Defective ion regulation in a class of membrane-excitation mutants in Paramecium.

The "paranoiac" mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na+, unlike wild-type paramecia, which jerk back and forth in Na+ solutions. The paranoiac mutants in Na+ solutions also show large losses of cellular K+ and large influxes of Na+. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na+ -dependent loss of cellular K+ and a much smaller Na+ influx. In K+ -containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K+ content. The Na+ influx, the K+ loss, and the duration of backward swimming are all proportional to the extracellular Na+ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na+ influxes and the large K+ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

Role of sodium ions in p-aminohippurate transport by newt kidney.

1. The effect of external Na+ concentration on p-aminohippurate uptake by isolated kidneys of newt (Triturus pyrrhogaster) was studied kinetically and electrophysiologically. 2. p-Aminohippurate uptake conformed to Michaelis-Menten type kinetics in regard to both p-aminohippurate and Na+ concentrations in the incubation medium. Kinetic studies revealed that reduction of Na+ concentration increased the values of Kt without altering the maximal rate (V) of p-aminohippurate uptake. The values of Kt were a linear function of the reciprocal of Na+ concentration. These results suggest the presence of interaction between p-aminohippurate and Na+ at the carrier level, i.e. Na+-coupled cotransport. 3. p-Aminohippurate had no effect on the electrical potential difference across the peritubular membrane in both 10 and 100 mM Na+ solutions, suggesting that p-aminohippurate is transported across the peritubular membrane in a form of electrically neutral carrier complex. This is consistent with the results of the kinetic studies. 4. p-Aminohippurate uptake was proportional to the electrochemical potential gradient of Na+ (delta mu Na) across the peritubular membrane. This result indicates that the maintenance of sufficient delta mu Na appears to be necessary for the accumulation of p-aminohippurate against its electrochemical potential gradient, supporting Na+ gradient hypothesis.     

Effects of changes in sodium electrochemical potential gradient on p-aminohippurate transport in newt kidney

1. The relation between p-aminohippurate uptake and the electrochemical potential gradient of Na+ (delta muNa+) across the peritubular membrane was examined in newt (Triturus pyrrhogaster) kidney. The delta muNa+ was modified by changing cellular Na+ concentration and/or lowering the electrical potential difference across the peritubular membrane (peritubular membrane potential) 2. Elevation of external K+ concentration or addition of alanine at 40 mM to the medium decreased the delta muNa+ mainly through the depolarization of the cells. Addition of 1 mM ouabain resulted in a decrease in the peritubular membrane potential and increase in cellular Na+ concentration, thus decrease in the delta muNa+. 3. p-Aminohippurate uptake decreased in proportion to the decrease in the delta muNa+ under all experimental conditions, indicating that the maintenance of the delta muNa+ is required for p-aminohippurate transport. 4. All three different experimental conditions, high medium K+ concentration, 40 mM alanine or 1 mM ouabain, increased the apparent Michaelis constant, Kt, without affecting the maximal uptake rate, V, for p-aminohippurate. These results suggests that the delta muNa+, largely the peritubular membrane potential, may affect the association and/or dissociation of p-aminohippurate and Na+ at both interfaces of the peritubular membrane of the proximal tubular cells.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

A bicarbonate-dependent process inhibitable by disulfonic stilbenes and a Na+/H+ exchange mediate 22Na+ uptake into cultured bovine corneal endothelium

22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10(-4)M) to inhibit active sodium extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10(-3)M) or by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-3)M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10(-4)M) and amiloride (10(-3)M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and sodium concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into sodium-depleted cells was reduced as opposed to sodium-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into sodium-free medium was less than efflux into sodium saline, indicating trans-stimulation by sodium. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into sodium-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10(-3), but not by 10(-5) M amiloride. It is concluded that bovine corneal endothelial cells possess at least two distinct pathways for sodium uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.     

Characterization of the myo-inositol transport system in Trypanosoma cruzi.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.     

Adriamycin-liposome interactions. A magnetic resonance study of the differential effects of cardiolipin on drug-induced fusion and permeability

L-Glutamate and L-aspartate transport into osmotically active intestinal brush border membrane vesicles is specifically increased by Na+ gradient (extravesicular greater than intravesicular) which in addition energizes the transient accumulation (overshoot) of the two amino acids against their concentration gradients. The "overshoot" is observed at minimal external Na+ concentration of 100 mM for L-glutamate and 60 mM for L-aspartate; saturation with respect to [Na+] was observed at a concentration near 100 mM for both amino acids. Increasing amino acid concentration, saturation of the uptake rate was observed for L-glutamate and L-aspartate in the concentration range between 1 and 2 mM. Experiments showing mutual inhibition and transtimulation of the two amino acids indicate that the same Na+ -dependent transport system is shared by the two acidic amino acids. The imposition of diffusion potentials across the membrane vesicles artificially induced by addition of valinomycin in the presence of a K+ gradient supports the conclusion that the cotransport Na+/dicarboxylic amino acid in rat brush border membrane vesicles is electroneutral.     

The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane

In vivo K+, Na+, Ca2+, Cl- and H+ activities in the cytosol and the contractile vacuole fluid, the overall cytosolic osmolarity, the fluid segregation rate per contractile vacuole and the membrane potential of the contractile vacuole complex of Paramecium multimicronucleatum were determined in cells adapted to 24 or 124 mosm l(-1) solutions containing as the monovalent cation(s): 1) 2 mmol l(-1) K+; 2) 2 mmol l(-1) Na+; 3) 1 mmol l(-1) K+ plus 1 mmol l(-1) Na+; or 4) 2 mmol l(-1) choline. In cells adapted to a given external osmolarity i) the fluid segregation rate was the same if adapted to either K+ or Na+, twice as high when adapted to solutions containing both K+ and Na+, and reduced by 50% or more in solutions containing only choline, ii) the fluid of the contractile vacuole was always hypertonic to the cytosol while the sum of the ionic activities measured in the fluid of the contractile vacuole was the same in cells adapted to either K+ or Na+, at least 25% higher in cells adapted to solutions containing both K+ and Na+, and was reduced by 55% or more in solutions containing only choline, iii) the cytosolic osmolarity was the same in cells adapted to K+ alone, to Na+ alone or to both K+ and Na+, whereas it was significantly lower in cells adapted to choline. At a given external osmolarity, a positive relationship between the osmotic gradient across the membrane of the contractile vacuole complex and the fluid segregation rate was observed. We conclude that both the plasma membrane and the membrane of the contractile vacuole complex play roles in fluid segregation. The presence of external Na+ moderated K+ uptake and caused the Ca2+ activity in the contractile vacuole fluid to rise dramatically. Thus, Ca2+ can be eliminated through the contractile vacuole complex when Na+ is present externally. The membrane potential of the contractile vacuole complex remained essentially the same regardless of the external ionic conditions and the ionic composition of the fluid of the contractile vacuole. Notwithstanding the large number of V-ATPases in the membrane of the decorated spongiome, the fluid of the contractile vacuole was found to be only mildly acidic, pH 6.4.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

A novel Na+/HCO3--codependent choline transporter in the syncytial epithelium of the cestode Hymenolepis diminuta

Absorption of exogenous choline by the cestode Hymenolepis diminuta was found to be both Na+- and HCO3--dependent and, at pH 6 to 7, accounted for up to 65% of the total choline uptake. Na+/HCO3- dependent choline uptake was activated at approximately 6 mM HCO3- (EC50 approximately 9 mM), and, above 100 mM Na+, the rate of uptake was directly proportional to the Na+ concentration. Atempts to uncouple Na+-dependent uptake from HCO3--dependent uptake were not successful: K+-depolarization was without effect on HCO3--dependent choline uptake, and use of valinoomycin to hyperpolarize the brush-border membrane resulted in inhibition of uptake. Na-/HCO3--dependent choline uptake was not associated with solvent drag. The Na+/HCO3--dependent choline uptake displayed a Q10 of 6.4 (27 degrees to 37 degrees) and a relatively high activation energy of 126 kJ x mol(-1). At pH 6.0 and 7.0, Na-/HCO3--dependent choline uptake rates were similar, but Na+/HCO3--dependent choline uptake was reduced at pH 5.0. The Na+/HCO3--dependent choline uptake, at pH 7.0, displayed a Kt of approximately 500 microM and a Vmax of 4.01 pmol x mg wet weight(-1) x min(-1). The Na+/HCO3--dependent choline uptake was hemicholinium-3 sensitive, but not significantly inhibited by 200 microM bumetanide, 100 microM amiloride, benzamil, or EIPA or by 1 mM 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) or 4-acetamido-4'-isothiocvanostilbene-2,2'-disulfonic acid (SITS). Although it remains to be shown that HCO3- uptake is coupled directly to both choline and Na+ uptake, the data suggest that choline up take occurs via choline/Na+/HCO3--co-trans porter.     

Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli.

Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport.     

Sodium-amino acid cotransport by type II alveolar epithelial cells

Type II alveolar epithelial cell monolayers have been shown to actively transport sodium (Na+). Coupling to amino acid uptake could be an important mechanism for Na+ entry into these cells. This study demonstrates the presence of such a coupled cotransport mechanism in the plasma membrane of isolated type II cells by use of the nonmetabolizable amino acid analogue alpha-methylaminoisobutyric acid (MeAIB). Transport of MeAIB in 137 mM Na+ is saturable, with the uptake constant (Vmax) equaling 13.9 pmol X mg prot-1 X s-1 and the Michaelis-Menten constant (Km) equaling 0.13 mM. In the presence of Na+, MeAIB is accumulated against a concentration gradient. MeAIB uptake in the absence of Na+ is linear with MeAIB concentration, as expected for simple diffusion. The Hill coefficient for Na+-MeAIB cotransport is 1.11, suggesting a 1:1 stoichiometry. Proline inhibits Na+-MeAIB cotransport, with Ki equaling 0.5 mM. These findings suggest that Na+-amino acid cotransport may be an important pathway for Na+ (and/or amino acid) uptake into type II alveolar epithelial cells.     

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.     

The stimulation of Na+ uptake in frog skin by uranyl ions.

1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane.     

Preservation of sodium-dependent iodide transport activity by methimazole and mercaptoethanol in phospholipid vesicles containing thyroid plasma membranes: with evidence of difference in the action of perchlorate and thiocyanate

The effect of methimazole (MMI) and 2-mercaptoethanol (ME) on I-transport was studied using phospholipid vesicles (P-vesicles) made from porcine thyroid plasma membranes and soybean phospholipids by sonication. 1. When buffer solutions contained either 1 mM MMI or 2 mM ME, I-uptake by P-vesicles in the presence of external Na+ was apparently higher than that in the absence of external Na+. Na+-dependent I- uptake was inhibited by both C1O4- and SCN- added externally. 2. When PM was treated with 4 mM N-ethylmaleimide prior to preparation of P-vesicles, the activity of Na+-dependent I- transport was completely lost even when P-vesicles were incubated in the presence of ME. 3. When neither MMI nor ME was added to buffers, I- uptake in the presence of external Na+ was not at all higher than that in the absence of external Na+. In these instances, however, I- uptake was much higher compared than the baseline uptake in the presence of MMI or ME, and was inhibited by external SCN- and not by C1O4- without relation to external Na+. These data indicate that MMI or ME has two distinct effects on our model system of I- transport. The one is preservation of the Na+-dependent I- transport activity by protecting a sulfhydryl group, and the other is reduction of nonspecific I- binding to P-vesicles. In addition, C1O4- is a more specific inhibitor of thyroid I- transport than SCN-, when non-specific I- oxidation is imperfectly prevented.     

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.     

Differential responses of synaptosomal cation movements and respiration to the convulsants penicillin, pentylenetetrazol and 3-aminopyridine

The effect of three convulsants was tested in rat cortical synaptosomes by measuring Na+ and K+ movements and respiration. Penicillin (of its derivatives the 3-phenyl-5-methyl-4-isoxazolyl-penicillin, i.e. oxacillin was used) at 2 to 14 mM concentration prolonged the uptake phase of the K+-curve without altering final cation equilibrium. Pentylenetetrazol, at 10 to 50 mM exerted a concentration-dependent inhibitory effect on K+-uptake, while 3-aminopyridine was effective only at high concentrations (50 mM) preventing synaptosomal K+-uptake and increasing the synaptosomal Na+-content without, however, blocking respiration. The data suggest a transitional rise of K+-permeability of synaptosomal membrane in the presence of oxacillin, without lasting depolarization. Sufficient doses of 3-aminopyridine probably depolarize synaptosomes, the underlying mechanism probably being associated with the primary blocking of K+-channels and a subsequent increase in Na+ influx rather than with the inhibition of the Na+--K+-pump.