Contribution of the IsK (MinK) potassium channel subunit to regulatory volume decrease in murine tracheal epithelial cells

The cell volume regulatory response following hypotonic shocks is often achieved by the coordinated activation of K(+) and Cl(-) channels. In this study, we investigate the identity of the K(+) and Cl(-) channels that mediate the regulatory volume decrease (RVD) in ciliated epithelial cells from murine trachea. RVD was inhibited by tamoxifen and 1,9-dideoxyforskolin, two agents that block swelling-activated Cl(-) channels. These data suggest that swelling-activated Cl(-) channels play an important role in cell volume regulation in murine tracheal epithelial cells. Ba(2+) and apamin, inhibitors of K(+) channels, were without effect on RVD, while tetraethylammoniun had little effect on RVD. In contrast, clofilium, an inhibitor of the KvLQT/IsK potassium channel complex potently inhibited RVD, suggesting a role for the KvLQT/IsK channel complex in cell volume regulation by tracheal epithelial cells. To investigate further the role of KvLQT/IsK channels in RVD, we used IsK knock-out mice. When exposed to hypotonic solutions, tracheal cells from IsK(+/+) mice underwent RVD, whereas cells from IsK(-/-) failed to recover their normal size. These data suggest that the IsK potassium subunit plays an important role in RVD in murine tracheal epithelial cells.     

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K⁺ channel inhibitors Ba²⁺, quinine, and tetraethylammonium chloride, implicating K⁺ channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K⁺ channel, and also a dominant-negative NH₂-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K⁺ current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K⁺ secretion. potassium channel; volume regulation; mammary gland     

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.     

Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line

The cell regulatory volume decrease (RVD) response triggered by hypotonic solutions is mainly achieved by the coordinated activity of Cl- and K+ channels. We now describe the molecular nature of the K(+) channels involved in the RVD response of the human bronchial epithelial (HBE) cell line 16HBE14o-. These cells, under isotonic conditions, present a K+ current consistent with the activity of maxi K+ channels, confirmed by RT-PCR and Western blot. Single-channel and whole cell maxi K+ currents were readily and reversibly activated following the exposure of HBE cells to a 28% hypotonic solution. Both maxi K+ current activation and RVD response showed calcium dependency, inhibition by TEA, Ba2+, iberiotoxin, and the cationic channel blocker Gd3+ but were insensitive to clofilium, clotrimazole, and apamin. The presence of the recently cloned swelling-activated, Gd3+-sensitive cation channels (TRPV4, also known as OTRPC4, TRP12, or VR-OAC) was detected by RT-PCR in HBE cells. This channel, TRPV4, which senses changes in volume, might provide the pathway for Ca2+ influx under hypotonic solutions and, consequently, for the activation of maxi K+ channels.     

Effects of Phospholemman Expression on Swelling-Activated Ion Currents and Volume Regulation in Embryonic Kidney Cells

Phospholemman (PLM) is a 72-amino-acid phosphoprotein that is a major substrate for cAMP-dependent protein kinase, protein kinase C, and NIMA kinase. In lipid bilayers, PLM forms ion channels selective for Cl-, K+, and taurine. Effluxes of these abundant intracellular osmolytes play an important role in the control of dynamic cell volume changes in many cell types. We measured swelling-activated ion currents and regulatory volume decrease (RVD) in human embryonic kidney cells stably overexpressing canine cardiac PLM. In response to swelling, two clonal cell lines overexpressing PLM had increased swelling-activated ion current densities and faster and more extensive RVD. A third clonal cell line overexpressing mutant PLM showed reduced ion current densities and a diminished RVD response. These results suggest a role for PLM in the regulation of cell volume, perhaps as a modulator of an endogenous swelling-activated signal transduction pathway or possibly by participating directly in swelling-induced osmolyte efflux.     

Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.     

Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons

The majority of mammalian cells demonstrate regulatory volume decrease (RVD) following swelling caused by hyposmotic exposure. A critical signal initiating RVD is activation of nucleotide receptors by ATP. Elevated extracellular ATP in response to cytotoxic cell swelling during pathological conditions also may initiate loss of taurine and other intracellular osmolytes via anion channels. This study characterizes neuronal ATP-activated anion current and explores its role in net loss of amino acid osmolytes. To isolate anion currents, we used CsCl as the major electrolyte in patch electrode and bath solutions and blocked residual cation currents with NiCl(2) and tetraethylammonium. Anion currents were activated by extracellular ATP with a K(m) of 70 microM and increased over fourfold during several minutes of ATP exposure, reaching a maximum after 9.0 min (SD 4.2). The currents were blocked by inhibitors of nucleotide receptors and volume-regulated anion channels (VRAC). Currents showed outward rectification and inactivation at highly depolarizing membrane potentials, characteristics of swelling-activated anion currents. P2X agonists failed to activate the anion current, and an inhibitor of P2X receptors did not block the effect of ATP. Furthermore, current activation was observed with extracellular ADP and 2-(methylthio)adenosine 5'-diphosphate, a P2Y(1) receptor-specific agonist. Much less current activation was observed with extracellular UTP, suggesting the response is mediated predominantly by P2Y(1) receptors. ATP caused a dose-dependent loss of taurine and alanine that could be blocked by inhibitors of VRAC. ATP did not inhibit the taurine uptake transporter. Thus extracellular ATP triggers a loss of intracellular organic osmolytes via activation of anion channels. This mechanism may facilitate neuronal volume homeostasis during cytotoxic edema.     

Characterization of regulatory volume decrease in freshly isolated mouse cholangiocytes

Cell volume regulation plays a vital role in many cell functions. Recent study indicates that both K(+) and Cl(-) channels are important for the regulatory volume decrease (RVD) of cholangiocarcinoma cells, but its physiological significance is unclear due to the tumorous nature of the cells used. This present study reports the RVD of normal mouse cholangiocytes by using freshly isolated bile duct cell clusters (BDCC). A relatively simple and practical method of measuring the cross-sectional area of BDCCs by quantitative videomicroscopy was used to indirectly measure their volumes. Mouse cholangiocytes exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, DIDS, and glibenclamide, suggesting its dependence on certain chloride channels, such as volume-activated chloride channels. It is also inhibited by barium chloride but not by tetraethylammonium chloride, indicating its dependence on certain potassium channels. However, cAMP agonists had no significant effect on the RVD of BDCCs. This indirect method described can be used to study the RVD of cholangiocytes from normal as well as genetically altered mouse livers.     

A novel function of BCL-2 overexpression in regulatory volume decrease. Enhancing swelling-activated Ca(2+) entry and Cl(-) channel activity

The cellular function of the oncogene bcl-2, a key regulator of apoptosis, is still debated. The goal of this study was to explore the relationship between BCL-2 overexpression and cell volume regulation by using two independent models, Madin-Darby canine kidney (MDCK) cells stably transfected with BCL-2 and MDCK clones with inducible BCL-2 expression by the lac operator/repressor. BCL-2 overexpression enhanced the capability of regulatory volume decrease (RVD), a cellular defensive process against hypotonic stress. In various clones of MDCK cells, hypotonic stress induced an outwardly rectified Cl(-) current that was significantly up-regulated by BCL-2 overexpression. Other fundamental characteristics of this channel were similar among different MDCK clones, such as sensitivity to Cl(-) channel inhibitor, anion permeability, and time-dependent inactivation at more positive potential. Most importantly, BCL-2 overexpression up-regulates the swelling-activated Ca(2+) transient that is a critical signaling for normal RVD and the activation of swelling-activated Cl(-) channel in MDCK cells. BCL-2 overexpression also enhances the capacitative Ca(2+) entry that can be differentiated from the swelling-activated Ca(2+) transient by kinetic analysis and sensitivity to Gd(3+). Moreover, neutralization of endogenous BCL-2 by antibody blocks the normal RVD response and the activation of swelling-activated Cl(-) channel in human cervical cancer HT-3 cells. These results provide a new insight into the novel function of BCL-2 overexpression in the regulation of cell volume and ion flux.     

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.     

Regulation of volume-sensitive Cl- channels in multi-drug resistant MCF7 cells

The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR-), resistant (MDR+), and reversed resistant (MDR(REV)) phenotypes. Western blot analysis showed that incubation of cells with doxorubicin induced P-gp expression in a reversible manner. Exposure of MDR+ cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents (I(Cl-swell)) in MDR-, MDR+, and MDR(REV) MCF7 cells. While verapamil inhibited I(Cl-swell) in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I(Cl-swell) uniquely in MDR+. Intracellular dialysis of MDR+ cells with C219 anti-P-gp antibody abolished the sensitivity of I(Cl-swell) to doxorubicin and led to a response pattern very close to that of MDR- cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I(Cl-swell) in resistant MCF7.     

Calcium and cytoskeleton signaling during cell volume regulation in isolated nematocytes of Aiptasia mutabilis (Cnidaria: Anthozoa)

Cell volume regulation has not been completely clarified in Coelenterates. The present investigation focuses on cell volume regulation under anisosmotic conditions, both hyposmotic and hypertonic, and on the underlying signals in nematocytes isolated from the Coelenterate Aiptasia mutabilis living in sea water. Nematocytes, once isolated from acontia, that were submitted to either hyposmotic (35%) and hypertonic shock (45%) show RVD and RVI capabilities, respectively. In order to ascertain the role of Ca2+ in triggering such regulatory mechanisms and the possible involvement of cytoskeleton components, tests were performed by employing either Ca2+ free conditions, Gd3+ as Ca2+ channel blockers, TFP as calmodulin inhibitor, colchicine as microtubule inhibitor and cytochalasin B as microfilament polymerization inhibitor. Results show that isolated nematocytes of A. mutabilis can regulate their volume upon both hyposmotic and hypertonic challenge. Ca2+ both from external medium and from internal stores is needed to perform RVD mechanisms, whereas, intracellular Ca2+ seems to be mainly involved in RVI. Moreover cytoskeletal components may play an important role since a significant RVD and RVI inhibition was observed in treated cells. On the basis of our observations further studies are warranted to further verify the role of signals, including phosphatases and phosphorylases, in cell volume regulation of primitive eukaryotic cells.     

Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells

Volume regulation is essential for cell function, but it is unknown which channels are involved in a regulatory volume decrease (RVD) in human gastric epithelial cells. Exposure to a hypotonic solution caused the increase in AGS cell volume, followed by the activation of a current. The reversal potential of the swelling-induced current suggested that Cl- was the primary charge carrier. The selectivity sequence for different anions was I- > Br- > Cl- > F- > gluconate. This current was inhibited by flufenamate, DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)benzoate. Intracellular dialysis of three different anti-ClC-3 antibodies abolished or attenuated the Cl- current and disrupted RVD, whereas the current and RVD was unaltered by anti-ClC-2 antibody. Immunoblot studies demonstrated the presence of ClC-3 protein in Hela and AGS cells. RT-PCR analysis detected expression of ClC-3, MDR-1, and pICln mRNA in AGS cells. These results suggest a fundamental role of endogenous ClC-3 in the swelling-activated Cl- channels function and cell volume regulation in human gastric epithelial cells.     

Modulation of KCNQ4 channel activity by changes in cell volume

KCNQ4 channels expressed in HEK 293 cells are sensitive to cell volume changes, being activated by swelling and inhibited by shrinkage, respectively. The KCNQ4 channels contribute significantly to the regulatory volume decrease (RVD) process following cell swelling. Under isoosmotic conditions, the KCNQ4 channel activity is modulated by protein kinases A and C, G protein activation, and a reduction in the intracellular Ca2+ concentration, but these signalling pathways are not responsible for the increased channel activity during cell swelling.     

Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress

Membrane transport changes in human lens epithelial (HLE‐B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K_i, uptake of the K congener rubidium, Rb_i, and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein‐kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2‐fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% K_i loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K_i, and accompanying water, and Rb_i uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 µM STP exposure, the cells lost ～40% water and ～60% K_i, respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K_i loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4‐aminopyridine (4‐AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE‐B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro‐apoptotic STP‐activation of 4‐AP‐sensitive voltage‐gated K channels preceded or accompanied PS externalization before subsequent apoptosis. J. Cell. Physiol. 223: 110–122, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of hyposmotic stress on exocytosis in isolated turbot, Scophthalmus maximus, hepatocytes

The effect of hyposmotic shock on exocytosis was examined in isolated hepatocytes of turbot, a marine flatfish, using the molecular probe FM1-43. Sudden exposure to a reduced osmolality caused an increase in cell exocytic activity related to the osmotic gradient between intra- and extracellular fluids. Cytoskeletal microtubules could contribute to this hyposmotic-induced exocytosis since colchicine inhibited the process. Protein kinase C, phosphatidylinositol-3 kinase, phospholipases A2, C and D could constitute key enzymes in the mechanism since their inhibition by specific agents altered the hyposmotic-induced exocytic activity. Moreover, arachidonic acid and derivates from the 5-lipoxygenase pathway as well as calcium could participate in the process. As regulatory volume decrease (RVD) exhibited by turbot hepatocytes following hyposmotic stimulation involves similar features, a potential role of exocytosis in volume regulation is suggested. In particular, exocytosis could serve RVD by contributing to ATP release since this latter process similarly appeared to be phospholipase D-dependent and related to the osmotic gradient. This study provides the first evidence of a volume-sensitive exocytosis that could aim at volume constancy in a marine teleost fish cell type.     

Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells.

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.     

The role of anion channels and Ca2+ in addition to K+ channels in the physiological volume regulation of murine spermatozoa

Studies in the human, transgenic mice, and cattle indicate that sperm cell volume regulation plays an important role in male fertility as spermatozoa encounter a hypo-osmotic challenge upon ejaculation into the female tract. Physiological regulatory volume decrease (RVD) was examined using flow cytometry in murine sperm released into incubation medium mimicking uterine osmolality and including putative channel inhibitors. The involvement of K+ channels was indicated by the recovery of volume regulation by the K+ ionophore valinomycin in defective sperm from infertile transgenic mice, and from blockage of RVD by quinine in normal sperm. However, in neither case was the recovery complete. The involvement of volume-sensitive osmolyte and anion channels (VSOAC) were investigated using blockers effective in other cell types. NPPB (5-nitro-2(3-phenylpropylamino) benzoic acid) and tamoxifen inhibited RVD but SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) at 0.4 and 1 mM had no effect whereas DIDS (di-isothiocyanato-stilbene-2,2'-disulphonic acid) at 1 mM enhanced RVD. Verapamil, but not another P-glycoprotein antagonist cyclosporin, caused sperm swelling which persisted in the presence of valinomycin, in Ca2+-free medium and in the presence of thapsigargin, but swelling was abolished by the Ca2+ ionophore A23187. Nifedipine was slightly effective in blocking RVD. Analysis by Western blotting failed to reveal ClC-2 and ClC-3 members of the chloride channel family in murine or rat sperm proteins despite signal bands in positive tissue controls. These findings implicate the involvement of some unidentified VSOAC in sperm volume regulation, which is probably Ca+-dependent.     

Swelling-activated K+ efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.     

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell linesn

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intra-cellular calcium to be activated. © 1994 wiley-Liss, Inc.