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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.

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.

SUMMARY1. 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 K1 about 50 and 15 mm, respectively.2. The inhibitory effect of other monovalent anions on the chloride transport was investigated by measuring the 3601-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-< Cl1 < 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 0C.5. The membrane potential (Vm) was measured as a function of the concentration of external K+, substituting K+ for Na+. The transference number for 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 %t, GN., and GK are all about 14 ,US/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 Experiments were performed at both addresses.