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.

Aberrant 3H labelling of ATP during in vivo labelling of Ehrlich mouse ascites tumour cells with [2-3H]inositol is significant in the study of isomers of InsP3 and InsP4.

Neutralized perchloric acid extracts of intra-abdominally proliferating Ehrlich mouse ascites-tumour cells harvested after 24 h exposure to [2-3H]inositol were analysed by Mono Q HR5/5 anion-exchange h.p.l.c. using an ammonium formate/phosphoric acid gradient or by ambient-pressure small-column anion-exchange chromatography (Bio-Rad AG 1-X8, 200-400 mesh). The results showed that cellular ATP contained aberrant 3H label in excess of 3H in the isomers of InsP3 and InsP4. The putative ATP 3H radioactivity showed: (i) h.p.l.c. run time as the material causing the largest A254 peak traced, (ii) precise spiking with ATP and [14C]ATP and (iii) specific absorption to charcoal. Moreover, enzymic conversion of ATP into ADP changed putative ATP 3H into putative ADP 3H. In addition, aberrant 3H labelling of cellular ADP and GTP was detected, although at a lower level.

pHi regulation in Ehrlich mouse ascites tumor cells: role of sodium-dependent and sodium-independent chloride-bicarbonate exchange.

pHi recovery in acid-loaded Ehrlich ascites tumor cells and pHi maintenance at steady-state were studied using the fluorescent probe BCECF. Both in nominally HCO3(-)-free media and at 25 mM HCO3-, the measured pHi (7.26 and 7.82, respectively) was significantly more alkaline than the pHi value calculated assuming the transmembrane HCO3- gradient to be equal to the Cl- gradient. Thus, pHi in these cells is not determined by the Cl- gradient and by Cl-/HCO3- exchange. pHi recovery following acid loading by propionate exposure, NH4+ withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3(-)-free media, and in the presence of HCO3- (25 mM) by DIDS-sensitive, Na+)-dependent Cl-/HCO3- exchange. A significant residual pHi recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pHi regulation. pHi maintenance at steady-state involves both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange. Acute removal of external Cl- induces a DIDS-sensitive, Na(+)-dependent alkalinization, taken to represent HCO3- influx in exchange for cellular Cl-. Measurements of 36Cl- efflux into Cl(-)-free gluconate media with and without Na+ and/or HCO3- (10 mM) directly demonstrate a DIDS-sensitive, Na(+)-dependent Cl-/HCO3- exchange operating at slightly acidic pHi (pHo 6.8), and a DIDS-sensitive, Na+)-independent Cl-/HCO3- exchange operating at alkaline pHi (pHo 8.2).

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.

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.

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.

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.

Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells.

The divalent cation-selective ionophore A23187 has been used to characterize cytoplasmic Ca and Mg buffering, Ca2+-pump parameters and the properties of a Ca2+-activated K+-channel in intact red cells. A critical assumption in these studies has been that the ionophore causes a uniform increase in divalent cation-permeability in all the cells. This has now been tested directly in ATP-depleted human red cells by analysing the kinetics of ionophore-induced 45Ca-tracer and net Ca2+ fluxes. The experimental curves were all adequately fitted by single-exponentials at all ionophore concentrations tested. Moreover, statistical analysis of 61 individual tracer influx curves and of pooled data showed no trend towards fast second exponential components. These results demonstrate uniformity of ionophore distribution, ionophore-induced Ca2+-permeability, and cytoplasmic Ca-buffering among all the cells. Experiments involving mixing of cell suspensions with high and low original ionophore content, and involving ionophore extraction by albumin, demonstrate a rapid redistribution of ionophore among the cells, indicating that homogeneity of ionophoric effects is achieved through dynamic ionophore redistribution.

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.

Inorganic phosphate in Ehrlich ascites tumor cells and its distribution across the cell membrane.

A regulatory function of the cell membrane in controlling the cytoplasmic level of Pi has been proposed, and in Ehrlich ascites tumor cells an active influx of primary phosphate has been reported in the literature. In the present study, Ehrlich cells were incubated at 1.5--50 mM extracellular Pi at pH 7.4 (Pi mainly secondary phosphate) and at pH 6.0 (mainly primary phosphate), and the measured cell Pi was compared with the value expected from a passive distribution of Pi. At a low extracellular Pi concentration the cell Pi was 3--6 mumol/g or even more. It is suggested that a major part of this cell Pi can be accounted for by enzymic release of Pi during the sampling procedure. If this interpretation is correct, the present results show that both ionic species of Pi are in electrochemical equilibrium across the cell membrane at steady state. Moreover, in vivo the concentration of free Pi in the cytosol will presumably be maintained at a steady-state level of about 0.4 mM, one order of magnitude below the directly measured values. This implies that the ratio [ATP]/[ADP][Pi] which is important in the regulation of energy metabolism, is higher than reported in the literature.

The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation.

Intracellular potentials were measured, using a piezoelectric electromechanical transducer to impale Ehrlich ascites tumor cells with capillary microelectrodes. In sodium Ringer's, the potential immediately after the penetration was -24±7 mV, and decayed to a stable value of about -8 mV within a few msec. The peak potentials disappeared in potassium Ringer's and reappeared immediately after resuspension in sodium. Ringer's, whereas the stable potentials were only slightly influenced by the change of medium. The peak potential is in good agreement with the Nernst potential for chloride. This is also the case when cell sodium and potassium have been changed by addition of ouabain. It is concluded that the peak potentials represent the membrane potential of the unperturbed cell, and that chloride is in electrochemical equilibrium across the cell membrane.The membrane potential of about -11 mV previously reported corresponds to the stable potential in this study, and is considered as a junction potential between damaged cells and their environment. Similar potential differences were recorded between a homogenate of cells and Ringer's.The apparent membrane resistance of Ehrlich cells was about 70 Ωcm(2). This is two orders of magnitude less than the value calculated from(36)Cl fluxes, and may, in part, represent a leak in the cell membrane.For comparison, the influence of an eventual leak on measurements in red cells and mitochondria is discussed.