Effects of ouabain and osmolarity on bumetanide-sensitive potassium transport in simian virus-transformed 3T3 cells

Unidirectional potassium influx in simian virus-transformed 3T3 cells was dissected into a ouabain-inhibitable "pump' component, a bumetanide-sensitive and chloride-dependent "cotransport' component, and a residual "leak' flux. The bumetanide-sensitive component was stimulated 2-3-fold by a 60-min preincubation with ouabain. Subsequent washing of the cells and incubation in ouabain-free saline reversed both the inhibition of the Na+ pump and the stimulation of bumetanide-sensitive flux. Bumetanide-sensitive potassium influx was also stimulated by hypertonic cell shrinkage (induced by 0.1 M or 0.2 M sorbitol). This latter observation suggests that the bumetanide-sensitive system may play a role in cellular volume regulation.     

Basic characterization of an ouabain-resistant,bumetanide-sensitive K+ carrier-mediated transport system in J774.2 mouse macrophage-like cell line and in variants deficient in adenylate cyclase and cAMP-dependent protein kinase activities

⁸⁶Rb(K⁺) transport across the plasma membrane of macrophage-like cells was studied. The cells used were the wild-type J774.2 and its two variants, CT2 cells, deficient in adenylate cyclase, and J7H1 cells, deficient in cAMP-dependent protein kinase. In the three cell lines about 15% of the total ⁸⁶Rb(K⁺) influx is transported by the K⁺ carrier-mediated transport system. The ⁸⁶Rb(K⁺) efflux carried by the same transporter is negligible when measured in the absence of ouabain in the medium. Therefore this carrier conducts a net inward flux of K⁺ under the experimental conditions used. The transporter is sensitive to extracellular Na⁺ and inhibited by ‘loop’ diuretics; bumetanide inhibits ouabain-resistant ⁸⁶Rb(K⁺) influx with IC₅₀ of 0.1, 5.0, and 0.05 μM for J774.2, CT2 and J7H1 macrophages, respectively. The membrane potential of the three cells was measured, using the distribution of [³H]tetraphenylphosphonium ([³H]TPP⁺) across the plasma membrane, and found to be −80.1, −108.5 and −105.1 mV for J774.2, CT2 and J7H1 cells, respectively. The addition of bumetanide to the cell medium does not alter [³H]TPP⁺ uptake indicating that the transporter is electrically silent. It is concluded that despite the differences in cAMP metabolism by the three macrophages, the basic characteristics of K⁺ carrier-mediated transport system of the three cells are very similar.     

Racial differences in bumetanide-sensitive cotransport and N-ethylmaleimide-stimulated potassium efflux

Racial differences in erythrocyte potassium effluxes mediated by two loop-diuretic sensitive modes of cotransport were compared. In red cells loaded to contain approximately equimolar amounts of sodium and potassium, black subjects had lower bumetanide-sensitive sodium-dependent net potassium effluxes as compared to whites. In fresh, washed erythrocytes pretreated with N-ethylmaleimide (NEM), maximal net potassium efflux was greater in blacks than in whites. NEM-stimulated potassium efflux was partially inhibited by bumetanide but only at very high concentrations. The quantitative differences in these two modes of potassium efflux suggest that NEM-stimulated potassium efflux is not an altered mode of sodium-dependent potassium efflux.     

Presteady-state kinetics and carrier-mediated transport: A theoretical analysis

Summary Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or ratelimiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero-trans substrate uptake was thus derived and then extended to models withp intermediary steps. It is concluded that (p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models withp>5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms.     

Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides.

Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.     

Second system for potassium transport in Streptococcus faecalis

It has been reported that the accumulation of K+ by Streptococcus faecalis is mediated by a transport system which required both ATP and the proton motive force (Bakker and Harold, J. Biol. Chem. 255:433-440, 1980). My results indicate that S. faecalis has a second transport system for K+. The features of this system are as follows: (i) the system is driven by ATP (or a derivative of ATP) and does not require the proton motive force; (ii) the system is normally absent in the wild-type strain but can be derepressed by lowering rhe intracellular concentration of K+; (iii) the pH optimum of this system is about 8.5, and no detectable K+ is accumulated at pH values below 6.5; and (iv) the rate of Rb+ accumulation by this system is very low. These properties are quite different from those of the transport system described by Bakker and Harold. Therefore, I propose that S. faecalis has two K+ transport systems.     

Tetrodotoxin-sensitive protein in the extracts from excitable tissues

The sodium permeability of liposomes preincubated with the soluble fraction of brain and heart muscle homogenates was increased veratrine. The veratrine increment was decreased by tetrodotoxin. The effect was specific for the extracts from excitable tissues. Bovine serum and soluble fraction of liver homogenate induced neither veratrine- nor tetrodotoxin-sensitivity of the liposomes. Treatment of the excitable tissue extracts by pronase and heat denaturation caused their complete inactivation. Tetrodotoxin-sensitive factor could be fractionated by ammonium sulfate precipitation and by DEAE-Servacel chromatography. On a column of Sephadex G-200 it was eluted with the void volume. It is suggested that the tetrodotoxin-sensitive factor is a protein which could be a soluble precursor of the voltage-dependent sodium channels.     

Dual system for potassium transport in Saccharomyces cerevisiae.

In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.     

A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.     

Enzymatic reduction and methylation of folate following pH-dependent,carrier-mediated transport in rat jejunum

Intestinal transport of [³H]folate was studied using everted sacs of rat jejunum. The proximal small intestine transports folate against a concentration gradient by a system which is saturable, pH-dependent, energy-dependent, sodium-dependent, sensitive to temperature, and appears to be a common transport system for folate' compounds. Chromatographic analysis of folate compounds in the serosal compartment after a 60 min incubation with folate in the mucosal medium in sodium phosohate buffer indicated that metabolism of folate to 5-methyltetrahydrofolate was extensive at pH 6.0 and negligible at pH 7.5. The percent conversion of folate to 5-methyltetrahydrofolate at pH 6.0 was reduced by increasing the concentration of folate in the mucosal medium, thus indicating saturation of the reduction and methylation process. These findings indicate that folate transport in rat jejunum occurs by an energy-dependent, carried-mediated system and that both folate transport and intestinal conversion of folate to 5-methyltetrahydrofolate are pH-dependent.     

Enzymatic reduction and methylation of folate following pH-dependent, carrier-mediated transport in rat jejunum.

Intestinal transport of [3H] folate was studied using everted sacs of rat jejunum. The proximal small intestine transports folate against a concentration gradient by a system which is saturable, pH-dependent, energy-dependent, sodium-dependent, sensitive to temperature, and appears to be a common transport system for folate compounds. Chromatographic analysis of folate compounds in the serosal compartment after a 60 min incubation with folate in the mucosal medium in sodium phosohate buffer indicated that metabolism of folate to 5-methyltetrahydrofolate was extensive at pH 6.0 and negligible at pH 7.5. The percent conversion of folate to 5-methyltetrahydrofolate at pH 6.0 was reduced by increasing the concentration of folate in the mucosal medium, thus indicating saturation of the reduction and methylation process. These findings indicate that folate transport in rat jejunum occurs by an energy-dependent, carried-mediated system and that both folate transport and intestinal conversion of folate to 5-methyltetrahydrofolate are pH-dependent.     

Electrokinetic membrane processes in relation to properties of excitable tissues. I. Experiments on oscillatory transport phenomena in artificial membranes

An artificial system is studied consisting of salt solutions of different concentrations separated by a porous, "charged" membrane, through which a constant electric current is passed. Experiments on such systems demonstrate rhythmic variations of the transmembrane potential and the membrane resistance, which are concomitant with an oscillatory streaming of water solution across the membrane. The repetitive oscillations can be of a damped or undamped type dependent on the "stimulating" current density. A qualitative discussion of the mechanism of the oscillations is given. It centers around the periodic resistance changes in the membrane, which result from a complicated interplay between the driving forces present. The importance of electro-osmotic effects is emphasized. A few comparisons relating to possible electrophysiological implications are presented. In the metastable state of this membrane oscillator, "make" and "break" responses can be triggered by electric as well as by mechanical (pressure) "stimuli."     

A potassium transport mutant of Saccharomyces cerevisiae

A mutant in Saccharomyces cerevisiae required one hundred times more K⁺ than wild type for the same half maximal growth rate. Mutant cells and wild type cells grown at millimolar K⁺ did not show significant differences in Rb⁺ transport. In the mutant, a rapid K⁺ loss induced by azide or incubation (4 h) in K⁺-free medium decreased the Rb⁺ transport K _m by one half; in the wild type, those treatments decreased the Rb⁺ K _m twenty and one hundred times, respectively. Mutant and wild type did not show significant differences in Na⁺ transport and in the Na⁺ inhibition of Rb⁺ transport, either in normal-K⁺ cells or in K⁺-starved cells. The results suggest that either two systems or one system with two interacting sites mediate K⁺ transport in S. cerevisiae.Abbreviations YPD yeast-peptone-dextrose medium