alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

Proline uptake through the major transport system of Salmonella typhimurium is coupled to sodium ions.

Strains of Salmonella typhimurium deficient in one or more of the proline transport systems have been constructed and used to study the mechanism of energy coupling to transport. Proline uptake through the major proline permease (PP-I, putP) is shown to be absolutely coupled to Na+ ions and not to H+ ions as has previously been assumed. Transport through the minor proline permease (PP-II, proP), however, is unaffected by the presence or absence of Na+. The effect of Na+ on the kinetics of proline uptake shows that external Na+ increases the Vmax for transport. It seems probable that proline transport through PP-I is also coupled to Na+ ions in Escherichia coli.     

Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems

We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CHO-K1 pro- cells. Mutants alar2 and alar3, isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro-, stable, cross resistant to alpha-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake. Ethyl methane sulfonate (EMS) increases the frequency of pro- alar clones in the population by at least 50 times the spontaneous frequency. The increased velocity of proline transport by alar2 and alar3 can be attributable to the 1.5 to 3 times increase in velocity of transport of proline through systems A, ASC, and P. The Vmax for proline transport through the A system has increased two times for alar2 while the Km and Vmax for alar3 has increased by 1.4 and 2.3 times that of CHO-K1. There is a corresponding increase in Vmax of proline transport by alar2 through the P system. The P system is defined operationally as that portion of the Na+-dependent velocity that remains when the A, ASC, and glutamine-inhibitable fraction are eliminated. The system is concentrative. Proline appears to be the preferred substrate. Li+ cannot be substituted for Na+. The system is moderately dependent upon pH. It obeys Michaelis-Menten kinetics and is not derepressible by starvation. There is no evidence for an N system in CHO-K1.     

Identification of mammalian proline transporter SIT1 (SLC6A20) with characteristics of classical system imino

Amino acid homeostasis depends on specific amino acid transport systems, many of which have been characterized at the molecular level. However, the classical System IMINO, defined as the Na+-dependent proline transport activity that escapes inhibition by alanine, had not been identified at the molecular level. We report here the functional characteristics and tissue distribution of Sodium/Imino-acid Transporter 1 (SIT1), which exhibits the properties of classical System IMINO. SIT1, the product of the slc6a20 gene, is a member of the SLC6 Na+- and Cl--dependent neurotransmitter transporter family whose function has remained unknown. When expressed in Xenopus oocytes, rat SIT1 mediated the uptake of imino acids such as proline (K0.5 approximately 0.2 mM) and pipecolate, as well as N-methylated amino acids (e.g. MeAIB, sarcosine). SIT1-mediated proline transport was pH-independent and insensitive to inhibition by alanine or lysine. Proline transport was Na+-dependent, Cl--stimulated, and voltage-dependent. Li+, but not H+, could substitute for Na+. Human SIT1 also functioned as a Na+-dependent proline transporter. Rat SIT1 mRNA was expressed in epithelial cells of duodenum, jejunum, ileum, stomach, cecum, colon, and kidney proximal tubule S 3 segments. SIT1 mRNA was also expressed in the choroid plexus, microglia, and meninges of the brain and in the ovary. Previous reports have documented the marked urinary hyperexcretion of proline in newborn rodents and man. We found that SIT1 was dramatically up-regulated in the kidneys of 3-day-old mice, accounting for the maturation of proline reabsorption in the mouse. The human slc6a20 gene coding SIT1 is an appropriate target for investigation of hereditary forms of iminoaciduria in man.     

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H(+)/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5' and 3' untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na(+)-independent uptake of proline, glycine, l-alanine and alpha-(methylamino)isobutyric acid. Proline uptake was maximal at pH 5.0 (K(m) 2.2+/-0.7 mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na+ but in the presence of an inwardly directed H+ gradient. In the presence of Na+ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

Active potassium transport coupled to active sodium transport in vesicles reconstituted from purified sodium and potassium ion-activated adenosine triphosphatase from the rectal gland of Squalus acanthias.

Vesicles containing a purified shark rectal gland (sodium + potassium)-activated adenosine triphosphatase-(NaK ATPase) were prepared by dialyzing for 2 days egg lecithin, cholate, and the NaK ATPase purified from the rectal gland of Squalus acanthias. These vesicles were capable of both Na+ and K+ transport. Studies of K+ transport were made by measuring the ATP-stimulated transport outward of 42K+ or 86Rb+. Vesicles were preloaded with isotope by equilibration at 4 degrees for 1 to 3 days. Transport of 42K+ or 86Rb+ was initiated by addition of MgATP to the vesicles. The ATP-dependent exit of either isotope was the same. Experiments are presented which show that this loss of isotope was not due to changes in ion binding but rather due to a loss in the amount of ion trapped in the vesicular volume. The transport of K+ was dependent on external Mg2+. CTP was almost as effective as ATP in stimulating K+ transport, while UTP was relatively ineffective. These effects of nucleotides parallel their effects on Na+ accumulation and their effectiveness as substrates for the enzyme. Potassium transport was inhibited by ouabain and required the presence of Na+. The following asymmetries were seen: (a) addition of external Mg2+ supported K+ transport; (b) ouabain inhibited K+ transport only if it was present inside the vesicles; (c) addition of external Na+ to the vesicles stimulated K+ transport. External Li+ was ineffective as a Na+ substitute. The specific requirement of external Na+ for K+ transport indicates that K+ exit is coupled to Na+ entry. Changes in the internal vesicular ion concentrations were studied with vesicles prepared in 20 mM NaCl and 50 mM KCl. After 1 hour of transport at 25 degrees, a typical Na+ concentration in the vesicles in the presence of ATP was 72 mM. A typical K+ concentration in the vesicles was 10 mM as measured with 42K+ or 6 mM as measured with 86Rb+. The following relationships have been calculated for Na+ transport, K+ transport and ATP hydrolysis: Na+/ATP = 1.42, K+/ATP =1.04, and Na+/K+ = 1.43. The ratio of 2.8 Na+ transported in to 2 K+ transported out is very close to the value reported for the red cell membrane. Potassium-potassium exchange similar to that observed in the red cell membrane and attributed to the Na+-K+ pump (stimulated by ATP and orthophosphate and inhibited by ouabain) was observed when vesicles were prepared in the absence of Na+. The results reported in this paper prove that the shark rectal gland NaK ATPase, which is 90 to 95% pure, is the isolated pump for the coupled transports of Na+ and K+.     

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.     

Influence of proliferative rates and a system substrate availability on proline transport in primary cell cultures of the R3230AC mammary tumor

Regulation of A system amino acid transport was studied in primary cultures of the R3230AC mammary adenocarcinoma. Higher rates of carrier-mediated Na⁺-dependent proline transport, v^c, was decreased and was attributed to a two-fold decrease in Vmax and a two-fold increase in Km. When compared to cells grown in standard media (Eagle's minimal essential medium, MEM), cells grown in media supplemented with A system substrates (alanine, serine, glycine, and proline) demonstrated adaptive decreases in proline transport; the decrease was due to two-fold reduction in Vmax, with no change in Km for proline. Even in the presence of preferred substrates for the A system, a density-dependent decrease in proline transport was manifested. Both fast- and slow-growing cultures maintained in MEM exhibited rapid increases in proline transport when switched to buffers devoid of amino acids; two-fold increases in Vmax were seen within 4 hr, but Km was unchanged. This starvation-induced adaptation was completely prevented by inclusion in the buffer of 10 mM proline, 0.1 mM -(methylamino)-isobutyric acid (MetAIB) or 10 mM serine, whereas inclusion of the poorer A system substrate, phenylalanine (10 mM), had no effect. The effects of MetAIB to prevent starvation-induced increases in proline transport were dose-related, rapid, and reversible. Amino acid starvation-induced increases in proline transport were partially blocked by cycloheximide or actinomycin D. Data were obtained demonstrating a temporal relationship between increasing intracellular [proline] and decreasing v^c for proline uptake. In addition, efflux of proline from preloaded cells preceded the increase in initial rates of proline entry. Taken together, we concluded that: (1) A system transport in primary cultures of this mammary adenocarcinoma is regulated by cell density as well as by availability of A system substrates, but these two types of regulation are kinetically distinct; and (2) starvation-induced enhancement of proline transport appears to be due to release from transinhibition, but may also involve a derepression-repression type of mechanism.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles.

The sodium-dependent entry of proline and glycine into rat renal brush-border membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry.     

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

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.     

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.     

Saturable ammonium ion transport in myeloma and hybridoma cells is mediated by the Na+K+2Cl--cotransporter

The total entry of ammonium ions into Sp2/0-Ag14 myeloma cells and hybridoma cells consists of a saturable and a non-saturable component. The plasma membrane Na+K+2Cl--cotransporter was identified as the saturable ammonium ion transporter in both cell lines, and the non-saturable entry was due to simple diffusion of ammonium ions. The theoretical maximum transport rate via the Na+K+2Cl--cotransporter was identical in the two cell lines, but the ammonium ion diffusion rate was considerably higher in the hybridoma cells. We speculate that this is an effect of different membrane properties caused by dissimilar expression of tumour characteristics.     

Electrophysiology of L-lysine entry across the brush-border membrane of Necturus intestine

Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.     

L-threonine transport in pig jejunal brush border membrane vesicles. Functional characterization of the unique system B in the intestinal epithelium.

Uptake and inhibitory kinetics of [3H]L-threonine were evaluated in preparations of pig jejunal brush border membrane vesicles. Uptake of [3H]L-threonine under O-trans, Na+ gradient, and O-trans, Na(+)-free conditions was best described by high affinity transport (Km < 0.01 mM) plus a nonsaturable component. The maximal velocity of transport was 3-fold greater under Na+ gradient conditions. 100 mM concentrations of all of the dipolar amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid caused complete inhibition of [3H]L-threonine transport under Na+ gradient and Na(+)-free conditions. Imino acids, anionic amino acids, cationic amino acids, and methylamino-isobutyric acid caused significant partial inhibition of L-threonine uptake. Inhibitor concentration profiles for proline and lysine were consistent with low affinity competitive inhibition. The Ki values of alanine and phenylalanine approximated 0.2 and 0.5 mM, respectively, under both Na+ gradient and Na(+)-free conditions. These data indicate that the transport system available for L-threonine in the intestinal brush border membrane (system B) is functionally distinct from other amino acid transport systems. Comparison of kinetics parameters in the presence and absence of a Na+ gradient suggests that both partially and fully loaded forms of the carrier can function to translocate substrate and that Na+ serves to accelerate L-threonine transport by a mechanism that does not involve enhanced substrate binding.     

Phosphorylation of Na,K-ATPase by acetyl phosphate and inorganic phosphate. Sidedness of Na+, K+ and nucleotide interactions and related enzyme conformations.

The effects of K+, Na+ and nucleotides (ATP or ADP) on the steady-state phosphorylation from [32P]Pi (0.5 and 1 mM) and acetyl [32P]phosphate (AcP) (5 mM) were studied in membrane fragments and in proteoliposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20 degrees C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K+ and to 20% by 10 mM K+ plus 1 mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K+ but was 30% increased by 50 mM Na+. In proteoliposomes E-P formation from Pi was (i) not influenced by 5-10 mM K+cyt or 100 mM Na+ext, (ii) about 50% reduced by 5, 10 or 100 mM K+ext and (iii) completely prevented by 50 mM Na+cyt. Enzyme phosphorylation from AcP was 30% increased by 10 mM K+cyt or 50 mM Na+cyt; these E-P were 50% reduced by 10-100 mM K+ext. However, E-P formed from AcP without K+cyt or Na+cyt was not affected by extracellular K+. Fluorescence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E2 state in the presence of 10 mM Na+ or 2 mM K+ but to an E1 state in the absence of both cations. With pNPP, the data indicated an E1 state in the absence of Na+ and K+ and also in the presence of 20 mM Na+, and an E2 form in the presence of 5 mM K+. These results suggest that, although with some similarities, the reversible Pi phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.     

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.     

Phosphorylation of Na+, K(+)-ATPase by ATP in the presence of K+ and dimethylsulfoxide but in the absence of Na+

Purified Na+, K(+)-ATPase was phosphorylated by [gamma-32P]ATP in a medium containing dimethylsulfoxide and 5 mM Mg2+ in the absence of Na+ and K+. Addition of K+ increased the phosphorylation levels from 0.4 nmol phosphoenzyme/mg of protein in the absence of K+ to 1.0 nmol phosphoenzyme/mg of protein in the presence of 0.5 mM K+. Higher velocities of enzyme phosphorylation were observed in the presence of 0.5 mM K+. Increasing K+ concentrations up to 100 mM lead to a progressive decrease in the phosphoenzyme (EP) levels. Control experiments, that were performed to determine the contribution to EP formation from the Pi inevitably present in the assays, showed that this contribution was of minor importance except at high (20-100 mM) KCl concentrations. The pattern of EP formation and its KCl dependence is thus characteristic for the phosphorylation of the enzyme by ATP. In the absence of Na+ and with 0.5 mM K+, optimal levels (1.0 nmol EP/mg of protein) were observed at 20-40% dimethylsulfoxide and pH 6.0 to 7.5. Addition of Na+ up to 5 mM has no effect on the phosphoenzyme level under these conditions. At 100 mM Na+ or higher the full capacity of enzyme phosphorylation (2.2 nmol EP/mg of protein) was reached. Phosphoenzyme formed from ATP in the absence of Na+ is an acylphosphate-type compound as shown by its hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the alpha-subunit of the Na+, K(+)-ATPase as demonstrated by acid polyacrylamide gel electrophoresis followed by autoradiography.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH4+ transport

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium- sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)- NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.