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1. Slices of rat cerebral cortex incubated aerobically at 37 degrees C in Krebs-Ringer-bicarbonate solution accumulated 3,4-L-dihydroxyphenylalanine (L-DOPA) against its concentration gradient. With 1 mM L-DOPA in the medium, tissue-water/medium concentration ratios of about 6 : 1 are reached, which are modified by the presence of other amino acids in the medium. 2. Kinetic analysis suggested that L-DOPA influx into brain cells occurred by at least two saturable processes, which show apparent Km values in the range of 10(-3) M and 10(-5) M, respectively. 3. Prior incubation of the slices in Na+-free (choline-containing) medium at 37 degrees C depressed their subsequent uptake of L-DOPA in normal Na+-containing medium; this inhibition did not appear when the preincubation was carried out at 0-4 degrees C. Besides this effect of preincubation, most of L-DOPA influx into brain slices was independent of the actual concentration of Na+ in the medium; the two saturable processes described in this article behaved similarly in this respect. 4. Most of L-DOPA uptake by the high-Km process is mediated by an agency that resembles the Na+-independent L system described in Ehrlich cells (Oxender, D. L. and Christensen, H. N. (1963) J. Biol. Chem. 238, 2686-2699), both in its specificity and in its participation in exchange phenomena. A lesser component of uptake by a type A mediation is also suggested as contributing to the high-Km process . 5. The kinetic and specificity properties of the low-Km process of L-DOPA uptake suggest a similarity between its mediation and that of the high-affinity systems for L-tyrosine and L-tryptophan found in brain tissue preparations (Belin, M. F. and Pujol, J. F. (1973) Experientia 29, 411-413; Bauman, A., Bourgoin, S., Benda, P., Glowinski, J. and Hamon, M. (1971 Brain Res. 66, 253-263).

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.

Pb2+ modified the apparent threshold sensitivity to Ca2+ of individual K+ channels with a biphasic time-course. At first, the sensitivity to Ca2+ was lowered with the result of a decrease of the fraction of activated vesicles at a given Ca2+ concentration. Later, Pb2+ increased the sensitivity to Ca2+ and the fraction of activated vesicles. The increase of Pb2+ concentration increased the extent of the initial inhibition but decreased its duration. The inhibitory effect was not observed when the addition of Ca2+ preceded the addition of Pb2+. The presence of Mg2+ in the incubation medium was also required. In the absence of Mg2+, Pb2+ decreased the rate of uptake of 86Rb, but no decrease in the fraction of activated vesicles could be demonstrated.

The all or nothing behaviour of Ca2+-dependent K+ channels has been analyzed in one-step inside-out vesicles. There is a threshold for Ca2+ below which the K+ channels remain silent, and which ranges between the 10(-6) and 10(-8) M for different vesicles under the experimental conditions tested, in the absence of Mg2+. The increase of Ca2+ concentration within this range recruits a larger fraction of the vesicles to the active (permeable to 86Rb+) state. The apparent rate of 86Rb+ transport through each individual channel was found to increase, however, with Ca2+ concentration. This finding is not an artefact due to size heterogeneity of the vesicle population, and it is consistent with the variations of the mean open time of the channels with Ca2+ concentration reported previously in patch-clamp experiments. The electron donor system ascorbate + phenazine-methosulphate increases the rate of 86Rb+ transport through the channels whereas oxidized cytochrome c has the opposite effect.

We studied the transport of tryptophan through transport system T in the human red cell, the Ehrlich ascites-tumour cell and in everted sacs of rat intestine. In red cells we confirmed earlier results on Na+-independence and aromatic amino acid specificity (Rosenberg, R., Young, J.D. and Ellory, J.C. (1980) Biochim. Biophys. Acta 598, 375-384). In addition we observed that N-methylation or N-acetylation did not reduce the affinity of the substrates for system T, hydroxylation could increase or decrease substrate affinity, and system T was insensitive to pH changes in the medium. These results characterized reactive differences between system T and other known amino acid transport systems. We also found that D-isomers were about 1/3 as effective as L-isomers to inhibit L-tryptophan uptake. D-Tryptophan competitively inhibited L-tryptophan uptake, but was not taken up by system T. L-Tryptophan produced trans-stimulation of the uptake (influx) and trans-inhibition of the release (efflux) of L-[3H]tryptophan; D-tryptophan produced trans-inhibition of the efflux but did not affect significantly the uptake. These results show that in red cells the transport properties of transport system T are asymmetric. Transport system T seems to be absent in the other two preparations studied, the Ehrlich ascites-tumour cell and the rat intestine.

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5 X 10(-8) M) while oxidized cytochrome c decreases the sensitivity to about 1/3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.

The effects of variations of the NAD+/NADH quotient on the uptake of 86Rb by human red cells loaded by non-disruptive means with the chelator Benz2 and different amounts of 45Ca has been examined. The NAD+/NADH quotient was modified by the addition of pyruvate and/or lactate or xylitol. It was found that the uptake of 86Rb at a given intracellular Ca2+ concentrations was faster in the reduced state (lactate or xylitol added). Metabolic changes were associated with variations of the redox state. However, glycolitic intermediates did not significantly modify the apparent affinity for Ca2+ of the Ca2+-dependent K+ channel in one-step inside-out vesicles prepared from the erythrocyte membrane. Taken together, these results suggest that modifications of the cytoplasmic redox potential could modulate the sensitivity to Ca2+ of the Ca2+-dependent K+ channel in the human red cells under physiological conditions. This conclusion is consistent with previous findings in inside-out vesicles of human erythrocytes using artificial electron donors.

The activation of Ca2+ -dependent K+ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na+ -dependent transport of alpha-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K+ conductance. The same treatment does not modify the Na+ -independent uptake of the norbornane amino acid.

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca2+-induced loss of KC1 from human red cells. However, the Ki values for this effect did not bear good correlation with the Ki values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KC1 fluxes were also affected. Added calmodulin did not have any effect on Ca2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported Ki for calmodulin-dependent systems. These results suggest that Ca2+-dependent K+ channels of red cells are not regulated by calmodulin.

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K+ permeability had been increased, either by valinomycin or by activation of Ca2+-dependent K+ channels, in medium containing different K+ concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na+-dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2-3.1-times for each 60 mV-hyperpolarization. The Na+-dependent system ASC was insensitive to membrane potential. The Na+-independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.

When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.

Ca2+-dependent K+ transport and plasma membrane NADH dehydrogenase activities have been studied in several 'high-K+' (human, rabbit and guinea pig) and 'low-K+' (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca2+-dependent K+ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome c reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca2+-dependent K+ channel activities in the species studied. Nor were any of the above activities correlated with (Na+ + K+)-ATPase activity.