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.

Regulatory volume decrease in cultured astrocytes. II. Permeability pathway to amino acids and polyols

1994 ◽  
Vol 266 (1) ◽  
pp. C172-C178 ◽  
Author(s):  
H. Pasantes-Morales ◽  
R. A. Murray ◽  
R. Sanchez-Olea ◽  
J. Moran
Keyword(s):  
Amino Acids ◽  
Regulatory Volume Decrease ◽  
Anion Channel ◽  
Inhibitory Effect ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Gamma Aminobutyric Acid ◽  
Extracellular Concentration ◽  
Cultured Astrocytes ◽  
Volume Decrease

The permeability of the hyposmolarity-activated pathway to amino acids and polyols in cultured astrocytes was examined following the change in rate and direction of regulatory volume decrease (RVD) when the extracellular concentration of the osmolytes was increased to reverse their intracellular-extracellular concentration gradient. Activation of the pathway by swelling would allow those permeable osmolytes to enter the cell and inhibit RVD. The pathway was found to be permeable to neutral amino acids, with beta-amino acids (beta-alanine = taurine > gamma-aminobutyric acid) more permeable than alpha-amino acids. Glycine, alanine, threonine, phenylalanine, and asparagine, but not glutamine, were permeable through this pathway. Aspartate was more permeable than glutamate, and K+ and not Na+ must be the accompanying cation. Basic amino acids were excluded. The dimension of the amino acid pore activated by hyposmolarity seems to be at the limit of glutamate-glutamine size. Influx rather than efflux of amino acids was observed when extracellular concentration was greater than intracellular concentration, with differences in the amount accumulated by cells correlating with their efficiency as RVD blockers. Influx of taurine (as representative of permeable amino acids) was inhibited by the Cl- channel blockers/exchangers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (40%) and dipyridamole (85%) , and it is suggested that amino acids permeate through an anion channel. Sorbitol and mannitol, but not inositol, exhibited a small inhibitory effect on the later phase of RVD, whereas inositol slightly accelerated RVD.

Separate K+ and Cl- transport pathways are activated for regulatory volume decrease in jejunal villus cells

1991 ◽  
Vol 260 (3) ◽  
pp. G405-G415 ◽  
Author(s):  
R. J. MacLeod ◽  
J. R. Hamilton
Keyword(s):  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Transport Pathways ◽  
Kg Medium ◽  
Cell Sizing ◽  
Rate Limiting ◽  
Li Substitution ◽  
Hypotonic Swelling ◽  
Volume Decrease

We assessed ion transport mechanisms operative during regulatory volume decrease (RVD) in jejunal villus enterocytes, isolated in suspension from guinea pig jejunum and examined with electronic cell sizing. Immediately after reduction of osmolarity (153 mosmol/kg medium) enterocytes swelled, but within 5 min they shrank by 50%. This RVD, which was complete by 20 min, was unaffected by Li+ substitution for Na+ or by Na(+)-free (N-methyl-D-glucose, NMDG+) medium. Passive loss of K+ is required for RVD because both the magnitude and direction of RVD changed when external [K+] varied. Increasing K+ permeability with gramicidin (0.5 microM) accelerated RVD in NMDG+ medium (10.0 +/- 0.8 vs. 6.2 +/- 0.4% min-1, P less than 0.01) suggesting that K+ loss is rate limiting for RVD. Inhibition of K(+)- and Ca2(+)-activated K+ conductance with Ba2+ (5 mM, P less than 0.005), quinine (100 microM, P less than 0.005), or apamin (1 microM, P less than 0.005) prevented RVD. Inhibition of Cl- conductance with 9-anthracenecarboxylic acid (100 microM, P less than 0.005) or dipyridamole (75 microM, P less than 0.005) also prevented RVD. In isotonic HCO3(-)-buffered medium, the addition of gramicidin to cells generated conditions in which anion permeability was rate limiting for cell swelling. This swelling was inhibited 97% by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In K(+)-free, HCO3(-)-buffered medium containing DIDS and gramicidin hypotonic swelling resulted in continued (secondary) swelling (rel vol 1.19 +/- 0.01 vs. 1.25 +/- 0.02, P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Human Platelets Exposed to Mechanical Stresses Express a Potent Lipoxygenase Product

1992 ◽  
Vol 68 (05) ◽  
pp. 589-594 ◽  
Author(s):  
Alon Margalit ◽  
Avinoam A Livne
Keyword(s):  
Regulatory Volume Decrease ◽  
Human Platelets ◽  
Mechanical Stresses ◽  
Arterial Stenosis ◽  
Platelet Suspension ◽  
Suspension Flows ◽  
Lipoxygenase Product ◽  
Pathological Conditions ◽  
K Permeability ◽  
Volume Decrease

SummaryHuman platelets exposed to hypotonicity undergo regulatory volume decrease (RVD), controlled by a potent, yet labile, lipoxygenase product (LP). LP is synthesized and excreted during RVD affecting selectively K+ permeability. LP is assayed by its capacity to reconstitute RVD when lipoxygenase is blocked. Centrifugation for preparing washed platelets (1,550 × g, 10 min) is sufficient to express LP activity, with declining potency in repeated centrifugations, indicating that it is not readily replenish-able. When platelet suspension flows in a vinyl tubing (1 mm i.d.), at physiological velocity, controlled at 90–254 cm/s, LP formation increases as a function of velocity but declines as result of increasing the tubing length. Stirring the platelets in an aggregometer cuvette for 30 s, yields no LP unless the stirring is intermittent. No associated platelet lysis or aggregation are observed following the mechanical stress applications. These results demonstrate that although mechanical stresses result in LP production, the mode of its application plays a major role. These results may indicate that LP is synthesized under pathological conditions and could be of relevance to platelets behavior related to arterial stenosis.

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.

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.

Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. C820-C832 ◽  
Author(s):  
Peter K. Lauf ◽  
Sandeep Misri ◽  
Ameet A. Chimote ◽  
Norma C. Adragna
Keyword(s):  
Epithelial Cells ◽  
Regulatory Volume Decrease ◽  
Anion Channel ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
K Channel ◽  
Channel Blockers ◽  
Rt Pcr ◽  
Cell Water ◽  
Human Lens Epithelial Cells

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain ± bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media ∼90% of the total Rb influx occurred through the Na-K pump and NKCC and ∼10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased Kc by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 ∼25 μM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.

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.

Regulatory volume decrease in HL-60 cells: importance of rapid changes in permeability of Cl- and organic solutes

1994 ◽  
Vol 267 (4) ◽  
pp. C1045-C1056 ◽  
Author(s):  
K. R. Hallows ◽  
P. A. Knauf
Keyword(s):  
Regulatory Volume Decrease ◽  
Organic Osmolytes ◽  
Diffusion Kinetic ◽  
Nernst Potential ◽  
Ehrlich Ascites ◽  
Rapid Changes ◽  
36Cl Fluxes ◽  
Free Media ◽  
Rate Limiting ◽  
Volume Decrease

Results obtained through the use of inhibitors and isotope flux and equilibration techniques indicate that the regulatory volume decrease (RVD) response of human promyelocytic leukemic HL-60 cells occurs largely through the efflux of K+ and Cl- through separate conductive membrane pathways. These "channels" differ pharmacologically and in their modes of activation from those described in lymphocytes and Ehrlich ascites tumor cells. With use of measured 86Rb+ and 36Cl- fluxes, together with a diffusion kinetic model, the membrane potential (Em) and apparent K+ and Cl- permeabilities (PK and PCl) were estimated under various isotonic and hypotonic conditions. Under isotonic (300 mosM) conditions, Em is close to the Nernst potential for K+ and PCl is < 0.1 PK. Rapid and steeply graded increases in the measured Cl- efflux rate and calculated PCl occur with decreasing tonicity, with the largest increases at tonicities < 80% of isotonic. K+ efflux and the apparent PK increase only modestly with decreasing tonicity. At 50% tonicity, PCl rises to nearly 10 times PK, which should cause substantial membrane depolarization, with Em approaching the Nernst potential for Cl-. Gramicidin treatment markedly accelerates the rate of RVD and net 36Cl- efflux in hypotonic Na(+)-and Cl(-)-free media, providing further evidence that PK is rate limiting during RVD. K+ loss exceeds Cl- loss during RVD, and the total loss of K+ and Cl- is insufficient to account for the observed degree of volume recovery in 50% tonicity media, indicating that other (organic) osmolytes must take part in the HL-60 cell RVD response.

Control of intracellular pH during regulatory volume decrease in HL-60 cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1057-C1066 ◽  
Author(s):  
K. R. Hallows ◽  
D. Restrepo ◽  
P. A. Knauf
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Regulatory Volume Decrease ◽  
Transport Systems ◽  
Disulfonic Acid ◽  
Volume Dependence ◽  
Supportive Role ◽  
22Na Uptake ◽  
Volume Decrease ◽  
Stimulation Of

Intracellular pH (pHi) homeostasis was investigated in human promyelocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pHi is regulated and which transport systems are involved. Cells suspended in hypotonic (50-60% of isotonic) media undergo a small (< 0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pHi is not significantly different from the initial pHi in 20 mM HCO3- medium and is significantly higher in HCO3(-)-free medium. Experiments performed in media with or without 150 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate that the anion exchanger (AE) mediates a net Cl- influx, with compensating HCO3- efflux, during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pHi. We found that inhibition of Na+/H+ exchange (NHE) with 12.5 microM ethylisoproplamiloride (EIPA) causes pHi to fall significantly by the end of 30 min of RVD. As assessed by EIPA-sensitive 22Na+ uptake measurements, NHE, largely dormant under resting isotonic conditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pHi and cell volume to near-normal levels. Thus a shift in the normal pHi dependence and/or volume dependence of NHE activity must occur during RVD under hypotonic conditions. In contrast, H(+)-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [14C]lactate efflux during RVD.

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.

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