Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

Taurine and β-alanine uptake in primary astrocytes differentiating in culture: Effects of ions

The effects of ions on taurine and -alanine uptake were studied in astrocytes during cellular differentiation in primary cultures. The uptakes were strictly Na⁺-dependent and also inhibited by the omission of K⁺ and in the presence of ouabain suggesting that their transport is fuelled mainly by these cation gradients. Two sodium ions were associated in the transport of one taurine and -alanine molecule across cell membranes. A reduction in Cl^– concentration also markedly inhibited the uptake of both amino acids, indicating that this anion is of importance in the transport processes. The similar ion dependency profiles of taurine and -alanine uptake corroborate the assumption that the uptake of these amino acids in astrocytes is mediated by the same carrier. In Na⁺- and K⁺-free media both taurine and -alanine uptakes were reduced significantly more in 14-day-old or older than in 7-day-old cultures. No significant changes occurred in the coupling ratio between Na⁺ and taurine or -alanine as a function of spontaneous cellular differentiation or upon dBcAMP treatment. These results suggest that the uptake systems of these structurally related amino acids in astrocytes have reached a relatively high degree of functional maturity by two weeks in culture.     

Different properties of two brain extracts separated in sephadex G-50 that modify synaptosomal ATPase activities

We have previously reported that Na⁺,K⁺-ATPase of nerve ending membranes is stimulated by catecholamines only in the presence of a brain soluble fraction. The filtration of this soluble fraction through Sephadex G-50 permitted the separation of two extracts of maximal UV absorbance (peaks I and II) which showed different effects on ATPases. Peak I stimulated both Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities and peak II inhibited Na⁺,K⁺-ATPase activity. We have now studied the activity of ATPases in the presence of the whole eluate obtained from the Sephadex G-50 column. It was observed that maximal effects on ATPases were obtained with peaks I and II. Peak I and peak II fractions were unable to modify the activity of acetylcholinesterase or 5-nucleotidase present in the synaptosomal membranes. The stimulatory effect of peak I on ATPases was concentration dependent (up to 1100), it was stable at different pHs and it was reverted by catecholamines. The inhibitory effect of peak II on Na⁺,K⁺-ATPase was concentration dependent (up to 150,000), it was stable only at acid pH, and it was partially reverted by catecholamines. These findings indicate that the factors responsible for the effects of peaks I and II have different properties and that their actions on ATPases show enzyme specifity.     

Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells

Summary. The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B^0,+ and y⁺) were evaluated in cells treated with IL-1β or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y⁺ and B^0,+ respectively, were examined. L-arginine transport was estimated with ³H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1β depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]_i < 10 μM) was 41.1 ± 18 μM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y⁺ and B^0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1β or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48 h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools. Received February 7, 1999; Accepted June 21, 1999     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Protein kinase C activates the renal apical membrane Na+/H+ exchanger

Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na⁺/H⁺ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm ²²Na⁺ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of²²Na⁺ taken as a measure of the activity of the Na⁺/H⁺ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of²²Na⁺ uptake TPA did not affect the amiloride-insensitive component of²²Na⁺ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na⁺/H⁺ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na⁺/H⁺ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[³²P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

Na-ATPase in spontaneous hypertensive rats: Possible AT1 receptor target in the development of hypertension

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na⁺-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na⁺-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10^− 12 to 10^− 6 mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT₂ receptor, PD123319 (10^− 8 mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT₂-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4-14) prevents the increase in Na⁺-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT₁ receptor activation in SHR and increased ouabain-insensitive Na⁺-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.     

Taurine transport by brush border membrane vesicles isolated from the flounder kidney

Summary Taurine transport was investigated in brush border membrane vesicles isolated from renal tubules of the winter flounder (Pseudopleuronectes americanus). Taurine uptake by the vesicles was greater in the presence of NaCl as compared to uptake in KCl. The Na⁺-dependent taurine transport was electrogenic and demonstrated tracer replacement and inhibition by -alanine and HgCl₂, indicating the presence of Na⁺-dependent, carrier-mediated taurine transport. In contrast to Na⁺-dependent taurine transport across the basolateral membrane, there was not a specific Cl^– dependency for transport in the brush border membrane. No evidence was obtained for Na⁺-independent carrier-mediated taurine transport. The possible involvement of the brush border Na⁺-dependent transport system in the net secretion of taurine from blood to tubular lumen in vivo (Schrock et al. 1982) is discussed.     

Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status

The characteristics of valine uptake by isolated microcolonies of Galaxea fascicularis (Linnaeus 1758) were studied under various conditions including light, dark and feeding. The results demonstrated the presence of: (1) a linear component which might represent either a diffusional transport or a low-affinity carrier-mediated transport (apparent carrier affinity >250 mol·l^–1), and (2) a high-affinity active carrier-mediated transport (apparent carrier affinity about 5 mol·l^-1). The latter is mediated by two different systems: (i) a Na⁺-dependent carrier, stimulated by light and operative in both fed and unfed polyps, and (ii) a Na⁺-independent carrier, light insensitive and present only in unfed polyps. Competition experiments with other amino acids show that the Na⁺-dependent carrier is highly specific for neutral amino acids, as indicated by the high inhibition constants of basic and acidic amino acids. Our results suggest that the energy supplied by zooxanthellae photosynthates is necessary for the process of amino acid uptake, and that the Na⁺-dependent carrier responsible for valine uptake by G. fascicularis is similar to the B^0,+ system.Abbreviations AA amino acid(s) - AC/HC ratio autotrophic/heterotrophic carbon - ASW artificial sea water - DOM dissolved organic material - HPLC high performance liquid chromatography - K ₁ apparent inhibition constant - K _m apparent affinity of the carrier - SE standard error - V _max maximal rate of absorption     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Wirkung von K+ auf die Fluxe und den Transport von Na+ in Gerstenwurzeln,K+-stimulierter Na+-Efflux in der Wurzelrinde

Summary Barley roots grown on a nutrient solution containing 1 mM Na⁺ but no K⁺ are capable of a considerable Na⁺ transport via the symplasm of the root and the xylem vessels. K⁺ added to the medium surrounding the root cortex severely inhibits this transport after a lag period at a high rate constant (Fig. 3).It is likely that the fluxes of Na⁺ are changed drastically during this transition from low to high K⁺ status. Although originally limited to steady state fluxes, the extended method of efflux analysis for excised roots (Pitman, 1971) has been applied to the non-steady fluxes which occur upon the addition of K⁺ to the roots. It is shown that besides other changes the efflux of ²²Na⁺ through the cortex of barley roots is stimulated instantaneously (Fig. 5) by the addition of K⁺ and presumably by an influx of K⁺ ions. From this a transient, K⁺-stimulated Na⁺ efflux at the plasmalemma of the cortical cells can be estimated. It amounts to 10.9 moles/g fw · h compared to the control efflux of 3.3 moles/g fw · h without K⁺.The stimulated efflux is attributed to a Na⁺ efflux pump at the plasmalemma and is thus related to the K-Na-selectivity of barley plants. The inhibition of the Na⁺ transport by K⁺ is probably a consequence of this increased efflux of Na⁺ from the symplasm through the root cortex.