Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H⁺ exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na⁺ >Li⁺ >K⁺, Rb⁺, Cs⁺, or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca²⁺, and loss of activity on aging which can be related to loss of endogenous K⁺. The reaction is nearly insensitive to either addition of exogenous Mg²⁺ or removal of membrane Mg²⁺ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl^?/OH^? exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li⁺ and Na⁺ supporting higher rates of swelling at higher efficiency than K⁺, Rb⁺, and Cs⁺. The high rates of respiration-dependent swelling in Li⁺ and Na⁺ are inhibited by low levels of exogenous Mg²⁺ (K_i of 5–10 μm), but a significant swelling with almost no cation selectivity persists in the presences of 2 mm Mg²⁺. Removal of membrane Mg²⁺ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li⁺ concentration to a hyperbolic one with a Km of about 14 mm Li⁺. The cation selectivity and Mg²⁺ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg²⁺-sensitive uniport with high activity for Na⁺ and Li⁺ and a Mg²⁺-insensitive pathway with little cation discrimination are available in the membrane.     

Effects of quinine on K+ transport in heart mitochondria

Quinine inhibits the respiration-dependent extrusion of K⁺ from Mg²⁺-depleted heart mitochondria and the passive osmotic swelling of these mitochondria in K⁺ and Na⁺ acetate at alkaline pH. These observations concur with those of Nakashima and Garlid (J. Biol. Chem. 257, 9252, 1982) using rat liver mitochondria. Quinine also inhibits the respiration-dependent contraction of heart mitochondria swollen passively in Na⁺ or K⁺ nitrate and the increment of elevated respiration associated with the extrusion of ions from these mitochondria. Quinine, at concentrations up to 0.5 mM, inhibits the respiration-dependent⁴²K⁺/K⁺ exchange seen in the presence of mersalyl, but higher levels of the drug produce increased membrane permeability and net K⁺ loss from the matrix. These results are all consistent with an inhibition of the putative mitochondrial K⁺/H⁺ antiport by quinine. However, quinine has other effects on the mitochondrial membrane, and possible alternatives to this interpretation are discussed.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

Novel interactions of CLN3 protein link Batten disease to dysregulation of fodrin-Na+, K+ ATPase complex

Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) is the most common progressive neurodegenerative disorder of childhood. CLN3, the transmembrane protein underlying JNCL, is proposed to participate in multiple cellular events including membrane trafficking and cytoskeletal functions. We demonstrate here that CLN3 interacts with the plasma membrane-associated cytoskeletal and endocytic fodrin and the associated Na⁺, K⁺ ATPase. The ion pumping activity of Na⁺, K⁺ ATPase was unchanged in Cln3^−/− mouse primary neurons. However, the immunostaining pattern of fodrin appeared abnormal in JNCL fibroblasts and Cln3^−/− mouse brains suggesting disturbances in the fodrin cytoskeleton. Furthermore, the basal subcellular distribution as well as ouabain-induced endocytosis of neuron-specific Na⁺, K⁺ ATPase were remarkably affected in Cln3^−/− mouse primary neurons. These data suggest that CLN3 is involved in the regulation of plasma membrane fodrin cytoskeleton and consequently, the plasma membrane association of Na⁺, K⁺ ATPase. Most of the processes regulated by multifunctional fodrin and Na⁺, K⁺ ATPase are also affected in JNCL and Cln3-deficiency implicating that dysregulation of fodrin cytoskeleton and non-pumping functions of Na⁺, K⁺ ATPase may play a role in the neuronal degeneration in JNCL.     

A study of sodium release in the course of ATP hydrolysis by membrane ATPase

Summary With the aid of sodium-sensitive glass electrodes, changes in sodium ion activity were studied in the course of subsequent additions of components required for ATP hydrolysis provided by Na⁺–K⁺-dependent membrane ATPase. Membrane ATPase was obtained from guinea pig kidney cortex. In the presence of ATP, Mg⁺⁺ and Na⁺ in media, the addition of K⁺ caused an increase in Na⁺ activity. The omission of ATP or its substitution by ADP as well as the addition of Ca⁺⁺ to the media eliminated the above-mentioned increase of Na⁺ activity. Quabain did not affect Na⁺ release caused by the addition of K⁺, although it significantly inhibited ATPase activity of the preparation. The data obtained were considered to be a direct indication of ion exchange during the course of membrane ATPase reaction. This ion-exchange stage of the reaction is not inhibited by ouabain. The ratio of sodium ions released per one inorganic phosphate formed in the course of the reaction was found to be much higher than that established for transporting membranes of intact cells. A possible cause of this difference is discussed.     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.     

Mechanisms by which Li+ stimulates the (Na++K+)-dependent ATPase

The addition of LiCl stimulated the (Na⁺+K⁺)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na⁺/low K⁺ media and at low Mg. ATP concentrations. Apparent affinities for Li⁺ were estimated at the α-sites (moderate-affinity sites for K⁺ demonstrable in terms of activation of the associated K⁺-dependent phosphatase reaction), at the β-sites (high-affinity sites for K⁺ demonstrable in terms of activation of the overall ATPase reaction), and at the Na⁺ sites for activation. The relative efficacy of Li⁺ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li⁺ was substituted for K⁺, and also in terms of the relative effect of Li⁺ on the apparent K_M for Mg· ATP. With these data, and previously determined values for the apparent affinities of K⁺ and Na⁺ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li⁺ stimulates by relieving inhibition due to K⁺ and Na⁺ (i) by competing with K⁺ for the α-sites on the enzyme through which K⁺ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na⁺ at low-affinity inhibitory sites, which may represent the external sites at which Na⁺ is discharged by the membrane NA⁺/K⁺ pump that this enzyme represents. Both these sites of action for Li⁺ would thus lie, in vivo, on the cell exterior.     

Energy-dependent contraction of swollen heart mitochondria--activation by butacaine.

Butacaine and certain other local anesthetics markedly stimulate the rate, extent, and efficiency of respiration-dependent contraction of heart mitochondria in nitrate salts at alkaline pH. The local anesthetics also induce respiratory control associated with contraction (i.e., the elevated rate of respiration during contraction declines to a State 4-like controlled rate when contraction is complete) so that the reaction at alkaline pH closely resembles the rapid and highly efficient process seen at neutral pH. Respiration-dependent contraction appears to be an osmotic response to cation extrusion on an endogenous cation/H⁺ exchanger (G. P. Brierley, M. Jurkowitz, E. Chavez, and D. W. Jung, 1977, J. Biol. Chem.252, 7932–7939). At alkaline pH, net ion extrusion is slow and inefficient due to the elevated permeability of the membrane to monovalent cations through a putative uniport pathway. Butacaine and other local anesthetics seem to decrease influx-efflux cycling of cations at alkaline pH by restricting cation influx through this uniport. Passive swelling at pH 8.3 in nitrate salts indicates that the uniport reaction is sensitive to Ca²⁺ and has a cation-selectivity of Na⁺ > K⁺ > Li⁺. Butacaine does not inhibit passive swelling under these conditions but produces effects identical to those of classical uncouplers and consistent with increased H⁺ conductance and accelerated influx of cations by cation/H⁺ exchange in nonrespiring mitochondria. However, since contraction in respiring mitochondria is inhibited by uncouplers but stimulated by butacaine, it is apparent that butacaine is not an effective proton conductor in energized mitochondria.     

Transport ATPase: further studies on the properties of the thimerosal-treated enzyme.

Our previous studies showed that when ethylmercurithiosalicylate (thimerosal) interacts with the transport ATPase of the guinea pig kidney under specified conditions, the Na⁺ + K⁺-dependent ATPase activity is inhibited, while the Na⁺-dependent ATPase, the Na⁺ + ATP-dependent phosphorylation of the enzyme, and the K⁺-dependent discharge of the phosphoenzyme seem to be unaffected. Here we describe other properties of the thimerosal-treated enzyme: Na⁺-dependent ADP-ATP exchange, Na⁺-dependent UTPase, and K⁺-dependent p-nitrophenylphosphatase activities of the modified enzyme are not inhibited. Kinetics of the Na⁺ effect on the UTPase activities of the native and the modified enzyme are the same. However, K⁺ has a greater inhibitory effect on the Na⁺-UTPase of the modified enzyme than on the Na⁺-UTPase of the native enzyme. The increase in the apparent affinity of the thimerosal-treated enzyme for K⁺ is also evident from the kinetics of the K⁺ effect on p-nitrophenylphosphatase. Neither the native enzyme nor the modified enzyme catalyzes a P₁-ATP exchange. The uninhibited activities of the thimerosal-treated enzyme are sensitive to ouabain. These data provide further support for those reaction mechanisms in which the existence of two ATP sites within the enzyme is assumed.     

Harmaline: A competitive inhibitor of Na Ion in the (Na++K+)-ATPase system

Summary Experimental evidence is given that the hallucinogen harmaline (HME) behaves as an inhibitor of the (Na⁺+K⁺)-ATPase system, specifically in the Na⁺-dependent phosphorylation reaction. HME at 0.3 to 3mm inhibited several membrane ATPase preparations such as those from human erythrocytes, rat brain and squid retinal axons. The same concentration blocked Na⁺ outflow from squid giant axons. The behavior of several harmane derivatives such as harmine, harmalol and harmaline demonstrated that certain groups influenced the concentration for 50% inhibition of the ATPase system. The following evidence demonstrated that HME blocked the formation of the phosphorylated intermediate by competition with Na ions in the (Na⁺+K⁺)-ATPase reaction in rat brain. (1) The HME effect on the overall (Na⁺+K⁺)-ATPase reaction showed a fully competitive inhibition with respect to Na ion concentration. (2) The inhibition of the Na⁺-stimulated phosphorylation by HME was fully competitive with respect to Na ions, with or without oligomycin present. (3) HME inhibited the effect of ADP on the phosphorylation reaction using³²P-ATP. (4) HME did not accelerate the rate of membrane dephosphorylation by means of³²P-ATP and cold ATP.From the behavior of HME as a competitive inhibitor at Na ion sites of the (Na⁺+K⁺)-ATPase reactions one may gain information about (a) The chemical nature of Na⁺ sites which may be responsible for the selectivity of this cation, and (b) The sequence of Na⁺ and ATP entrance into the Na⁺-dependent phosphorylation reaction. The experimental evidence supports the hypothesis that the entrance of Na⁺ into the enzyme system may precede the formation of the phosphorylated intermediate.     

Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine

The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K⁺/H⁺ antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K⁺/H⁺ exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K⁺/H⁺ antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane.     

Na+ and K+ transport in Nitrosomonas europaea and Nitrobacter agilis

Potassium-depleted cells of Nitrosomonas europaea and Nitrobacter agilis were prepared by diethanolamine treatment and contained less than 5 mM intracellular K⁺. The addition of K⁺ to K⁺-depleted cells of N. europaea and N. agilis resulted in a depolarization of membrane potential (ΔΨ) by about 5 and 10 mV, respectively. This depolarization was, however, compensated by an equivalent increase in transmembrane pH gradient (ΔpH), so that the total proton-motive force (Δp) remained constant, indicating that K⁺ transport was electrogenic in both bacteria. Using ²²Na⁺-loaded cells, it is shown that both bacteria lack a respiration-dependent Na⁺ pump; however, antiporters for Na⁺/H⁺, K⁺/Na⁺ and K⁺/H⁺ were detected. Of these, at least the K⁺/Na⁺ antiporter required an electrochemical gradient for its operation. It is also shown that the unprotonated form of NH₄⁺ is transported into these bacteria by a simple diffusion mechanism.     

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.     

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 ± 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K⁺/Na⁺ permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na⁺ accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K⁺/Na⁺ permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K⁺/Cl⁻ was greater than 50:1. The K⁺/Na⁺ permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca²⁺ concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na⁺ had no significant effect on the K⁺/Na⁺ permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K⁺/Na⁺ selectivity of these channels.     

Swelling and contraction of potato mitochondria

Mitochondria isolated from potato tubers fail to undergo passive osmotic swelling when suspended in isotonic Na⁺ acetate or phosphate, in NaCl following addition of tripropyltin, or in Na⁺ nitrate following addition of an uncoupler. Swelling under each of these conditions in mitochondria from other sources has been attributed to the inward movement of Na⁺ on an endogenous Na⁺/H⁺ exchanger. Such a monovalent cation/H⁺ exchanger has also been implicated in respiration-dependent cation extrusion and contraction of swollen mitochondria. Potato mitochondria swollen in chloride and nitrate salts extrude ions and contract when respiration is initiated. The contraction reaction is slower and less efficient than that in beef heart mitochondria, but like the latter, is sensitive to uncouplers and stimulated by nigericin, butacaine, and Mg²⁺. These comparative studies suggest that a cation⁺/H⁺ exchanger is present in potato tuber mitochondria, but that it functions exclusively as a cation-extruding mechanism. They further suggest that cation⁺/H⁺ exchange activity is not identical in mitochondria from different sources and that these exchange components may have a directionality and regulatory features which differ with the metabolic needs of the source tissue.     

Effects of beta-ecdysone and juvenile hormone on the Na+/K+-dependent ATPase in imaginal disks of Drosophila melanogaster.

The evagination of imaginal disks of Drosophila melanogaster is induced in vitro by β-ecdysone and inhibited by juvenile hormone. The possibility that these hormones act by changing intracellular Na⁺ and K⁺ levels was investigated by studying their effects on the sodium-potassium dependent adenosinetriphosphatase (NaK ATPase), an enzyme with a major rôle in regulating Na⁺ and K⁺ levels in cells. We find that β-ecdysone has no effect on this enzyme and can induce evagination even when intracellular Na⁺ concentrations are increased 2 to 3 fold by ouabain. Juvenile hormone stimulates the enzyme, but still acts to inhibit evagination when NaK ATPase activity is inhibited by ouabain. We conclude that the actions of β-ecdysone and juvenile hormone on imaginal disk evagination do not directly involve the NaK ATPase or require specific changes in Na⁺ and K⁺ concentrations.     

Effect of corpus cardiacum extracts on the ATPase activity of locust rectum

The Mg²⁺ dependent and Na⁺K⁺-activated ATPase activities of microsomal preparations from the rectum of Locusta migratoria were both stimulated, to varying extents, by crude extracts of the corpora cardiaca of this species. Mg²⁺ ATPase activity increased by approximately 549% whereas the hormonal stimulation of Na⁺K⁺-activated ATPase depended upon the concentration of sodium and potassium ions. At 100 mM Na⁺ and 20 mM K⁺, conditions which approximate to optimum for this enzyme system, Na⁺K⁺-activated ATPase activity increased by about 14%. At sub-optimum concentrations of these ions, i.e. 50 and 5 mM Na⁺ and K⁺ respectively, the increase in Na⁺K⁺-activated ATPase activity was about 205%. Ouabain at a concentration of 10^?3 M completely abolished this stimulated activity and was consistently effective in partially reducing the stimulation of Mg²⁺ ATPase activity by corpora cardiaca extracts.     

Ageing in the honey-bee, Apis mellifera, as related to brain ATPases and their DDT sensitivity

The adenosine triphosphatase (ATPase) system in worker honey-bee brains showed an increased activity of 57 per cent in Na⁺K⁺ATPase and 63 per cent in Mg²⁺ATPase from adult emergence to 7 days post-emergence. Mg²⁺ATPase activity remained about the same throughout the remainder of adult life, while Na⁺K⁺ATPase remained the same until the sixth week, when a decline occurred. The percentage mortality of the bees exceeded 90 per cent at the time of decline of Na⁺K⁺ATPase. The in vitro inhibition of Mg²⁺ATPase and Na⁺K⁺ATPase by 10 μM DDT was between 40 and 50 per cent and about 20 per cent, respectively. A somewhat greater sensitivity to DDT was determined in brains of older honey-bees.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.