the activation of nonselective cation channels. In FDLE cells treated with terbutaline, which is a beta 2-adrenergic receptor agonist, or forskolin, these currents were detected in the pipette solution containing 20 mM Cl- but were suppressed with time when the pipette solution contained 50 or 145 mM Cl-. It seems likely that maintenance of [Cl-]i at the lowered level is an important requirement for the FDLE cells to activate the amiloride-blockable whole cell currents. It is proposed that cellular mechanisms, such as cell shrinkage, exist to reduce the [Cl-]i in response to cAMP.

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