Effects of P-glycoprotein expression on cyclic AMP and volume-activated ion fluxes and conductances in HT-29 colon adenocarcinoma cells

The tissue distribution of P-glycoprotein (Pgp) and the structurally related cystic fibrosis transmembrane conductance regulator (CFTR) is apparently mutually exclusive, particularly in epithelia; where one protein is expressed the other is not. To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT-29 colon adenocarcinoma cells, which constitutively express CFTR, were pharmacologically adapted to express the classical multidrug resistance (MDR) phenotype (Pgp⁺). Concomitant with the appearance of Pgp and MDR phenotype (drug resistance, reduced drug accumulation and increased drug efflux), CFTR levels and cAMP-stimulated Cl conductances were markedly decreased compared to wild-type HT-29 (Pgp^?) cells (as shown using the whole cell patch clamp technique). Removal of drug pressure led to the gradual decrease in Pgp levels and MDR phenotype, as evidenced by increased rhodamine 123 accumulation (Pgp-Rev). Concomitantly, CFTR levels and cAMP-stimulated Cl^? conductances incresed. The cell responses of Pgp/Rev cells were heterogeneous with respect to both Pgp and CFTR functions. We also studied the possible contribution of Pgp to hypotonically activated (HCS) ion conductances. K⁺ and Cl^? effluxes from Pgp^? cells were markedly increased by HCS. This increase was twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl^? channel blocker DIDS (4,4′-disothiocyano-2,2′-disulfonic stilbene) and required extracellular Ca²⁺. In Pgp⁺ cells, the HCS-induced fluxes were not significantly different from those of Pgp^? cells. Verapamil (10 μM), which caused 80% reversal of Pgp-associated drug extrusion, failed to inhibit the HCS-evoked Cl^? efflux of Pgp⁺ cells. Similarly, HCS increased Cl^? conductance to the same extent in Pgp^?, Pgp⁺ and Pgp-Rev cells. Verapamil (100 μM), but not 1,9-dideoxyforskolin (50 and 100 μM), partially inhibited the HCS-evoked whole cell current (WCC) in all three lines. Since the inhibition by verapamil was not detected in the presence of the K⁺ channel blocker Ba²⁺ (3 mM), it is suggested that verapamil affects K⁺ and not Cl^? conductance. We conclude that hypotonically activated Cl^? and K⁺ conductances are similar in HT-29 cells irrespective of Pgp expression. Expression of high levels of Pgp in HT-29 cells confers no physiologically significant capacity for cell volume regulation. © 1994 Wiley-Liss, Inc.     

The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells in vitro almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

Volume regulation by human lymphocytes: Characterization of the ionic basis for regulatory volume decrease

The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5–0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K⁺ decreased during RVD, while Na⁺ content remained unchanged. Similarly, the efflux of ⁸⁶Rb (used as a K⁺ analog) increased upon dilution, whereas ²²Na efflux was not altered. ⁸⁶Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na⁺- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K⁺-free media, and also in Na⁺-free media when Li⁺, choline⁺, or Tris⁺ were the substitutes. In high K⁺ or Rb⁺ hypotonic media shrinking was absent and a second swelling phase was observed. Cs⁺ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K⁺ concentration was varied and, with 50 mM K⁺, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K⁺, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na⁺ entry into the cell, but this activation is not essential for RVD.     

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD

Background

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids.

Methods

Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry.

Results

There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10^-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all).

Conclusions

Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.     

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Summary Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K⁺ (and Cl^–) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K⁺-free media a net influx of Na⁺ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K⁺ and triphenylmethyl phosphonium⁺, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time⁸⁶Rb⁺ fluxes were measured:⁸⁶Rb⁺ influx was not affected by hypotonicity, implying an unchanged operation of the Na⁺–K⁺-ATPase. On the other hand,⁸⁶Rb⁺ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.Abbreviations and symbols DW dry weight - E extracellular (polyethylene glycol) space - E Nernst potential - H₂O_e H₂O_i tissue water, extra- and intracellular - TPMP ⁺ triphenyl methyl phosphonium salt - WW wet weight     

The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells

p-Cresol that is produced by the intestinal microbiota from the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite on colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc^–/+ cell line, we found that 0.8 mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the S phase and with a slight increase of cell detachment without necrotic effect. At this concentration, p-cresol inhibited oxygen consumption in HT-29 Glc^–/+ cells. In rat normal colonocytes, p-cresol also inhibited respiration. Pretreatment of HT-29 Glc^–/+ cells with 0.8 mM p-cresol for 1 day resulted in an increase of the state 3 oxygen consumption and of the cell maximal respiratory capacity with concomitant increased anion superoxide production. At higher concentrations (1.6 and 3.2 mM), p-cresol showed similar effects but additionally increased after 1 day the proton leak through the inner mitochondrial membrane, decreasing the mitochondrial bioenergetic activity. At these concentrations, p-cresol was found to be genotoxic toward HT-29 Glc^–/+ and also LS-174T intestinal cells. Lastly, a decreased ATP intracellular content was observed after 3 days treatment. p-Cresol at 0.8 mM concentration inhibits colonocyte respiration and proliferation. In response, cells can mobilize their “respiratory reserve.” At higher concentrations, p-cresol pretreatment uncouples cell respiration and ATP synthesis, increases DNA damage, and finally decreases the ATP cell content. Thus, we have identified p-cresol as a metabolic troublemaker and as a genotoxic agent toward colonocytes.     

Rheological behavior of rat mesangial cells during swelling in vitro

The response of cells to mechanical forces depends on the rheological properties of their membranes and cytoplasm. To characterize those properties, mechanical and electrical responses to swelling were measured in rat mesangial cells (MC) using electrophysiologic and video microscopic techniques. Ion transport rates during hyposmotic exposures were measured with whole-cell recording electrodes. Results showed that cell swelling varied nonlinearly with positive internal pressure, consistent with a viscoelastic cytoplasm. The extrapolated area expansivity modulus for small deformations was estimated to be 450 dyne/cm. Cell swelling, caused either by positive pipet pressure or hyposmotic exposure (40–60 mOsm Kg⁻¹), rapidly induced an outwardly rectifying membrane conductance with an outward magnitude 4–5 times the baseline conductance of 0.9±0.5 nS (p<.01). Swelling-induced (SI) current was weakly selective for K⁺ over Na⁺, partially reversed upon return to iso tonicity, and was antagonized by 0.5 mM GdCl₃ (p < 0.02; n = 6). Isolated cells treated with GdCl₃ rapidly lysed after hypotonic exposure, in contrast to untreated cells that exhibited regulatory volume decrease (RVD). Our results indicate that volume regulation by MC depends upon a large swelling-induced K⁺ efflux, and suggest that swelling in MC is a viscoelastic process, with a viscosity dependent on the degree of swelling.     

Inhibition of human HT-29 colon carcinoma cell adhesion by a 4-fluoro-glucosamine analogue

Cell surface glycoconjugates play an important role in cellular recognition and adhesion. Modification of these structures in tumour cells could affect tumour cell growth and behaviour, including metastasis. 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro--D-glycopyranose (4-F-GlcNAc) was synthesized as a potential inhibitor and/or modifier of tumour cell glycoconjugates. The effect of this sugar analogue on the adhesive properties of human colon carcinoma HT-29 cells was evaluated. Treatment of HT-29 cells with 4-F-GlcNAc led to reduced cell surface expression of terminal lactosamine, sialyl-Le^x and sialyl-Le^a, as determined by Western blotting and flow cytometry. The aberrant expression of these oligosaccharide structures on the HT-29 cell surface resulted in: (1) decreased E-selectin mediated adhesion of human colon cells to human umbilical cord endothelial cells (HUVEC); (2) impaired adhesion of HT-29 cells to -galactoside binding lectin, galectin-1; and (3) reduced ability to form homotypic aggregates. After exposure to 4-F-GlcNAc, lysosomal associated membrane proteins (lamp) 1 and 2, and carcinoembryonic antigen (CEA) detected in HT-29 cells were of lower molecular weight, probably due to impaired glycosylation. These results strongly suggest that modification of tumour cell surface molecules can alter tumour cell adhesion and that tumour cell surface oligosaccharides may be suitable targets for therapeutic exploitation.Abbreviations 4-F-GlcNAc 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro--glucopyranose - GlcNAc N-acetylglucosamine - s-Le^x sialyl-Lewis^x - s-Le^a sialyl-Lewis^a - lamp-1 and lamp-2 Lysosomal Associated Membrane Protein 1 and 2 - CEA carcinoembryonic antigen - DMEM Dulbecco's Modified Eagle Medium - PBS Phosphate Buffered Saline (2.7 mM KCl, 1.5 mM KH₂PO₄, 137 mM NaCl, 6.5 mM Na₂HPO₄, pH 7.3) - BSA Bovine Serum Albumin - PMSF Phenylmethylsulfonylfluoride - TBS Tris Buffered Saline (10 mM Tris, 20 mM NaCl, pH 7.3) - TCA Trichloroacetic Acid - DSA Datura stramonium agglutinin     

Contribution of organic and inorganic osmolytes to volume regulation in rat brain cells in culture

In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, andd-aspartate in the two types of cells, -alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1–2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%–65% and 13%–31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions.⁸⁶Rb⁺, used as tracer for K⁺, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K⁺ and Cl^– in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.Special issue dedicated to Dr. Claude Baxter.     

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443–450, 2009), we demonstrated that the activity of K–Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K⁺ influx was estimated as ⁸⁶Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K⁺ efflux was determined as the difference between K⁺ loss by the cells incubated in parallel in isotonic or hypotonic K⁺-free Cl⁻- and NO₃ ⁻-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K⁺ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 μM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 μM), reduced KCC activity by 25–70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 μM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.     

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells

Summary When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na⁺ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na⁺-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K⁺ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.     

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na⁺ from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na⁺-free conditions was inhibited by a Na⁺/H⁺ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Temperature dependence of membrane permeability in cultured cells exposed to benzene and phenol

The effects of benzene (20mM) and phenol (20mM) on the passive membrane permeability of cultured cells of neuronal (N1E115), glial (138MG), muscle (L6) and liver (BRL123) origin were studied. Permeability changes were assayed as alterations in the release of radioactivity from cells preloaded with [³H]2-deoxy-D-glucose. Temperature plots of the efflux revealed that benzene and phenol caused altered flux rates in N1E115 cells without affecting the activation energy. Also in 138 MG cells the activation energy was unaffected by both compounds, while phenol decreased the flux rates. Benzene had no effect on the efflux in L6 cultures, whereas a low activation energy efflux was induced by phenol. In BRL123 cells both benzene and phenol induced a dual (high and low) activation energy efflux.     

KCl loss and cell shrinkage in the ehrlich ascites tumor cell induced by hypotonic media, 2-deoxyglucose and propranolol

Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca²⁺ ionophore, A23187, plus Ca²⁺ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO₃^? does not substitute for Cl^?; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl^? -Cl^? exchange, a net loss of K⁺ and a net gain of Na⁺ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca²⁺-activated, quinine-sensitive K⁺ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K⁺ through the Ca²⁺ stimulated pathway appears to be limited by Cl^? conductive movements; for when NO₃^?, an anion demonstrated here to have a higher conductive movement than Cl^?, is substituted for Cl^?, the cells will shrink when the Ca²⁺-stimulated K⁺ pathway is activated.     

Ion transport and regulation ofTetrahymena pyriformis in isotonic and hypotonic media

Summary The ion and volume regulatory mechanisms ofTetrahymena pyriformis were studied in normal or hypotonic nutrient media and in isotonic inorganic media with different Na/K ratios, in conjunction with the effects of a general metabolic inhibitor (low temperature) and a specific inhibitor (iodoacetate). For K two mechanisms of active influx were found: The first is sensitive to IAc and seems to be the basic mechanism for the maintenance of the K_i/K_o gradient. The second is sensitive to cooling and related to the function of the contractile vacuole; it is also responsible for the high intracellular levels of K. The passive K efflux seems to be a basic factor for volume regulation, together with the contractile vacuole. It increases in hypotonic media and this seems to be related to structural changes of the membranes occurring in hypotonic media. For Na two mechanisms of active transport were also found: One for active Na efflux with highK _m, which is associated with the contractile vacuole and another, for active Na influx with lowK _m, which is inhibited by high levels of intracellular K.The electrochemical potentials of Na and K and the membrane potential (Cl Nernst potential) were also studied in isotonic inorganic media. The membrane is negatively polarized, except if Na_o<5 mM when it becomes positive. In normal conditions, Na is transported outwards actively and leaks passively, while K is transported inwards actively and leaks 56 times more rapidly than Na ions.A model for the overall transport and regulation of ions inTetrahymena is proposed.Abbreviations IAc iodoacetate - PCV packed cell volume - Na _i,K _i,Cl intracellular concentrations ofNa ⁺,K ⁺,Cl ^–, respectively - Na _o,K _o, Cl_o extracellular concentrations of Na⁺, K⁺, Cl^–, respectively - DR distribution ratio - HyN hypotonic nutrient medium - IsN isotonic nutrient medium - HyS IsS hypotonic, and isotonic salt medium, respectively     

Rat liver membrane preparations

The quality of rat liver plasma membranes was examined using three different methods of preparation. Hypotonic Ca⁺⁺-free preparation media were compared with hypotonic Ca⁺⁺ media and with isotonic Ca⁺⁺ media. The plasma membranes prepared in hypotonic Ca⁺⁺ free media were as pure or better than the other two preparation types, while the (Na⁺K⁺)-MgATPase, MgATPase, and the ADPase specific activities were higher. The AMPase specific activities were of similar value for all three types of preparation.