Na+ transport by human placental brush border membranes: Are there several mechanisms?

Na⁺ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na⁺ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na⁺ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na⁺ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na⁺ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na⁺ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na⁺ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na⁺ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na⁺. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na⁺ uptake, whereas benzamil, the Na⁺ channel blocker, had no effect, it was concluded that the amiloride sensitive Na⁺ uptake was predominantly or exclusively due to a Na⁺-H⁺ exchanger activity. K⁺ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na⁺ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10⁻⁷ M insulin, 10⁻¹⁰ M angiotensin II, or 10⁻⁸ M human parathyroid hormone (PTH) did not influence Na⁺ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na⁺-H⁺ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na⁺-H⁺ exchange is probably the unique mechanism of Na⁺ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Activation of Na+,H+-Exchange in Erythrocytes of the River Lamprey Lampetra fluviatilis

To study H⁺ transport, the lamprey red blood cells were acidified to pH 6.0 by a pretreatment with an ionophore, nigericin. Incubation of the acidified cells in NaCl-medium at pH 8.0 was accompanied by a rapid H⁺ efflux from the erythrocytes. There was a tenfold decrease of the H⁺ efflux rate on addition to NaCl-medium of dimethylamiloride or on replacing Na⁺ in the medium (KCl-medium, pH 8.0). A high rate of Na+ influx into the acidified erythrocytes occurred only in the presence of H⁺ gradient (pH medium 8.0), but not in its absence (pH medium 6.0). The Na⁺-dependent H⁺ efflux from the cells and H⁺-dependent Na⁺ influx into the cells were quantitatively similar (about 700 mmol/l cells/h). A rapid elevation of the intracellular Na⁺ concentration as measured by flame photometry was also observed during incubation of the acidified cells in NaCl-medium (pH 8.0). The H⁺-dependent Na⁺ influx and an increase of the Na⁺ content in the acidified cells were significantly inhibited by amiloride. The data obtained for the first time prove with certainty the presence of the Na⁺/H⁺ exchanger in erythrocytes of the river lamprey.     

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the β-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na⁺ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K⁺ or Li⁺ do not increase taurine accumulation more than Na⁺-free mannitol, except that the combination of external K⁺ and Na¹ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN⁻ and NO₃⁻) or less permeant (SO₄²⁻), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl⁻ stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO₃ and LiSO₄. Internal LiCl, regardless of the final Na⁺ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO₃ or LiSO₄ with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl⁻ in taurine uptake in addition to Na⁺ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H⁺ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na⁺/H⁺ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na⁺ concentrations. Taurine uptake is blocked only by other β-amino acids and in a competitive fashion. d-glucose and p-aminohippurate at high concentrations (> 10⁻³ M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of d-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal β-amino acid transport system in brush-border vesicles and indicate a role for external Cl in this uptake system.     

Nature of the light-induced h efflux and na uptake in cyanobacteria

We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H⁺ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C_i). Light and C_i stimulated the uptake of sodium, but the stimulation by C_i was observed only when C_i was present at the time sodium was provided. Amiloride, a potent inhibitor of Na⁺/H⁺ antiport and Na⁺ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na⁺/H⁺ antiport and Na⁺ uniport.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Amiloride inhibits protein synthesis in isolated rat hepatocytes

The effects of amiloride on Na⁺ ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of ²²Na⁺ uptake and the amount of ²²Na⁺ taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1?¹⁴C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [¹⁴C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na⁺ in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na⁺, and that amiloride is an efficient inhibitor of protein synthesis.     

Reversal of Na+-dependent glycine transport in sheep reticulocyte membrane vesicles

Inside-out membrane vesicles have been prepared from sheep reticulocytes. With these vesicles, Na⁺-dependent glycine uptake and net accumulation have been demonstrated to occur in reverse, i.e., from extravesicular (normal cytoplasmic) to intravesicular (normal extravesicular) surface. Uptake and accumulation are inhibited by energization of the sodium pump by ATP whereby the Na⁺ electrochemical gradient is dissipated. Glycine-dependent Na⁺ uptake was also observed, providing evidence that Na⁺-dependent glycine influx into these vesicles, equivalent to normal efflux, is characterized by Na⁺-glycine co-transport.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+-and H+-gradients

In the presence of a Na⁺-gradient (out > in), l-glutamic acid and l-and d-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na⁺ by K⁺. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na⁺-dependent uphill transport of l-glutamic acid was stimulated by K⁺ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K⁺-gradient (in > out). Quantitative and qualitative differences in the K⁺ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in $\text{Na}{}^{\mathrm{+}}\text{-(out > in) + K}{}^{\mathrm{+}}\text{-(in > out)}$ gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na⁺-gradient and the Na⁺ + K⁺-gradient-dependent transport of l-glutamic acid. However, pH- or K⁺-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of l-glutamic acid transport in which Na⁺ and K⁺ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na⁺ in the protonated or unprotonated forms, respectively. We also propose that K⁺ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.     

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

Summary Dicyclohexylcarbodiimide (DCCD) and the 5-ethylisopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na⁺/H⁺ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na^–/H⁺ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na⁺, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na⁺-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na⁺/H⁺ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na⁺ and Li⁺ protected. [¹⁴C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na⁺ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [¹⁴C]-DCCD that was identified as the -subunit of the Na⁺, K⁺-ATPase. [¹⁴C]-Br-EIPA was mainly incorporated into a brushborder membrane polypeptide with apparent molecular weight of 65,000. Na⁺ decreased the labeling of this protein. Similar to the Na⁺/H⁺ exchanger this Na⁺-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na⁺/H⁺ exchange.     

Collapse of ATP-Induced pH Gradient by Sodium Ions in Microsomal Membrane Vesicles Prepared from Atriplex gmelini Leaves: Possibility of Na/H Antiport

Sealed microsomal membrane vesicles were prepared from leaves of a 250 millimolar NaCl-grown halophyte (Atriplex gmelini C. A. Mey). The vesicles exhibited ATP-dependent proton-transporting activity which was inhibited 60% by NO₃⁻ (50 millimolar) but not by vanadate (100 micromolar) and 23% by oligomycin (10 micrograms per milliliter), suggesting that tonoplast-derived vesicles were the major constituents of the preparation. The pH gradient established by the vesicles by ATP in the presence of oligomycin collapsed upon the addition of Na⁺ salts. The vesicles took up Na⁺ ions in the presence of ATP and this activity was canceled by gramicidin. These results suggest that Na⁺ ions were taken up by the vesicles via a Na⁺-specific uptake system, possibly a Na⁺/H⁺ antiport.     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Sulfate transport by mouse renal cortical slices does not represent uptake by brush-border membrane

We measured uptake of isotopically ³⁵S-labelled sulfate anion by slices and by brush-border membrane vesicles prepared from mouse renal cortex to identify: (i) whether metabolic incorporation of anion influences net transport; (ii) which membrane is primarily exposed in the renal cortex slice. Slices accumulated sulfate without significant incorporatoin into metabolic pools. Net uptake of sulfate at 0.1 mM by the slice occurred against an electrochemical gradient as determined by mesurement of free intracellular sulfate concentration, the isotopic distribution ratio at steady-state, and the distribution of lipophilic ions (TPP⁺ and SCN^?). Carrier mediation of sulfate transport in the slice was confirmed by observing concentration-dependent saturation of net uptake and counter-transport stimulation of efflux. Anion uptake was Na⁺-independent, K⁺- and H⁺-stimulated, and inhibited by disulfonated stilbenes. Brush-border membrane vesicles accumulated sulfate by a saturable mechanism dependent on a Na⁺ gradient (outside > inside); others have shown that uptake of sulfate by brush-border membrane vesicles is insensitive to inhibition by disulfonated stilbenes. These findings indicate that different mechanisms serve sulfate transport in renal cortex slice and brush-border membrane vesicle preparations. They also imply that the slice exposes an epithelial surface different from the brush-border, presumably the basolateral membrane, or its equivalent, since sulfate transport by slices resembles that obserbed with isolated basolateral membrane vesicles.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Destruction of highly purified cytochromes P-450 associated with metabolism of fluorinated ether anesthetics

The effects of ethanol and acetaldehyde on rat intestinal microvillus membrane integrity and glucose transport function were examined in vitro with purified membrane vesicles. Ethanol could influence glucose transport function by alterations in the conformation of the carrier, the lipid environment surrounding the carrier, or in the transport driving force (Na⁺ electrochemical gradient). Due to the rapid nature of glucose uptake, transport was assayed with the use of an apparatus that permitted uptake measurements as early as 1 s. Ethanol (340 mm) partially and acetaldehyde (44 mm) completely inhibited concentrative glucose uptake throughout the 1-min time course. Their inhibitory effects were reversible and irreversible, respectively. Kinetic measurements made during the initial rate of uptake (at 2 s) with various concentrations of glucose (0.05–8 mm) showed that ethanol and acetaldehyde both caused a decrease in V. Although ethanol did not substantially alter the transport K_m, acetaldehyde increased the K_m almost 50%. To determine whether ethanol or acetaldehyde directly interfered with glucose carrier function, uptake was measured in the presence of equilibrated Na⁺. Only acetaldehyde had a significant inhibitory effect under these conditions. Membrane permeability, as determined by efflux of preloaded 6-carboxyfluorescein dye, increased upon exposure of the vesicles to ethanol or acetaldehyde. Membrane fluidity measurements by fluorescence polarization showed that only acetaldehyde had a significant fluidizing effect. These results indicate that ethanol and acetaldehyde acted to perturb membrane integrity and inhibited glucose uptake indirectly by allowing the Na⁺ gradient to dissipate. Acetaldehyde, which had a stronger inhibitory effect than ethanol, appeared also to directly inhibit carrier function.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na⁺ gradient ([Na⁺]outside > [Na⁺]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.     

Transport of β-alanine in renal brush border membrane vesicles

The findings that the equilibrium uptake of β-alanine decreased with increasing medium osmolarity and preincubation with β-alanine increased uptake of the amino acid indicate that the uptake of β-alanine by rabbit renal brush border membranes represents transport into membrane vesicles. A Na⁺ electrochemical gradient (extravesicular > intravesicular) stimulated the initial rate of β-alanine uptake about three times and effected a transient accumulation of the amino acid twice the equilibrium value. Stimulation of the uptake was specific for Na⁺. Gramicidin abolished the overshoot, presumably by dissipating the gradient by accelerating the electrogenic entrance of Na⁺ into the vesicle via a pathway not coupled to uptake of β-alanine. In K⁺-loaded vesicle, valinomycin enhanced the Na⁺ gradient-dependent uptake of β-alanine. These findings indicate that the Na⁺ gradient-dependent transport of β-alanine is an electrogenic process and suggest that the membrane potential is a determinant of β-analine transport. Uptake of β-aniline, at a given concentration, reflected the sum of contributions from Na⁺ gradient-dependent and -independent transport systems. The dependent system saturated at 100 μM. The independent system did not saturate. At physiological concentrations the rate of the Na⁺ gradient-dependent uptake was four times that in the absence of the gradient. The Na⁺ gradient-dependent rate of β-alanine uptake was strongly inhibited by taurine, suggesting that β-amino acids have a common transport system, α-Amino acids, i.e. l-arginine, l-glutamate, l-proline, and glycine, representing previously reported specific α-amino acid transport systems in the brush border membrane, did not inhibit the uptake of β-alanine. These findings indicate that the brush border membrane has a distinct transport system for β-amino acids.