22Na+ fluxes in thymic lymphocytes. I. Na+/Na+ and Na+/H+ exchange through an amiloride-insensitive pathway

The Na+ transport pathways of normal rat thymocytes were investigated. Na+ conductance was found to be lower than K+ conductance, which is consistent with reported values of membrane potential. In contrast, the isotopically measured Na+ permeability was greater than 10-fold higher than that of K+, which indicates that most of the flux is electroneutral. Cotransport with Cl- (or K+ and Cl-) and countertransport with Ca2+ were ruled out by ion substitution experiments and use of inhibitors. Countertransport for Na+ or H+ through the amiloride-sensitive antiport accounts for only 15-20% of the resting influx. In the presence of amiloride, 22Na+ uptake was increased in Na+-loaded cells, which suggests the existence of Na+/Na+ countertransport. Cytoplasmic pH determinations using fluorescent probes indicated that under certain conditions this amiloride-resistant system will also exchange Na+ for H+, as evidenced by an internal Na+- dependent acidification is proportional to internal [Na+] but inversely related to extracellular [Na+]. Moreover, 22Na+ uptake is inhibited by increasing external [H+]. The results support the existence of a substantial amiloride-insensitive, electroneutral cation exchange system capable of transporting Na+ and H+.     

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na+-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convoluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na+-dependent, but electrically silent transport of propionate is saturable (Km = 10.9 +/- 1.1 mM and Vmax = 3.6 +/- 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of L- and D-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na+-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K+ diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na+-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of L- and D-lactate in this region of rabbit kidney proximal tubule.     

Theoretical effects of transmembrane electroneutral exchange on membrane potential

Transmembrane electroneutral transport mechanisms [e.g., Na/H exchange, Cl/HCO3 exchange, (K + Cl) cotransport] have recently been identified in a wide variety of cell types. If these exchanges sum to give a net electroneutral Na/K exchange, they may hyperpolarize the membrane potential beyond the value calculated from the Mullins-Noda equation, provided the cell maintains steady state intracellular ionic concentrations. In extreme circumstances, the membrane potential could hyperpolarize beyond the potassium reversal potential. This effect is mediated by the electrogenic Na/K pump. If either Na or K exchanges electroneutrally against a third ion (e.g., Na/Ca exchange), then the exchange may depolarize the membrane potential.     

Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.     

Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

Short-term stimulation of Na+-dependent amino acid transport by dibutyryl cyclic AMP in hepatocytes. Characteristics and partial mechanism.

The short-term protein-synthesis-independent stimulation of alanine transport in hepatocytes was further investigated. Cyclic AMP increased the Vmax. of alanine transport. Amino acid transport via systems A, ASC and N was stimulated. A good correlation was found between the initial rate of transport and the cell membrane potential as calculated from the distribution of Cl-. Cyclic AMP increased the rate of alanine transport, stimulated Na+/K+ ATPase (Na+/K+-transporting ATPase) activity and caused membrane hyperpolarization. The time courses and cyclic AMP dose-dependencies of all three effects were similar. Ouabain abolished the effect of cyclic AMP on Cl- distribution and on transport of alanine. The effect of cyclic AMP on alanine transport and Cl- distribution was mimicked by the antibiotic nigericin; the effect of nigericin was also abolished by ouabain. It is concluded that the effect of cyclic AMP on transport is mediated via membrane hyperpolarization. It is suggested that the primary action of cyclic AMP is to increase the activity of an electroneutral Na+/K+-exchange system in the liver cell plasma membrane, thus hyperpolarizing the membrane by stimulating the electrogenic Na+/K+ ATPase.     

Direct measurement of the membrane potential of Ehrlich ascites tumor cells: lack of effect of valinomycin and ouabain.

The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+, K+ and Cl- showed that valinomycin induced net losses of K+ and Cl- and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.     

The monovalent cation "leak" transport in human erythrocytes: an electroneutral exchange process.

The mechanism of the "ground permeability" of the human erythrocyte membrane for K+ and Na+ was investigated with respect to a possible involvement of a previously unidentified specific transport pathway, because earlier studies showed that it cannot be explained on the basis of simple electrodiffusion. In particular, we analyzed and described the increase in the (ouabain+bumetanide+EGTA)-insensitive unidirectional K+ and Na+ influxes as well as effluxes (defined as "leak" fluxes) observed in erythrocytes suspended in low-ionic-strength media. Using a carrier-type model and taking into account the influence of the ionic strength on the outer surface potential according to the Gouy-Chapman theory (i.e., the ion concentration near the membrane surface), we are able to describe the altered "leak" fluxes as an electroneutral process. In addition, we can show indirectly that this electroneutral flux is due to an exchange of monovalent cations with protons. This pathway is different from the amiloride-sensitive Na+/H+ exchanger present in the human red blood cell membrane and can be characterized as a K+(Na+)/H+ exchanger.     

Chloride transport in control and cystic fibrosis human skin fibroblast membrane vesicles.

Plasma membrane vesicles were isolated from either cystic fibrosis (CF) or non-CF cultured fibroblasts derived from skin biopsies of either foetus, child or adolescent human donors. The total membrane yield was essentially identical for either CF or control membranes. By using a rapid filtration technique, 36Cl uptake by these vesicles was quantitated in the absence and presence of alkali-metal ion-, electrical- and/or pH gradients. In the absence of a pH gradient (pHout = pHin = 7.5), Cl uptake took place downhill in both cases. Either cis K+, cis Na+ or an equimolar mixture of cis Na+ plus K+ caused Cl uptake activation. In the presence of an alkaline-inside pH gradient (pHout/pHin = 5.5/7.5), Cl uptake exhibited an apparent overshoot independently of the presence or absence of any metal-ion gradient. The observed potassium-, sodium- and proton-dependent Cl influx rates were all unaffected by voltage clamping, indicating the existence in these vesicles of electroneutral symport systems of the type Cl-/H+, Cl-/K+ and/or Cl-/Na+; but not 2 Cl-/Na+/K+. In the presence of an inward-directed K+ gradient, valinomycin further increased Cl uptake, both in the presence and in the absence of a pH gradient, indicating the presence of a rheogenic Cl uniport. In absolute quantitative terms, the two different modes (rheogenic and electroneutral) of Cl transport evinced in these vesicles were about 45% lower in CF than in control skin fibroblasts. However, qualitatively, there was no difference between normal and CF cells. The evidence obtained indicates that the CF defect, which is expressed in fibroblast plasma membranes, does not affect specifically either the rheogenic or the electroneutral Cl transport systems. Rather, the CF cells appear to give a smaller yield of closed, functional vesicles, reflected by a significantly smaller apparent intravesicular volume. Because it also affects the transport of D-glucose and L-alanine, this anomaly could be the consequence of a generalized membrane defect characterizing CF fibroblasts.     

The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.     

Proton pathways in rat renal brush-border and basolateral membranes

The quenching of acridine orange fluorescence was used to monitor the formation and dissipation of pH gradients in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. The fluorescence changes of acridine orange were shown to be sensitive exclusively to transmembrane delta pH and not to membrane potential difference. In brush-border membrane vesicles, an Na+ (Li+)-H+ exchange was confirmed. At physiological Na+ concentrations, 40-70% of Na+-H+ exchange was mediated by the electroneutral Na+-H+ antiporter; the remainder consisted of Na+ and H+ movements through parallel conductive pathways. Both modes of Na+-H+ exchange were saturable, with half-maximal rates at about 13 and 24 mM Na+, respectively. Besides a Na+ gradient, a K+ gradient was also able to produce an intravesicular acidification, demonstrating conductance pathways for H+ and K+ in brush-border membranes. Experiments with Cl- or SO2-4 gradients failed to demonstrate measurable Cl--OH- or SO2-4-OH- exchange by an electroneutral antiporter in brush-border membrane vesicles; only Cl- conductance was found. In basolateral membrane vesicles, neither Na+(Li+)-H+ exchange nor Na+ or K+ conductances were found. However, in the presence of valinomycin-induced K+ diffusion potential, H+ conductance of basolateral membranes was demonstrated, which was unaffected by ethoxzolamide and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid. A Cl- conductance of the membranes was also found, but antiporter-mediated electroneutral Cl--OH- or SO2-4-OH- exchange could not be detected by the dye method. The restriction of the electroneutral Na+-H+ exchanger to the luminal membrane can explain net secretion of protons in the mammalian proximal tubule which leads to the reabsorption of bicarbonate.     

Characterization of the ion transport activity of the budding yeast Na+/H+ antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H+ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na+/H+ but not K+/H+ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na+ and K+ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb+ and Li+. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na+/H+ antiporters, whereas other known eukaryotic Na+/H+ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na+/H+ antiporters.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.     

Sodium pump hyperpolarization-relaxation in rat caudal artery

Electrogenic ion transport contributes vitally to the Em in vascular muscle and thus is an important influence on contraction and relaxation. Agents that act on membrane ion transport will cause depolarization or hyperpolarization of sufficient magnitude to cause contraction or relaxation, respectively. In the caudal artery of the rat, the principal ion involved appears to be Na+. The transport process appears to be the Na+, K+-ATPase, which is ouabain sensitive, rather than other possible candidates such as the Na+-Ca2+ countertransport mechanism. The hyperpolarization and parallel relaxation found in caudal artery on return to K+ provide unequivocal evidence for an electrogenic Na+ pump. In contrast, the lack of a contraction on transition to O Na+ suggests that the caudal artery does not show an Na+-K+ countertransport system. Although other ion transport systems might be established later for caudal artery and other kinds of vascular muscle, it now appears that the electrogenic Na+ pump is the main ion transport system controlling contraction through a continuous contribution to Em.     

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.     

Effectors of amino acid transport processes in animal cell membranes

Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells. Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions. Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it. Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl-. Both H+ and K+ have been found to be inhibitory in other systems. Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior). In some cell systems an inwardly directed Cl- and H+ gradient or an outwardly directed K+ gradient can energize transport. Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient. Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+ gradient or interact with the substrate at the carrier level either allosterically or at a common site. Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport. Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis. Microtubules may be involved in the regulation of transport by insulin or glucagon. Some reports also suggest that insulin has a direct effect on membranes. In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis. Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone. These agents appear to function at the level of protein synthesis. While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+- and K+-dependent uridine transport in rat renal brush-border membrane vesicles

The transport of uridine into rat renal brush-border membrane vesicles was investigated using an inhibitor-stop filtration method. Uridine was not metabolized under these conditions. The rapid efflux of intravesicular uridine was prevented by adding 1 mM phloridzin to the ice-cold stop solution. In the presence of inwardly directed gradients of either Na+ or K+, zero-trans uridine uptake exhibited a transient overshoot phenomenon indicating active transport. The overshoot was much more pronounced with Na+ than K+ and it was not observed when either Na+ or K+ was at equilibrium across the membrane. The K+-induced overshoot was not due to the presence of a membrane potential alone, as an inwardly directed gradient of choline chloride failed to produce it. The amplitude of the overshoot was increased by raising either the Na+ or K+ concentration outside the membrane or by using more lipophilic anions (reactive order was NO3- greater than SCN- greater than Cl- greater than SO4(2-). Zero-trans efflux studies showed that the uridine transport is bidirectional. Li+ could substitute poorly for Na+ but not at all for K+. Stoichiometries of 1:1 and greater than 1:1 were observed for Na+: uridine and K+: uridine coupling, respectively. A preliminary analysis of the interactions between Na+ and K+ for uridine uptake showed complex interactions which can best be explained by the involvement of two different systems for nucleoside transport in the rat renal brush-border membrane, one requiring Na+ and the other K+ as transport coupler.