Pflügers Archiv - European Journal of Physiology, 2008
The expression of the H-ras oncogene increases the migratory activity of many cell types and ther... more The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild-type fibroblasts. This increase in VRAC sensitivity is accompanied by increased migratory activity of H-ras fibroblasts. Moreover, the high-affinity VRAC blocker NS3728 inhibits migration of H-ras fibroblasts dose-dependently by up to about 60%, whereas migration of wild-type fibroblasts is reduced by only about 35%. Consistent with higher VRAC activity in H-ras than in wild-type fibroblasts, more VRAC blocker is needed to achieve a comparable degree of inhibition of migration. We suggest that H-ras modulates the volume set point of VRAC and thus facilitates transient changes of cell volume required for faster cell migration.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2007
This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascit... more This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K + -and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na + -K + -2Cl − cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl − -secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na + /K + ATPase, NKCC1 and a K + channel on the basolateral side. Osmotic control of Cl − secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.
Several types of K + channels have been identified in epithelial cells. Among them high conductan... more Several types of K + channels have been identified in epithelial cells. Among them high conductance Ca 2+activated K + channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological and morphometric analysis on the intact tissue. BK Ca channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral) by increasing intracellular Ca 2+ concentration with the Ca 2+ ionophore ionomycin (1 µM). BK Ca channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BK Ca inhibitor) abolished the Regulatory Volume Decrease (RVD) of the intestinal cells after hypotonic swelling. In conclusion, our results demonstrated the molecular and functional expression of high conductance Ca 2+ -activated K + channels in eel intestine; the physiological role of these channels is mainly related to the RVD response of the epithelial cells following hypotonic swelling.
Pfl�gers Archiv - European Journal of Physiology, 2004
Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an oblig... more Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na + /H + -exchanger (NHE1) and Na + , K + ,2Cl − -cotransporter (NKCC1) to RVI. Hypertonic stress induced caspase-3 activity in WEHI cells and iMEFs, an effect potentiated by Hsp70 in WEHI cells but inhibited by Hsp70 in iMEFs. Osmotic shrinkage-induced PCD was associated with Hsp70-inhibitable cysteine cathepsin release in iMEFs and attenuated by caspase and cathepsin inhibitors in WEHI cells. Treatment with TNF-α or the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced the viability of WEHI cells further under isotonic and mildly, but not severely, hypertonic conditions. Thus, it is concluded that shrinkage-induced PCD involves both caspase-and cathepsin-dependent death mechanisms and is potently counteracted by Hsp70.
The ability to control cell volume is fundamental for proper cell function. This review highlight... more The ability to control cell volume is fundamental for proper cell function. This review highlights recent advances in the understanding of the complex sequences of events by which acute cell volume perturbation alters the activity of osmolyte transport proteins in cells from vertebrate organisms. After cell swelling, the main effectors in the process of regulatory volume decrease are swelling-activated K(+) and Cl(-) channels, a taurine efflux pathway, and KCl cotransport. After cell shrinkage, the main effectors in the process of regulatory volume increase are Na(+)/H(+) exchange, Na(+), K(+), 2Cl(-) cotransport, and in some cells, shrinkageactivated Na(+) channels. All of these proteins are regulated in a unique manner by cell volume perturbations. The molecular identity of most, although not all, of these transport pathways is now known. Among other important advances, this has lead to the identification of transporter binding partners such as protein kinases and phosphatases, cy...
The importance of the TRPV4 channel for human physiology has been highlighted in recent years wit... more The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure-function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5-6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants - F617L and Y621L - showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and a...
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 19, 2014
Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MD... more Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MDR can develop by numerous mechanisms including decreased drug uptake, increased drug efflux and the failure to undergo drug-induced apoptosis. Evasion of drug-induced apoptosis through modulation of ion transporters is the main focus of this paper and we demonstrate how pro-apoptotic ion channels are downregulated, while anti-apoptotic ion transporters are upregulated in MDR. We also discuss whether upregulation of ion transport proteins that are important for proliferation contribute to MDR. Finally, we discuss the possibility that the development of MDR involves sequential and localized upregulation of ion channels involved in proliferation and migration and a concomitant global and persistent downregulation of ion channels involved in apoptosis.
The review describes molecular and functional properties of the volume regulated anion channel an... more The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-dependent Cl(-) channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl(-) channels in various diseases.
American journal of physiology. Cell physiology, Jan 15, 2015
The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompl... more The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/5...
The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits... more The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.
Pflügers Archiv - European Journal of Physiology, 2008
The expression of the H-ras oncogene increases the migratory activity of many cell types and ther... more The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild-type fibroblasts. This increase in VRAC sensitivity is accompanied by increased migratory activity of H-ras fibroblasts. Moreover, the high-affinity VRAC blocker NS3728 inhibits migration of H-ras fibroblasts dose-dependently by up to about 60%, whereas migration of wild-type fibroblasts is reduced by only about 35%. Consistent with higher VRAC activity in H-ras than in wild-type fibroblasts, more VRAC blocker is needed to achieve a comparable degree of inhibition of migration. We suggest that H-ras modulates the volume set point of VRAC and thus facilitates transient changes of cell volume required for faster cell migration.
Pfl�gers Archiv European Journal of Physiology, 1998
Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in E... more Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2',7',-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca2+]i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na+/H+ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca2+]i. In contrast, a smaller, slower increase in [Ca2+]i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na+/H+ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca2+]i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na+/H+ exchanger. Thus, activation of the exchanger by the Ca2+-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH4Cl-induced intracellular alkalinization resulted in an increase in [Ca2+]i, apparently via stimulation of Ca2+ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca2+ influx. Thus, cell shrinkage appears to inhibit the Ca2+ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca2+ influx was inhibited by cell shrinkage.
Pfl�gers Archiv European Journal of Physiology, 1999
Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isol... more Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.
The Ehrlich ascites tumor cell has been used as a model of an unspecialized mammalian cell, in an... more The Ehrlich ascites tumor cell has been used as a model of an unspecialized mammalian cell, in an attempt to disclose the mechanisms involved in the regulation of cellular water and salt content. In hypotonic medium Ehrlich cells initially swell as nearly perfect osmometers, but subsequently recover their volume within about 10 min with an associated net loss of KCl, amino acids, taurine and cell water. The net loss of KCl takes place mainly via separate, conductive K+ and Cl- transport pathways, and the net loss of taurine through a passive leak pathway. Ca2+ and calmodulin appear to be involved in the activation of the K+ and Cl- channels, as well as the taurine leak pathway. In hypertonic medium Ehrlich cells initially shrink as osmometers, but subsequently recover their volume with an associated net uptake of KCl and water. In this case, the net uptake of KCl is the result of the activation of an electroneutral, Na+- and Cl- -dependent cotransport system with subsequent replacement of cellular Na+ by extracellular K+ via the Na+/K+ pump. In the present review we describe the ion and taurine transporting systems which have been identified in the plasma membrane of the Ehrlich ascites tumor cell. We have emphasized the selectivity of these transport pathways and their activation mechanisms. Finally, we propose a model for the activation of the conductive K+ and Cl- transport pathways in Ehrlich cells which includes Ca2+, leukotrienes, and inositol phosphate as intracellular second messengers.
Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich asci... more Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized, Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na,lCl,ltaurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the teukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A2 is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD4. The swelling-induced taurine efflux is also inhibited by addition of the 5-1ipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-1ipoxygenase pathway. LTD4 seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the C1 channels, i,e., via a depolarization of the cell membrane.
A Ca 2+ -activated (I Cl,Ca ) and a swellingactivated anion current (I Cl,vol ) were investigated... more A Ca 2+ -activated (I Cl,Ca ) and a swellingactivated anion current (I Cl,vol ) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca 2+ ] i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl − concentration. I Cl,Ca current density increased with increasing [Ca 2+ ] i , and this current was abolished by lowering [Ca 2+ ] i to <1 nM using 1,2-bis-(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetra-acetic acid (BAPTA). In contrast, activation of I Cl,vol did not require an increase in [Ca 2+ ] i . The kinetics of I Cl,Ca and I Cl,vol were different: at depolarized potentials, I Cl,Ca as activated in a [Ca 2+ ] i -and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I Cl,vol exhibited time-and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I Cl,vol was dependent on the extracellular Mg 2+ concentration. The anion permeability sequence for both currents was I − > Cl − > gluconate. I Cl,Ca was inhibited by niflumic acid (100 M), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 M) and 4,4Ј-diisothiocyano-2,2Ј-stilbenedisulfonic acid (DIDS, 100 M), niflumic acid being the most potent inhibitor. In contrast, I Cl,vol was unaffected by niflumic acid (100 M), but abolished by tamoxifen (10 M). Thus, in Ehrlich cells, separate chloride currents, I Cl,Ca and I Cl,vol , are activated by an increase in [Ca 2+ ] i and by cell swelling, respectively.
Ehrlich ascites tumor cells, loaded with 3 Hlabeled arachidonic acid and 14 C-labeled stearic aci... more Ehrlich ascites tumor cells, loaded with 3 Hlabeled arachidonic acid and 14 C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of 3 Hlabeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of 14 C-stearic acidlabeled lysophosphatidylcholine. This indicates that a phospholipase A 2 is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in 14 C-labeled stearic acid release nor in the synthesis of phosphatidyl 14 C-butanol in the presence of 14 C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of 14 C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A 1 , C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of 3 Hlabeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP  S or with AACOCF 3 , an inhibitor of the 85 kDa, cytosolic phospholipase A 2 . Based on these results we propose that cell swelling activates a phospholipase A 2 -perhaps the cytosolic 85 kDa type-by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.
Stimulation with leukotriene D 4 (LTD 4 ) (3-100 nM) induces a transient increase in the free int... more Stimulation with leukotriene D 4 (LTD 4 ) (3-100 nM) induces a transient increase in the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) in Ehrlich ascites tumor cells. The LTD 4 -induced increase in [Ca 2+ ] i is, however, significantly reduced in Ca 2+ -free medium (2 mM EGTA), and under these conditions stimulation with a low LTD 4 concentration (3 nM) does not result in any detectable increase in [Ca 2+ ] i . Addition of LTD 4 (3-100 nM) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD 4 -induced (100 nM) acceleration of the RVD response is also seen in Ca 2+free medium and also at 3 nM LTD 4 , indicating that LTD 4 can open K + -and Cl − -channels without any detectable increase in [Ca 2+ ] i . Buffering cellular Ca 2+ with BAPTA almost completely blocks the LTD 4 -induced (100 nM) acceleration of the RVD response. Thus, the reduced [Ca 2+ ] i level after BAPTA-loading or buffering of [Ca 2+ ] i seems to inhibit the LTD 4 -induced stimulation of the RVD response even though the LTD 4 -induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca 2+ ] i . The LTD 4 receptor antagonist L649,923 (1 M) completely blocks the LTD 4 -induced increase in [Ca 2+ ] i and inhibits the RVD response as well as the LTD 4 -induced acceleration of the RVD response. When the LTD 4 receptor is desensitized by preincubation with 100 nM LTD 4 , a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD 4 plays a role in the activation of the RVD response. LTD 4 seems to activate K + and Cl − channels via stimulation of a LTD 4 receptor with no need for a detectable increase in [Ca 2+ ] i .
Amiloride-sensitive, Na + -dependent, DIDSinsensitive cytoplasmic alkalinization is observed afte... more Amiloride-sensitive, Na + -dependent, DIDSinsensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2Ј,7Ј-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na + influx is also observed. This indicates that hypertonic challenge activates a Na + /H + exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH i , but the overall picture confirms the results from cell suspensions.
Pflügers Archiv - European Journal of Physiology, 2008
The expression of the H-ras oncogene increases the migratory activity of many cell types and ther... more The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild-type fibroblasts. This increase in VRAC sensitivity is accompanied by increased migratory activity of H-ras fibroblasts. Moreover, the high-affinity VRAC blocker NS3728 inhibits migration of H-ras fibroblasts dose-dependently by up to about 60%, whereas migration of wild-type fibroblasts is reduced by only about 35%. Consistent with higher VRAC activity in H-ras than in wild-type fibroblasts, more VRAC blocker is needed to achieve a comparable degree of inhibition of migration. We suggest that H-ras modulates the volume set point of VRAC and thus facilitates transient changes of cell volume required for faster cell migration.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2007
This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascit... more This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K + -and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na + -K + -2Cl − cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl − -secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na + /K + ATPase, NKCC1 and a K + channel on the basolateral side. Osmotic control of Cl − secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.
Several types of K + channels have been identified in epithelial cells. Among them high conductan... more Several types of K + channels have been identified in epithelial cells. Among them high conductance Ca 2+activated K + channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological and morphometric analysis on the intact tissue. BK Ca channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral) by increasing intracellular Ca 2+ concentration with the Ca 2+ ionophore ionomycin (1 µM). BK Ca channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BK Ca inhibitor) abolished the Regulatory Volume Decrease (RVD) of the intestinal cells after hypotonic swelling. In conclusion, our results demonstrated the molecular and functional expression of high conductance Ca 2+ -activated K + channels in eel intestine; the physiological role of these channels is mainly related to the RVD response of the epithelial cells following hypotonic swelling.
Pfl�gers Archiv - European Journal of Physiology, 2004
Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an oblig... more Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na + /H + -exchanger (NHE1) and Na + , K + ,2Cl − -cotransporter (NKCC1) to RVI. Hypertonic stress induced caspase-3 activity in WEHI cells and iMEFs, an effect potentiated by Hsp70 in WEHI cells but inhibited by Hsp70 in iMEFs. Osmotic shrinkage-induced PCD was associated with Hsp70-inhibitable cysteine cathepsin release in iMEFs and attenuated by caspase and cathepsin inhibitors in WEHI cells. Treatment with TNF-α or the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced the viability of WEHI cells further under isotonic and mildly, but not severely, hypertonic conditions. Thus, it is concluded that shrinkage-induced PCD involves both caspase-and cathepsin-dependent death mechanisms and is potently counteracted by Hsp70.
The ability to control cell volume is fundamental for proper cell function. This review highlight... more The ability to control cell volume is fundamental for proper cell function. This review highlights recent advances in the understanding of the complex sequences of events by which acute cell volume perturbation alters the activity of osmolyte transport proteins in cells from vertebrate organisms. After cell swelling, the main effectors in the process of regulatory volume decrease are swelling-activated K(+) and Cl(-) channels, a taurine efflux pathway, and KCl cotransport. After cell shrinkage, the main effectors in the process of regulatory volume increase are Na(+)/H(+) exchange, Na(+), K(+), 2Cl(-) cotransport, and in some cells, shrinkageactivated Na(+) channels. All of these proteins are regulated in a unique manner by cell volume perturbations. The molecular identity of most, although not all, of these transport pathways is now known. Among other important advances, this has lead to the identification of transporter binding partners such as protein kinases and phosphatases, cy...
The importance of the TRPV4 channel for human physiology has been highlighted in recent years wit... more The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure-function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5-6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants - F617L and Y621L - showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and a...
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 19, 2014
Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MD... more Multi-drug resistance (MDR) to chemotherapy is the major challenge in the treatment of cancer. MDR can develop by numerous mechanisms including decreased drug uptake, increased drug efflux and the failure to undergo drug-induced apoptosis. Evasion of drug-induced apoptosis through modulation of ion transporters is the main focus of this paper and we demonstrate how pro-apoptotic ion channels are downregulated, while anti-apoptotic ion transporters are upregulated in MDR. We also discuss whether upregulation of ion transport proteins that are important for proliferation contribute to MDR. Finally, we discuss the possibility that the development of MDR involves sequential and localized upregulation of ion channels involved in proliferation and migration and a concomitant global and persistent downregulation of ion channels involved in apoptosis.
The review describes molecular and functional properties of the volume regulated anion channel an... more The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-dependent Cl(-) channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl(-) channels in various diseases.
American journal of physiology. Cell physiology, Jan 15, 2015
The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompl... more The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/5...
The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits... more The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.
Pflügers Archiv - European Journal of Physiology, 2008
The expression of the H-ras oncogene increases the migratory activity of many cell types and ther... more The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild-type fibroblasts. This increase in VRAC sensitivity is accompanied by increased migratory activity of H-ras fibroblasts. Moreover, the high-affinity VRAC blocker NS3728 inhibits migration of H-ras fibroblasts dose-dependently by up to about 60%, whereas migration of wild-type fibroblasts is reduced by only about 35%. Consistent with higher VRAC activity in H-ras than in wild-type fibroblasts, more VRAC blocker is needed to achieve a comparable degree of inhibition of migration. We suggest that H-ras modulates the volume set point of VRAC and thus facilitates transient changes of cell volume required for faster cell migration.
Pfl�gers Archiv European Journal of Physiology, 1998
Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in E... more Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2&#39;,7&#39;,-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca2+]i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na+/H+ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca2+]i. In contrast, a smaller, slower increase in [Ca2+]i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na+/H+ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca2+]i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na+/H+ exchanger. Thus, activation of the exchanger by the Ca2+-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH4Cl-induced intracellular alkalinization resulted in an increase in [Ca2+]i, apparently via stimulation of Ca2+ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca2+ influx. Thus, cell shrinkage appears to inhibit the Ca2+ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca2+ influx was inhibited by cell shrinkage.
Pfl�gers Archiv European Journal of Physiology, 1999
Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isol... more Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.
The Ehrlich ascites tumor cell has been used as a model of an unspecialized mammalian cell, in an... more The Ehrlich ascites tumor cell has been used as a model of an unspecialized mammalian cell, in an attempt to disclose the mechanisms involved in the regulation of cellular water and salt content. In hypotonic medium Ehrlich cells initially swell as nearly perfect osmometers, but subsequently recover their volume within about 10 min with an associated net loss of KCl, amino acids, taurine and cell water. The net loss of KCl takes place mainly via separate, conductive K+ and Cl- transport pathways, and the net loss of taurine through a passive leak pathway. Ca2+ and calmodulin appear to be involved in the activation of the K+ and Cl- channels, as well as the taurine leak pathway. In hypertonic medium Ehrlich cells initially shrink as osmometers, but subsequently recover their volume with an associated net uptake of KCl and water. In this case, the net uptake of KCl is the result of the activation of an electroneutral, Na+- and Cl- -dependent cotransport system with subsequent replacement of cellular Na+ by extracellular K+ via the Na+/K+ pump. In the present review we describe the ion and taurine transporting systems which have been identified in the plasma membrane of the Ehrlich ascites tumor cell. We have emphasized the selectivity of these transport pathways and their activation mechanisms. Finally, we propose a model for the activation of the conductive K+ and Cl- transport pathways in Ehrlich cells which includes Ca2+, leukotrienes, and inositol phosphate as intracellular second messengers.
Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich asci... more Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized, Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na,lCl,ltaurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the teukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A2 is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD4. The swelling-induced taurine efflux is also inhibited by addition of the 5-1ipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-1ipoxygenase pathway. LTD4 seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the C1 channels, i,e., via a depolarization of the cell membrane.
A Ca 2+ -activated (I Cl,Ca ) and a swellingactivated anion current (I Cl,vol ) were investigated... more A Ca 2+ -activated (I Cl,Ca ) and a swellingactivated anion current (I Cl,vol ) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca 2+ ] i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl − concentration. I Cl,Ca current density increased with increasing [Ca 2+ ] i , and this current was abolished by lowering [Ca 2+ ] i to <1 nM using 1,2-bis-(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetra-acetic acid (BAPTA). In contrast, activation of I Cl,vol did not require an increase in [Ca 2+ ] i . The kinetics of I Cl,Ca and I Cl,vol were different: at depolarized potentials, I Cl,Ca as activated in a [Ca 2+ ] i -and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I Cl,vol exhibited time-and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I Cl,vol was dependent on the extracellular Mg 2+ concentration. The anion permeability sequence for both currents was I − > Cl − > gluconate. I Cl,Ca was inhibited by niflumic acid (100 M), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 M) and 4,4Ј-diisothiocyano-2,2Ј-stilbenedisulfonic acid (DIDS, 100 M), niflumic acid being the most potent inhibitor. In contrast, I Cl,vol was unaffected by niflumic acid (100 M), but abolished by tamoxifen (10 M). Thus, in Ehrlich cells, separate chloride currents, I Cl,Ca and I Cl,vol , are activated by an increase in [Ca 2+ ] i and by cell swelling, respectively.
Ehrlich ascites tumor cells, loaded with 3 Hlabeled arachidonic acid and 14 C-labeled stearic aci... more Ehrlich ascites tumor cells, loaded with 3 Hlabeled arachidonic acid and 14 C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of 3 Hlabeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of 14 C-stearic acidlabeled lysophosphatidylcholine. This indicates that a phospholipase A 2 is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in 14 C-labeled stearic acid release nor in the synthesis of phosphatidyl 14 C-butanol in the presence of 14 C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of 14 C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A 1 , C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of 3 Hlabeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP  S or with AACOCF 3 , an inhibitor of the 85 kDa, cytosolic phospholipase A 2 . Based on these results we propose that cell swelling activates a phospholipase A 2 -perhaps the cytosolic 85 kDa type-by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.
Stimulation with leukotriene D 4 (LTD 4 ) (3-100 nM) induces a transient increase in the free int... more Stimulation with leukotriene D 4 (LTD 4 ) (3-100 nM) induces a transient increase in the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) in Ehrlich ascites tumor cells. The LTD 4 -induced increase in [Ca 2+ ] i is, however, significantly reduced in Ca 2+ -free medium (2 mM EGTA), and under these conditions stimulation with a low LTD 4 concentration (3 nM) does not result in any detectable increase in [Ca 2+ ] i . Addition of LTD 4 (3-100 nM) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD 4 -induced (100 nM) acceleration of the RVD response is also seen in Ca 2+free medium and also at 3 nM LTD 4 , indicating that LTD 4 can open K + -and Cl − -channels without any detectable increase in [Ca 2+ ] i . Buffering cellular Ca 2+ with BAPTA almost completely blocks the LTD 4 -induced (100 nM) acceleration of the RVD response. Thus, the reduced [Ca 2+ ] i level after BAPTA-loading or buffering of [Ca 2+ ] i seems to inhibit the LTD 4 -induced stimulation of the RVD response even though the LTD 4 -induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca 2+ ] i . The LTD 4 receptor antagonist L649,923 (1 M) completely blocks the LTD 4 -induced increase in [Ca 2+ ] i and inhibits the RVD response as well as the LTD 4 -induced acceleration of the RVD response. When the LTD 4 receptor is desensitized by preincubation with 100 nM LTD 4 , a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD 4 plays a role in the activation of the RVD response. LTD 4 seems to activate K + and Cl − channels via stimulation of a LTD 4 receptor with no need for a detectable increase in [Ca 2+ ] i .
Amiloride-sensitive, Na + -dependent, DIDSinsensitive cytoplasmic alkalinization is observed afte... more Amiloride-sensitive, Na + -dependent, DIDSinsensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2Ј,7Ј-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na + influx is also observed. This indicates that hypertonic challenge activates a Na + /H + exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH i , but the overall picture confirms the results from cell suspensions.
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