Extravesicular pH Research Articles

Expression of plasma membrane calcium ATPases confers Ca2+/H+ exchange in rodent synaptic vesicles

Ca2+ transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca2+] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca2+/H+ antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca2+ ATPases (PMCAs) are responsible for both the Ca2+/H+ exchange activity and Ca2+ uptake into SVs. The Ca2+/H+ exchange activity monitored by acidification assay exhibited high affinity for Ca2+ (Km ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca2+ transporters on SVs, since both Ca2+/H+ exchange activity and Ca2+ transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca2+ homeostasis and the modulation of H+ gradient in SVs.

Real-Time Recording of the Cellular Effects of the Anion Transporter Prodigiosin

Summary The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field.

A new use of β-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide β-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH6.6 and different concentrations of β-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the β-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations >100μM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100μM β-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of β-Ala-Lys (AMCA) at 20min showed that the Km and Vmax were 783.7±115.7μM and 2191.2±133.9ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at Km=93.6±21.9μM and Vmax=935.8±50.2ΔF/min/mg. These findings demonstrate the applicability of β-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.

Tuning biomimetic membrane barrier properties by hydrocarbon, cholesterol and polymeric additives

The barrier properties of cellular membranes are increasingly attracting attention as a source of inspiration for designing biomimetic membranes. The broad range of potential technological applications makes the use of lipid and lately also polymeric materials a popular choice for constructing biomimetic membranes, where the barrier properties can be controlled by the composition of the membrane constituent elements. Here we investigate the membrane properties reported by the light-induced proton pumping activity of bacteriorhodopsin (bR) reconstituted in three vesicle systems of different membrane composition. Specifically we quantify how the resulting proton influx and efflux rates are influenced by the membrane composition using a variety of membrane modulators. We demonstrate that by adding hydrocarbons to vesicles with reconstituted bR formed from asolectin lipids the resulting transmembrane proton fluxes changes proportional to the carbon chain length when compared against control. We observe a similar proportionality in single-component 1,2-Dioleoyl-sn-glycero-3-phosphocholine model membranes when using cholesterol. Lastly we investigate the effects of adding the amphiphilic di-block co-polymer polybutadiene–polyethyleneoxide (PB12–PEO10) to phospholipid membranes formed from 1,2-Dioleoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-Dioleoyl-sn-glycero-3-phosphatidylserine. The proton pumping activity of bR (measured as a change in extra-vesicular pH) in mixed lipid/PB12–PEO10 lipid systems is up to six-fold higher compared to that observed for bR containing vesicles made from PB12–PEO10 alone. Interestingly, bR inserts with apparent opposite orientation in pure PB12–PEO10 vesicles as compared to pure lipid vesicles. Addition of equimolar amounts of lipids to PB12–PEO10 results in bR orientation similar to that observed for pure lipids. In conclusion our results show how the barrier properties of the membranes can be controlled by the composition of the membrane. In particular the use of mixed lipid-polymer systems may pave the way for constructing biomimetic membranes tailored for optimal properties in various applications including drug delivery systems, biosensors and energy conservation technology.

Mechanistic model and analysis of doxorubicin release from liposomal formulations

Reliable and predictive models of drug release kinetics in vitro and in vivo are still lacking for liposomal formulations. Developing robust, predictive release models requires systematic, quantitative characterization of these complex drug delivery systems with respect to the physicochemical properties governing the driving force for release. These models must also incorporate changes in release due to the dissolution media and methods employed to monitor release. This paper demonstrates the successful development and application of a mathematical mechanistic model capable of predicting doxorubicin (DXR) release kinetics from liposomal formulations resembling the FDA-approved nanoformulation DOXIL® using dynamic dialysis. The model accounts for DXR equilibria (e.g. self-association, precipitation, ionization), the change in intravesicular pH due to ammonia release, and dialysis membrane transport of DXR. The model was tested using a Box–Behnken experimental design in which release conditions including extravesicular pH, ammonia concentration in the release medium, and the dilution of the formulation (i.e. suspension concentration) were varied. Mechanistic model predictions agreed with observed DXR release up to 19h. The predictions were similar to a computer fit of the release data using an empirical model often employed for analyzing data generated from this type of experimental design. Unlike the empirical model, the mechanistic model was also able to provide reasonable predictions of release outside the tested design space. These results illustrate the usefulness of mechanistic modeling to predict drug release from liposomal formulations in vitro and its potential for future development of in vitro – in vivo correlations for complex nanoformulations.

Oriented Reconstitution of a Membrane Protein in a Giant Unilamellar Vesicle: Experimental Verification with the Potassium Channel KcsA

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.

Characteristics of cefdinir uptake by rabbit small intestinal brush-border membrane vesicles.

Aminocephalosporins with peptide-like structures have been shown to be absorbed by the intestinal peptide carrier. We investigated the transport mechanism of cefdinir, an oral monocarboxylic acid cephalosporin, using rabbit small intestinal brush-border membrane vesicles. Transport of cefdinir showed a slow and almost linear uptake rate for concentrations up to 30 mM with and without and inward H+ gradient. No overshoot phenomenon was observed in the presence of an inward H+ gradient. The uptake rate increased only slightly with decreasing extravesicular pH, and a protonophore had little effect on the uptake. Aminocephalosporins such as cephalexin only slightly inhibited cefdinir uptake even in the presence of an inward H+ gradient, and vice-versa. Monocarboxylic acids such as acetic acid and salicylic acid had little effect on cefdinir uptake. These findings suggest that in contrast with other oral cephalosporins cefdinir uptake through the brush-border membrane is slow and involves a mechanism similar to passive diffusion.

Roles of K+, H+, H2O, and ΔΨ in Solute Transport Mediated by Major Facilitator Superfamily Members ProP and LacY

H (+)-solute symporters ProP and LacY are members of the major facilitator superfamily. ProP mediates osmoprotectant (e.g., proline) accumulation, whereas LacY transports the nutrient lactose. The roles of K (+), H (+), H 2O, and DeltaPsi in H (+)-proline and H (+)-lactose symport were compared using right-side-out cytoplasmic membrane vesicles (MVs) from bacteria expressing both transporters and proteoliposomes (PRLs) reconstituted with pure ProP-His 6. ProP activity increased as LacY activity decreased when osmotic stress (increasing osmolality) was imposed on MVs. The activities of both transporters decreased to similar extents when Na (+) replaced K (+) in MV preparations. Thus, K (+) did not specifically control ProP activity. As with LacY, an increasing extravesicular pH stimulated ProP-mediated proline efflux much more than ProP-mediated proline exchange from de-energized MVs. In contrast to that of LacY, ProP-mediated exchange was only 2-fold faster than ProP-mediated efflux and was inhibited by respiration. In the absence of the protonmotive force (Deltamu H (+) ), efflux of lactose from MVs was much more sensitive to increasing osmolality than lactose exchange. Thus, H 2O may be directly involved in proton transport via LacY. In the absence of Deltamu H (+) , proline efflux and exchange from MVs were osmolality-independent. In PRLs with a DeltapH of 1 (lumen alkaline), ProP-His 6 was inactive when the membrane potential (DeltaPsi) was zero, was active but insensitive to osmolality when DeltaPsi was -100 mV, and became osmolality-sensitive as DeltaPsi increased further to -137 mV. ProP-His 6 had the same membrane orientation in PRLs as in cells and MVs. ProP switches among "off", "on", and "osmolality-sensitive" states as the membrane potential increases. Kinetic parameters determined in the absence of Deltamu H (+) represent a ProP population that is predominantly off.

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 Charge Transfer Pathway in Proton Transport of Gastric H+,K+-ATPase

Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.

Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and P(i). In rat, biliary P(i) concentration (0.01 mM) is approximately 100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary P(i). We aimed to functionally characterize canalicular P(i) reabsorption in rat liver and to identify the involved P(i) transport system(s). P(i) transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative P(i) transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent P(i) uptake. Initial P(i) uptake rates were saturable with increasing P(i) concentrations, exhibiting an apparent K(m) value of approximately 11 muM. P(i) transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of P(i) from primary hepatic bile and thus might play an important role in the regulation of biliary P(i) concentration.

Involvement of OCTN1 (SLC22A4) in pH-Dependent Transport of Organic Cations

OCTN1 (SLC22A4) transports cationic compounds such as tetraethylammonium in a pH-sensitive and sodium-independent manner in cultured cells, and is expressed in wide variety of tissues, including kidney, muscle, placenta, heart, and others. This study focused on the clarification of its subcellular distribution in kidney and on its driving force to throw light on the pharmacological and physiological roles of OCTN1. Uptake of [14C]tetraethylammonium by membrane vesicles prepared from HEK293 cells stably transfected with human OCTN1 cDNA was osmolarity-sensitive, and the Km of tetraethylammonium was 1.28 mM at intravesicular and extravesicular pH values of 6.0 and 7.4, respectively. Tetraethylammonium uptake was pH-dependent, and overshoot uptake was observed in the presence of an outwardly directed proton gradient. A protonophore and membrane potential affected the overshoot uptake. Furthermore, preloading tetraethylammonium in the vesicles significantly increased the rate of uptake of [14C]tetraethylammonium. In mouse kidney, OCTN1 was expressed predominantly at the apical membrane of cortical proximal tubular epithelial cells. It was concluded that OCTN1 is involved in renal excretion of organic cations across the apical membrane in a pH-dependent, membrane potential-sensitive manner and is affected significantly by the organic cations on the trans side, showing counter transport activity.

Lactate efflux from sarcolemmal vesicles isolated from rainbow trout Oncorhynchus mykiss white muscle is via simple diffusion.

Lactic acid is produced as an end product of glycolysis in rainbow trout white muscle following exhaustive exercise. The metabolically produced lactic acid causes an intramuscular acidosis that must be cleared, either via net transport out of the muscle or by conversion to glycogen, thereby replenishing the muscle energy store. Trout muscle has been shown to retain lactate and utilise it as a substrate for in situ glycogen resynthesis. The giant sarcolemmal vesicle preparation was used to characterise the potential for lactate loss from white muscle of rainbow trout. Minimal lactate loss was expected due to the requirement within the intramuscular compartment of lactate for glycogen resynthesis. The sarcolemma was found to be highly resistant to lactate loss, with efflux rates approximately 500-fold lower than influx rates [0.049+/-0.006 nmol mg(-1) min(-1) (N=21) versus 26.4+/-6.3 nmol mg(-1) min(-1) (N=5), respectively, at 25 mmol l(-1) lactate concentration]. Lactate efflux was linear over the range 10-250 mmol l(-1) lactate, and greatest under conditions when intravesicular pH was lower than extravesicular pH, but was unaffected by alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. These results suggest that lactate is relatively impermeant to the trout white muscle membrane and any lactate loss occurs via passive diffusion. This resistance to lactate diffusion can explain why trout muscle retains lactate post-exercise, despite transmembrane gradients that should favour net efflux.

PH dependence of Na+/myo-inositol cotransporters in rat thick limb cells

To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo-inositol (MI). Expression of Na+-MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na+-MI cotransporter in MTAL cells. Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [3H]myo-inositol ([3H]MI) was used to assess Na+-MI cotransport activity. In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na+-gradient versus tetramethylammonium (TMA) stimulated the initial [3H]MI uptake in BLMV and LMV. Phlorizin inhibited Na+ gradient-dependent initial [3H]MI uptake in both preparations, with IC50 values of 565 and 29 micromol/L in BLMV and LMV, respectively. 2-0,C-methylene myo-inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na+-MI cotransporter. Phlorizin-sensitive Na+ gradient-dependent initial [3H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar Vmax but different Km values of 51 and 107 micromol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na+-MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pHi) was decreased from 8.0 to 6.0 at extravesicular pH (pHe) 8.0, and as pHe was increased from 6.0 to 8.0 at pHi 6.0. Maximal activation was observed at pHi 6.5 and pHe 7.5. In rat MTAL cells, Na+-MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na+-MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pHe and pHi.

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior–middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K +, a low-affinity saturable transport of arginine in both AM- and P-BBMV ( K m 1.01 mM, V max 4.07 nmol/7s/mg protein and K m 1.38 mM, V max 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Δ ψ) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N G-monomethylarginine, N G-nitroarginine>lysine≫ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K +, the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter ( K m 0.035 mM, V max 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Δ ψ and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N G-monomethylarginine≫cysteine, lysine≫ N G-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K +/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH out) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles.

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.

Characterization of Aspartate Transport Across the Symbiosome Membrane in Pea Root Nodules

Summary Aspartate transport was studied in symbiosome membrane (SM) vesicles, facing bacteroid-side-out, purified from pea (Pisum sativum L.) root nodules. An intravesicular alkaline pH gradient (ΔpH) and an intravesicular negative membrane potential (Δψ) were imposed to energize the vesicles. Energized vesicles were capable of accumulating aspartate from the extravesicular medium against a concentration gradient. The uptake of aspartate in SM vesicles was electrogenic and the transport capacity was influenced by both ΔpH and Δψ. Furthermore, vesicle uptake of aspartate had an extravesicular pH optimum at 6. The uptake of aspartate driven by the energization of SM vesicles was biphasic showing saturation kinetics in the range of 0.01 to 1 mmol/L aspartate. Two protein modifying reagents, p-chloromercuribenzenesulphonic acid and phenylglyoxal, inhibited the uptake of aspartate in energized vesicles. The data are consistent with H+/aspartate symport(s) transporting aspartate from the bacteroid side of the SM to the plant cytosol.

Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe.

As a major Ca exit system in myocytes, the electrogenic Na+-Ca2+ exchange is exposed to rapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation-contraction coupling. The dynamic aspects of the exchanger response to regulatory factors have not been resolved in the past due to technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activity of Na+-Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dye Merocyanine-540 (M540). The M540 signal of Nao-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 microM Cafree. This signal is inhibited by a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler (valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-state burst (210 s-1), followed by slow steady-state phase (</=5 s-1). Extravesicular (cytosolic) Ni inhibits both phases with an IC50 of 0.80 +/- 0.24 mM. At an extravesicular pH of 6.0, the Nao-dependent Ca efflux is able to generate the M540 signal, thereby supporting the idea that the stoichiometry of Na+-Ca2+ exchange is not altered at low pH [Khanashvili, D., et al. (1995) Biochemistry 34, 10290-10297]. The M540 signal of Nao-dependent Ca efflux is lost when the extravesicular Cafree concentration drops to 0.2 microM. This effect cannot be explained by a lack of Ca access to extravesicular (cytosolic) transport sites, because the reaction of Nao-dependent Ca efflux utilizes intravesicular Ca as a substrate. These data suggest that in sarcolemma vesicles a regulatory cytosolic Ca site controls the exchanger activity. The properties of this putative regulatory site do not resemble the properties of the "slow" Ca regulatory mode, observed in electrophysiological studies. Under saturating ionic conditions, the Nao-dependent Ca efflux generates the initial rates of 21 mV/ms in the vesicles with a diameter of 3000-5000 A. If a site density of 300-400 exchangers/micrometer2 and a vesicular surface of 0.5 micrometer2 are assumed, each vesicle may contain 150-200 exchanger molecules with a maximal turnover rate of 4000-5000 s-1. This upper limit for turnover (no matter what the site density is) may put considerable restrictions on the exchanger capacity to mediate Ca entry in the cell under physiologically related conditions.

PH-dependent inhibitory effects of angiotensin-converting enzyme inhibitors on cefroxadine uptake by rabbit small intestinal brush-border membrane vesicles and their relationship with hydrophobicity and the ratio of zwitterionic species.

The inhibitory effects of five angiotensin-converting enzyme (ACE) inhibitors on the uptake of an aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush-border membrane vesicles were examined in the presence of an inward H+ gradient. Dixon plot analysis showed that all these ACE inhibitors inhibited the uptake of cefroxadine, which is transported by a H+/oligopeptide transporter in the membrane, in the order of enalapril<quinapril, benazepril<temocapril, trandolapril at extravesicular pH 6.0. These drugs, except for enalapril, which are relatively hydrophobic, exhibited a mix of competitive and noncompetitive inhibition. We also examined the inhibitory effects of quinapril and temocapril at extravesicular pH 5.5, at which the ratio of the zwitterionic species of the drugs increased. The inhibition occurred in a nearly competitive manner at this pH and the inhibitory effects were stronger than at pH 6.0. Regression analysis of the inhibitory effects suggested that the affinity of these ACE inhibitors for the transporter was regulated by the hydrophobicity of these ACE inhibitors and the ratio of the zwitterionic species of the drugs.

Riboflavin transport by rabbit renal basolateral membrane vesicles

The present study examined riboflavin (RF) uptake by isolated rabbit renal basolateral membrane (BLM). RF uptake was linear during the initial 10 seconds and leveled off thereafter with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the BLM RF uptake was the results of transport (∼45%) into the intravesicular space as well as binding (∼55%) to membrane surfaces. The RF binding to BLM was Na +-dependent so that replacement of Na + by other cations eliminated the binding component of RF uptake. The process of BLM RF uptake was saturable as a function of substrate concentration and was significantly inhibited by cis-addition of its structural analogs, lumiflavin and lumichrome, indicating the involvement of a carrier-mediated process. The BLM RF uptake was affected by changes in extravesicular pH so that, as compared to pH 7.5, RF uptake was lower at pH 6.5 and higher at pH 8.5. The effect of extravesicular pH persisted when the transmembrane H + gradient was dissipated by FCCP, indicating the direct effect of pH on BLM RF uptake. The BLM RF uptake was not affected by alterations of the transmembrane electrical potential, induced by either the presence of anions with different membrane permeability (Cl −= NO − 3 > SO − 4 >gluconate −) or using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K + gradient. The BLM RF uptake was, however, inhibited by probenecid and p-aminohippurate, and was enhanced by trans-RF. In summary, these results demonstrate the existence of a Na +-dependent BLM binding of RF and a membrane-associated carrier system for RF uptake by renal BLM.

Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate.

1. Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence of the RT-PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT-PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well-characterized human and pig colonic lumenal membrane vesicles (LMV). 2. Properties of the colonic lumenal membrane L-lactate transporter were studied by the uptake of L-[U-14C]lactate into human and pig colonic LMV. L-Lactate uptake was stimulated in the presence of an outward-directed anion gradient at an extravesicular pH of 5.5. Transport of L-lactate into anion-loaded colonic LMV appeared to be via a proton-activated, anion exchange mechanism. 3. L-Lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl- and SO4(2-). The uptake of L-lactate was inhibited by phloretin, mercurials and alpha-cyano-4-hydroxycinnamic acid (4-CHC), but not by the stilbene anion exchange inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS). 4. The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of L-lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.