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I H Lambert

Publications and source records attributed to I H Lambert.

At least 19 recordsLinked to original sources
PubMed1997

Cell swelling activates phospholipase A2 in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells, loaded with 3H-labeled arachidonic acid and 14C-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 3H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of 14C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A2 is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in 14C-labeled stearic acid release nor in the synthesis of phosphatidyl 14C-butanol in the presence of 14C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of 14C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A1, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of 3H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP beta S or with AACOCF3, an inhibitor of the 85 kDa, cytosolic phospholipase A2. Based on these results we propose that cell swelling activates a phospholipase A2--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.

Arachidonic Acid↗
PubMed1997

On the role of calcium in the regulatory volume decrease (RVD) response in Ehrlich mouse ascites tumor cells.

The putative role for Ca2+ entry and Ca2+ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P3 content, as measured in extracts from [2-3H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca2+-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca2+-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca2+ (1% only at 0.1-mM Ca2+ and in Ca2+-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca2+ upon the volume-induced K+ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca2+ chelator BAPTA when the BAPTA-loading is performed in K+ equilibrium medium; (vi) an inhibition of the swelling-induced K+ loss (about 50%) at 1-mM Ba2+, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 microM), reported inhibitors of the K+ loss induced by Ca2+-mobilizing agonists. Thus, Ca2+signaling by Ca2+ release or Ca2+ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells.

Animals↗
PubMed1997

Leukotriene D4-induced Ca2+ mobilization in Ehrlich ascites tumor cells.

Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1-100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+]i. The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+]i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD4 (100 nm), the highest measured increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-containing medium is 260 +/- 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nM. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+]i. Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+]i in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4, 5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4, 5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+]i, indicating that a U73122-sensitivity phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores.

Animals↗
PubMed1996

Phosphorylation is involved in the regulation of the taurine influx via the beta-system in Ehrlich ascites tumor cells.

The role of 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the beta-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the beta-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the beta-system is stimulated by an increased level of cAMP or following addition of N6,2'-O-dibutyryl-3',5'-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the beta-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the beta-system is in State II.

Animals↗
PubMed1996

Role of LTD4 in the regulatory volume decrease response in Ehrlich ascites tumor cells.

Stimulation with leukotriene D4 (LTD4) (3-100 nM) induces a transient increase in the free intracellular Ca2+ concentration ([Ca2+]i) in Ehrlich ascites tumor cells. The LTD4-induced increase in [Ca2+]i is, however, significantly reduced in Ca2+-free medium (2 mM EGTA), and under these conditions stimulation with a low LTD4 concentration (3 nM) does not result in any detectable increase in [Ca2+]i. Addition of LTD4 (3-100 nM) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD4-induced (100 nM) acceleration of the RVD response is also seen in Ca2+-free medium and also at 3 nM LTD4, indicating that LTD4 can open K+- and Cl--channels without any detectable increase in [Ca2+]i. Buffering cellular Ca2+ with BAPTA almost completely blocks the LTD4-induced (100 nM) acceleration of the RVD response. Thus, the reduced [Ca2+]i level after BAPTA-loading or buffering of [Ca2+]i seems to inhibit the LTD4-induced stimulation of the RVD response even though the LTD4-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca2+]i. The LTD4 receptor antagonist L649, 923 (1 microM) completely blocks the LTD4-induced increase in [Ca2+]i and inhibits the RVD response as well as the LTD4-induced acceleration of the RVD response. When the LTD4 receptor is desensitized by preincubation with 100 nM LTD4, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD4 plays a role in the activation of the RVD response. LTD4 seems to activate K+ and Cl- channels via stimulation of a LTD4 receptor with no need for a detectable increase in [Ca2+]i.

Animals↗
PubMed1994

Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells.

The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl- but inhibited by addition of MK196 (anion channel blocker) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl-, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a "Mini Cl- channel." The Cl- efflux via this Cl- channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS and inhibited by arachidonic acid and oleic acid. It is suggested that the swelling-activated "Mini Cl- channel" and the swelling-activated taurine channel in the Ehrlich cell represent two distinct types of channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

On the similarity between the small Cl- channel and the taurine channel activated after cell swelling in Ehrlich ascites tumor cells.

The swelling-activated Cl- efflux, mediated by the mini Cl- channel (3-7 pS), and the swelling-activated taurine efflux in Ehrlich cells are both inhibited by the anion channel blocker MK196 (indacrinone). The swelling-activated taurine efflux is also inhibited by DIDS and following depolarization of the cell membrane, but it is significantly stimulated by arachidonic acid in the micromolar range. The swelling-activated Cl- efflux is, in contrast, only slightly affected by DIDS, unaffected by the membrane potential, and completely blocked by arachidonic acid in the micromolar range. It is suggested that the swelling-activated mini Cl- channel and the taurine channel in the Ehrlich cells represent two distinct types of channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl- efflux.

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, 36Cl- efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl- channels. This activation of Cl- channels depends on extracellular as well as on intracellular Ca2+. The swelling-induced Cl- efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl-/HCO3- exchange because it is independent of the external Cl- concentration.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Relation between cytoskeleton, hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells.

Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca2+ or Ca(2+)-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca2+ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K+ ionophore valinomycin.

Actin Cytoskeleton↗
PubMed1993

Regulation of taurine transport in Ehrlich ascites tumor cells.

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,1C1,1taurine 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 leukotriene 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-lipoxygenase 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-lipoxygenase 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 Cl- channels, i.e., via a depolarization of the cell membrane.

Animals↗
PubMed1989

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells.

The nature of the leukotriene-D4 (LTD4) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD4 treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD4 also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO3. On the other hand, LTD4 fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD4 in Ehrlich cells induces Cl-independent K loss through the Ca2+-dependent K channels. However, the effect of physiological doses of LTD4 on cell volume seems not to be as potent in Cl-free, NO3 cells when compared to Cl-containing cells, indicating that LTD4 in Ehrlich cells also provokes Cl-dependent K loss. LTD4 seems not to produce K loss through an electroneutral K+/H+ exchange system. LTD4 still produces Cl-independent K loss and cell shrinkage in the presence of the anti-calmodulin drug pimozide but not in the presence of the LTD4 receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD4-induced shrinkage. It is suggested that the LTD4-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD4 might exert its effect through another lipoxygenase product. The Ca2+-calmodulin complex is not involved in the LTD4-induced activation of K and Cl transporting systems.

Animals↗
PubMed1989

Membrane potential, anion and cation conductances in Ehrlich ascites tumor cells.

The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 microS/cm2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Mercuric chloride activates latent, anion-dependent cation transport systems in the plasma membrane of Ehrlich ascites tumour cells.

Electronic cell sizing of Ehrlich ascites tumour cells is presented as a biological test system for assessment of membrane associated effects of toxic compounds. Ehrlich ascites tumour cells readjust their cell volume after osmotic swelling in hypotonic media. This regulatory process (Regulatory Volume Decrease, RVD) involves a net loss of KCl from the cells. Addition of HgCl2 (1 microM) results in a Cl- -dependent acceleration of RVD in hypotonic medium. Cells in isotonic Cl- -containing medium shrink upon addition of HgCl2 due to a Cl- -dependent net loss of K+. In addition, a Cl- -dependent net uptake of Na+ was also seen in the presence of HgCl2. It is concluded that HgCl2 activates a latent K+, Cl- cotransport as well as Na+, Cl- cotransport in Ehrlich cells.

Animals↗
PubMed1988

Na+/H+ exchange in Ehrlich ascites tumor cells: activation by cytoplasmic acidification and by treatment with cupric sulphate.

Exposure of Ehrlich cells to isotonic Na+-propionate medium induces a rapid cell swelling. This treatment is likely to impose an acid load on the cells. Cell swelling is absent in K+-propionate medium but may be induced by the ionophore nigericin, which mediates K+/H+ exchange. Cell swelling in Na+-propionate medium is blocked by amiloride, but an alternative pathway is introduced by addition of the ionophore monensin, which mediates Na+/H+ exchange. Consequently, swelling of Ehrlich cells in Na+-propionate medium is due to the operation of an amiloride-sensitive, Na+-specific mechanism. It is concluded that this mechanism is a Na+/H+ exchange system, activated by cytoplasmic acidification. We have previously demonstrated that the heavy metal salt CuSO4 in micromolar concentrations inhibits regulatory volume decrease (RVD) of Ehrlich cells following hypotonic swelling. The present work shows that CuSO4 inhibits RVD as a result of a net uptake of sodium, of which the major part is sensitive to amiloride. Measurements of intracellular pH show that CuSO4 causes significant cytoplasmic alkalinization, which is abolished by amiloride. Concomitantly, CuSO4 causes an amiloride-sensitive net proton efflux from the cells. The combined results confirm that a Na+/H+ exchange system exists in Ehrlich cells and demonstrate that the heavy metal salt CuSO4 activates this Na+/H+ exchange system.

Amiloride↗
PubMed1987

Effect of arachidonic acid, fatty acids, prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells.

Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE2 inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE2-induced Na uptake. PGE2 has no effect on the volume-activated K and Cl permeabilities. LTB4, LTC4 and LTE4 inhibit RVD insignificantly in hypotonically swollen cells. LTD4, moreover, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD4 even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.

Animals↗
PubMed1987

Role of prostaglandins and leukotrienes in volume regulation by Ehrlich ascites tumor cells.

PGE2 and LTC4 syntheses in Ehrlich ascites cells were measured by radioimmunoassay. Hypotonic swelling results in stimulation of the leukotriene synthesis and a concomitant reduction in the prostaglandin synthesis. If the cells have access to sufficient arachidonic acid there is a parallel increase in the synthesis of both leukotrienes and prostaglandins following hypotonic exposure. PGE2 significantly inhibits regulatory volume decrease (RVD) following hypotonic swelling in Na-containing medium but not in Na-free media, supporting the hypothesis that the effect of PGE2 is on the Na permeability. PGE2 also had no effect on RVD in Na-free media in the presence of the cation ionophore gramicidin. Since the Cl permeability becomes rate limiting for RVD in the presence of gramicidin, whereas the K permeability is rate limiting in its absence, it is concluded that PGE2 neither affects Cl nor K permeability. Addition of LTD4 accelerates RVD and since the K permeability is rate limiting for RVD this shows that LTD4 stimulates the K permeability. Inhibition of the leukotriene synthesis by nordihydroguaiaretic acid inhibits RVD even when a high K conductance has been ensured by the presence of gramicidin. It is, therefore, proposed that an increase in leukotriene synthesis after hypotonic swelling is involved also in the activation of the Cl transport pathway.

Animals↗
PubMed1986

Separate, Ca2+-activated K+ and Cl- transport pathways in Ehrlich ascites tumor cells.

The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl- transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl- transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl- conductance is increased 14- and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl- conductance is rate limiting. An A23187-induced activation of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: inhibited by quinine which blocks the Ca2+-activated K+ channel, unaffected by substitution of NO-3 or SCN- for Cl-, and inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl- conductance. The Cl- conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl- transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl- transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl- transport pathway. The activated anion transport pathway is unselective, carrying both Cl-, Br-, NO-3, and SCN-. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl- transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN-) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from 42K flux measurements at high external K+, and from net K+ flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Animals↗