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E K Hoffmann

Publications and source records attributed to E K Hoffmann.

At least 19 recordsLinked to original sources
PubMed1992

Activation of the Na+/K+/Cl- cotransport system by reorganization of the actin filaments in Ehrlich ascites tumor cells.

Reorganization (disassembly) of the actin filaments in Ehrlich ascites tumor cells, either by hypotonic treatment in the presence of Ca2+ or by addition of cytochalasin B, results in activation of the Na+/K+/Cl- cotransport system. However, other regulatory processes, some of which may be dependent on an intact filament system, are responsible for the activation of the Na+/K+/Cl- cotransport system after cell shrinkage.

Actins↗
PubMed1992

Cell swelling activates K+ and Cl- channels as well as nonselective, stretch-activated cation channels in Ehrlich ascites tumor cells.

Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba2+ and hence presumably to Ca2+. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca2+ permeability, as part of the volume-activated signal system. In isolated inside-out patches a Ca(2+)-dependent, inwardly rectifying K+ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mM K+ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K+ channel takes place after an increase in Ca2+ from 10(-7) to 10(-6) M which is in the physiological range. Patch-clamp studies in cell-attached mode show K+ channels with spontaneous activity and with characteristics similar to those of the K+ channel seen in excised patches. The single-channel conductance for outward current at 5 mM external K+ is estimated at about 7 pS. A K+ channel with similar properties can be activated in the cell-attached mode by addition of Ca2+ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K+ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K+ channels following addition of Ca2+ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K+ channels in the presence of 3 mM Ca2+ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca2+ and could not be activated by membrane stretch (suction). In cell-attached mode single-channel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca2+ plus ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Cell swelling and volume regulation.

The extracellular space in the brain is typically 20% of the tissue volume and is reduced to at least half its size under conditions of neural insult. Whether there is a minimum size to the extracellular space was discussed. A general model for cell volume regulation was presented, followed by a discussion on how many of the generally involved mechanisms are identified in neural cells and (or) in astrocytes. There seems to be clear evidence suggesting that parallel K+ and Cl- channels mediate regulatory volume decrease in primary cultures of astrocytes, and a stretch-activated cation channel has been reported. The role of the different channels was discussed. A taurine leak pathway is clearly activated after cell swelling both in astrocytes and in neurones. The relations between the effect of glutamate and cell swelling were discussed. Discussion on the clearance of potassium from the extracellular space was continued. Neither the spatial buffering mechanism nor the role of the Na+/K+ pump was discussed here, but additional possibilities for K+ removal were discussed. At a high extracellular K+ concentration there is a possibility of channel-mediated KCl uptake by the glia cells, and a Na-K-2Cl cotransport system is present in astrocytes and seems to play a role at relatively low extracellular K+ concentrations (6-20 mM). The cotransport system is likely to be regulated, but little is known about its regulation in glia cells.

Animals↗
PubMed1990

Cytoplasmic acidification and activation of Na+/H+ exchange during regulatory volume decrease in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells undergoing regulatory volume decrease (RVD) exhibit cytoplasmic acidification as measured by an intracellular fluorescent pH indicator. The acidification results in an activation of the Na+/H+ exchanger. The intracellular pH 'set point' for the activation is estimated to be around 7.0. The activation of the Na+H+ exchanger leads to an incomplete RVD. In support of this conclusion, amiloride and Na+-free medium, known to limit the Na+/H+ exchange, indeed enhance the RVD response. Intracellular acidification and activation of Na+/H+ exchange may be a general response of cells undergoing RVD.

Amiloride↗
PubMed1990

Inhibition of Na-K-C1 cotransport in Ehrlich ascites cells by antiserum against purified proteins of the cotransporter.

Two proteins were purified earlier from solubilized membranes of Ehrlich ascites cells by using a bumetanide-Sepharose affinity column. These proteins were proposed to be constituents of the Na-K-C1 cotransporter. However, the specificity of binding of bumetanide to the cotransporter was insufficient evidence for this proposal. We now have direct evidence that the purified protein contains components of the cotransporter. Antiserum raised against the bumetanide-binding proteins strongly inhibits Na-K-C1 cotransport measured by two independent methods. Cotransport was induced by hypertonic challenge and was measured as the bumetanide-sensitive portion of unidirectional C1 influx and as regulatory cell volume increase. In both assays, cotransport was strongly inhibited by the antiserum. Fab fragments of the antibodies inhibited cotransport to a similar extent.

Animals↗
PubMed1989

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells.

Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, non-denaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K+ transport activity of these vesicles was assayed by measurement of 86Rb+ uptake against a large opposing K+ gradient. The reconstituted system was found to contain a K+ transporting protein, which is sensitive to Ba2+ like the K+ channel previously demonstrated to be activated in intact cells after cell swelling.

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↗
PubMed1988

Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column.

Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4'-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4'-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized with n-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were approximately 76 kDa and 38-39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a "control" column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.

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

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

Anion transport systems in the plasma membrane of vertebrate cells.

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)

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↗
PubMed1986

Identification of the anion exchange protein of Ehrlich cells: a kinetic analysis of the inhibitory effects of 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) and labeling of membrane proteins with 3H-DIDS.

In Ehrlich ascites tumor cells 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibits the chloride exchange both reversibly and irreversibly. The reversible inhibition is practically instantaneous and of a competitive nature with Ki about 2 microM at zero chloride concentration. This is succeeded by a slow irreversible binding of DIDS to the transporter, with a chloride dependence suggesting binding to the same site as for reversible DIDS binding/inhibition. To identify the membrane protein involved in anion exchange, cells were labeled with 3H-DIDS. Incubation of cells for 10 min with 25 microM DIDS at pH 8.2 leads to more than 95% inhibition of the DIDS-sensitive chloride exchange flux when the chloride concentration is low (15 mM). This condition was used for the 3H-DIDS-labeling experiments. After incubation the cells were disrupted, the membranes isolated and solubilized, and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The distribution of the 3H-activity in the gel showed only one major peak, which could be related to protein with a mol wt of about 30,000 Daltons. The number of transport sites was estimated at about 400,000 per cell, and from the DIDS-sensitive chloride flux under steady-state conditions we calculate a turnover number of 340 ions per sec per site.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with [3H]bumetanide.

Simultaneous measurements were made of net Cl influxes and [3H]bumetanide binding to Ehrlich ascites tumor cells in which the chloride-cation cotransport pathway had been activated by hypertonic challenge. There was a good linear correlation between inhibition of Cl influx during regulatory volume increase and numbers of bumetanide molecules bound per cell, consistent with high specificity of bumetanide binding to cotransport sites. The extrapolation to the number of bumetanide binding sites per cell at maximal inhibition of Cl transport thus gives the number of cotransport sites per cell as 2.0 X 10(6). From this, and the fluxes measured (not necessarily maximum fluxes), the turnover number is calculated at 50 Cl ions per site per second. Unstimulated cells in isotonic medium, with negligible bumetanide-inhibitable fluxes, have the same number of bumetanide binding sites as the activated cells undergoing volume regulation.

Animals↗
PubMed1986

Activation of Cl-dependent K transport in Ehrlich ascites tumor cells.

N-ethylmaleimide (NEM) treatment of steady-state Ehrlich cells induces a substantial net loss of cellular KCl and cell shrinkage. The majority of the initial K loss is Cl dependent. From estimates of membrane potential it is concluded that the NEM-induced KCl loss is electroneutral. The effect of NEM on H extrusion by cells in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-containing medium showed that only an insignificant part of the K loss could be attributed to an activation of a K-H exchange system. Consequently, NEM appears to activate a K-Cl cotransport, which causes cell shrinkage. The anion preference of the K loss is Cl greater than Br much greater than SCN = NO3. NEM also seems to inhibit a Cl-dependent Na uptake previously described in shrunken cells. Addition of NEM to cells undergoing regulatory volume decrease after swelling in hyposmotic media results in a Cl-dependent acceleration of cell shrinkage, suggesting that a Cl-dependent component of K efflux is induced by NEM also in swollen cells. A Cl-dependent K efflux is also activated in Ca-depleted cells or at reduced extracellular pH after cell swelling. Under isotonic conditions activation of Cl-dependent K flux after Ca depletion or pH reduction could not be demonstrated. The combined results show that Ehrlich cells possess a latent K-Cl cotransport that becomes active after changes in the state of SH groups, regardless of the initial cell volume. A similar K-Cl cotransport is activated in hypotonically swollen cells after Ca depletion or after reduction of the extracellular pH.

Animals↗
PubMed1985

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells.

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.

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