The preferential interaction of L-threonine with transport system ASC in cultured human fibroblasts.

The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts.     

Characterization of neutral and cationic amino acid transport in Xenopus oocytes

Amino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+-dependent and saturable Na+-independent processes. Na+-dependent transport of 2-aminoisobutyric acid (AIB) was insensitive to cis- or trans-inhibition by the System A-defining substrate 2-(methylamino)-isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+-dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+-dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo,+ was obtained through inhibition analysis of Na+-dependent arginine transport as well arginine inhibition of Na+-dependent threonine uptake. The Na+-independent transport of leucine was subject to trans-stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2-amino-(2,2,1)-bicycloheptane-2-carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+-independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.     

Stereoselective transport of histidine in rat lung microvascular endothelial cells

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na(+)-K(+)-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na(+) with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma-monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na(+). Moreover, the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na(+) removal, indicating facilitated diffusion by a Na(+)-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na(+)-dependent system N and Na(+)-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na(+) replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma-monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.     

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.     

Neutral amino acid transport in isolated rat pancreatic islets

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.     

Differential effects of transmembrane potential on two Na+-dependent transport systems for neutral amino acids

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K+ permeability had been increased, either by valinomycin or by activation of Ca2+-dependent K+ channels, in medium containing different K+ concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na+-dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2-3.1-times for each 60 mV-hyperpolarization. The Na+-dependent system ASC was insensitive to membrane potential. The Na+-independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.     

Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Mechanism of polyamine spermidine uptake by Xenopus laevis oocytes

In this study, the mechanisms of polyamine spermidine (Spd) uptake were investigated in Xenopus laevis oocytes. Spd uptake followed a sigmoidal kinetics with [S]90/[S]10 = 3 microM and Hill interaction coefficient (n) = 2. The order of magnitude of uptake and efflux was similar (t1/2 = 45 min). The equilibrium potential for Spd, calculated by Nenrst equation, was 90.78 mV. Free energy change for the uptake (delta G) was found to be 2.31 Kcal/mole of Spd. During efflux, Spd was not converted into putrescine or spermine. It seems that there are two types of Spd uptake pathways: Na(+)-dependent and Na(+)-independent since replacement of Na+ from incubation medium did not completely abolish the Spd uptake. The Na(+)-dependent component of Spd uptake was shared neither by system A nor by system ASC amino acids.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

Characterization of D-Aspartic Acid Uptake by Rat Hippocampal Slices and Effect of Ischemic Conditions

The cellular uptake of D-aspartic acid (D-Asp) as a model compound for glutamic acid transport was studied in rat hippocampal slices. D-Asp is accumulated by both Na(+)-dependent and Na(+)-independent processes in hippocampal slices, and both processes are dependent on temperature. The Na(+)-dependent uptake is assumed to be high in affinity (apparent Km = 0.17 mM), but low in capacity, whereas the Na(+)-independent uptake is much lower in affinity (Km = 2.86 mM), but higher in capacity. L-Aspartic acid, L-glutamic acid, dihydrokainic acid, and threo-3-hydroxy-DL-aspartic acid markedly inhibited the uptake of D-Asp with Na+ in the medium, whereas D-glutamic acid, glycine, and L-lysine had no significant effect. The Na(+)-dependent uptake of D-Asp was significantly reduced under "hypoglycemic," "anoxic," and "ischemic" conditions, whereas the Na(+)-independent uptake was unaffected. Metabolic inhibitors such as NaCN and ICH2COOH significantly inhibited the Na(+)-dependent uptake, but not the Na(+)-independent uptake. These results suggest that the Na(+)-dependent component of D-Asp transport in rat hippocampal cells is inactivated under ischemic conditions, whereas the Na(+)-independent component is unaffected.     

Neutral amino acid transport in embryonal carcinoma cells

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.     

Heterogeneity of transport systems for L-glutamine in mouse mammary gland

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Topographical similarities between harmaline inhibition sites on Na+-dependent amino acid transport system ASC in human erythrocytes and Na+-independent system asc in horse erythrocytes

Na+-dependent system ASC and Na+-independent system asc are characterized by a common selectivity for neutral amino acids of intermediate size such as L-alanine and by their interactions with dibasic amino acids. For system ASC, the positive charge on the dibasic amino acid side chain is considered to occupy the Na+-binding site on the transporter. We report here the use of harmaline (a Na+-site inhibitor in some systems) as a probe of possible structural homology between these two classes of amino acid transporter. Harmaline was found to inhibit human erythrocyte system ASC noncompetitively with respect to L-alanine concentration, but approximated competitive inhibition with respect to Na+ concentration (apparent Ki = 2.0 and 0.9 mM, respectively). Similarly, harmaline noncompetitively inhibited L-alanine uptake by horse erythrocyte systems asc1 and asc2 (apparent Ki = 2.0 and 1.9 mM, respectively). In contrast, harmaline functioned as a competitive inhibitor of L-lysine uptake by system asc1 (apparent Ki = 2.6 mM). It is concluded that harmaline competes with Na+ for binding to system ASC and that a topographically similar harmaline inhibition site is present on system asc. This site does not however bind Na+, the asc1 transporter exhibiting normal L-alanine and L-lysine influx kinetics in the total absence of extracellular cations.     

Inhibition of transport system b0,+ in blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor site

The most conspicuous, Na(+)-independent amino acid transport process in preimplantation mouse blastocysts was provisionally designated system b0,+ because it accepts some cationic and zwitterionic amino acids about equally well as substrates. Although system b0,+ is not Na(+)-stimulated, it has not been determined if it is inhibited by Na+, or if its activity is affected by most other ions. Therefore, we measured uptake of amino acids by blastocysts in isotonic solutions of different ionic and nonionic osmolites. Na(+)-independent L-leucine uptake was unaffected by the ion concentration, but L-lysine transport was several-fold faster in isotonic solutions of non-electrolytes than in similar solutions of inorganic and organic salts or zwitterions. The Km value for 'Na(+)-independent' L-lysine transport was about 10-fold higher in isotonic salt solutions than in solutions of nonionic osmolites, whereas the Km value for L-leucine transport was about the same in either type of solution. Therefore, inorganic and organic cations and the cationic portions of zwitterions appear to compete with cationic but not zwitterionic amino acids for system b0,+ receptor sites. The cation, harmaline, was a particularly strong competitive inhibitor of 'Na(+)-independent' L-lysine uptake by system b0,+, even in isotonic salt solutions, whereas it inhibited L-leucine uptake noncompetitively. Moreover, harmaline appeared to compete with inorganic cations for the lysine receptor sites of system b0,+. Harmaline also has been found by other investigators to competitively inhibit L-lysine uptake by the Na(+)-independent system asc1 in horse erythrocytes, whereas it noncompetitively inhibits alanine uptake by the same system. Similarly, harmaline noncompetitively inhibits L-alanine uptake by the Na(+)-dependent system ASC in human erythrocytes, but it appears to compete for binding with L-alanine's cosubstrate, Na+. In addition, others have found that the positively-charged side chains of cationic amino acids seem to take the place of Na+ needed near side chains in order for zwitterionic amino acids to be transported by systems ASC and y+. We conclude that system b0,+ may be similar to systems asc1, ASC and y+, and that each of these systems may be a variant of the same ancestral transport process. We speculate that since it appears to accept a broader scope of substrates and to interact with a wider variety of cations than do systems asc1, ASC or y+, system b0,+ may more closely resemble the proposed ancestral transport process than any of the other contemporary systems.     

Glycine transport by cultured human fibroblasts

The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na+-dependent and Na+-independent mechanisms. Na+-independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na+-dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.     

Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes

The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na(+)-dependent and a Na(+)-independent system. The Na(+)-dependent system was inhibited by aspartate and was consistent with the commonly occurring system designated system X-AG. The Na(+)-independent system was inhibited by cystine and was consistent with system x-c described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na(+)-independent glutamate uptake. In C-6 cells, the glutamate uptake via systems X-AG and x-c accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes, the glutamate uptake via system X-AG was very potent, whereas the uptake via system xc- was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system xc- was necessary for the uptake of cystine, another substrate of system xc-. Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.     

Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A,ASC, and l transport activities

Amino acid transport in Madin-Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na⁺-independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na⁺-dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na⁺-dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na⁺-dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na⁺-dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE₁, and was elevated 3–7 fold in the transformed cell line, MDCK-T₁. The activity of the ASC system was slightly stimulated by insulin and by PGE₁, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.