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E K Hoffmann

Publications and source records attributed to E K Hoffmann.

88 records · Page 5Linked to original sources
PubMed1984

Volume-induced increase of K+ and Cl- permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+.

Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers , but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl-, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca2+- activated K+ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca2+ ionophore A23187 in the presence of external Ca2+, and also by a sudden increase in external Ca2+. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca2+ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl- permeability of the cell membrane there was no change in cell volume or Cl- content when the K+ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca2+ ionophore A23187 in isotonic medium promoted Cl- loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl- permeability was increased in parallel with and even more than the K+ permeability. It is proposed that separate conductive K+ and Cl- channels are activated during regulatory volume decrease by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin.

Animals↗
PubMed1983

Na+,Cl- cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase).

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Amino acid transport and cell volume regulation in Ehrlich ascites tumour cells.

Cellular and extracellular concentrations of amino acids were measured in Ehrlich ascites cells by amino acid analysis and by distribution of radioactive amino acids between cells and medium. Dilution of the medium results in a reduction in the cellular concentration of non-essential amino acids and taurine and an equivalent increase in the extracellular content of these amino acids. The membrane potential and the electrochemical gradient of sodium were measured. The decrease in the cellular to extracellular gradient of taurine and glycine is not a consequence of a decrease in the sodium gradient but is caused by the changes in osmolarity and cell volume. The unidirectional influx and efflux of glycine and taurine were measured as the initial influx of [14C]glycine and [35S]taurine, using a rapid filter technique. For both amino acids the rate constants for influx are decreased and the rate constants for efflux increased following a reduction in osmolarity. The taurine and glycine fluxes were analysed as a simple pump and leak system. Reduction in osmolarity increases the leak permeability to both taurine and glycine. The results for glycine are also discussed in relation to a Na+-glycine co-transport model, where reduction in osmolarity increases the leak permeability to glycine. The cellular permeability to taurine and glycine increases as a function of increasing cell volume. The taurine permeability increases 7-fold and the glycine permeability 1.5-fold with an increase in cell volume of 30%. The absolute increase in permeability is equal for both taurine and glycine.

Amino Acids↗
PubMed1983

Doxorubicin (Adriamycin) transport in Ehrlich ascites tumour cells: comparison with transport in human red blood cells.

The doxorubicin (Adriamycin) transport was investigated in murine Ehrlich ascites tumour cells by measuring the initial rate of cellular net uptake in vitro at 37 degrees C (pH 7.3). Transport characteristics were compared with previously published data on doxorubicin transport in human red blood cells. The apparent permeability coefficient in ascites cells (2.4 X 10(-5) cm sec-1) and in red cells was of the same order of magnitude when calculated from the initial influx into cells suspended in a salt solution (37 degrees C, pH 7.3). Doxorubicin was strongly adsorbed to the cell surface of ascites cells in contrast to the doxorubicin adsorption to red cells when the cells were suspended in a salt solution. The adsorbed doxorubicin could be removed by washing the ascites cells either with DNA or with human albumin salt solutions indicating that the adsorption to cell surface components was reversible. Cell membrane modifiers, 1-alcohols, local anaesthetics, phloretin, HgCl2, para-chloromercuribenzoate, affected doxorubicin transport in ascites tumour cells and red cells in the same manner as these modifiers affected the transport of other lipophilic compounds. Ehrlich ascites tumour cells and human red blood cells appeared to represent two extremes with regard to doxorubicin adsorption to cell surfaces but doxorubicin seems to pass through the rate limiting barrier of the cell membrane by simple diffusion of the uncharged doxorubicin molecule in both cell types.

1-Octanol↗
PubMed1982

Anion exchange and anion-cation co-transport systems in mammalian cells.

Electroneutral anion transfer in the Ehrlich ascites tumour cell has been found to occur by two separate mechanisms. One is an exchange diffusion system with many similarities to that found in erythrocytes, e.g. saturation kinetics with 'self-inhibition', a relatively pronounced temperature dependence, competitive interactions of Br-, NO3- and SCN-, and a low conductive PCl- of 4 x 10(-8) cm s-1. The main differences are that the Cl- flux in Ehrlich cells at 38 degrees C is one thousandth of the flux in red cells, and that the specificity of the system is less pronounced. It is suggested that the density of anion exchange sites in Ehrlich cells could be the same as in red blood cells, but with a lower turnover rate. The other system is an anion-cation co-transport system capable of mediating a secondary active Cl- influx. This system has a volume-regulatory function and is activated by a reduction in cell volume and intracellular [Cl-]. The two transport systems can be separated by using DIDS as an inhibitor of anion exchange and bumetanide as an inhibitor of co-transport. Under normal steady-state conditions Cl- flux is dominated by the exchange system. It is suggested that intracellular pH regulation can be achieved by the two systems operating in parallel, because the chloride disequilibrium maintained by the co-transport system can drive an influx of bicarbonate through the exchange mechanism.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Identification of membrane proteins related to anion transport in Ehrlich ascites tumor cells.

DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) can interact covalently with membrane sites, resulting in an inhibition of anion exchange. This inhibition varies from reversible to irreversible, depending on the length of times DIDS interacts with the membranes. During the reversible phase, a kinetic analysis of the nature of its inhibitory effect on C1 self-exchange can be performed. The effect of variations in the chloride concentration on the inhibitory potency of DIDS is consistent with the concept that C1 and DIDS compete for the transport site of the anion exchange system in Ehrlich cells. The value of Ki for DIDS inhibition is approx. 0.5 microM. Since the reversible binding may be specific to the anion recognition site in the transport system, it is likely that the subsequent covalent reaction (which is very slow in the Ehrlich cell) involves a nucleophilic group in the membrane close to the transport site. In this case DIDS can be used as a covalent label for the transport protein. We have synthesized a 3-H labelled DIDS with a high specific activity (0.62 X 10(8) cpm/mumol). Using the labelled DIDS, it has been possible to demonstrate that in low C1 medium, only one membrane protein seems to be labelled, and that the number of binding sites per cell is 7 X 10(7). This value is very close to the known density of binding sites in the red blood cell.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1979

Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells.

1. The steady-state tracer exchange flux of chloride was measured at 10-150 mM external chloride concentration, substituting either lactate or sucrose for chloride. The chloride flux saturates in both cases with a K 1/2 about 50 and 15 mM, respectively. 2. The inhibitory effect of other monovalent anions on the chloride transport was investigated by measuring the 36Cl- efflux into media where either bromide, nitrate, or thiocyanate had been substituted for part of the chloride. The sequence of increasing affinity for the chloride transport system was found to be: Br- less than Cl- less than SCN- = NO3-. 3. The chloride steady-state exchange flux in the presence of nitrate can be described by Michaelis-Menten kinetics with nitrate as a competitive inhibitor of the chloride flux. 4. The apparent activation energy (EA) was determined to be 67 +/- 6.2 kJ/mole, and was constant between 7 and 38 degrees C. 5. The membrane potential (Vm) was measured as a function of the concentration of external K+, substituting K+ for Na+. The transference number of K+ (tK) was estimated from the slope of Vm vs. log10 (K+)e, and tCl and tNa were calculated, neglecting current carried by ions other than Cl-, K+, and Na+. The diffusional net flux of K+ was calculated from the steady-state exchange flux of 42K+, assuming the flux ratio equation to be valid. From this value the K+ conductance and the Na+ and Cl- conductances were calculated. The experiments showed that GCl, GNa, and GK are all about 14 muS/cm2. 6. The net (conductive) chloride permeability derived from the chloride conductance was 4 x 10(-8) cm/sec compared with the apparent permeability of 6 x 10(-7) cm/sec as calculated from the chloride tracer exchange flux. These data suggest that about 95% of the chloride transport is mediated by an electrically silent exchange diffusion. 7. Comparable effects of phloretin (0.25 mM) on the net (conductive) permeability and the apparent permeability to chloride (about 80% inhibition) may indicate that the chloride exchange and conductance pathways are not completely separate and distinct modes of transport, but may involve common elements. The reduced chloride permeability in the presence of phloretin is estimated to be two orders of magnitude larger than the ground permeability of the cell membrane.

Animals↗
PubMedDate not supplied

Swelling-activated ion channels: functional regulation in cell-swelling, proliferation and apoptosis.

Cell volume regulation is one of the most fundamental homeostatic mechanisms and essential for normal cellular function. At the same time, however, many physiological mechanisms are associated with regulatory changes in cell size meaning that the set point for cell volume regulation is under physiological control. Thus, cell volume is under a tight and dynamic control and abnormal cell volume regulation will ultimately lead to severe cellular dysfunction, including alterations in cell proliferation and cell death. This review describes the different swelling-activated ion channels that participate as key players in the maintenance of normal steady-state cell volume, with particular emphasis on the intracellular signalling pathways responsible for their regulation during hypotonic stress, cell proliferation and apoptosis.

Animals↗
PubMedDate not supplied

Mechanisms in volume regulation in Ehrlich ascites tumor cells.

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.

Animals↗