Characterization of glutamine uptake in mouse two-cell embryos and blastocysts.

Mouse two-cell embryos and blastocysts take up [3H]glutamine in vitro at a constant rate for at least 15 min, depending on the concentration of glutamine and developmental stage of the embryo. Uptake by two-cell embryos can be resolved into two saturable components. The major contributing system is Na+ independent, inhibited by alanine, methionine, 2-amino-2-norbornanecarboxylic acid (BCH) or leucine and has a Km of 3856 +/- 672 mumols l-1 and Vmax of 436 +/- 58 fmol per embryo per 10 min. These features are characteristics of the ubiquitous system L transporter. The second component is Na+ dependent with Km of 1064 +/- 914 mumols l-1 and Vmax 107 +/- 47 fmol per embryo per 10 min. Similar Vmax and inhibition of this component by glycine suggest a low reactivity with the gly-system. Blastocyst uptake of glutamine is mainly by a Na(+)-dependent saturable mechanism with Km of 524 +/- 75 mumols l-1 and Vmax of 1264 +/- 101 fmol per embryo per 10 min which is inhibited by alanine, isoleucine, leucine and BCH, features characteristic of the system B0,+. The increase in uptake capacity as a consequence of the appearance of the system B0,+ may be related to increased metabolic requirements for glutamine, in the rapidly expanding blastocyst.     

Glycine transport in mouse eggs and preimplantation conceptuses

At least two Na+-dependent systems for glycine transport became detectable, while another became undetectable during preimplantation development of mouse conceptuses. Glycine was taken up by a process in eggs and cleavage-stage conceptuses which closely resembles system Gly. Mediated transport at these stages was more rapid at higher Cl- concentrations, sigmoidally related to the exogenous Na+ concentration, and strongly inhibited by sarcosine but not by amino acids with larger side chains. Moreover, neither Li+ nor choline could substitute for Na+ in stimulating glycine transport. System Gly was the only mediated process detected for glycine uptake in unfertilized and fertilized eggs and two-cell conceptuses, but two, less conspicuous, sarcosine-resistant, Na+-dependent components of transport also appeared to be present in eight-cell conceptuses. One of the latter components seemed to remain relatively inconspicuous when conceptuses formed blastocysts, while system Gly became undetectable. In contrast, the other less conspicuous component in eight-cell conceptuses appeared to become the most conspicuous transport process in blastocysts. The latter process, previously designated system B0,+, was shown here also to interact strongly with a broad scope of zwitterionic and cationic amino acid structures. Moreover, transport of glycine via system B0,+ was more rapid at higher Cl- concentrations, and this Na+-dependent process as well as Na+-independent leucine uptake were inhibited by choline. Furthermore, Na+-dependent amino acid transport in two-cell conceptuses and blastocysts was inhibited by 1.0 or 10 mM ouabain, but the inhibition was incomplete at both concentrations. Since Na+/K+-ATPase has not been detected in two-cell conceptuses, inhibition of amino acid transport by ouabain may not have been due solely to an effect on this enzyme. The level of system Gly activity decreased during the development of eight-cell conceptuses from eggs, and this decrease could contribute to an associated decline in intracellular glycine. Since other amino acids begin to compete strongly with glycine for transport when system B0,+ replaces system Gly in conceptuses, this qualitative change in transport activity may help account for a further decrease in the glycine content of conceptuses, reported elsewhere to occur after they form blastocysts.     

A possible effect of the Na+ concentration in oviductal fluid on amino acid uptake by cleavage-stage mouse embryos

Two- and four-cell mouse embryos exhibited both Na+-dependent and Na+-independent components of zwitterionic alpha-amino acid transport, which we tentatively ascribe to the A and L amino acid transport systems, respectively. Uptake of taurine was virtually all Na+-dependent and is probably via the beta system. Na+-independent L-lysine uptake by two-cell embryos may have been via system y+. The small amount of lysine transport which was Na+-dependent (30% of the total) could not be attributed to any well known transport system and may have been due to the early ontogenetic expression of a newly described transport system which predominates in preimplantation blastocysts. We conclude that the rate of Na+-dependent amino acid transport in two-cell mouse embryos could be significantly affected in situ by changes in the [Na+] which are known to occur in oviductal fluid.     

Polyamine transport systems in the LLC-PK1 renal epithelial established cell line

LLC-PK1 cells were brought to a quiescent state by treatment with DL-2-difluoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamine synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous putrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependent and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vmax values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent Km = 4.7 +/- 0.7 microM) than the Na+-independent transporter (apparent Km = 29.8 +/- 3.5 microM). As a consequence, although the latter transporter had a higher Vmax, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the antileukemic agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+-dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK1 are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.     

Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons

Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.     

Development of system B0,+ and a broad-scope Na(+)-dependent transporter of zwitterionic amino acids in preimplantation mouse conceptuses

The nature and ontogeny of Na(+)-dependent L-alanine transport was examined in mouse eggs and preimplantation conceptuses. Mediated L-alanine uptake was not detected in fertilized or unfertilized eggs, but a small amount of Na(+)-dependent L-alanine transport was detected in 2-cell conceptuses. Na(+)-dependent alanine transport was more rapid at the 8-cell stage of development, and more than 10-fold faster in blastocysts than in 8-cell conceptuses. Analog inhibition analyses were consistent with the interpretation that L-lysine-sensitive and L-lysine-resistant components of transport were present at the 2-cell, 8-cell and blastocyst stages of development. The range of amino acids and their analogs that inhibited the most conspicuous component of alanine transport in blastocysts was consistent with the conclusion that system B0,+ is largely responsible for L-alanine uptake in these conceptuses. Moreover, system B0,+, but not other known systems in blastocysts, became susceptible to activation as these conceptuses approached the time of implantation, so this activation could be involved in implantation. Although the data are consistent with the possibility that system B0,+ is also present in 2-cell and 8-cell conceptuses, the relatively slow L-alanine transport in conceptuses at these earlier stages of development precluded more detailed study of their ability to take up alanine. Similarly, the less conspicuous L-lysine-resistant component of L-alanine transport in blastocysts also may be present in conceptuses as early as the 2-cell stage. The L-lysine-resistant component of L-alanine transport could not be attributed to residual system B0,+ activity, however, because it was inhibited more strongly by trans-OH-L-proline than L-arginine, whereas the reverse was the case for system B0,+. Similarly, L-tryptophan and L-leucine each inhibited system B0,+ more strongly than L-serine or L-cysteine, whereas all four of these amino acids inhibited the L-lysine-resistant component equally well. Moreover, a Hofstee plot for L-alanine influx was consistent with the interpretation that at least two mediated components of Na(+)-dependent L-alanine transport are present in blastocysts. The less conspicuous component of L-alanine transport in blastocysts was relatively susceptible to inhibition by L-leucine and L-tryptophan, but it resisted inhibition by the 'model' system A substrate, MeAIB, and the system ASC inhibitors, L-penicillamine and cationic amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional changes in cation-preferring amino acid transport during development of preimplantation mouse conceptuses

In a previous study, a Na(+)-independent, cation-preferring amino acid transport system was detected in preimplantation mouse blastocysts. The system resisted Na(+)-dependent inhibition by homoserine and so resembled the lysosomal system c more than it resembled the plasmalemmal system y+. We now report the presence of a cation-preferring system in unfertilized and fertilized eggs and cleavage-state conceptuses which also resists Na(+)-dependent inhibition by homoserine. The systems in 1-cell conceptuses and blastocysts are, however, insensitive to changes in pH in the interval of 6.0 to 8.0 and, thus, different from the pH-sensitive system c. Moreover, the relative strengths of the interactions of a variety of basic amino acids with the systems in conceptuses do not correspond well with the relative strengths of their interactions with either system c or system y+. Similarly, the system in 1-cell conceptuses can be distinguished from the system in blastocysts because L-arginine interacts about equally well with each of these systems, whereas the system in 1-cell conceptuses is inhibited more strongly than the system in blastocysts by most other basic amino acids. In addition, inhibition of the system in 1-cell conceptuses by some basic amino acids is Na(+)-stimulated, whereas Na+ does not affect inhibition of the system in blastocysts. Finally, L-tryptophan inhibits the system in blastocysts better than L-histidine or D-arginine do, but the reverse is true for the system in 1-cell conceptuses. Therefore, the relative activities of at least two forms of a novel, cation preferring amino acid transport process change during development of blastocysts from fertilized eggs. For convenience, the forms of the cation-preferring transport processes that seem to predominate at the 1-cell and blastocysts stages are provisionally designated systems b+1 and b+2, respectively, although these two systems need not represent entirely different gene products.     

Betaine is a highly effective organic osmolyte but does not appear to be transported by established organic osmolyte transporters in mouse embryos

Betaine protects early preimplantation mouse embryos against increased osmolarity in vitro, functioning as an organic osmolyte. Betaine is effective at very low external concentrations, with half-maximal protection of 1-cell embryo development to blastocysts at approximately 50 microM, making it one of the best osmoprotectants for mouse preimplantation embryos. We performed studies designed to determine whether known high-affinity organic osmolyte transporters could account for the ability of betaine to act as an organic osmolyte in preimplantation embryos. We found no evidence in 1-cell embryos of transport by a betaine/GABA transporter (BGT1), the osmoregulated betaine transporter found in a number of cell types, as betaine and GABA did not inhibit each other's transport. Instead, all saturable GABA transport in embryos was apparently via the beta-amino acid transporter. We also found that the glycine transporter, GLY, which mediates osmoprotective transport of glycine in early preimplantation embryos, does not appear to transport betaine. Finally, increased osmolarity did not induce any detectable System A amino acid transporter activity, which is osmotically-inducible in other cells and can transport betaine. There does appear, however, to be a saturable betaine transporter in 1-cell mouse embryos, as considerable 14C-betaine transport was measured which was substantially inhibited by excess unlabeled betaine. Our data imply that betaine functions as an organic osmolyte in embryos due to its saturable transport via a mechanism distinct from known osmolyte transporters. We propose that an unidentified high-affinity betaine transporter may be expressed in early embryos and mediate transport of betaine as an organic osmolyte.     

Changes in the activities of amino acid transport systems b0,+ and L during development of preimplantation mouse conceptuses

Uptake of leucine, lysine, and arginine was predominantly Na(+)-independent in mouse conceptuses through the 8-cell stage of development, and two components of saturable transport were detected for each of these amino acids. Uptake of cationic substrates from solutions near 1 microM was inhibited most strongly by bulky cationic and zwitterionic amino acids whose carbon skeletons do not branch at the alpha or beta positions. By this criterion, system b0,+ accounted for most of the Na(+)-independent arginine and lysine transport in eggs and conceptuses throughout preimplantation development. A small, leucine-resistant, cation-preferring component of amino acid transport was also detected in these cells. Leucine uptake was inhibited most strongly by bicyclic, branched-chain or benzenoid, zwitterionic amino acids in eggs and conceptuses prior to formation of blastocysts. Therefore, it appeared to be taken up mainly by system L, while system b0,+ accounted for a smaller portion of leucine uptake during this developmental period. In blastocysts, in contrast, system L was less conspicuous, and system b0,+ was primarily responsible for Na(+)-independent leucine uptake. The Vmax values for transport of amino acids by system b0,+ increased by up to 30-fold in conceptuses between the 1-cell and blastocyst stages. In contrast, the Vmax value for leucine transport via system L decreased while the Km value increased between these two developmental stages. Although several explanations for these changes are possible, we favor the hypothesis that the density of system L transport sites in plasma membranes decreases while the number of system b0,+ sites increases during development of blastocysts from 1-cell conceptuses.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.     

Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Uptake of [(3)H]glycine by synaptosomes of channel

It is known that channel catfish erythrocytes can take up glycine by several distinct transport systems. Further, glycine is an inhibitory neurotransmitter in mammalian brain and spinal cord. Consequently, the uptake of [(3)H]glycine by catfish brain was investigated and found to be a saturable process, dependent on the presence of Na(++) and Cl(--) and sensitive to temperature. A kinetic analysis of transport was performed at 22C. This showed that a high-affinity system existed which exhibited a K(m) of 5.1 (+/- 2. 1) microM. Several structural analogues of glycine were capable of inhibiting uptake in a competitive manner. The most effective inhibitor was sarcosine (IC(50) 5 36 microM). Uptake was also able to be inhibited by harmaline, a drug known to interfere with Na(+)-dependent transport processes. It is concluded that glycine transport by channel catfish brain has much in common with transport by mammalian nervous tissue which is carried out by the membrane carriers GLYT1 and GLYT2. On the other hand, synaptosomal transport differs somewhat from glycine transport by channel catfish erythrocytes.     

Characterization of Xylamine Binding to Proteins of PC12 Pheochromocytoma Cells

PC12 pheochromocytoma cells take up 3,4-dihydroxyphenylethylamine (dopamine) and norepinephrine by a Na+-dependent, cocaine-sensitive system. The kinetics suggest that the same transporter functions for both substrates. Xylamine, a nitrogen mustard that blocks catecholamine uptake into neurons, irreversibly inhibited norepinephrine uptake into PC12 (IC50 = 15 microM). Pretreatment with 10 microM xylamine did not inhibit norepinephrine transport if 10 microM cocaine or 100 microM norepinephrine was also present during the pretreatment period or if Na+ was absent. These results indicate that xylamine must interact with the norepinephrine transporter to inhibit norepinephrine uptake. PC12 accumulated [3H]xylamine; this uptake had Na+-dependent and Na+-independent components. The Na+-dependent uptake was saturable (Km = 13 microM), and it was inhibited by cocaine (IC50 = 0.6 microM), desipramine (IC50 less than 1 nM), and norepinephrine (IC50 = 1 microM). Several proteins became prominently labeled when intact PC12 cells were incubated with [3H]xylamine; these proteins were enriched in a plasma membrane fraction and have molecular weights of 17,000, 24,000, 31,000, 33,000, 41,000, 42,000, 52,000, and 80,000. Other proteins were labeled less prominently. The labeling of all proteins was markedly decreased when the incubation with [3H]xylamine occurred in the presence of cocaine, desipramine, gramicidin D, or in a Na+-free buffer. These results indicate that xylamine must be transported into the cells for covalent binding to proteins to occur. [3H]Xylamine labeled essentially the same proteins when incubated with cell homogenates, but competition experiments with bretylium, desipramine, and cocaine failed to reveal which of the [3H]xylamine-labeled proteins is associated with the norepinephrine transporter.     

Na+-dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na+-dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na+ dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na+-dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid (Ki approximately 1.0 mM) and L-valine (Ki approximately 0.10 mM) appeared to inhibit Na+-dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, we conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system.     

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.     

Na+-Ca2+ exchange activity in rabbit lymphocyte plasma membranes

Plasma membranes of rabbit thymus lymphocytes accumulated Ca2+ when a Na+ gradient (intravesicular greater than extravesicular) was formed across the membranes. Dissipation of the Na+ gradient by the addition of Na+ to the external medium decreased Ca2+ uptake. Ca2+ preloaded into the lymphocytes was extruded when Na+ was added to the external medium. The Ca2+ uptake decreased at acidic pH but increased at alkaline pH (above 8) and the activity was saturable for Ca2+ (apparent Km for Ca2+ was 61 microM and apparent Vmax was 11.5 nmol/mg protein per min). Na+-dependent uptake of Ca2+ was inhibited by tetracaine and verapamil, and partially inhibited by La3+. The uptake was not influenced by orthovanadate.     

Pharmacological properties of glycine uptake in the developing rat retina

A pharmacological characterization of glycine transport was performed in the rat retina at different postnatal ages. The uptake of 3H-glycine increased during the first 2 weeks of postnatal age, reaching maximum values at 12 days; then it decreased sharply to the adult values. We found a Na+ -dependent and high-affinity transport system with a Km of 100 microM. The Na+ Hill coefficient for glycine uptake was 1.76 +/- 0.07. Although glycine uptake was insensitive to staurosporine and phorbol ester, it was reduced 40-50% by sarcosine and ALX5407. Besides, amoxapine inhibited glycine uptake by 40 and 70% in adult and immature retina, respectively. These results suggest that the Glyt1 transporter was concentrated in the nerve terminals. In addition to the presence of Glyt1 in the retina, our results provided evidence of the occurrence of Glyt2 and/or another isoform of glycine transporter, which might have had a role in the retina development.     

alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

Involvement of cytoskeletal structures in nerve-growth-factor-mediated induction of ornithine decarboxylase.

Taurine transport in isolated brush-border-membrane vesicles from rat kidney is concentrative and it is driven by the Na+ gradient and transmembrane potential difference; binding is not a significant component of net uptake. The Na+-dependent component of net uptake is saturable with an apparent Km of 17 microM. The taurine-transport mechanism is selective for beta-amino compounds.