Intravesicular Space Research Articles

Carrier-mediated transport system for choline and its related quaternary ammonium compounds on rat intestinal brush-border membrane

The characteristics of the intestinal transport system for choline were investigated using isolated brush-border membrane vesicles from rat small intestine. In spite of the diminutive lipid solubility, the uptake of choline by membrane vesicles reflected smooth permeation into intravesicular space rather than the binding to the membrane surface. Physiological conditions, present in the intact intestine, such as an inward-directed Na + or H + gradient and inside negative membrane potentials, didn't directly involve in choline transport across the brush-border membrane. Moreover, an outward-directed H + gradient had no significant effect on the time course of choline transport. However, in the absence of a driving-force, the initial uptake of choline exhibited a saturable manner. A kinetic analysis of the initial uptake rate gave an apparent K m of 159 μM. Furthermore, unlabeled choline caused both cis-inhibition and trans-stimulation for labeled choline transport, suggesting the existence of a carrier-mediated transport system for choline, classified as so-called ‘facilitated diffusion’. Since tetramethylammonium, acetylcholine, and N 1-methylnicotinamide caused both cis-inhibition and trans-stimulation, they appear to be accepted as the substrate of choline carrier. On the other hand, quaternary ammonium compounds (QACs) such as those which possessed hydrophobic parts in their molecules exhibited only cis-inhibition. They also inhibited Na +-dependent d-glucose transport, indicating that they influenced various carrier-mediated transport systems non-specifically due to interaction with the membrane. These findings strongly suggest that the choline transport system on the brush-border membrane of rat intestine recognizes only small molecular QACs as its substrate.

Transport of methotrexate in basolateral membrane vesicles from rat liver

Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by initial uptake studies and by varying medium osmolarity with increasing concentrations of sucrose. Methotrexate transport was linear for the first 20 s of incubation. Transport was not affected by imposition of a Na + gradient across the vesicular membrane. Transport of methotrexate displayed a broad pH optimum: at an intravesicular pH of 7.5, the initial rate of uptake was not significantly different at extravesicular pH values ranging from 5.5 to 7.5, but uptake was less at extravesicular pH of 5.0 or 8.0. Methotrexate transport was saturable: K m = 0.15 ± 0.05 μM and V max = 11.4 ± 1.1 pmol 10 s −1 mg −1 protein. Methotrexate uptake into BLMVs was not inhibited by 5-methyltetrahydrofolate nor by 5-formyltetrahydrofolate but was weakly inhibited by folic acid in a concentration-dependent manner. Uptake was also inhibited by the anion-exchange inhibitor 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid (DIDS), and by the structurally unrelated anions ATP, ADP, Cl −, SO 4 2−, and oxalate 2−. Adenosine (no negative charge) had no effect on transport. When vesicles were preloaded with anions (ADP, SO 4 2−, oxalate 2−) such that an anion gradient existed from the intra- to the extravesicular compartment, and methotrexate uptake was measured, no stimulation of uptake was seen. Methotrexate uptake into rat liver BLMVs was electrogenic as shown by stimulation of the initial rate of uptake by a valinomycin-imposed K + diffusion potential across the vesicular membrane. These results suggest that methotrexate is transported into the hepatocyte across the basolateral membrane by an electrogenic, multispecific anion carrier system.

Permeability of rat liver microsomal membrane to glucose 6-phosphate.

Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.

Modulation of dihydropyridine-sensitive gastric mucosal calcium channels by GM 1-ganglioside

1. 1. A dihydropyridine-sensitive calcium channel complex was solubilized from gastric mucosal cell membranes and purified by affinity chromatography on wheat germ agglutinin. 2. 2. The calcium channel complex labeled with [ 3H]PN200-110, when reconstituted into phosphatidylcholine vesicles, exhibited active 45Ca 2+ uptake into intravesicular space as evidenced by La 3+ displacement and osmolarity studies. The channel complex responded in a dose-dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 μM exerted maximal inhibitory effect of 66% in 45Ca 2+ uptake. 3. 3. The uptake of 45Ca 2+ into vesicle-reconstituted gastric mucosal calcium channel complex was inhibited by GM 1-ganglioside. Maximum inhibitory effect was achieved at 10–15 nM gm 1, at which point a 74% decrease in 45Ca 2+ uptake occurred. Furthermore, gm 1 also inhibited dihydropyridine binding to gastric mucosal membranes, indicating the extracellular orientation of calcium channel domains for gm 1. 4. 4. The ability of GM 1 to modulate the intracellular calcium levels may be an important feature in gastric mucosal protection by this ganglioside.

Presence of a sodium-translocating ATPase in membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii.

Inverted membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii catalyzed the hydrolysis of ATP with a rate of 100-150 nmol.min-1.mg protein-1. The ATPase was stimulated 1.4-1.6-fold by NaCl and inhibited by N,N'-dicyclohexylcarbodiimide tributyltin or azide. The degree of inhibition caused by F0-directed but not F1-directed inhibitors was affected by the Na+ concentration in the medium. These experiments indicated the presence of a sodium-translocating ATPase. This was verified by transport studies. Upon addition of ATP to inverted vesicles, 22Na+ was actively transported into the intravesicular space up to a 24-fold accumulation. Na+ transport was inhibited by the sodium ionophore N,N,N',N',-tetracyclohexyl-1,2-phenyl-enedioxydiacetamide but stimulated by valinomycin with potassium whereas the protonophore 3,5,-di-tert-butyl-4-hydroxybenzylidenemalonitrile was without effect. N,N'-dicyclohexylcarbodiimide and tributyltin inhibited 22Na+ transport. These experiments are in accordance with a primary electrogenic Na+ transport as catalyzed by a F1F0-ATPase.

Demonstration of bicarbonate secretion in rat duodenal brush border membrane vesicles.

This study was designed to characterize the mechanisms available for bicarbonate transport across the apical membrane using rat duodenal brush border membrane vesicles (BBMVs), including the morphological findings. BBMVs were purified using a double Mg2+ precipitation technique. Electron microscopy of the final pellet showed only membrane material without contaminating organelles of cytoskeletal aggregates. BBMVs were enriched 15.1 +/- 3.2-fold in A-lp and 12.5 +/- 2.4-fold in LAP. The time course of 36Cl uptake into BBMVs demonstrated a good response curve and equilibrated to the intravesicular space over 120 min. In this present study, these BBMVs were purified and showed a stabilized structure. Therefore, these results suggest that this model was useful for studying HCO3- secretion and ulcer etiology.

Transport of 5-methyltetrahydrofolate in basolateral membrane vesicles of rat liver

We examined 5-methyltetrahydrofolate transport across the basolateral membrane (BLM) of rat liver using isolated membrane vesicles. Uptake of 5-methyltetrahydrofolate by BLM vesicles (BLMV) was found by osmolarity and initial uptake studies to be mostly the result of transport of the substrate into an active, intravesicular space with some binding (approximately 25%) to membrane surfaces. Uptake was similar in the presence of an inwardly directed Na+ or K+ gradient, indicating that transport is not dependent on Na+. Uptake of 5-methyltetrahydrofolate was linear for the first 30 s and was more rapid when an initial pH gradient was imposed across the vesicular membrane [extravesicular pH (pHo) = 5.0, intravesicular pH (pHi) = 7.5]. Under pH gradient conditions, uptake at 3-5 min was about twofold higher than at equilibrium (60 min). The initial rate of uptake at pHo = pHi = 5.0 was more rapid than at pHo = pHi = 7.5, but in neither case was uptake above equilibrium observed. Uptake under pH gradient conditions and at pH 5.0 (no pH gradient) was saturable (apparent Michaelis constants of 0.58 and 0.36 microM, respectively; maximum uptake rates of 1.25 and 0.79 pmol.10 s-1.mg protein-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Movement of biotin across the rat intestinal basolateral membrane. Studies with membrane vesicles

The characteristics of the transport process of the water-soluble vitamin biotin across the basolateral membrane (BLM) of rat intestine, i.e. the exit of biotin from rat enterocyte, were examined using established BLM vesicles (BLMV) techniques. Results of osmolarity and temperature studies have indicated that the majority of biotin taken up by these vesicles is the result of transport of the vitamin into the intravesicular spaces, with little membrane binding. Transport of biotin in these vesicles was found to be: (1) higher in BLMV of the jejunum than those isolated simultaneously from the ileum, (2) Na(+)-independent in nature, (3) saturable as a function of concentration, with an apparent Km of 3.51 microM and a Vmax. of 16.49 pmol/10 s per mg of protein, (4) inhibited by high concentrations of unlabelled biotin and its related compounds, but not by the unrelated taurocholate, (5) sensitive to the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2'-disulphonic acid (SITS), and (6) not affected by imposing a positive or a negative intravesicular space with the use of a valinomycin-induced K+ diffusion potential. Based on these findings, it is concluded that biotin transport across the rat intestinal BLM is mediated via a specialized carrier system which is Na(+)-independent, transports the vitamin by a process which is sensitive to the effects of anion transport inhibitors, and is electroneutral in nature.

ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles

We conducted an experimental study to examine the possibility that ATP is involved in the mechanism by which bile acids are excreted through the liver canalicular membrane in opposing the concentration gradient. Canalicular membrane vesicles were purified from the livers of Sprague-Dawley rats, and the uptake of tritiated sodium taurocholate into canalicular membrane vesicles was determined by rapid filtration technique. Vesicle-associated sodium taurocholate was increased in the presence of ATP and ATP-regenerating system. This was also observed at a voltage-clamped condition. ATP-dependent uptake into the osmotically reactive intravesicular space was saturated with increasing concentrations of sodium taurocholate (Km = 47 μmol/L, Vmax = 270 pmoles/mg protein · 20s). ATP-dependent uptake increased to the point of saturation when the sodium taurocholate concentration was 50 μmol/L and the ATP concentration was increased from 0 to 1 mmol/L (Km = 64 μmol/L). Among the several nucleotides used, ATP was a potent stimulator of transport, whereas a nonhydrolyzable analogue (i.e., adenosine 5′-[β,γ-imino]triphosphate) showed no effect. In addition, ATP-dependent transport was inhibited by vanadate in a dose-dependent manner. From these results it was concluded that the primary active transport of sodium taurocholate is present in hepatocellular canalicular membranes. This transport is directly dependent on ATP, and hydrolysis of γ-phosphate of ATP is required. (Hepatology 1991;14:655-659.)

An Na+-dependent and an Na+-independent system for glutamine transport in rat liver basolateral membrane vesicles

In the present study the transport of glutamine across rat liver basolateral membrane was examined with special emphasis on the existence of an Na+-independent system and on the characteristics of the Na+-dependent system with respect to stoichiometry of glutamine to Na+. Well-validated and purified liver basolateral membrane vesicles were used in the study. Results of studies on the effect of incubation medium osmolarity and incubation temperature indicated that glutamine uptake by liver basolateral membrane vesicles is largely the result of transport of the substrate into the intravesicular compartment with little binding to basolateral membrane vesicles. Transport of glutamine with time was Na+ gradient dependent (out > in) with a distinct “overshoot” phenomenon. Replacing Na+ with an equivalent concentration of K+, NH4+, choline, or mannitol caused significant inhibition of the initial rate of glutamine transport; on the other hand, Li+ could partially substitute for Na+. The initial rate of transport of glutamine as a function of concentration (0.05–12 mmol/L) was saturable both in the presence and in the absence of an inwardly directed Na+ gradient. Apparent Km values of 2.95 and 3.35 mmol/L and Vmax values of 11,565 and 6663 pmol · mg protein−1 · 10 s−1 were calculated in the presence and absence of a Na+ gradient, respectively. Both in the presence and absence of an Na+ gradient (out > in), transport of [3H]glutamine was significantly inhibited by the addition to the incubation medium of unlabeled glutamine as well as histidine, asparagine, and serine. Transport of glutamine by the Na+-dependent process was significantly inhibited or stimulated, respectively, by inducing a relatively positive or negative intravesicular space. On the other hand, glutamine transport by the Na+-independent process was not affected by changes in transmembrane electrical potential. Using the “activation method,” the stoichiometry of glutamine Na+ transport was found to be 1:1. These results show that glutamine transport in rat liver basolateral membrane vesicles is carrier mediated both in the presence and absence of an Na+ gradient. Furthermore, the Na+-dependent process is electrogenic in nature (net positive) and cotransports one glutamine molecule with one Na+. Transport of glutamine by the Na+-independent system, on the other hand, is electroneutral in nature.

ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles*1, *2

We conducted an experimental study to examine the possibility that ATP is involved in the mechanism by which bile acids are excreted through the liver canalicular membrane in opposing the concentration gradient. Canalicular membrane vesicles were purified from the livers of Sprague-Dawley rats, and the uptake of tritiated sodium taurocholate into canalicular membrane vesicles was determined by rapid filtration technique. Vesicle-associated sodium taurocholate was increased in the presence of ATP and ATP-regenerating system. This was also observed at a voltage-clamped condition. ATP-dependent uptake into the osmotically reactive intravesicular space was saturated with increasing concentrations of sodium taurocholate (Km = 47 mumol/L, Vmax = 270 pmoles/mg protein.20s). ATP-dependent uptake increased to the point of saturation when the sodium taurocholate concentration was 50 mumol/L and the ATP concentration was increased from 0 to 1 mmol/L (Km = 64 mumol/L). Among the several nucleotides used, ATP was a potent stimulator of transport, whereas a nonhydrolyzable analogue (i.e., adenosine 5'-[beta,gamma-imino]triphosphate) showed no effect. In addition, ATP-dependent transport was inhibited by vanadate in a dose-dependent manner. From these results it was concluded that the primary active transport of sodium taurocholate is present in hepatocellular canalicular membranes. This transport is directly dependent on ATP, and hydrolysis of gamma-phosphate of ATP is required.

Carrier-mediated L-lactate transport in brush-border membrane vesicles from rat placenta during late gestation.

The mechanism for L-lactate transport across microvillous membrane vesicles prepared from rat placenta was examined. Uptake of L-lactate into these vesicles was mainly the result of transport into the intravesicular (osmotically active) space. The initial rate of L-lactate uptake was not affected by the presence of an inward gradient of either Na+ or K+. In the presence of an inward-directed proton gradient, L-lactate uptake was markedly stimulated, accumulating at concentrations 6-7-fold higher than the equilibrium. Lower transmembrane pH gradients were associated with slower initial uptakes and smaller overshoots. L-Lactate uptake determined under an inside-directed pH gradient was strongly inhibited by p-chloromercuriphenylsulphonic acid, a protein-thiol oxidizing agent. L-Lactate uptake was: (1) saturable as a function of the concentration of L-lactate, (2) inhibited by monocarboxylic acids such as pyruvate, D-lactate, beta-hydroxybutyrate and alpha-cyano-4-hydroxycinnamic acid, and (3) temperature-dependent. When present inside the vesicles, L-lactate, pyruvate and beta-hydroxybutyrate caused trans-stimulation of L-lactate uptake both in the presence and in the absence of an inside-directed pH gradient, indicating that L-lactate transport is a reversible process that can be shared by other monocarboxylic acids. There were no significant changes in maximal initial rate or in the kinetic parameters of L-lactate transport during the last 3 days of gestation.

Spermine uptake by rat intestinal brush-border membrane vesicles

The uptake of spermine by isolated rat intestinal brush-border membrane vesicles was studied. Uptake was biphasic, with an initial rapid uptake followed by a prolonged slower phase. Spermine uptake was not affected by a Na + electrochemical gradient. The equilibrium uptake of spermine was considerably dependent upon the medium pH. At pH 7.5 the degree of uptake was higher than that at pH 6.5 and was inversely proportional to the extravesicular osmolarity with a relatively high binding, which was estimated by extraporation to infinite extravesicular osmolarity (zero intravesicular space), while the uptake at pH 6.5 was not altered under the various medium osmolarities. A kinetic analysis of the initial uptake rate of spermine at 37°C gave a K m of 24.2 μM and V max of 206.1 pmol/mg protein per min. Furthermore, the uptake at 4° C was nonlinear, providing evidence for saturability. These findings suggest that spermine was associated with intestinal brush-border membrane vesicles in two ways, by binding to the outside and inside of membrane vesicles. The interaction of spermine and the apical membrane can be contributory factor in the accumulation of this polyamine in the intestine of the intact animal.

Increased Na+-H+ exchange in jejunal brush border membrane vesicles of spontaneously hypertensive rats

The spontaneously hypertensive rats and their genetically matched controls, Wistar-Kyoto, serve as models of essential hypertension. The present study was undertaken to determine whether brush border membrane vesicles obtained from jejunal enterocytes of spontaneously hypertensive rats show increased Na+-H+ exchange as part of a generalized membrane disorder. Brush border membrane vesicles were prepared from the jejunum of adult spontaneously hypertensive rats and Wistar-Kyoto rats using an Mg2+/ethylene glycol tetraacetic acid precipitation method. Uptake of 22Na by these vesicles was found to be into an osmotically sensitive intravesicular space rather than mere binding. Initial Na+ uptake by brush border membrane vesicles was greater in spontaneously hypertensive rats than in Wistar-Kyoto rats (P < 0.05). Higher total and amiloride-sensitive Na+ uptake in spontaneously hypertensive rats occurred in the presence of an outwardly directed pH gradient, and uptake became statistically similar to that of Wistar-Kyoto rats in the absence of a pH gradient. Moreover, amiloride-insensitive Na+ uptake under an outwardly directed pH gradient did not differ significantly between the two groups. The enhanced Na+-H+ activity in spontaneously hypertensive rats is not due to altered membrane permeability to protons, as is shown by acridine orange-quenching studies. Kinetic studies for amiloride-sensitive Na+ uptake showed a greater Vmax in spontaneously hypertensive rats compared with Wistar-Kyoto rats (1.46 ± 0.05 vs. 1.08 ± 0.08 nmol · mg protein−1 · 7 s−1) but the Km values were similar in the two groups. These findings, along with similar findings previously reported in vascular smooth muscle and renal tissue of SHR, strongly suggest that an increased Na+-H+ exchange is related to the development of hypertension.

Use of membrane vesicles to estimate the numbers of system y+ and system L amino acid transporters in human erythrocytes

We have used equilibrium values for L-leucine and L-lysine uptake by right-side-out vesicles to estimate the membrane abundance (sites/cell) of Na(+)-dependent amino acid transport systems L and y+ in human erythrocytes. All of the intravesicular space was accessible to L-leucine, as judged by comparisons with uridine uptake via the equilibrative nucleoside transporter (10(4) sites/cell). In contrast, only 28% of the total intravesicular space was accessible to L-lysine uptake via system y+. Since human erythrocyte membranes generate an average of approximately 1000 vesicles/cell, these data provide evidence that system L is a relatively high-abundance membrane transport protein in human erythrocytes, while system y+ is present in smaller amounts (approximately 300 copies/cell). Calculated turnover numbers for L-lysine transport by system y+ at 37 degrees C are 24 s-1 for zero-trans influx and 150 s-1 for equilibrium-exchange influx.

Carrier-mediated mechanism for biotin transport in rabbit intestine: studies with brush-border membrane vesicles

Simple diffusion has been reported as the mechanism of biotin transport in rabbit intestine. In this study, we reevaluated this concept by examining biotin transport in rabbit intestine using optimal experimental conditions and a well-established brush-border membrane vesicles (BBMV) technique. Uptake of biotin by rabbit intestinal BBMV was found by an osmolarity study to be mostly the result of transport of the vitamin into an osmotically sensitive intravesicular space with little binding to membrane surfaces. Biotin transport in rabbit intestinal BBMV was 1) Na+ gradient dependent (out greater than in) with a clear "overshoot" phenomenon, indicating the accumulation of the substrate against a concentration gradient; 2) initial rate of biotin transport by the Na+ gradient-dependent component was saturable as a function of substrate concentration with apparent Km and maximum velocity (Vmax) values of 6.7 microM and 10.7 pmol.mg protein-1 x 10 s-1, respectively; 3) inhibited by high concentrations of unlabeled biotin and its related compounds desthiobiotin and thioctic acid in the presence, but not absence, of a Na+ gradient; and 4) not affected by inducing a relatively positive or negative intravesicular space with the use of valinomycin-induced K+ diffusion potential. These findings indicate that the biotin transport mechanism in rabbit intestine is carrier mediated in nature. Furthermore, this mechanism is Na+ gradient dependent, capable of accumulating the substrate against a concentration gradient and transport the vitamin via an electroneutral process.

Acetate uptake by intestinal brush border membrane vesicles.

The mechanism of acetate absorption in the small intestine is not yet established. One possible mechanism is by carrier mediated Na(+)-acetate cotransport since acetate, like glucose, stimulates intestinal Na+ and water absorption in vivo. Uptake of radioactive carbon acetate by small intestinal brush border membrane vesicles was not saturable or Na+ dependent and did not respond to osmotic shrinkage of the vesicles. This suggests that acetate binds to the membranes but is not transported into the intravesicular space and argues against carrier mediated Na+ acetate cotransport. These results are consistent with acetate absorption by a non-mediated diffusion and suggest that the stimulation of water and Na+ absorption by acetate in vivo is largely due to osmotic forces and solvent drag.

Analysis of the pH Dependence of Folate Binding and Transport by Rat Kidney Brush Border Membrane Vesicles

Folate reabsorption by the mammalian kidney occurs following a tight binding reaction with the renal brush border membrane. Previous studies have shown that transport of folic acid (PteGlu) by rat kidney brush border membrane vesicles occurs maximally at pH 5.6 via a saturable system that is associated with a binding component. The present studies have shown that the pH dependency of transport was due to the development of the transmembrane pH gradient (7.3 in/5.6 out), not to the acidic pH per se. The pH gradient-mediated transport was stimulated by an inwardly directed ionic gradient, either of NaCl or choline chloride. These gradients also stimulated the membrane binding of PteGlu suggesting that NaCl and choline chloride may have increased PteGlu transport by altering binding to the brush border membrane. Renal brush border membrane vesicular transport of PteGlu was not affected by induction of a relatively positive intravesicular space. Transport was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, an anion exchange inhibitor. The results suggest that rat kidney brush border membrane transport of PteGlu is initiated by association with a specific membrane protein, followed by transfer of folate across the membrane. The overall activity is influenced by a transmembrane pH gradient.

Na(+)-independent Cl(-)-HCO3- exchange in sarcolemmal vesicles from vascular smooth muscle

Intracellular pH (pHin) affects vascular smooth muscle function, but the mechanisms that control pHin in this tissue are not well understood. These studies were performed to determine whether sarcolemmal vesicles from bovine superior mesenteric artery (SMA) contain a Na(+)-independent Cl(-)-HCO3- exchanger and, if so, to determine its sensitivity to membrane voltage and inhibitors. 36Cl- was taken up by vesicles into an osmotically active intravesicular space. In Na(+)-free media, an outwardly or inwardly directed HCO3- gradient stimulated 36Cl- transport in the opposite direction. An outwardly directed unlabeled Cl- gradient stimulated 36Cl- uptake by a mechanism that was inhibited by external HCO3-. HCO3- or Cl- gradient-stimulated 36Cl- uptake was not due to voltage coupling between ions. In the nominal absence of HCO3-, a threefold outwardly directed OH- gradient did not affect 36Cl- uptake. Total 36Cl- uptake was stimulated by an inside-positive voltage, but the HCO3- gradient-stimulated component of 36Cl- uptake was insensitive to a change in membrane voltage. Finally, HCO3- gradient-stimulated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and furosemide, with 50% inhibitory concentration values equalling approximately 1.0 and 0.5 mM, respectively. These data indicate that sarcolemmal vesicles from bovine SMA contain a Na(+)-independent Cl(-)-HCO3- exchanger. This transport system is probably electroneutral and is inhibitable by DIDS and furosemide. A conductive pathway for Cl- is present in the vesicles, but Cl(-)-OH- exchange activity was not observed.

Transport of riboflavin in human intestinal brush border membrane vesicles

The transport of riboflavin across the brush border membrane of human intestine was examined using the established brush border membrane vesicle technique. Both osmolarity and temperature studies have concluded that the uptake of riboflavin by these vesicles is mostly the result of transport of riboflavin into an active intravesicular space with less binding to membrane surfaces. When an inwardly directed Na+ gradient was imposed, transport of riboflavin was linear with time for approximately 20 seconds of incubation and was significantly higher than in the presence of an identical K+ gradient. Initial rate of transport of riboflavin as a function of concentration was found to include a saturable component in the presence of an inwardly directed Na+ gradient but was linear in the presence of an identical K+ gradient. The apparent Km and Vmax of the Na+ stimulated transport process were found to be 7.26 (μmol/L and 0.97 pmol/mg protein per 10 seconds, respectively. The addition of high concentrations of unlabeled riboflavin and its structural analogue lumiflavin to the incubation medium caused significant inhibition in the transport of 3H-riboflavin in the brush border membrane vesicle incubated in the presence of an inwardly directed Na+ gradient but not in vesicles incubated in the presence of an identical K+ gradient. Inducing a relatively positive intravesicular space with the use of valinomycin and an inwardly directed K+ gradient caused significant inhibition in the transport of riboflavin. On the other hand, inducing a relatively negative intravesicular space with the use of anions of different lipid permeabilities caused significant stimulation in the transport of riboflavin. These results demonstrate that riboflavin transport in human intestinal brush border membrane vesicle is through a carrier-mediated system. This system functions in the presence of a Na+-gradient and seems to transport the substrate by an electrogenic process.