65Zn2+ Transport by lobster hepatopancreatic lysosomal membrane vesicles

In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.     

Presence of 5-hydroxytryptamine (serotonin) transport coupled with vacuolar-type H(+)-ATPase in neurosecretory granules from bovine posterior pituitary

Electrogenic H(+)-ATPase was found in neurosecretory granules from bovine posterior pituitary. This enzyme was sensitive to bafilomycin, a specific inhibitor of vacuolar H(+)-ATPase, and was inactivated completely by cold treatment in the presence of MgATP and NaNO3. Immunoblot analysis showed the presence of immunologically identical polypeptides (72, 57, and 34 kDa) in the ATPases of the neurosecretory granules and chromaffin granules. The granules showed MgATP-dependent activity for 5-hydroxytryptamine (serotonin) uptake. This uptake was temperature-dependent and showed saturation kinetics (apparent Km for 5-hydroxytryptamine, 2 microM) and counter-flow. Reserpine and tetrabenazine at 1 microM inhibited the uptake, whereas imipramine at 2 microM had no effect. Dopamine, epinephrine and norepinephrine were also inhibitory. The uptake was abolished by various treatments that dissipated the electrochemical H+ gradient or inhibited the H(+)-ATPase. These results indicate that a vacuolar type H(+)-ATPase in the neurosecretory granules forms an electrochemical H+ gradient that drives 5-hydroxytryptamine uptake by a specific transport system. A similar granular fraction from the anterior pituitary had no ATP-dependent activity for 5-hydroxytryptamine uptake.     

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.     

Flux coupling in the human serotonin transporter

The serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) catalyzes the movement of 5HT across cellular membranes. In the brain, SERT clears 5HT from extracellular spaces, modulating the strength and duration of serotonergic signaling. SERT is also an important pharmacological target for antidepressants and drugs of abuse. We have studied the flux of radio-labeled 5HT through the transporter stably expressed in HEK-293 cells. Analysis of the time course of net transport, the equilibrium 5HT gradient sustained, and the ratio of the unidirectional influx to efflux of 5HT indicate that mechanistically, human SERT functions as a 5HT channel rather than a classical carrier. This is especially apparent at relatively high [5HT](out) (> or =10 microM), but is not restricted to this regime of external 5HT.     

Further characterization of adenosine transport in renal brush-border membranes

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.     

Electrogenic glutamine uptake by Peptostreptococcus anaerobius and generation of a transmembrane potential.

Peptostreptococcus anaerobius converted glutamine stoichiometrically to ammonia and pyroglutamic acid, and the Eadie-Hofstee plot of glutamine transport was biphasic. High-affinity, sodium-dependent glutamine transport (affinity constant [Kt] of 1.5 microM) could be driven by the chemical gradient of sodium, and more than 20 mM sodium was required for half-maximal velocity. High-affinity glutamine transport was not stimulated or inhibited by a membrane potential (delta psi). Low-affinity glutamine transport had a rate which was directly proportional to the external glutamine concentration, required less than 100 microM sodium, and was inhibited strongly by a delta psi. Cells which were treated with N,N-dicyclohexylcarbodiimide to inhibit the F1F0 ATPase still generated a delta psi but did so only if the external glutamine concentration was greater than 15 mM. Low-affinity glutamine uptake could not be saturated by as much as 200 mM glutamine, but glutamine-1 accounts for only a small fraction of the total glutamine at physiological pH values (pH 6 to 7). On the basis of these results, it appeared that the low-affinity glutamine transport was an electrogenic mechanism which was converting a chemical gradient of glutamine-1 into a delta psi. Other mechanisms of delta psi generation (electrogenic glutamine-pyroglutamate or -ammonium exchange) could not be demonstrated.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Characterization of sodium-dependent nucleoside transport in rabbit intestinal brush-border membrane vesicles

The characteristics of uridine transport were studied in rabbit intestinal brush-border membrane vesicles. Uridine was taken up into an osmotically active space in the absence of metabolism and there was no binding of uridine to the membrane vesicles. Uridine uptake was markedly enhanced by sodium, but showed no significant stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of uridine flux indicated a single system obeying Michaelis-Menten kinetics (Km value of 6.4 +/- 1.4 microM with a Vmax of 9.1 +/- 3.6 pmol/mg protein per s as measured under zero-trans conditions with a 100 mM NaCl gradient at 24 degrees C). A variety of purine and pyrimidine nucleosides were able to inhibit sodium-dependent uridine transport, suggesting that these nucleosides are also permeants for the same system. Consistent with this suggestion was the finding that these nucleosides also stimulated uridine efflux from the brush-border membrane vesicles. The sodium: uridine coupling stoichiometry was found to be 1:1 as measured by the activation method. From these results it is concluded that a broad specificity sodium-dependent nucleoside transporter is present at the brush-border membrane surface of rabbit enterocytes.     

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.     

Synthesis and transport characteristics of photoaffinity probes for the hepatocyte bile acid transport system

In an effort to characterize the hepatocyte bile acid transport system, a photoreactive derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid (7-ADTC) has been synthesized and its transport properties compared to those of the natural substrate. Both the bile acid and its synthetic analog were shown to be transported against an electrochemical gradient as well as a chemical gradient. Transport as a function of concentration and the presence of sodium indicated that both substrates were taken up by a sodium-dependent and a sodium-independent route. Taurocholate had Km values of 26 and 57 microM and Vmax values of 0.77 and 0.15 nmol/mg of protein/min, respectively. In comparison, 7-ADTC had very similar kinetic properties with Km values of 25 and 31 microM and Vmax values of 1.14 and 0.27 nmol/mg of protein/min. Each compound was shown to inhibit competitively the transport of the other, suggesting that these substrates utilized a common membrane carrier. The transport properties of the photoreactive anion transport inhibitor, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) were also characterized in the hepatocyte system. Transport occurred via a sodium-dependent and a sodium-independent route with Km values of 210 and 555 microM and Vmax values of 0.57 and 1.62 nmol/mg of protein/min. As in the case of 7-ADTC, NAP-taurine and taurocholate were also shown to be mutual competitive inhibitors. In the absence of light, 7-ADTC was a reversible inhibitor of taurocholate uptake. Upon irradiation, irreversible photoinactivation of the taurocholate uptake system was observed. These results indicate that 7-ADTC and NAP-taurine can be utilized as photoaffinity probes for the identification of the bile acid carrier protein(s) in hepatocyte plasma membranes.     

Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain

Efflux of L-glutamic acid from synaptic plasma membrane vesicles requires external potassium. This requirement is saturated by concentrations of about 15 mequiv/L potassium. In the absence of potassium, L-glutamic acid can be released from the vesicles in the presence of external L-glutamic acid. This stimulation does not require external sodium but is dependent on the external concentration of L-glutamic acid. Half-maximal effects are obtained by concentrations of about 1 microM which are very similar to the apparent Km for L-glutamic acid influx. Efflux of labeled glutamate driven by external sodium plus glutamate requires internal sodium. These findings suggest that the transporter displays an asymmetric behavior toward sodium. This ion dissociates much more slowly than L-glutamic acid on the external surface of the membrane but not on the internal surface. Furthermore, it appears that the transporter translocates potassium in a step distinct from the L-glutamic acid translocation step. The simplest explanation is that upon translocation of sodium and L-glutamic acid and their release to the inside, potassium binds to the transporter, enabling it to return to the outside to allow initiation of a new transport cycle.     

Calcium transport mechanisms in dog red blood cells studied from measurements of initial flux rates

Ca2+ efflux from dog red blood cells loaded with Ca2+ using the A23187 ionophore could be separated into two main components: (1) Mg- and ATP-dependent (active transport) and (2) dependent on external Na (K1/2 around 15 mM); at 80 microM internal free Ca the relative magnitudes of these fluxes were 70% and 30% respectively. The Na-dependent Ca2+ efflux had the following additional properties: (i) it was partially inhibited by ATP depletion or preincubation with vanadate, but it was not affected by Mg2+ depletion; (ii) it failed to be stimulated by external monovalent cations other than Na: (iii) it was stimulated by reduction in the internal Na+ concentration. Both active and Na-dependent Ca2+ efflux remained unchanged in hypotonic solutions or in solutions with alkaline pH (8.5). In cells containing ATP and Mg2+, external Ca2+ inhibited Ca2+ efflux (K1/2 around 1 mM); on the other hand, in Mg-free dog red cells external Ca2+ stimulated Ca2+ efflux (K1/2 about 30 microM). In Mg-depleted red cells incubated in the absence of external Na2+, Ca2+ influx as a function of external Ca2+ followed a monotonically saturable function (K1/2 around 20 microM): addition of Na resulted in (i) inhibition of Ca2+ influx and (ii) a sigmoid relationship between flux and external Ca2+. Intracellular Ca2+ stimulated the external Na-dependent Ca2+ efflux along a sigmoid curve (K1/2 around 30 microM); on the other hand the Ca pump had a biphasic response to internal Ca2+: stimulation at low internal Ca2+ (K1/2 between 1 and 10 microM), followed by a decline at internal Ca2+ concentrations higher than 50 microM.     

Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex

Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport.     

Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus

The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time-dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier-mediated transport system. The second component, which accounted for about 10-30% of the DA uptake at 2 microM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier-mediated system. The saturable, Na-dependent component had an apparent Km(DA) of about 0.5 microM. The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier-mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.     

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.     

Proton-symport of L-valine in plasma membrane vesicles isolated from leaves of the wild-type and the Val(r)-2 mutant of Nicotiana tabacum L

Transport of amino acids across the plasma membranes of various cell types is a key process in controlling the nitrogen balance of leaves. We studied the transport of the neutral amino acid L-valine into plasma membrane vesicles obtained by aqueous polymer two-phase partitioning of a microsomal fraction isolated from leaves of the wild-type and the Val(r)-2 mutant of tobacco (Nicotiana tabacum L.). Initial influxes were determined after the imposition of a pH-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. The initial magnitudes of the imposed gradients were DeltapH=2 and Deltapsi=-68 mV. In vesicles from the wild-type, the DeltapH-dependent valine influx could be analysed into a high-affinity (Km approximately 20 microM) and a low-affinity (Km approximately 3 mM) component. The influx of valine by the low-affinity system was stimulated about twofold, and that by the high-affinity system more than sixfold by the imposition of Deltapsi. This strong stimulation of the high-affinity system may indicate that it transports 2H+/amino acid. In the Val(r)-2 mutant the high-affinity component appeared to be completely absent.     

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.     

Characterization of L-carnitine transport into rat skeletal muscle plasma membrane vesicles.

Transport of L-carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient, L-carnitine transport showed a clear overshoot. The uptake of L-carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However, L-carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential-dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport of L-carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of the L-carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis-Menten kinetics with an apparent Km of 13.1 microM and a Vmax of 2.1 pmol.(mg protein-1).s-1. L-Carnitine transport could be trans-stimulated by preloading the vesicles with L-carnitine but not with the carnitine precursor butyrobetaine, and was cis-inhibited by L-palmitoylcarnitine, L-isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush-border membrane vesicles and OCTN2, a sodium-dependent high-affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, the Km values are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle.     

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

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

Acyclovir transport into human erythrocytes

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.