Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells

1997 ◽  
Vol 273 (3) ◽  
pp. C999-C1007 ◽  
Author(s):  
A. Stutzin ◽  
A. L. Eguiguren ◽  
L. P. Cid ◽  
F. V. Sepulveda
Keyword(s):  
Hela Cells ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Common Channel ◽  
Inward Current ◽  
Cl Channel ◽  
Organic Osmolyte ◽  
Efflux Pathway ◽  
Taurine Efflux

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.

The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding

1994 ◽  
Vol 267 (5) ◽  
pp. C1203-C1209 ◽  
Author(s):  
P. S. Jackson ◽  
R. Morrison ◽  
K. Strange
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Pipette Solution ◽  
Organic Osmolyte ◽  
Atp Levels ◽  
Patch Pipette ◽  
Taurine Efflux

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.

Anion channel blockers inhibit swelling-activated anion, cation, and nonelectrolyte transport in HeLa cells

1996 ◽  
Vol 271 (2) ◽  
pp. C579-C588 ◽  
Author(s):  
J. A. Hall ◽  
J. Kirk ◽  
J. R. Potts ◽  
C. Rae ◽  
K. Kirk
Keyword(s):  
Hela Cells ◽  
Anion Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Influx Rate ◽  
Cation Permeability ◽  
Transport Component ◽  
Selective Channel ◽  
Substrate Concentrations

The effect of osmotic cell swelling on the permeability of HeLa cells to a range of structurally unrelated solutes including taurine, sorbitol, thymidine, choline, and K+ (96Rb+) was investigated. For each solute tested, reduction in the osmolality of the medium from 300 to 200 mosmol/kgH2O caused a significant increase in the unidirectional influx rate. In each case, the osmotically activated transport component was nonsaturable up to external substrate concentrations of 50 mM. Inhibitors of the swelling-activated anion channel of HeLa cells [quinine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, niflumate, 1,9-dideoxyforskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and tamoxifen] blocked the osmotically activated influx of each of the different substrates tested, as well as the osmotically activated efflux of taurine and I-. Tamoxifen and NPPB were similarly effective at blocking the osmotically activated efflux of 96Rb+. The simplest of several hypotheses consistent with the data is that the osmotically activated transport of the different solutes tested here is via a swelling-activated anion-selective channel that has a significant cation permeability and a minimum pore diameter of 8-9 A.

Osmolyte channel regulation by ionic strength in skate RBC

2000 ◽  
Vol 279 (1) ◽  
pp. R69-R76 ◽  
Author(s):  
Kathleen A. Wittels ◽  
Elise M. Hubert ◽  
Mark W. Musch ◽  
Leon Goldstein
Keyword(s):  
Enzyme Activity ◽  
Ionic Strength ◽  
Blood Cells ◽  
Electrolyte Concentration ◽  
Cell Swelling ◽  
Channel Activity ◽  
Channel Regulation ◽  
Taurine Efflux ◽  
Stimulation Of ◽  
In Cells

The aim of this study was to determine whether the opening of the osmolyte channel in skate red blood cells (RBC) is regulated by intracellular electrolyte concentration and conductivity. Consistent with previous studies, experiments with hyperosmotic preincubation before cell swelling or swelling with an isosmotic electrolyte (e.g., ammonium chloride) showed that an increase in ionic strength inhibits the opening of the taurine channel. However, a decrease in intracellular ionic strength did not always stimulate taurine efflux to the same degree. Whereas hyposmotic swelling caused a large increase in taurine efflux, swelling induced by treatment with isosmotic nonelectrolytes produced much smaller stimulation. Results with assays for band 3 phosphorylating enzymes were consistent with those from the taurine efflux studies; stimulation of enzyme activity was lower in cells that were swollen with isosmotic nonelectrolyte media than in cells swollen in hyposmotic media. These results indicate that a decrease in ionic strength is not the only signal for the opening of the taurine channel in skate RBC. Ionic strength does affect channel activity, but there must also be some other regulator.

Prostaglandin E2 increases 7-pS Cl- channel density in the apical membrane of A6 distal nephron cells

1997 ◽  
Vol 273 (2) ◽  
pp. C548-C557 ◽  
Author(s):  
K. E. Kokko ◽  
P. S. Matsumoto ◽  
Z. R. Zhang ◽  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Prostaglandin E2 ◽  
Channel Blocker ◽  
Short Circuit ◽  
Apical Cell ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Distal Nephron ◽  
Open Probability ◽  
Channel Density ◽  
Cl Channel

In A6 distal nephron cells, short-circuit current (Isc) was increased by basolateral exposure to prostaglandin E2 (PGE2; peak response at 1 microM). The effect was only partially abolished by either apical amiloride, an Na+ channel blocker, or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl- channel blocker. In apical cell-attached patches, we observed a 7-pS Cl- channel with a linear current-voltage relationship, a reversal potential near resting membrane potential, and open probability > 0.5. The channel was blocked by diphenylamine-2-carboxylate, glibenclamide, and NPPB but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The frequency of observed Cl- channel activity increased 7-fold with 10-min exposure to PGE2 and 3.7-fold with longer (10-50 min) exposure to PGE2. The PGE2-induced increase in Cl- channel activity was due primarily to an increase in the number of functional channels. The following conclusions were made: 1) activation of apical, 7-pS Cl- channels in A6 cells accounts for the PGE2-induced increase in the amiloride-insensitive Isc, and 2) 7-pS Cl- channel activation was mediated via an increase in channel density without substantial effects on channel kinetics.

Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells

1991 ◽  
Vol 261 (4) ◽  
pp. C658-C674 ◽  
Author(s):  
C. K. Solc ◽  
J. J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Current Density ◽  
Single Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Whole Cell ◽  
Cl Channels ◽  
Single Channel Currents

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

Taurine transport in skate hepatocytes. II. Volume activation, energy, and sulfhydryl dependence

1992 ◽  
Vol 262 (3) ◽  
pp. G451-G460 ◽  
Author(s):  
N. Ballatori ◽  
J. L. Boyer
Keyword(s):  
Plasma Membrane ◽  
Concentration Gradient ◽  
Regulatory Volume Decrease ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Organic Osmolytes ◽  
Taurine Transport ◽  
Taurine Concentration ◽  
Taurine Efflux

Isolated skate (Raja erinacea) hepatocytes swollen in hypotonic media exhibited a regulatory volume decrease (RVD) that was associated with only a small increase in K+ or 86Rb+ efflux but a substantial increase in the release of taurine, an amino acid found in high concentrations in skate hepatocytes. Taurine efflux was stimulated in media made hypotonic by addition of H2O or removal of NaCl, as well as in cells swollen in isotonic media containing rapidly penetrating solutes (202 mM ethylene glycol or 202 mM additional urea substituted for 101 mM NaCl), suggesting that cell swelling rather than hyposmolarity is the stimulus for the activation of taurine release. In contrast, release of glutathione, L-[14C]alanine and other alpha-amino acids (e.g., threonine, serine, glutamate, glutamine, glycine, or valine) was unaffected by dilution with 40% H2O. Taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ and was only slightly diminished by replacing Cl- with NO3-. Addition of 50 mM taurine or hypotaurine to the incubation media also had no effect on volume-stimulated [14C]taurine efflux, suggesting that the taurine concentration gradient across the plasma membrane is not the driving force. Volume-stimulated taurine transport was temperature sensitive, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibitable (0.5 mM), and nearly completely blocked by metabolic inhibitors (2,4-dinitrophenol, KCN, sodium azide, oligomycin, carbonyl cyanide m-chlorophenylhydrazone, and antimycin A), suggesting an active energy-dependent process. Sulfhydryl-reactive reagents (N-ethylmaleimide, diamide, iodoacetate, tert-butyl hydroperoxide, and mercury) also blocked volume-stimulated taurine efflux, whereas efflux was unaffected by Ca2+ ionophore, phorbol ester, dibutyryl-adenosine 3',5'-cyclic monophosphate, vasopressin, or pretreatment with ouabain or furosemide. N-ethylmaleimide, diamide, 2,4-dinitrophenol, and iodoacetate plus KCN also inhibited the RVD. These findings suggest that, in contrast to hepatocytes from most vertebrate species, RVD in skate hepatocytes is associated with the release of only a small fraction of intracellular K+ but a substantial fraction of intracellular taurine and perhaps other organic osmolytes. This volume-activated taurine transport mechanism is energy and sulfhydryl group dependent and is not related to the taurine concentration gradient across the skate hepatocyte plasma membrane.

A novel cGMP-activated Cl- channel in renal proximal tubules

1995 ◽  
Vol 268 (2) ◽  
pp. F323-F329 ◽  
Author(s):  
N. Darvish ◽  
J. Winaver ◽  
D. Dagan
Keyword(s):  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Open Probability ◽  
Cl Channel ◽  
Bath Application ◽  
Cl Channels ◽  
Dose Dependent

Cl- channels activated by natriuretic peptides were detected in cultured rat proximal convoluted tubule (PCT) cells with the use of patch-clamp methodology. Bath application of atrial natriuretic peptide (ANP) activates a 150-pS Cl- channel with the open probability (Po) of the channel increasing from 0.0008 +/- 0.0003 to 0.021 +/- 0.008. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a membrane-permeable analogue of cGMP, increased channel activity in the on-cell mode. In inside-out patches the channel was activated by cGMP in a dose-dependent manner. Channel activity decreased after washing out and increased on reapplication of cGMP. A similar activation was observed also in presence of either of two protein kinase inhibitors, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride or KT5823, or a phosphatase inhibitor. Bath application of urodilatin mimicked the action of ANP. Po of the channel was found to be independent of both voltage and Ca2+, and gating activity could be blocked by the stilbene, 4,4-dinitrostilbene-2,2-disulfonic acid. These results demonstrate a Cl- conductance in PCT cells modulated by ANP and urodilatin via their second messenger, cGMP.

G protein-regulated large-conductance chloride channels in freshly isolated fetal type II alveolar epithelial cells

1993 ◽  
Vol 265 (4) ◽  
pp. L323-L329 ◽  
Author(s):  
P. J. Kemp ◽  
G. G. MacGregor ◽  
R. E. Olver
Keyword(s):  
Epithelial Cells ◽  
G Protein ◽  
Alveolar Epithelial Cells ◽  
Disulfonic Acid ◽  
Single Channels ◽  
Type Ii ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Alveolar Epithelial ◽  
Cl Channel

Using the patch-clamp technique, we have recorded single channels in cell-attached and inside-out excised patches from the plasma membrane of type II alveolar epithelial cells freshly isolated from fetal guinea pig lung by elastase digestion and differential filtration. In cell-free patches the channels were highly selective for Cl- (PCl:Pcat = 9:1), had a large unitary conductance (375 pS +/- 23 pS), and current reversal of 0 mV in either symmetrical Na(+)-rich solutions or when the inner membrane leaflet was bathed in a K(+)-rich solution. The large-conductance Cl- channel exhibited little or no voltage inactivation at positive potentials, remained open for a significant amount of time at potentials negative to -40 mV, and was blocked at all potentials by 0.1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid. Channel activity was independent of intracellular calcium concentration. Bath addition of the nonmetabolizable analogue of GTP, GTP gamma S (0.1 mM), caused a voltage-dependent inhibition of channel activity [open probability (Po) plot was shifted by at least +25 mV]. Smaller channels (25 +/- 3 pS) were recorded in the cell-attached configuration with a current-voltage (I-V) relationship which was compatible with a Cl- conductance. On excision, the patches previously containing small-conductance channels exhibited only large-conductance Cl- channel behavior. These large-conductance, G protein-regulatable Cl- channels may provide a route for alveolar cell Cl- exit and as such may be an integral part of the mechanism responsible for secretion of fetal lung fluid.

Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling

1992 ◽  
Vol 262 (2) ◽  
pp. C501-C509 ◽  
Author(s):  
R. E. Yantorno ◽  
D. A. Carre ◽  
M. Coca-Prados ◽  
T. Krupin ◽  
M. M. Civan
Keyword(s):  
Regulatory Volume Decrease ◽  
Membrane Conductance ◽  
Cell Swelling ◽  
K Channel ◽  
Ciliary Epithelium ◽  
Channel Blockers ◽  
Channel Activity ◽  
Patch Clamping ◽  
Whole Cell ◽  
Cl Channel

Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.

Sign in / Sign up

Export Citation Format

Share Document