ATP Released From Astrocytes During Swelling Activates Chloride Channels

2003 ◽  
Vol 89 (4) ◽  
pp. 1870-1877 ◽  
Author(s):  
Mark Darby ◽  
J. Brent Kuzmiski ◽  
William Panenka ◽  
Denise Feighan ◽  
Brian A. MacVicar
Keyword(s):  
Chloride Channels ◽  
Purinergic Receptors ◽  
Neuronal Excitability ◽  
Regulatory Volume Decrease ◽  
Atp Release ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
P2x7 Receptors ◽  
Whole Cell Patch Clamp ◽  
Cultured Astrocytes

ATP release from astrocytes contributes to calcium ([Ca2+]) wave propagation and may modulate neuronal excitability. In epithelial cells and hepatocytes, cell swelling causes ATP release, which leads to the activation of a volume-sensitive Cl− current ( I Cl,swell) through an autocrine pathway involving purinergic receptors. Astrocyte swelling is counterbalanced by a regulatory volume decrease, involving efflux of metabolites and activation of I Cl,swell and K+currents. We used whole cell patch-clamp recordings in cultured astrocytes to investigate the autocrine role of ATP in the activation of I Cl,swell by hypo-osmotic solution (HOS). Apyrase, an ATP/ADP nucleotidase, inhibited HOS-activated I Cl,swell, whereas ATP and the P2Y agonists, ADPβS and ADP, induced Cl− currents similar to I Cl,swell. Neither the P2U agonist, UTP nor the P2X agonist, α,β-methylene ATP, were effective. BzATP was less effective than ATP, suggesting that P2X7 receptors were not involved. P2 purinergic antagonists, suramin, RB2, and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) reversibly inhibited activation of I Cl,swell, suggesting that ATP-activated P2Y1 receptors. Thus ATP release mediates I Cl,swell in astrocytes through the activation of P2Y1-like receptors. The multidrug resistance protein (MRP) transport inhibitors probenicid, indomethacin, and MK-571 all potently inhibited I Cl.swell. ATP release from astrocytes in HOS was observed directly using luciferin-luciferase and MK-571 reversibly depressed this HOS-induced ATP efflux. We conclude that ATP release via MRP and subsequent autocrine activation of purinergic receptors contributes to the activation of I Cl,swell in astrocytes by HOS-induced swelling.

Activation of Cl-dependent K transport in Ehrlich ascites tumor cells

1986 ◽  
Vol 251 (3) ◽  
pp. C369-C379 ◽  
Author(s):  
B. Kramhoft ◽  
I. H. Lambert ◽  
E. K. Hoffmann ◽  
F. Jorgensen
Keyword(s):  
Regulatory Volume Decrease ◽  
Extracellular Ph ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Cell Shrinkage ◽  
Ehrlich Cells ◽  
Ehrlich Ascites ◽  
Dependent Component ◽  
Ca Depletion ◽  
Volume Decrease

N-ethylmaleimide (NEM) treatment of steady-state Ehrlich cells induces a substantial net loss of cellular KCl and cell shrinkage. The majority of the initial K loss is Cl dependent. From estimates of membrane potential it is concluded that the NEM-induced KCl loss is electroneutral. The effect of NEM on H extrusion by cells in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-containing medium showed that only an insignificant part of the K loss could be attributed to an activation of a K-H exchange system. Consequently, NEM appears to activate a K-Cl cotransport, which causes cell shrinkage. The anion preference of the K loss is Cl greater than Br much greater than SCN = NO3. NEM also seems to inhibit a Cl-dependent Na uptake previously described in shrunken cells. Addition of NEM to cells undergoing regulatory volume decrease after swelling in hyposmotic media results in a Cl-dependent acceleration of cell shrinkage, suggesting that a Cl-dependent component of K efflux is induced by NEM also in swollen cells. A Cl-dependent K efflux is also activated in Ca-depleted cells or at reduced extracellular pH after cell swelling. Under isotonic conditions activation of Cl-dependent K flux after Ca depletion or pH reduction could not be demonstrated. The combined results show that Ehrlich cells possess a latent K-Cl cotransport that becomes active after changes in the state of SH groups, regardless of the initial cell volume. A similar K-Cl cotransport is activated in hypotonically swollen cells after Ca depletion or after reduction of the extracellular pH.

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.

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.

Regulatory volume decrease in cultured astrocytes. I. Potassium- and chloride-activated permeability

1994 ◽  
Vol 266 (1) ◽  
pp. C165-C171 ◽  
Author(s):  
H. Pasantes-Morales ◽  
R. A. Murray ◽  
L. Lilja ◽  
J. Moran
Keyword(s):  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
K Channel ◽  
Channel Blockers ◽  
Rate Limiting Step ◽  
Cultured Astrocytes ◽  
Cerebellar Astrocytes ◽  
Rate Limiting ◽  
K Permeability ◽  
Volume Decrease

Regulatory volume decrease (RVD) in detached cerebellar astrocytes in culture after acute exposure to hyposmolarity was characterized in this and the accompanying paper [H. Pasantes-Morales, R. A. Murray, R. Sanches-Olea, and J. Moran. Am. J. Physiol. 266 (Cell Physiol. 35): C172-C178, 1994]. RVD was independent of extracellular calcium, was accelerated at pH 8-9 and retarded at pH 6, and was reduced at temperatures < 18 degrees C. The cationic pathway activated by hyposmolarity was specific for K+ and Rb+, since RVD was abolished and secondary swelling occurred when these ions replaced Na+. However, Li+, choline, tris(hydroxymethyl)aminomethane, and glucosamine, all as Cl- salts, did not affect RVD. The anion pathway was unselective, since RVD was inhibited when NaCl was replaced by anion K+ salts with a permeability rank of SCN- = I- > NO3- > Cl- > benzoate > acetate >> SO3- > gluconate. RVD was unaffected by bumetanide (50 microM) and weakly inhibited by furosemide (2 mM). Quinidine but not other K+ channel blockers inhibited RVD, and its effect was reversed by gramicidin. RVD was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dipyridamole but not by diphenylamine-2-carboxylate or anthracene-9-carboxylate. These results suggest that diffusion possibly via channels rather than cotransporters is involved in the swelling-activated K+ and Cl- fluxes. Gramicidin did not change astrocyte volume in isosmotic conditions, but greatly accelerated RVD, suggesting that low Cl- permeability in isosmotic conditions markedly increases by swelling, thus making K+ permeability the rate-limiting step for RVD.

Anion exchanger AE1 as a candidate pathway for taurine transport in rat erythrocytes

1997 ◽  
Vol 272 (5) ◽  
pp. C1457-C1464 ◽  
Author(s):  
C. Conejero
Keyword(s):  
Anion Exchanger ◽  
Chloride Channels ◽  
Regulatory Volume Decrease ◽  
Cell Types ◽  
Band 3 ◽  
Disulfonic Acid ◽  
Rat Erythrocytes ◽  
Taurine Transport ◽  
Uptake Process ◽  
Taurine Uptake

Taurine has been shown to act as an osmolyte during the regulatory volume decrease process in a variety of cell types. The nature of the taurine efflux carrier is thought to consist of a diffusional pathway with pharmacological properties similar to a chloride channel or through an anion exchanger. We propose that taurine is a substrate of the anion exchanger AE1, also called band 3. Experiments were performed in rat-erythrocytes, which express large amounts of band 3. Taurine uptake and efflux transport experiments were determined in the presence of inhibitors of anion carriers and chloride channels. Both taurine uptake and efflux were inhibited by band 3 inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), niflumic acid, or furosemide. Moreover, DIDS competes with taurine at a common binding site in the uptake process. Specific inhibitors of the electroneutral cotransport as well as inhibitors of the chloride channels were ineffective in blocking taurine transport. Thus we suggest that band 3 may be the protein responsible for taurine transport in rat erythrocytes.

Propionate activates multiple ion transport mechanisms in the HT29-18-C1 human colon cell line

1993 ◽  
Vol 265 (3) ◽  
pp. G564-G571 ◽  
Author(s):  
W. A. Rowe ◽  
D. L. Blackmon ◽  
M. H. Montrose
Keyword(s):  
Cell Line ◽  
Cellular Response ◽  
Regulatory Volume Decrease ◽  
Fold Increase ◽  
Human Colon ◽  
Short Chain Fatty Acids ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Epithelial Cell Line ◽  
Colon Cell

Short-chain fatty acids (SCFAs) are the major solutes and the major anions in the colonic lumen. We studied the response of suspended HT29-18-C1 cells (an epithelial cell line derived from a human colon carcinoma) to SCFA exposure. Cellular response was evaluated by measurement of cell volume (Coulter counter), intracellular pH [pHi; measured fluorometrically with 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF)], and intracellular Na+, K+, and Cl- content (flame photometry and chloride titrator). Exposure to 130 mM propionate in isosmotic medium causes a rapid decrease in pHi and activates pHi recovery via amiloride-sensitive Na-H exchange. In the presence of propionate, Na-H exchange also causes cell swelling to a peak volume 11% above control cells and causes a 2.8-fold increase in intracellular Na+ content. After peak swelling, a regulatory-volume decrease (RVD) significantly reduced volume and intracellular Na+ returned to baseline. Other SCFAs (acetate, butyrate, and valerate) also elicit swelling and RVD. Activation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) is required to return Na+ to normal levels and to indirectly provide ion gradients required for propionate-induced RVD, but Na(+)-K(+)-ATPase activity does not directly mediate RVD. When 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) is added in the presence of propionate, RVD was inhibited and cell Na+ content increased. Cl- depletion inhibited propionate-induced RVD and diminished the effect of SITS.

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.

On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells

2013 ◽  
Vol 304 (10) ◽  
pp. C1013-C1026 ◽  
Author(s):  
María V. Espelt ◽  
Felicitas de Tezanos Pinto ◽  
Cora L. Alvarez ◽  
Germán Sanchez Alberti ◽  
Jeremías Incicco ◽  
...  
Keyword(s):  
Regulatory Volume Decrease ◽  
Atp Release ◽  
Hepatoma Cells ◽  
Cell Swelling ◽  
Pcr Analysis ◽  
Human Hepatoma ◽  
Rt Pcr ◽  
Human Hepatoma Cells ◽  
Volume Regulatory Decrease ◽  
Hypotonic Treatment

Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD40 (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATPe) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na+-K+-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD40 by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD40 ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5′-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na+-K+-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y13. RT-PCR and Western blot analysis confirmed the presence of P2Y13. Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATPe accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATPe to ADPe by ENTPDase 2 activity facilitates the accumulated ADPe to activate P2Y13 receptors, which mediate complete RVD.

scholarly journals Regulation of ClC-2 chloride channels in T84 cells by TGF-α

2001 ◽  
Vol 280 (6) ◽  
pp. C1588-C1598 ◽  
Author(s):  
Moëz Bali ◽  
Joanna Lipecka ◽  
Aleksander Edelman ◽  
Janine Fritsch
Keyword(s):  
Growth Factor ◽  
Chloride Channels ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Cell Swelling ◽  
T84 Cells ◽  
Transforming Growth Factor Α ◽  
Whole Cell Patch Clamp ◽  
Phosphoinositide 3 Kinase ◽  
Factor Α

The almost ubiquitously expressed ClC-2 chloride channel is activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein tyrosine phosphorylation and activation of these channels. This study examines the possibility that transforming growth factor-α (TGF-α) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-α (0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a superimposed reversible activation of the current was observed at 8.3 nM TGF-α. Both effects required activation of the intrinsic epidermal growth factor receptor (EGFR) tyrosine kinase activity, of phosphoinositide 3-kinase, and of protein kinase C. With microspectrofluorimetry of the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-α was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-α-induced alkalinization activates ClC-2 current. This study indicates that ClC-2 channels are targets for EGFR signaling in epithelial cells.

Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye

1995 ◽  
Vol 269 (4) ◽  
pp. C1063-C1072 ◽  
Author(s):  
J. Arreola ◽  
K. R. Hallows ◽  
P. A. Knauf
Keyword(s):  
Chloride Channels ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Low Concentrations ◽  
Potent Inhibition ◽  
Voltage Dependent ◽  
Cl Channels

When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K+ and Cl-. This is primarily caused by a dramatic increase in Cl- permeability, which may reflect the opening of volume-sensitive channels (11). To test this hypothesis, we measured volume-activated Cl- currents in HL-60 cells using the patch-clamp technique. The whole cell Cl- conductance (in nS/pF at 100 mV) increased from 0.09 +/- 0.06 to 1.15 +/- 0.19 to 1.64 +/- 0.40 as the tonicity (in mosmol/kgH2O) of the external medium was decreased from 334 to 263 to 164, respectively. Cl- currents showed no significant inactivation during 800-ms pulses. Current-voltage curves exhibited outward rectification and were identical at holding potentials of 0 or -50 mV, suggesting that the gating of the channels is voltage independent. The selectivity sequence, based on permeability ratios (PX/PCl) calculated from the shifts of the reversal potentials, was SCN- > I- approximately NO3- > Br- > Cl- >> gluconate. 4-Acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with approximately 10-fold increased affinity at potentials greater than +40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of the membrane potential. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [diBA-(5)-C4] with a concentration that gives half inhibition (IC50) of 1.8 microM at room temperature. A similar apparent IC50 value (1.2 microM) was observed for net 36Cl- efflux into a Cl(-)-free hypotonic medium at 21 degrees C. It seems likely, therefore, that the volume-activated Cl- channels are responsible for the net Cl- efflux during RVD. These Cl- channels have properties similar to the “mini-Cl-” channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.

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