The isolation of a canavanine-resistant mouse embryonal carcinoma cell line which has reduced arginine transport

A mouse embryonal carcinoma cell line resistant to the toxic arginine analogue -canavanine has been isolated. Kinetic studies of the transport of arginine in the canavanine-sensitive parental cell indicate that there are two arginine uptake systems which operate at different substrate concentrations. The canavanine-resistant variant shows a reduction in the rate at which it can transport arginine at all substrate concentrations. This is not, however, due to the complete loss of either uptake system. The observation that the rate of arginine transport at high substrate concentrations is reduced in the variant can be explained, at least in part, by an increase in chromosome number and cell volume. This is not true of the reduction in the low substrate concentration uptake system. The observation that the reductions in the two uptake systems can be dissociated in this way provides support for the conclusion, based on the kinetic data from the parental cell, that there are two independent arginine transport systems in this mouse embryonal carcinoma cell line.     

Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity

Pertussis toxin (PTX) induces activation of l-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on l-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in l-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main l-arginine transporter in PAEC. Unlike holotoxin, the beta-oligomer-binding subunit of PTX did not affect l-arginine transport in PAEC, suggesting that Galpha(i) ribosylation is an important step in the activation of l-arginine transport by PTX. An activator of adenylate cyclase, forskolin, and an activator of protein kinase A (PKA), Sp-cAMPS, did not affect l-arginine transport in PAEC. In addition, inhibitors of PKA or adenylate cyclase did not change the activating effect of PTX on l-arginine uptake. Long-term treatment with PTX (18 h) induced a 40% decrease in protein kinase C (PKC)-alpha but did not affect the activities of PKC-epsilon and PKC-zeta in PAEC. An activator of PKC-alpha, phorbol 12-myristate 13-acetate, abrogated the activation of l-arginine transport in PAEC treated with PTX. Incubation of PTX-treated PAEC with phorbol 12-myristate 13-acetate in combination with an inhibitor of PKC-alpha (Go 6976) restored the activating effects of PTX on l-arginine uptake, suggesting PTX-induced activation of l-arginine transport is mediated through downregulation of PKC-alpha. Measurements of nitric oxide (NO) production by PAEC revealed that long-term treatment with PTX induced twofold increases in the amount of NO in PAEC. PTX also increased l-[(3)H]citrulline production from extracellular l-[(3)H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of l-arginine transport in PAEC.     

Transport of l-arginine in brush border vesicles derived from rabbit kidney cortex

The uptake of l-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37 °C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K_m values of 0.07 and 3.5 mm and V_max values of 1.5 and 8 nmol/mg protein × 15 s, respectively. For the inhibitors of l-[¹⁴C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mm in a mannitol medium) the following sequence of inhibitory strength was established: l-arginine, l-ornithine, l-cystine, l-lysine, d-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K⁺ in the presence of gramicidin D, an overshoot of l-arginine uptake was observed. Initial uptake of l-arginine was slightly faster in the presence of a Na⁺ gradient (outside to inside) than under a K⁺ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, l-arginine uptake in the first 30 s was faster in the NaCl than in the KCl medium. These experiments indicate, beside a major ion-independent l-arginine transport, the presence of a transport stimulated by Na⁺ in isolated brush border vesicles.     

Arginine uptake by rabbit spermatozoa

The transport of l-arginine by rabbit spermatozoa was found to proceed by saturable, chemically specific mechanisms. Kinetic analysis of initial rates of transport at substrate concentrations from 1.0 μm to 1.0 μm indicate the presence of two saturable transport components. A low-affinity component has an apparent K_m of 0.64 μm and an apparent V of 43.4 nmol/10⁸ cells/30 s. A second, high-affinity component has an apparent K_m of 4.0 μm and an apparent V of 425 pmol/10⁸ cells/30 s. Rabbit spermatozoa actively transported l-arginine in a range of pH values from 6.5 to 10.5 with a pH optimum for the low-affinity component of 7.2–7.6 and a pH optimum for the high-affinity component of 7.8–8.0. Inhibitor studies indicate that the energization for transport may be dependent on ATP rather than on a pH gradient or transmembrane potential. Competition experiments with arginine analogs and amino acids suggest that the high- and low-affinity components may recognize the terminal guanidino group.     

Arginine homeostasis in J774.1 macrophages in the context of Mycobacterium bovis BCG infection

The competition for L-arginine between the inducible nitric oxide synthase and arginase contributes to the outcome of several parasitic and bacterial infections. The acquisition of L-arginine, however, is important not only for the host cells but also for the intracellular pathogen. In this study we observe that strain AS-1, the Mycobacterium bovis BCG strain lacking the Rv0522 gene, which encodes an arginine permease, perturbs l-arginine metabolism in J774.1 murine macrophages. Infection with AS-1, but not with wild-type BCG, induced l-arginine uptake in J774.1 cells. This increase in L-arginine uptake was independent of activation with gamma interferon plus lipopolysaccharide and correlated with increased expression of the MCAT1 and MCAT2 cationic amino acid transport genes. AS-1 infection also enhanced arginase activity in resting J774.1 cells. Survival studies revealed that AS-1 survived better than BCG within resting J774.1 cells. Intracellular growth of AS-1 was further enhanced by inhibiting arginase and ornithine decarboxylase activities in J774.1 cells using L-norvaline and difluoromethylornithine treatment, respectively. These results suggest that the arginine-related activities of J774.1 macrophages are affected by the arginine transport capacity of the infecting BCG strain. The loss of Rv0522 gene-encoded arginine transport may have induced other cationic amino acid transport systems during intracellular growth of AS-1, allowing better survival within resting macrophages.     

Multiple pathways for cationic amino acid transport in rat seminiferous tubule cells

Arginine and ornithine are known to be important for various biological processes in the testis, but the delivery of extracellular cationic amino acids to the seminiferous tubule cells remains poorly understood. We investigated the activity and expression of cationic amino acid transporters in isolated rat Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids. We assessed the l-arginine uptake kinetics, Na(+) dependence of transport, profiles of cis inhibition of uptake by cationic and neutral amino acids, and sensitivity to trans stimulation of cationic amino acid transporters, and studied the expression of the genes encoding them by RT-PCR. Our data suggest that l-arginine is taken up by Sertoli cells and peritubular cells, principally via system y(+)L (SLC3A2/SLC7A6) and system y(+) (SLC7A1 and SLC7A2), with system B(0+) making a minor contribution. By contrast, system B(0+), associated with system y(+)L (SLC3A2/SLC7A7 and SLC7A6), made a major contribution to the transport of cationic amino acids in pachytene spermatocytes and early spermatids. Sertoli cells had higher rates of l-arginine transport than the other seminiferous tubule cells. This high efficiency of arginine transport in Sertoli cells and the properties of the y(+)L system predominating in these cells strongly suggest that Sertoli cells play a key role in supplying germ cells with l-arginine and other cationic amino acids. Furthermore, whereas cytokines induce nitric oxide (NO) production in peritubular and Sertoli cells, little or no upregulation of arginine transport by cytokines was observed in these cells. Thus, NO synthesis does not depend on the stimulation of arginine transport in these somatic tubular cells.     

Detrimental effect of oxidized LDL on endothelial arginine metabolism and transportation

The action of oxidatively modified low-density lipoprotein on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. However, the biochemical mechanism for such action is not clear. We have previously shown that arginine uptake and metabolism are major determinants of endothelial function in heart failure and hypertension. In the present study we therefore aimed to assess the effects of oxidized LDL, a major pro-atherogenic molecule, on endothelial l-arginine metabolism and its uptake. Endothelial cells were exposed to oxidized LDL or native LDL for 24h, and the resultant effects on (1) the intracellular content of arginine and its major metabolites including citrulline, N(G)-hydroxy-l-arginine, asymmetric dimethylarginine, symmetric dimethylarginine and ornithine, (2) [3H]-l-arginine uptake and, (3) the pattern of distribution of cationic amino acid transporter 1, the principal l-arginine transporter, by confocal microscopy. Oxidized LDL (100 microg/mL) reduced intracellular arginine and N(G)-hydroxy-l-arginine contents by 56 and 71% (P<0.05), respectively, with a concomitant 205% increase in ADMA (P<0.05). In conjunction, oxidized LDL reduced endothelial uptake of [3H]-arginine by 60%. Furthermore, incubation of endothelial cells with oxLDL led to internalization of cationic amino acid transporter 1. We demonstrate a novel mechanism, reduced l-arginine transport, by which oxidized LDL impairs the ability of the endothelium to generate nitric oxide.     

Histidine Uptake in Strains of Neurospora crassa with Normal and Mutant Transport Systems

Kinetic parameters for three systems of active histidine uptake by germinated conidia of Neurospora crassa have been measured. Each system appears to follow typical Michaelis-Menten kinetics when studied separately from the other systems. Under the conditions studied, the general amino acid transport system was found to account for the major portion of histidine uptake from low concentrations. Three types of transport mutants with altered growth inhibition patterns were selected in a histidine auxotroph. Growth of one mutant, type bas(a), could be inhibited by the addition of methionine to a histidine-supplemented medium, and another type, neu(a), could be inhibited by the addition of arginine. These mutants were shown to be lacking active histidine uptake by the basic amino acid and neutral amino acid transport systems, respectively. Another type of double mutant (his-3, neu(r)) could be inhibited only by the addition of very high concentrations of methionine in the presence of arginine and histidine, and the mutation appeared to have altered the specificity of the neutral amino acid permease.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Independent regulation of transport and biosynthesis of arginine in Escherichia coli K-12.

From an arginine auxotrophic strain, a mutant was isolated which is able to utilize d-arginine as a source of l-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to five-fold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component of the arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.     

Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation.

The zero-trans uptake of purines and pyrimidines was measured in suspensions of Novikoff rat hepatoma, mouse L, P388 mouse leukemia, and Chinese hamster ovary cells by a rapid kinetic technique which allows the determination of uptake time points in intervals as short as 1.5 s. Kinetic parameters for purine/pyrimidine transport were determined by measuring substrate influx into cells in which substrate conversion to nucleotides was negligible either due to lack of the appropriate enzymes or to depletion of the cells of ATP (5'-phosphoribosylpyrophosphate), and by computer fitting exact, integrated rate equations derived for various carrier-mediated transport models directly to zero-trans influx data. The results indicate that different carriers function in the transport of hypoxanthine/guanine, adenine, and uracil with substrate:carrier association constants (K) at 24 degrees C of 300 to 400 muM, 2 to 3 mM, and about 14 mM, respectively, for Novikoff cells. K and Vmax for hypoxanthine transport by L and P388 cells are similar to those for Novikoff cells, but the transport capacity of Chinese hamster ovary cells is much lower and K = 1500 muM. All transport systems are completely symmetrical. Hypoxanthine transport is so rapid that an intracellular concentration of free hypoxanthine (90%) close to that in the medium is attained within 20 to 50 s of incubation at 24 degrees C, at least at extracellular concentrations below K. In cells in which conversion to nucleotides is not blocked free hypoxanthine accumulates intracellularly to steady state levels with equal rapidity and thereafter the rate of hypoxanthine uptake into total cell material is strictly a function of the rate of phosphoribosylation. The low Km systems for hypoxanthine (1 to 9 muM) and adenine (0.2 to 40 muM) uptake detected previously in many types of cells reflect the substrate saturation of the respective phosphoribosyltransferases rather than of the transport system.     

Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter.

Enterococcus faecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway, uptake of arginine and excretion of ornithine are mediated by an arginine-ornithine antiport system. The translocation of agmatine was studied in whole cells grown in the presence of arginine, agmatine, or glucose. Rapid uncoupler-insensitive uptake of agmatine was observed only in agmatine-grown cells. A high intracellular putrescine pool was maintained by these cells, and this pool was rapidly released by external putrescine or agmatine but not by arginine or ornithine. Kinetic analysis revealed competitive inhibition for uptake between putrescine and agmatine. Agmatine uptake by membrane vesicles was observed only when the membrane vesicles were preloaded with putrescine. Uptake of agmatine was driven by the outwardly directed putrescine concentration gradient, which is continuously sustained by the metabolic process. Uptake of agmatine and extrusion of putrescine by agmatine-grown cells of E. faecalis appeared to be catalyzed by an agmatine-putrescine antiporter. This transport system functionally resembled the previously described arginine-ornithine antiport, which was exclusively induced when the cells were grown in the presence of arginine.     

Involvement of transport in Rhodobacter sphaeroides chemotaxis.

The chemotactic response to a range of chemicals was investigated in the photosynthetic bacterium Rhodobacter sphaeroides, an organism known to lack conventional methyl-accepting sensory transduction proteins. Strong attractants included monocarboxylic acids and monovalent cations. Results suggest that the chemotactic response required the uptake of the chemoeffector, but not its metabolism. If a chemoeffector could block the uptake of another attractant, it also inhibited chemotaxis to that attractant. Sodium benzoate was not an attractant but was a competitive inhibitor of the propionate uptake system. Binding in an active uptake system was therefore insufficient to cause a chemotactic response. At different concentrations, benzoate either blocked propionate chemotaxis or reduced the sensitivity of propionate chemotaxis, an effect consistent with its role as a competitive inhibitor of uptake. Bacteria only showed chemotaxis to ammonium when grown under ammonia-limited conditions, which derepressed the ammonium transport system. Both chemotaxis and uptake were sensitive to the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, suggesting an involvement of the proton motive force in chemotaxis, at least at the level of transport. There was no evidence for internal pH as a sensory signal. These results suggest a requirement for the uptake of attractants in chemotactic sensing in R. sphaeroides.     

Characteristics and energy requirements of an alpha-aminoisobutyric acid transport system in Streptococcus lactis.

Galactose-grown cells of Streptococcus lactis ML3 acculated alpha-aminoisobutyric acid (AIB) by using energy derived from glycolysis and arginine catabolism. The transport system displayed low-affinity Michaelis-Menten saturation kinetics. Using galactose or arginine as energy sources, similar V max and K m values for AIB entry were obtained, but on prolonged incubation the intracellular steady-state concentration of AIB in cells metabolizing arginine was only 65 to 70% that attained by glycolyzing cells. Efflux of AIB FROM PRELOADED CElls was temperature dependent and exhibited the characteristics of a first-order reaction. The rate of AIB exit was accelerated two- to threefold in the presence of metabolizable energy sources. Metabolic inhibitors including p-chloromercuribenzoate, dinitrophenol, azide, arsentate, and N, N'-dicyclohexylcarbodiimide either prevented or greatly reduced AIB uptake. Fluoride, iodoacetate and N-ethylmaleimide abolished galactose-dependent, but not arginine-energized, AIB uptake. K+ and Rb+ reduced the steady-state intracellular AIB concentration by approximately 40%, and these cations also induced rapid efflux of solute from actively transporting cells. Equivalent concentrations (10 mM) of Na+, Li+, or NH4+ were much less inhibitory. The proton-conducting ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenlyhydrazone abolished uptake and induced AIB efflux even though glycolysis and arginine catabolism continued at 60 and 140%, respectively, of control rates. A proton motive force is most likely involved in the active transport of AIB, whereas data from efflux studies suggest that energy is coupled to AIB exit in cells of S. lactis ML3.     

Energetics of arginine and lysine transport by whole cells and membrane vesicles of strain SR, a monensin-sensitive ruminal bacterium.

Strain SR, a monensin-sensitive, ammonia-producing ruminal bacterium, grew rapidly on arginine and lysine, but only if sodium was present. Arginine transport could be driven by either an electrical potential or a chemical gradient of sodium. Arginine was converted to ornithine, and it appeared that ornithine efflux created a sodium gradient which in turn drove arginine transport. There was a linear decline in arginine transport as pH was decreased from 7.5 to 5.5, and the cells did not grow at a pH less than 6.0. The Eadie-Hofstee plot was biphasic, and arginine could also be taken by a high-capacity diffusion mechanism. Because arginine was a strong inhibitor of lysine transport and lysine was a weak inhibitor of arginine transport, it appeared that both lysine and arginine were taken up by an arginine-lysine carrier which had a preference for arginine. The rate of lysine fermentation was always proportional to the extracellular lysine concentration, and facilitated diffusion was the dominant mechanism of lysine transport. When SR was grown in continuous culture on arginine or lysine, the theoretical maximal growth yield was similar (13 g of cells per mol of ATP), but the apparent maintenance energy requirement for arginine was greater than lysine (9.4 versus 4.4 mmol of ATP per g of cells per h). On the basis of differences in yield and maintenance energy, it appeared that active arginine transport accounted for approximately 40% of the total ATP.     

Energetics of arginine and lysine transport by whole cells and membrane vesicles of strain SR, a monensin-sensitive ruminal bacterium.

Strain SR, a monensin-sensitive, ammonia-producing ruminal bacterium, grew rapidly on arginine and lysine, but only if sodium was present. Arginine transport could be driven by either an electrical potential or a chemical gradient of sodium. Arginine was converted to ornithine, and it appeared that ornithine efflux created a sodium gradient which in turn drove arginine transport. There was a linear decline in arginine transport as pH was decreased from 7.5 to 5.5, and the cells did not grow at a pH less than 6.0. The Eadie-Hofstee plot was biphasic, and arginine could also be taken by a high-capacity diffusion mechanism. Because arginine was a strong inhibitor of lysine transport and lysine was a weak inhibitor of arginine transport, it appeared that both lysine and arginine were taken up by an arginine-lysine carrier which had a preference for arginine. The rate of lysine fermentation was always proportional to the extracellular lysine concentration, and facilitated diffusion was the dominant mechanism of lysine transport. When SR was grown in continuous culture on arginine or lysine, the theoretical maximal growth yield was similar (13 g of cells per mol of ATP), but the apparent maintenance energy requirement for arginine was greater than lysine (9.4 versus 4.4 mmol of ATP per g of cells per h). On the basis of differences in yield and maintenance energy, it appeared that active arginine transport accounted for approximately 40% of the total ATP.     

Transport of 5-hydroxytryptamine by dense granules from porcine platelets

A method is described for the isolation of a homogeneous preparation of dense granules from procine platelets. The purified dense granule fraction contained approximately 400 nmol of 5-hydroxytryptamine/mg of protein and appeared to be homogeneous when examined by electron microscopy. Isolated dense granules transport exogenously added 5-hydroxytryptamine via two mechanisms: 1) a carrier-mediated process predominating at low substrate concentrations and 2) a diffusion-controlled process predominating at high substrate concentrations. Temperature studies revealed an apparent energy of activation of 14.9 kcal/mol for the carrier-mediated transport. Kinetic data yielded a Km of 3.3 micron and a Vmax of 0.79 nmol/min/mg of protein for the mediated transport process. Steady state uptake was sensitive to changes in medium osmotic pressure and a decline in uptake below 300 mosM was correlated with release of endogenous 5-hydroxytryptamine. The transport was inhibited by a number of structural analogs of 5-hydroxytryptamine. These results demonstrate the existence of a carrier-mediated transport system for 5-hydroxytryptamine in the membranes of the platelet dense granules.     

Carrier-dependent and carrier-independent uptake of myo-inositol in cultured retinal pigment epithelial cells: activation by heat and concentration

Transport of myo-inositol (MI) was studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. At low external concentrations (0.01-1 mM), uptake appeared to follow saturation kinetics, although the reciprocal forms of the rate equations did not fit either Lineweaver-Burk or Eadie-Hofstee plots. Increasing external concentrations dramatically changed the pattern of MI entry. At two to three orders of magnitude higher than physiological concentrations, a second saturation occurred (pseudo saturation). Cells incubated with 20 microM [3H]MI for 60 min had a ratio of intracellular to extracellular radioactivity greater than or equal to 8, indicating active transport. MI transport reduction by Na+ replacement or inhibitors (phlorizin, ouabain, amiloride, KSCN, iodoacetamide, MI analogues) was greater when RPE cells were incubated with low (20-400 microM) than with high (10-20 mM) MI concentrations. Cells incubated with 20 microM MI at 53 or 65 degrees C showed increased transport (up to 560%) compared with cells at 22 degrees C. The effect on MI uptake (20 microM) of Na+ replacement also was reduced at 53 degrees C. The uptake of MI involved at least two transport systems. The major mechanism at low external MI concentrations (physiological levels) was a carrier-mediated active process. At high external MI levels, uptake occurred by a diffusion process. A lipotropic effect of MI may be responsible for this increased rate of diffusion.