Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells

1995 ◽  
Vol 268 (3) ◽  
pp. C721-C731 ◽  
Author(s):  
J. S. Adorante ◽  
P. M. Cala
Keyword(s):  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Ciliary Epithelium ◽  
Channel Blockers ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Osmotic Swelling ◽  
Ion Efflux ◽  
Volume Recovery ◽  
Volume Decrease

To study the net solute and water efflux pathways of the ciliary epithelium we employed a cultured human NPE cell line. Because of the possible relationship between transepithelial ion and water flux and cell volume regulation, the ion efflux pathways mediating regulatory volume decrease (RVD) were investigated. Osmotic swelling of NPE cells was followed by a volume recovery. Volume recovery was K+ dependent and inhibited by K+ channel blockers such as quinine (1 mM). After osmotic swelling, a Cl(-)-dependent membrane depolarization occurred that was inhibited by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) or Ca2+ chelators such as ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2.0 mM). Cell swelling was also accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) of approximately 200 nM. The swelling-induced rise in [Ca2+]i and RVD were diminished in the presence of 10 microM La3+, 50 nM 12-O-tetradecanoylphorbol 13-acetate, and nominally Ca(2+)-free medium. Near total blockage of RVD occurred after pretreatment of NPE cells with Ca(2+)-free EGTA-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester-containing solutions. The inhibition of RVD by EGTA-BAPTA treatment was overcome by increasing K+ conductance with gramicidin. The above findings indicate that RVD in NPE cells is mediated by separate K+ and Cl- conductances (channels). These data also show that swelling-induced increases in [Ca2+]i help modulate net ion efflux during regulation.

Swelling-activated K+ transport via two functionally distinct pathways in eel erythrocytes

1996 ◽  
Vol 270 (1) ◽  
pp. R61-R70 ◽  
Author(s):  
J. D. Bursell ◽  
K. Kirk
Keyword(s):  
Regulatory Volume Decrease ◽  
Anguilla Anguilla ◽  
Cell Swelling ◽  
European Eel ◽  
Pharmacological Properties ◽  
Osmotic Swelling ◽  
Diverse Range ◽  
K Transport ◽  
Volume Decrease ◽  
In Cells

Following osmotic swelling, erythrocytes from the European eel, Anguilla anguilla, underwent a regulatory volume decrease. This was prevented by replacement of Na+ with K+ in the suspending medium, consistent with a role for the (normally outward) electrochemical K+ gradient in the volume-regulatory response. The effect of cell swelling on K- transport in these cells was investigated using 86Rb+ as a tracer for K+. Osmotic swelling resulted in an increase in ouabain-insensitive K+ transport that was highest for cells in Cl- and Br- media but which was also significant in I- and NO3- media. Treatment of eel erythrocytes suspended in isotonic Cl- or Br- (but not I- or NO3-) media with the sulfhydryl reagent N-ethylmaleimide (NEM) resulted in a large increase in K+ transport. A quantitative comparison of the pharmacological properties of the “Cl(-)-dependent” NEM-activated pathway with those of the “Cl(-)-independent” pathway mediating swelling-activated K+ transport in cells in Cl(-)-free (NO3- containing) media showed there to be significant differences between them. By contrast, the pharmacological properties of the Cl(-)-independent swelling-activated K+ pathway were indistinguishable from those of the pathway responsible for the swelling-activated transport of taurine, the major organic osmolyte in these cells. A pharmacological analysis of ouabain-insensitive K+ transport in cells swollen in a hypotonic Cl(-)-containing medium showed there to be two components, one with the characteristics of the NEM-activated system, the other showing the characteristics of the Cl(-)-independent swelling-activated pathway. The data are consistent with the presence of two functionally distinct swelling-activated K+ transport mechanisms in eel erythrocytes: a KCl cotransporter that is activated under isotonic conditions by NEM and a Cl(-)-independent, broad-specificity channel that accommodates a diverse range of organic and inorganic solutes.

Effect of osmotic swelling on K+ conductance in jejunal crypt epithelial cells

1992 ◽  
Vol 262 (6) ◽  
pp. G1021-G1026 ◽  
Author(s):  
R. J. MacLeod ◽  
P. Lembessis ◽  
J. R. Hamilton
Keyword(s):  
Epithelial Cells ◽  
Regulatory Volume Decrease ◽  
Channel Blockers ◽  
Osmotic Swelling ◽  
Hypotonic Stress ◽  
Cell Sizing ◽  
Hypotonic Swelling ◽  
Cl Conductance ◽  
Volume Decrease ◽  
Crypt Cells

To further elucidate differences in ion transport properties between jejunal crypt and villus cells, we compared the responses of purified cell suspensions to hypotonic stress using electronic cell sizing to evaluate volume changes and 86Rb and 36Cl efflux. After hypotonic swelling, villus enterocytes undergo a regulatory volume decrease (RVD) due to the loss of K+ and Cl- through volume-activated conductances. After 0.6x isotonic challenge in Na(+)-free medium, crypt cells exhibited only partial RVD, with t1/2 congruent to 15 min. The addition of a cation ionophore, gramicidin (0.25 microM), to hypotonically swollen crypt cells caused an accelerated RVD, which was complete with t1/2 congruent to 5 min. Crypt epithelial cells showed no volume-activated 86Rb efflux, but villus enterocytes had an increased rate of 86Rb efflux after hypotonic dilution (P less than 0.001). Gramicidin added to hypotonically diluted crypt cells greatly increased the rate of 86Rb efflux compared with controls. Both villus (30 s; P less than 0.005) and crypt (2 min; P less than 0.001) cells exhibited volume-activated 36Cl efflux in absence of gramicidin. Cl- channel blockers anthracene-9-carboxylate (9-AC, 300 microM) and indanyloxyacetic acid (IAA-94, 100 microM) prevented crypt RVD (P less than 0.001) in the presence of gramicidin. Ouabain (P less than 0.001) or K(+)-free Na(+)-containing medium, but not Ba2+ (5 mM) or quinine (100 microM), prevented crypt partial RVD. We conclude that crypt cells lack volume-activated K+ conductance. The RVD exhibited by crypt cells, although partial, was due to Cl- loss through a volume-activated Cl- conductance and Na+ loss via Na(+)-K(+)-ATPase.

Cell volume regulation by the mouse zygote: mechanism of recovery from a volume increase

1997 ◽  
Vol 272 (6) ◽  
pp. C1854-C1861 ◽  
Author(s):  
D. G. Seguin ◽  
J. M. Baltz
Keyword(s):  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Volume Increase ◽  
K Channels ◽  
Cl Channel ◽  
Cl Channels ◽  
Mouse Zygotes ◽  
Volume Decrease

Mouse zygotes regulate their volumes after cell swelling. This regulatory volume decrease (RVD) is rapid and complete. RVD in zygotes was inhibited by K+ or Cl- channel blockers, indicating the participation of such channels in volume recovery. The channels are separate entities, as indicated by the ability of the cation ionophore gramicidin to restore RVD when K+ channels are blocked but not when Cl- channels are blocked. Intracellular Ca2+ concentration increased with cell swelling. Nevertheless, RVD occurred normally in zygotes loaded with the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which prevented Ca2+ from increasing above its normal resting concentration. Thus an increase in intracellular Ca2+ is not necessary for zygote RVD; consistent with this, inhibitors of Ca(2+)-activated K+ channels had little or no effect on RVD. RVD in zygotes was also completely inhibited by millimolar amounts of extracellular ATP. ATP has been shown to inhibit current passed by the volume-sensitive organic osmolyte-Cl- channel in other cells, and thus zygotes may have such a channel participating in RVD.

scholarly journals Regulatory volume decrease in carp red blood cells: mechanisms and oxygenation-dependency of volume-activated potassium and amino acid transport

1995 ◽  
Vol 198 (1) ◽  
pp. 155-165 ◽  
Author(s):  
F Jensen
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Osmotic Swelling ◽  
Original Cell ◽  
Sufficient Stimulus ◽  
K Release ◽  
Volume Decrease

Hypo-osmotic swelling of carp red blood cells (RBCs) induced a regulatory volume decrease (RVD), which restored the original cell volume within 140 min in oxygenated RBCs, whereas volume recovery was incomplete in deoxygenated RBCs. The complete RVD in oxygenated RBCs resulted from a sustained volume-activated release of K+, Cl- and amino acids (AAs). In the absence of ouabain, the cells also lost Na+ as released K+ was partially regained via the Na+/K+ pump. Inorganic osmolytes contributed approximately 70 %, and organic osmolytes approximately 30 %, to the RVD of oxygenated RBCs. Oxygenation in isotonic medium per se activated a K+ efflux from the RBCs. Hypo-osmotic cell swelling stimulated an additional K+ release. The oxygenation-activated and the volume-activated K+ efflux were both inhibited by DIDS and by the replacement of Cl- with NO3-, showing that both types of K+ efflux were Cl--dependent and probably occurred via the same K+/Cl- cotransport mechanism. Once activated by oxygenation, the K+/Cl- cotransport was further stimulated by cell swelling. Deoxygenation inactivated the oxygenation-induced Cl--dependent K+ release and cell swelling was not a sufficient stimulus to reactivate it significantly. In deoxygenated RBCs, the volume-induced K+ release was transient and primarily Cl--independent and, in the absence of ouabain, the cell K+ content recovered towards control values via the Na+/K+ pump. The Cl--independent K+ efflux seemed to involve K+/H+ exchange, but other transport routes also participated. Swelling-activated AA release differed in kinetics between oxygenated and deoxygenated RBCs but was important for RVD at both oxygenation degrees. Approximately 70 % of the AA release was inhibited by DIDS and substitution of NO3- for Cl- produced a 50 % inhibition, suggesting that the AA permeation was partly Cl--dependent. In oxygenated RBCs, a reduction in pH lowered the volume-activated Cl--dependent K+ efflux but not the AA efflux. In deoxygenated RBCs, the acute volume-stimulated K+ and AA release were both increased by acidification. The data are discussed in relation to possible transducer mechanisms and physiological implications.

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.

Ca(2+)-mobilizing hormones potentiate hypotonicity-induced activation of ionic conductances in Intestine 407 cells

1994 ◽  
Vol 267 (5) ◽  
pp. C1271-C1278 ◽  
Author(s):  
B. C. Tilly ◽  
M. J. Edixhoven ◽  
N. van den Berghe ◽  
A. G. Bot ◽  
H. R. de Jonge
Keyword(s):  
Tyrosine Phosphorylation ◽  
Regulatory Volume Decrease ◽  
Cell Types ◽  
Cell Swelling ◽  
Protein Tyrosine Phosphorylation ◽  
Acetoxymethyl Ester ◽  
Protein Tyrosine ◽  
Ionic Conductances ◽  
Cl Channels ◽  
Volume Decrease

Human Intestine 407 cells respond to hyposmotic stimulation by activating the conductive efflux of both Cl- and K+ (regulatory volume decrease) through pathways involving protein tyrosine phosphorylation (Tilly, B. C., N. van den Berghe, L. G. J. Tertoolen, M. J. Edixhoven, and H. R. de Jonge. J. Biol. Chem. 268: 19919-19922, 1993). Stimulation of the cells with hormones linked to the phospholipase C signaling cascade (e.g., bradykinin, histamine, or thrombin) or with the phosphotyrosine phosphatase inhibitor vanadate, potentiated the osmosensitive anion efflux by two- to threefold but did not affect anion efflux under isotonic conditions. No substantial increase in intracellular Ca2+ concentration ([Ca2+]i) was observed on mild hypotonicity-induced cell swelling. In addition, loading the cells with the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane- N,N,N',N',-tetraacetic acid acetoxymethyl ester (BAPTA-AM) caused a partial reduction of the osmoshock-induced 125I- efflux but did not affect its potentiation by vanadate. In contrast, bradykinin transiently elevated [Ca2+]i, and its potentiation of the osmosensitive anion efflux was completely inhibited after BAPTA-AM loading. Both the Ca(2+)-mobilizing hormones as well as osmotic cell swelling rapidly triggered the phosphorylation of several proteins on tyrosine residues. However, the effects of the hormones, but not the effect of hypotonicity, on protein tyrosine phosphorylation was largely abolished in BAPTA-loaded cells. Taken together the results indicate a novel role for Ca(2+)-mobilizing hormones, although elevation of [Ca2+]i, in potentiating volume-sensitive ionic efflux even in cell types lacking the expression of Ca(2+)-activated Cl- channels in their plasma membrane.

scholarly journals Human trabecular meshwork cell volume regulation

2002 ◽  
Vol 283 (1) ◽  
pp. C315-C326 ◽  
Author(s):  
Claire H. Mitchell ◽  
Johannes C. Fleischhauer ◽  
W. Daniel Stamer ◽  
K. Peterson-Yantorno ◽  
Mortimer M. Civan
Keyword(s):  
Cell Volume ◽  
Trabecular Meshwork ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Channel Blockers ◽  
Uptake Mechanism ◽  
Fluid Uptake ◽  
Intracellular Ca ◽  
Predominant Effect ◽  
Volume Decrease

The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl−/HCO[Formula: see text]exchange, and K+-Cl− efflux mechanisms have on the volume of TM cells. Volume, Cl− currents, and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+channel blockers Ba2+ and tetraethylammonium, and the K+-Cl− symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+ exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl− symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl− channel displaying the permeability ranking Cl− > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl− channels, Na+/H+ antiports, and possibly K+-Cl− symports in addition to Na+-K+-2Cl− symports.

Dihydropyridine-sensitive cell volume regulation in proximal tubule: the calcium window

1990 ◽  
Vol 259 (6) ◽  
pp. F950-F960 ◽  
Author(s):  
N. A. McCarty ◽  
R. G. O'Neil
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Ca Channel ◽  
Hypotonic Solution ◽  
Dependent Manner ◽  
Intracellular Ca

The mechanism underlying the activation of hypotonic cell volume regulation was studied in rabbit proximal straight tubule (PST). When isolated non-perfused tubules were exposed to hypotonic solution, cells swelled rapidly and then underwent a regulatory volume decrease (RVD). The extent of regulation after swelling was highly dependent on extracellular Ca concentration ([Ca2+]o), with a half-maximal inhibition (K1/2) for [Ca2+]o of approximately 100 microM. RVD was blocked by the Ca-channel blockers verapamil, lanthanum, and the dihydropyridines (DHP) nifedipine and nitrendipine, implicating voltage-activated Ca channels in the RVD response. Using the fura-2 fluorescence-ratio technique, we observed that cell swelling caused a sustained rise in intracellular Ca ([Ca2+]i) only when [Ca2+]o was normal (1 mM) but not when [Ca2+]o was low (1-10 microM). Furthermore, external Ca was required early on during swelling to induce RVD. If RVD was initially blocked by reducing [Ca2+]o or by addition of verapamil during hypotonic swelling, volume regulation could only be restored by subsequently inducing Ca entry within the first 1 min or less of exposure to hypotonic solution. These data indicate a "calcium window" of less than 1 min, during which RVD is sensitive to Ca, and that part of the Ca-dependent mechanism responsible for achieving RVD undergoes inactivation after swelling. It is concluded that RVD in rabbit PST is modulated by Ca via a DHP-sensitive mechanism in a time-dependent manner.

Volume-activated K+ and Cl- pathways of dissociated epithelial cells (MDCK): role of Ca2+

1990 ◽  
Vol 258 (5) ◽  
pp. C827-C834 ◽  
Author(s):  
A. Rothstein ◽  
E. Mack
Keyword(s):  
Mdck Cells ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Ionophore A23187 ◽  
Madin Darby Canine Kidney ◽  
Osmotic Swelling ◽  
Volume Activation ◽  
Darby Canine Kidney ◽  
Volume Decrease

Osmotic swelling of dissociated Madin-Darby canine kidney (MDCK) cells in NaCl medium is followed by shrinking (regulatory volume decrease, or RVD) or in KCl medium by secondary swelling. The cation ionophore gramicidin has little effect on volumes of isotonic cells but accelerates volume-activated changes in either medium. Immediately after hypotonic exposure, the membrane becomes transiently hyperpolarized followed by depolarization. The depolarization phase is diminished by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Swelling is also associated with an almost immediate increase in Ca2+ influx and elevation of cytoplasmic Ca2+ ([Ca2+]i) preceding RVD. In Ca2(+)-free medium, [Ca2+]i rapidly declines to a low level. Osmotic swelling, under these circumstances, is associated with a small transient increase in [Ca2+]i, but RVD or secondary swelling (in KCl) are minimal. Under these conditions, addition of gramicidin or the Ca2(+)-ionophore A23187 induces significant volume changes, although not as large as those found in the presence of Ca2+. Quinine inhibits RVD in the absence of gramicidin, but not in its presence; oligomycin C, DIDS, and trifluoperazine, on the other hand, inhibit in the presence of the ionophore. These findings suggest that in MDCK cells RVD involves activation of distinct conductive K+ and Cl- pathways which allow escape of KCl and osmotically obligated water and that activation of both pathways is associated with elevated [Ca2+]i derived largely from volume activation of a Ca2(+)-influx pathway.

Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport

1996 ◽  
Vol 270 (3) ◽  
pp. C866-C877 ◽  
Author(s):  
C. C. Armsby ◽  
A. K. Stuart-Tilley ◽  
S. L. Alper ◽  
C. Brugnara
Keyword(s):  
Normal Cell ◽  
Genetic Locus ◽  
Regulatory Volume Decrease ◽  
Osmotic Fragility ◽  
Cell Swelling ◽  
K Channel ◽  
Cell Dehydration ◽  
Osmotic Lysis ◽  
Volume Decrease ◽  
Characteristic Resistance

The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis.

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