IK channels are involved in the regulatory volume decrease in human epithelial cells

2003 ◽  
Vol 284 (1) ◽  
pp. C77-C84 ◽  
Author(s):  
Jun Wang ◽  
Shigeru Morishima ◽  
Yasunobu Okada
Keyword(s):  
Epithelial Cells ◽  
Regulatory Volume Decrease ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Blockers ◽  
Hypotonic Stress ◽  
Human Epithelial Cells ◽  
Ik Channel ◽  
Inside Out ◽  
Volume Decrease

Parallel activation of Ca2+-dependent K+ channels and volume-sensitive Cl− channels is known to be responsible for KCl efflux during regulatory volume decrease (RVD) in human epithelial Intestine 407 cells. The present study was performed to identify the K+ channel type. RT-PCR demonstrated mRNA expression of Ca2+-activated, intermediate conductance K+(IK), but not small conductance K+ (SK1) or large conductance K+ (BK) channels in this cell line. Whole cell recordings showed that ionomycin or hypotonic stress activated inwardly rectifying K+ currents that were reversibly blocked by IK channel blockers [clotrimazole (CLT) and charybdotoxin] but not by SK and BK channel blockers (apamin and iberiotoxin). Inside-out recordings revealed the existence of CLT-sensitive single K+-channel activity, which exhibited an intermediate unitary conductance (30 pS at −100 mV). The channel was activated by cytosolic Ca2+ in inside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin or iberiotoxin. Thus we conclude that the IK channel is involved in the RVD process in these human epithelial cells.

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.

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.

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.

scholarly journals Regulatory volume decrease in isolated nematocytes is affected by crude venom from the jellyfish Pelagia noctiluca

2014 ◽  
Vol 87 (2) ◽  
Author(s):  
Rossana Morabito ◽  
Silvia Dossena ◽  
Giuseppa La Spada ◽  
Angela Marino
Keyword(s):  
Cultured Cells ◽  
Cell Model ◽  
Regulatory Volume Decrease ◽  
Inhibitory Effect ◽  
Crude Venom ◽  
Trypan Blue Exclusion ◽  
Hypotonic Stress ◽  
Pelagia Noctiluca ◽  
Volume Decrease

Crude venom from nematocysts of the Scyphozoan <em>Pelagia noctiluca</em> possesses hemolytic and cytotoxic power on cultured cells and elicits local and systemic inflammation reactions <em>in vivo</em>. The ability of regulating their volume after exposure to an anisosmotic solution is a fundamental feature common to cells from vertebrates and invertebrates, including Cnidarians. The aim of the present work i s to assay whether crude venom from <em>Pelagia noctiluca</em> may affect the regulatory volume decrease (RVD) of nematocytes isolated from the Anthozoan <em>Aiptasia mutabilis</em>, here employed as a cell model. For this purpose, nematocytes were isolated by 605 mM NaSCN plus 0.01 mM Ca2+ application on acontia of <em>Aiptasia mutabilis</em>, while crude venom was obtained by sonication of a population of, respectively, 10, 25 and 50 nematocysts/µL (n/µL). Isolated nematocytes were pre-treated for 30 min with crude venom, submitted to hypotonic stress and their osmotic response and RVD were measured optically. Our results show that, after exposure to crude venom, nematocytes were morphologically intact, as shown by the Trypan blue exclusion test, but did not exhibit RVD. This effect was dose-dependent and reversed by the ionopho re gramicidin. The last observation suggests an inhibitory effect of venom on cell membrane ion transport mechanisms involved in RVD. Further studies are needed to verify this hypothesis and ascertain if a similar effect could be observed in human cells.

scholarly journals Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin

2009 ◽  
Vol 297 (1) ◽  
pp. G159-G167 ◽  
Author(s):  
M. D. Perry ◽  
G. I. Sandle
Keyword(s):  
Tyrosine Phosphatase ◽  
Distal Colon ◽  
Human Colon ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Secretory Diarrhea ◽  
Inside Out ◽  
Protein Phosphatase Type

High-conductance apical K+(BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K+losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+enrichment. We found that PKA activation using 50 μmol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 μmol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 μmol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 μmol/l Na+orthovanadate and 10 μmol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

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