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.

Volume-associated osmolyte fluxes in cell lines with or without the anion exchanger

1995 ◽  
Vol 269 (5) ◽  
pp. C1280-C1286 ◽  
Author(s):  
R. Sanchez-Olea ◽  
C. Fuller ◽  
D. Benos ◽  
H. Pasantes-Morales
Keyword(s):  
Amino Acid ◽  
Cell Lines ◽  
Anion Exchanger ◽  
Chinese Hamster ◽  
Anion Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Cell Type ◽  
Amino Acid Efflux

To investigate the involvement of a red cell-type anion exchanger in the volume-sensitive amino acid release, the hyposmolarity-evoked release of D-[3H]aspartate and [3H]taurine was examined in three cell lines: 1) wild-type Chinese hamster ovary (CHO-K1) cells, expressing an anion exchanger activity (Cl-/SO4(2-)) functionally similar to the erythroid band 3; 2) a mutant CHO cell type (CHO 605) lacking this anion exchanger activity; and 3) 293 cells in which the Cl-/HCO3(-) anion exchanger is absent. All cell types accumulated D-[3H]aspartate and [3H]taurine under isosmotic conditions, and, similarly, in the three cell lines, cell swelling evoked by hyposmolarity induced a rapid and transient increase in the amino acid efflux. Blockers of the anion exchanger and/or Cl- channels [niflumic acid, dipyridamole, diphenylamine-2-carboxylate,5-nitro-2-(3-phenylpropylamino)-benzoi c acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid] were potent inhibitors of amino acid efflux in the three cell lines. 125I- efflux, used as a marker for Cl- fluxes, was also markedly increased in response to cell swelling in all cell lines, and this efflux was inhibited by the anion exchanger/Cl- channel blockers. These results do not support a role for an anion exchanger in the hyposmolarity-induced amino acid efflux and suggest that amino acids and Cl- may be transported by the same or a similar mechanism, presumably an anion channel-like structure.

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

2002 ◽  
Vol 283 (2) ◽  
pp. C569-C578 ◽  
Author(s):  
Alexander A. Mongin ◽  
Harold K. Kimelberg
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Atp Release ◽  
Anion Channel ◽  
P2y Receptors ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Medium Osmolarity ◽  
Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

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.

Mechanical strain-induced Ca2+waves are propagated via ATP release and purinergic receptor activation

2000 ◽  
Vol 279 (2) ◽  
pp. C295-C307 ◽  
Author(s):  
H. Sauer ◽  
J. Hescheler ◽  
M. Wartenberg
Keyword(s):  
Gap Junctions ◽  
Purinergic Receptor ◽  
Mechanical Strain ◽  
Atp Release ◽  
Anion Channel ◽  
Receptor Activation ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Junctional Coupling ◽  
Intracellular Ca

Mechanical strain applied to prostate cancer cells induced an intracellular Ca2+ (Cai 2+) wave spreading with a velocity of 15 μm/s. Cai 2+ waves were not dependent on extracellular Ca2+ and membrane potential because propagation was unaffected in high-K+ and Ca2+-free solution. Waves did not depend on the cytoskeleton or gap junctions because cytochalasin B and nocodazole, which disrupt microfilaments and microtubules, respectively, and 1-heptanol, which uncouples gap junctions, were without effects. Fluorescence recovery after photobleaching experiments revealed an absence of gap junctional coupling. Cai 2+ waves were inhibited by the purinergic receptor antagonists basilen blue and suramin; by pretreatment with ATP, UTP, ADP, UDP, 2-methylthio-ATP, and benzoylbenzoyl-ATP; after depletion of ATP by 2-deoxyglucose; and after ATP scavenging by apyrase. Waves were abolished by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid, tamoxifen, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, niflumic acid, and gadolinium. ATP release following strain was significantly inhibited by anion channel blockers. Hence, ATP is secreted via mechanosensitive anion channels and activates purinergic receptors on the same cell or neighboring cells in an autocrine and paracrine manner, thus leading to Cai 2+ wave propagation.

Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells

1997 ◽  
Vol 273 (1) ◽  
pp. C214-C222 ◽  
Author(s):  
V. G. Manolopoulos ◽  
T. Voets ◽  
P. E. Declercq ◽  
G. Droogmans ◽  
B. Nilius
Keyword(s):  
Endothelial Cells ◽  
Anion Channel ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Organic Osmolytes ◽  
Dependent Manner ◽  
Anion Channels ◽  
Whole Cell Patch Clamp ◽  
Osmolyte Efflux ◽  
Dose Dependent

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.

Sustained outward current observed after I(to1) inactivation in rabbit atrial myocytes is a novel Cl- current

1992 ◽  
Vol 263 (6) ◽  
pp. H1967-H1971 ◽  
Author(s):  
D. Y. Duan ◽  
B. Fermini ◽  
S. Nattel
Keyword(s):  
Reversal Potential ◽  
Outward Current ◽  
Cell Voltage ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
K Channel ◽  
Transient Outward Current ◽  
Channel Blockers ◽  
Atrial Myocytes ◽  
K Concentration

In rabbit atrial myocytes, depolarization of the membrane results in a rapidly activating transient outward current (I(to)) that then decays to a sustained level. The sustained current (Isus) remains constant for at least 5 s during continued depolarization. The present study was designed to identify the ionic mechanism underlying Isus with the use of whole cell voltage-clamp techniques. After exposure to 2 mM 4-aminopyridine (4-AP), the 4-AP-sensitive transient outward current (I(to1)) was abolished, but Isus was unaffected. Isus was not blocked by the K+ channel blockers tetraethylammonium chloride and Ba2+, was not changed by increasing superfusate K+ concentration, and was still present when K+ was replaced by Cs+ in both the superfusate and the pipette. Isus was significantly reduced by the Cl- transport blockers 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The current-voltage relations of Isus showed outward rectification, and the reversal potential of Isus shifted with changes in the transmembrane Cl- gradient in the fashion expected for a Cl- current. We conclude that Isus in rabbit atrium is due to a noninactivating Cl- current which, unlike previously described cardiac Cl- currents, is manifest in the absence of exogenous stimulators of adenosine 3',5'-cyclic monophosphate formation, cytosolic Ca2+ transients, or cell swelling.

Chloride current activated by swelling in retinal pigment epithelium cells

1993 ◽  
Vol 265 (4) ◽  
pp. C1037-C1045 ◽  
Author(s):  
L. M. Botchkin ◽  
G. Matthews
Keyword(s):  
Retinal Pigment Epithelium ◽  
Fluid Transport ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ionic Composition ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Subretinal Space ◽  
Channel Blockers ◽  
Membrane Stretch

A membrane conductance activated by cell swelling was characterized in cells of the retinal pigment epithelium (RPE). Manipulations of internal and external Cl concentration revealed that the conductance is permeable to Cl and somewhat permeable to the gluconate anion used for Cl substitution (ratio of gluconate to Cl permeability approximately 0.1). The conductance was blocked by the Cl channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid in a manner consistent with open-channel blockade. Both the onset and recovery of the Cl current following a transient increase in cell volume were slow. This suggests that activation of the current depends on some internal signal rather than directly on membrane stretch. Experiments to examine a possible role for intracellular Ca concentration ([Ca]i) in regulation of the current demonstrated that an increase in [Ca]i was not involved in the linkage between swelling and Cl current; activation of the current was unaffected by the calcium-buffering conditions, the current could not be activated by large increases in [Ca]i elicited by ionomycin, and no changes in [Ca]i were observed to be associated with swelling. RPE cells normally experience changes in the volume and ionic composition of the extracellular subretinal space during changes in illumination conditions; therefore, the volume-sensitive Cl conductance may play a role in volume regulation in the RPE in response to these extracellular changes and/or in transepithelial fluid transport.

Diverse modulations of chloride channels in renal proximal tubules

1994 ◽  
Vol 267 (5) ◽  
pp. F716-F724 ◽  
Author(s):  
N. Darvish ◽  
J. Winaver ◽  
D. Dagan
Keyword(s):  
Chloride Channels ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Cation Permeability ◽  
Voltage Dependent ◽  
Selective Channel ◽  
Cl Channels ◽  
The Rich ◽  
Apical Membranes ◽  
Inside Out

Cl- selective channels were detected and characterized in apical membranes of cultured rat renal proximal convoluted tubule cells (PCT) using patch-clamping methods. Subpopulations of Cl- channels modulated by cyclic nucleotides, Ca2+, or voltage were identified. Two different 30-pS, voltage-independent, Cl- channels modulated by adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ were seen most frequently. The cAMP-dependent channel was activated by membrane-permeable analogues of cAMP, dibutyryl-cAMP or 8-bromo-cAMP. Catalytic subunit of protein kinase A (PKA) applied to detached inside-out patches, activated the channel as well, suggesting activation via phosphorylation. Channel activity was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, by 4,4-dinitrostilbene-2,2-disulfonic acid, and by SCN-. Permeability sequence for different halides was Cl- > I > F with a Cl(-)-to-cation permeability ratio (PCl/Pcation) of 7:1. The Ca(2+)-sensitive channel was not activated by cAMP nor by PKA. A third anionic selective channel encountered infrequently is voltage dependent and has a unitary conductance of 145 pS, with a PCl/Pcation value of 9:1. This diversity of Cl- channels may underlie the rich repertoire of physiological functions attributed to Cl- channels.

Contribution of chloride channels to volume regulation of cortical astrocytes

2003 ◽  
Vol 284 (6) ◽  
pp. C1460-C1467 ◽  
Author(s):  
Kimberly A. Parkerson ◽  
Harald Sontheimer
Keyword(s):  
Volume Regulation ◽  
Chloride Channels ◽  
Brain Regions ◽  
Niflumic Acid ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Relative Contribution ◽  
Cortical Astrocytes ◽  
Hippocampal Astrocytes

The objective of this study was to determine the relative contribution of Cl− channels to volume regulation of cultured rat cortical astrocytes after hypotonic cell swelling. Using a Coulter counter, we showed that cortical astrocytes regulate their cell volume by ∼60% within 45 min after hypotonic challenge. This volume regulation was supported when Cl− was replaced with Br−, NO[Formula: see text], methanesulfonate−, or acetate− but was inhibited when Cl− was replaced with isethionate− or gluconate−. Additionally, substitution of Cl− with I−completely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl− channel blockers, including 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and niflumic acid. Surprisingly, the combination of Cd2+ with NPPB, DIDS, or niflumic acid inhibited regulation to a greater extent than any of these drugs alone. Volume regulation did not differ among astrocytes cultured from different brain regions, as cerebellar and hippocampal astrocytes exhibited behavior identical to that of cortical astrocytes. These data suggest that Cl− flux through ion channels rather than transporters is essential for volume regulation of cultured astrocytes in response to hypotonic challenge.

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.

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