Effect of osmolality and myo-inositol deprivation on the transport properties of myo-inositol in primary astrocyte cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na+ with a Km of 13-18 microM and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 microM with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na(+)-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

Biphasic L-arginine uptake by the isolated guinea-pig heart

L-Arginine is the physiological substrate for the formation of nitric oxide (NO) and accounts for the biological activity of endothelium-derived relaxing factor. We have studied L-arginine transport in the heart using a rapid dual-isotope dilution technique. The time course of L-[3H]arginine uptake (extraction) by the isolated perfused guinea-pig heart was found to occur in two phases. The first phase reached a plateau in 6.6 +/- 0.6 s and lasted 8.8 +/- 0.7 s, whereas the second phase developed a plateau after 16.3 +/- 0.8 s. The first phase of maximal uptake (Umax,1) accounted for 13.4 +/- 1.4% of the total uptake and the second (Umax,2) for 32.3 +/- 1.8%. The two phases of uptake were inhibited by unlabelled L-arginine in a dose-dependent manner, which suggests that both phases are carrier mediated. The degree of inhibition of Umax,1 and Umax,2 by unlabelled L-arginine was not significantly different. Studies of the kinetics of uptake of these processes revealed an apparent Km,1 of 183 +/- 10 microM with a Vmax,1 of 50 +/- 10 nmol min-1 g-1 for the first phase and Km,2 of 167 +/- 14 microM with a Vmax,2 of 93 +/- 13 nmol min-1 g-1 for the second phase of uptake. These results suggest a similar affinity for the receptors of both transport systems, but with different values for Vmax (P < 0.05). In contrast, 1 mM unlabelled D-arginine had no effect on either the first or second phase of uptake of L-[3H]arginine by the heart, which suggests that these processes are stereospecific. In the presence of the L-stereoisomer of nitro-arginine-mono-methyl ester (L-NAME), a potent inhibitor of NO synthesis, the Umax,1 was inhibited by about 60% while Umax,2 was inhibited by only 20%, which suggests that there is a difference in the effect of L-NAME on the two phases of L-arginine uptake. The first phase most probably represents uptake into the capillary wall, i.e. endothelium and smooth muscle, while the second phase represents entry into the extra-endothelial compartment, i.e. the cardiac myocytes and fibroblasts.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreases glutamate uptake in cultured astrocytes

The deleterious effect of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic neurons of the substantia nigra is well established. In addition, increased glutamatergic drive to basal ganglia output nuclei is considered a likely contributor to the pathogenesis of Parkinson's disease. One possibility for the increased excitatory tone may be related to an impairment in glutamate uptake. As astrocytes possess efficient transport mechanisms for both MPTP and glutamate, we have examined the effect of this agent on D-aspartate uptake into these cells. Treatment of cultures with 50 microM MPTP for 24 h decreased uptake by 39%. Kinetic analysis revealed that this effect was due to a 35% decrease in Vmax with no change in the Km. Treatment with deprenyl, a monoamine oxidase B inhibitor, produced a complete reversal of MPTP-induced uptake inhibition, but was ineffective following exposure of cells to the MPTP metabolite, 1-methyl-4-phenylpyridinium (MPP+). Removal of MPTP from cultures resulted in a complete restoration of glutamate uptake after 24 h. These results show that MPTP reversibly compromises glutamate uptake in cultured astrocytes, which is dependent on the conversion of MPTP to MPP+. Such findings suggest that the glutamate transporter in astrocytes plays an important role in MPTP-induced neurotoxicity and possibly in parkinsonism.     

Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors

N omega-Substituted analogues of L-arginine have proven useful as specific inhibitors of nitric oxide formation in various biological systems. In the present study we describe the characteristics of amino acid transporters that mediate uptake of N omega-methyl-L-arginine (L-NMA) and N omega-nitro-L-arginine (L-NNA) into cultured porcine aortic endothelial cells. The transport of L-[14C]NMA showed biphasic kinetics, with Km values of 4 and 368 microM, and was inhibited by L-arginine, L-homoarginine, L-lysine, and L-ornithine but not by L-leucine or L-isoleucine. Similar transport kinetics (Km values of 6 and 609 microM) and substrate specificities were obtained for L-[3H]arginine uptake, indicating that L-arginine and L-NMA are transported by the same system. In contrast to L-arginine and L-NMA transport, uptake of L-[3H]NNA was monophasic (Km = 617 microM) and was inhibited by L-leucine and L-isoleucine but not by L-arginine, L-homoarginine, L-NMA, L-lysine, or L-ornithine. Uptake studies with L-[3H]leucine revealed that the transport of this amino acid occurred in a manner very similar to that of L-[3H]NNA transport, suggesting that the uptake of both compounds may be mediated by the same system. In additional experiments, we determined the effects of L-NMA and L-NNA on the A23187-induced accumulation of intracellular cGMP, to establish to what extent these transport systems are involved in the actions of nitric oxide synthase inhibitors. L-Lysine and L-ornithine, which both inhibited L-NMA uptake, increased the IC50 of L-NMA from 7.8 microM to 57 microM but did not reduce the inhibitory effects of L-NNA. In the presence of L-leucine or L-isoleucine, however, which both inhibited L-NNA uptake, the IC50 of L-NNA was increased from 1.2 microM to 37 microM but the inhibitory actions of L-NMA remained unaffected. These data demonstrate that the endothelial transport systems for L-arginine and L-leucine mediate the biological effects of L-NMA and L-NNA, respectively.     

L-glutamine inhibits nitric oxide synthesis in bovine venular endothelial cells

This study was conducted to test the hypothesis that L-glutamine has differential effects on nitric oxide (NO) synthesis from L-arginine in bovine venular endothelial cells (EC) stimulated by A23187 (a Ca++ ionophore) and receptor-mediated vasodilators (bradykinin and substance P). EC were cultured at 37 degrees C for 24 h in the presence of 0.4 mM L-arginine and 0.0 to 2.0 mM L-glutamine with or without 1 microM A23187, 1 microM bradykinin or 10 microM substance P. The release of nitrite and nitrate by EC was used as an indicator of NO synthesis. A23187, bradykinin or substance P increased NO synthesis from L-arginine by EC in the presence or absence of L-glutamine. The addition of L-glutamine (0.5 and 2 mM) markedly increased intracellular concentrations of L-glutamine, L-glutamate and L-aspartate and decreased NO synthesis by EC in a concentration-dependent manner in the presence or absence of A23187, bradykinin or substance P. L-Glutamine had no effect on L-arginine uptake by EC or on intracellular L-arginine concentration. Neither L-glutamine nor its glutaminase metabolites (ammonia, L-glutamate and L-aspartate) had any effect on endothelial NO synthase activity. Taken together, these results suggest that the inhibition by L-glutamine of NO synthesis from L-arginine is unlikely to result from an effect of L-glutamine on L-arginine transport or NO synthase activity. Although the mechanism involved remains unknown, regulation of the arginine-NO pathway by L-glutamine may have pharmacologic and therapeutic implications in such conditions as inflammation and septic shock by inhibiting NO generation from L-arginine in endothelial cells.     

Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-14C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity Km of 0.66 mM and Vmax of 3.66 mM for pyruvate, and a low-affinity system with a Km of 9.9 mM for both lactate and pyruvate and Vmax values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM alpha-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of 14C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-14C]lactate by synaptosomes was saturable, and yielded a Km of 1.23 mM and a Vmax of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.     

The utilization of dipeptides containing L-arginine by chicken macrophages

L-Arginine is the precursor of NO, a cytotoxic agent of macrophages. Studies were carried out to determine whether dipeptides containing arginine can be utilized by lipopolysaccharide (LPS)-activated avian macrophages for NO production. A chicken macrophage cell line, the HD11 cell, was used in all experiments. Peptidase activities were observed in fetal bovine serum (FBS) and macrophage serum free medium (Mac-SFM). Therefore, the utilization of dipeptides by macrophages was examined using Dulbecco's modified Eagle medium (D-MEM), a chemically defined medium, in short-term culture without FBS. Nitrite accumulation in the culture medium was used as the indicator of NO production. At concentrations of 0.15 mM in the culture media, L-leucinyl-L-arginine was 89% as effective as L-arginine in providing substrate for NO production. L-Argininyl-L-leucine was 38% as effective as L-arginine. The effectiveness increased to 93 and 58%, respectively, when the concentrations of dipeptides and arginine were 1.0 mM. Both values were slightly higher in a second experiment (97 and 70%, respectively). L-Lysine (10 mM) inhibited nitrite formation from all three sources of L-arginine. In studies of initial rates of transport by HD11 cells in Hanks Balanced Salts solution (HBSS), both L-argininyl-L-leucine and L-leucinyl-L-arginine inhibited arginine uptake. As lysine and arginine share a common transporter for cationic amino acids and are known to compete for transport, these studies suggest that the peptides were hydrolyzed extracellularly, yielding arginine that was transported into the cell where it served as a substrate for NO synthesis.     

Interactions between transport of triiodothyronine and tryptophan in JAR cells

We studied the effect of a number of amino acids on uptake of L-triiodothyronine (T3) in the human choriocarcinoma cell line, JAR. Tryptophan inhibited saturable T3 uptake by about 57% without any significant effect on the non-saturable uptake. Michaelis constant (Km) for T3 uptake was 1.06 +/- 0.15 microM (n = 15) with the corresponding maximum velocity (Vmax) of 24.2 +/- 3.1 pmol/min/mg cellular protein. For tryptophan uptake the Km was 1.31 +/- 0.26 microM (n = 7) and Vmax was 166.4 +/- 35.7 pmol/min/mg protein. The kinetic parameters for both uptake processes were similar to those reported in normal placenta. Uptake of T3 was inhibited by tryptophan but not phenylalanine, but tryptophan uptake was inhibited both by T3 and phenylalanine. Inhibition of T3 uptake by tryptophan was dose dependent, with an inhibition constant (Ki) of 2.9 +/- 0.5 mM. Similarly, tryptophan uptake was inhibited by T3 and phenylalanine in a dose dependent way with Ki values of 4.9 +/- 0.5 microM and 15.6 +/- 4.8 microM respectively. Km for T3 uptake was significantly increased to 1.86 +/- 0.42 microM (n = 4) in the presence of 3 mM unlabelled tryptophan and, similarly, Km for tryptophan uptake was significantly increased to 9.91 +/- 2.57 microM (n = 3) in the presence of 5 microM unlabelled T3. Efflux of T3 was progressively inhibited by increasing concentrations of both ligands, i.e. was saturable. We conclude that there is mutual competitive inhibition between uptake systems for T3 and tryptophan in JAR cells, but the kinetic parameters of cross-inhibition of uptake by the substrates suggest that the carriers are distinct. T3 may be transported in JAR cells by at least two transport systems with differing substrate specificities. We also demonstrated the presence of a saturable membrane carrier mediating the efflux of T3 from the cells which was subject to trans-inhibition by T3 and tryptophan.     

In vitro modulation of L-arginine transport in trophoblast cells by glucose

BACKGROUND: The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. METHODS: This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. RESULTS: At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. CONCLUSION: These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.     

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.     

Peritubular transport of ochratoxin A by single rabbit renal proximal tubules

Epifluorescence microscopy was used to study peritubular transport of the fluorescent mycotoxin ochratoxin A (OTA) into single proximal tubule segments of the rabbit. Initial rates of OTA uptake into S2 segments were saturable and adequately described by Michaelis-Menten kinetics, with an apparent Km of 2.2+/-0.3 microM (SEM). Several lines of evidence indicated that peritubular uptake of OTA in S2 segments was effectively limited to the "classical" organic anion transporter. First, 5 mM p-aminohippurate (PAH) cis-inhibited the uptake of 1 microM OTA into tubules by 96%. Kinetic analysis of the inhibition of OTA uptake by PAH (100 microM to 5 mM) yielded an apparent Ki of 164 microM, similar to the 100 to 200 microM range of Km values previously reported for the peritubular uptake of PAH. Second, efflux of OTA from tubules was trans-stimulated 3.2-fold by the presence of 2.5 mM PAH in the uptake medium. Third, 100 microM alpha-ketoglutarate (alphaKG) trans-stimulated the uptake rate of 1 microM OTA by 1.8-fold. Fourth, besides PAH, other organic anions effectively cis-inhibited the uptake of 1 microM OTA into tubules (inhibitor, % inhibition): 1.5 mM alphaKG, 80%; 1 mM probenecid, 100%; 1 mM piroxicam, 100%; 1 mM octanoate, 100%. In contrast, 1.5 mM tetraethylammonium, an organic cation, blocked uptake of 1 microM OTA by only 7%. The inhibition of OTA uptake into S1 and S3 segments of the proximal tubule was qualitatively similar: 5 mM PAH cis-inhibited the uptake of 1 microM OTA by approximately 95% in both S1 and S3 segments. Thus, peritubular OTA uptake into all segments of the proximal tubule appears to be dominated by its interaction with the classical organic anion transporter. The high-affinity and relatively high capacity of this pathway for OTA suggest that peritubular uptake may be a significant avenue for the entry of this toxin into proximal tubule cells.     

Uptake of tyramine by rat hepatocytes

Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a Km of 39 microM and a Vmax of 270 pmol/min/10(5) cells. The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol. An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved. This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol. NO3-, SCN- and SO4(2-), which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane. Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier. Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine. It is possible that other amines can enter hepatocytes by using this transporter.     

Heavy metals mercury, cadmium, and chromium inhibit the activity of the mammalian liver and kidney sulfate transporter sat-1

Heavy metal intoxication leads to defects in cellular uptake mechanisms in the mammalian liver and kidney. We have studied the effects of several heavy metals, including mercury, lead, cadmium, and chromium (at concentrations of 1 to 1000 microM), on the activity of the mammalian sulfate transporter sat-1(2) in Xenopus oocytes. sat-1 encodes a sulfate/bicarbonate anion exchanger expressed in the rat liver and kidney. Mercury (10 microM) strongly inhibited sat-1 transport by reducing Vmax by eightfold but not its Km for inorganic sulfate (Si). Lead (up to 1 mM) was unable to significantly inhibit sat-1 transporter activity. Cadmium (500 microM) showed weak inhibition of sat-1 transport by decreasing only sat-1 Vmax. Chromium (100 microM) strongly inhibited sat-1 transport by reducing Km for Si by sevenfold, most probably by binding to the Si site, due to the strong structural similarity between the CrO2-4 and SO2-4 substrates. This study presents the first characterization of heavy metal inhibition of the hepatic and renal sulfate/bicarbonate transporter sat-1, through various mechanisms, which may lead to sulfaturia following heavy metal intoxication.     

Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular membranes

Driving forces and substrate specificity for transport of reduced glutathione (GSH) across rat liver cell canalicular membrane were examined in vesicles isolated from this plasma membrane domain. In contrast to previous studies indicating a single saturable component of canalicular GSH transport, the present results demonstrate the presence of both high and low affinity components with apparent Km values of 0.24 +/- 0.04 and 17.4 +/- 2.1 mM and Vmax values of 0.09 +/- 0.01 and 2.3 +/- 0.3 nmol.mg-1.20 s-1, respectively. The Km values in two previously published reports are discordant, 0.33 versus 16 mM, but are comparable with the two transport components identified in the present study. To further characterize these GSH transport mechanisms, [3H]GSH uptake by canalicular vesicles was measured at concentrations of 50 microM, where transport is expected to occur largely on the high affinity component, and at 5 mM, where the low affinity system should predominate. Neither component of GSH transport was affected by ATP or a Na+ gradient, but both were stimulated by a valinomycin-induced membrane potential, indicating electrogenic transport pathways. The high affinity component was cis-inhibited by glutathione S-conjugates (1 mM), other gamma-glutamyl compounds (5 mM), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (0.1 mM), whereas these agents had no effect on the low affinity component at similar inhibitor concentrations. Sulfobromophthalein (BSP, 0.1 mM) inhibited both GSH transport components. However, neither component was affected by taurocholate (0.5 mM) or L-glutamate (10 mM). The inhibition by S-butylglutathione, the GSH analogue ophthalmic acid, and by BSP was competitive in nature, although BSP also produced a slight decrease in Vmax, suggesting a mixed type of inhibition. Ophthalmic acid and some glutathione S-conjugates were also able to trans-stimulate high affinity GSH uptake. These results indicate the presence of at least two ATP-independent, electrogenic glutathione transport mechanisms on the canalicular membrane; the high affinity component may function to deliver some glutathione S-conjugates, gamma-glutamyl compounds, and other anions into bile, whereas the low affinity system probably functions as a high capacity transporter capable of delivering large amounts of GSH into bile.     

Nitric oxide modulates neuropeptide Y regulation of ion transport in mouse ileum

The possible involvement of nitric oxide in the regulation of intestinal ion transport induced by neuropeptide Y (NPY) was investigated by evaluating the effects of NG-methyl-L-arginine (L-NMA), L-arginine and S-nitroso-N-acetylpenicillamine (SNAP) on NPY activity in mouse ileum mounted in Ussing chambers in vitro. Serosal NPY (10 nM) produced a sustained decrease in basal transmural short circuit current (Isc) and potential difference without altering the tissue conductance. Pretreatment of tissues with L-arginine (3 mM), but not D-arginine (10 mM), blocked the NPY-mediated changes in Isc. This L-arginine effect on NPY activity was reversed by L-NMA (3 mM), and not by NG-methyl-D-arginine (10 mM). The L-arginine effect on NPY activity was concentration-related with an A50 (95% CL) value of 1.6 (0.9-2.3) mM. In contrast to L-arginine, L-NMA (1 mM) pretreatment of tissues produced an enhancement of NPY activity, resulting in a 3.8-fold leftward displacement of the NPY concentration-response curve; NG-methyl-D-arginine was without effect. The effect of L-NMA on NPY activity was concentration-related with an A50 (95% CL) value of 45.3 (23.2-68.8) microM. Serosal application of SNAP, a nitric oxide donor, produced a concentration-related decrease in basal Isc and potential difference without altering tissue conductance with an A50 (95% CL) value of 22.5 (11.1-40.5) microM. Pretreatment of tissue with SNAP (100 microM) reduced the NPY activity with rightward displacement of NPY concentration-response curve. Pretreatment of tissues with L-arginine also blocked the reduction of Isc by [D-Pen2, D-Pen5]enkephalin (10-30 nM), H2N-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (10-30 nM) and somatostatin (0.3-1.0 microM), but had no effect on norepinephrine (0.1-0.3 microM)-induced decrease in mouse ileal Isc. These results show that [fgc]l-arginine and SNAP block NPY-mediated changes in ion transport, suggesting that nitric oxide may play a role in the regulation of NPY-mediated ion transport in the mouse ileum.     

Nitric oxide exposure and sulfhydryl modulation alter L-arginine transport in cultured pulmonary artery endothelial cells

The effect of nitric oxide (NO) exposure and sulfhydryl-reactive chemicals on L-arginine transport in pulmonary artery endothelial cells was evaluated. Exposure of pulmonary artery endothelial cells to 7.5 ppm (0.4 microM) NO for 4 h resulted in a significant (p < 0.05) reduction of Na(+)-dependent but not Na(+)-independent L-arginine transport. More prolonged exposure for 12-24 h reduced both Na(+)-dependent and Na(+)-independent transport of L-arginine with maximal loss of transport after 18 h of exposure (p < 0.02 for both). Similarly, incubation of cells in the presence of 50-200 microM S-nitroso-acetyl-penicillamine (SNAP) (but not 500 microM each of nitrate or nitrite) for 2 h also reduced both the Na(+)-dependent and Na(+)-independent transport of L-arginine (p < 0.05 for all concentrations). The SNAP-induced reduction of L-arginine transport was blocked by the NO scavenger oxyhemoglobin. When cell monolayers were exposed to varying concentrations of the sulfhydryl reactive chemicals N-ethylmaleimide (NEM) and acrolein, a dose-dependent reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes was observed. Na(+)-dependent L-arginine transport was more susceptible to inhibition by exposure to NO and to sulfhydryl reactive chemicals. Incubation of cells with 0.5 mM of the thiol-containing agent N-acetyl-L-cysteine prior to and during NEM or acrolein exposure blocked NEM and acrolein-induced reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes. Similarly, NO-induced reductions of Na(+)-dependent and Na(+)-independent L-arginine transport were reversed to control levels 24 h after termination of NO exposure. Treatment with the disulfide reducing agent dithiothreitol after exposure to NO resulted in partial reversal of the decreases in L-arginine transport. These results demonstrate that exposure to exogenous NO is responsible for reversible reductions of plasma membrane-dependent L-arginine transport mediated by both the Na(+)-dependent (system Bo,+) and the Na(+)-independent (system y+) transport processes. Modulation of the sulfhydryl status of plasma membrane proteins involved in L-arginine transport, such as L-arginine transporters and/or Na+/K(+)-ATPase, may be responsible, at least in part, for reductions in overall L-arginine transport in pulmonary artery endothelial cells.     

Intestinal uptake of uridine in suckling rats: mechanism and ontogeny

Nucleosides, essential substrates for a variety of intracellular metabolic reactions, are obtained from dietary and endogenous sources. Nucleotides (which dephosphorylate to nucleosides prior to intestinal absorption) are present in milk and have trophic effects on the developing gastrointestinal tract. The mechanism of transport of nucleosides in the developing intestine of suckling rats is unknown. To address this issue, we therefore examined uridine uptake in rat everted intestinal sacs. In suckling rats (15-17 days old), tissue uptake of low (5-microM) and high (60 microM) concentrations of [3H]-uridine was linear for up to 2 min of incubation. Initial rate of uptake of [3H]-uridine was (i) not significantly different in the jejunum and the ileum; (ii) greater in the presence of Na+, than other cations; (iii) saturable as a function of concentration with a Vmax of 21,044 +/- 2,302 pmol/g tissue wet wt/30 sec and an apparent Km of 33.8 +/- 10.1 microM; (iv) inhibited by high concentration (500 microM) of unlabeled uridine and other nucleosides; (v) temperature-dependent; (vi) energy-dependent; and (vii) pH-sensitive. Developmental maturation was associated with a progressive decrease in the Vmax of the uridine transport process (21,044 +/- 2,302, 14,651 +/- 1,679, and 8,461 +/- 1,369 pmol/g tissue wet wt/30 sec for suckling, weanling, and adult rats, respectively) and a progressive increase in the apparent Km of the uptake system (33.8 +/- 10.1, 55.6 +/- 13.1, and 61.7 +/- 14.5 microM for suckling, weanling, and adult rats, respectively). We concluded that uptake of uridine by the developing intestine of suckling rats involves a carrier-mediated system, which is energy- and temperature-dependent, and requires extracellular sodium. Furthermore, the uptake process was found to undergo clear ontogenic changes with maturation.     

Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity

Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO from L-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 +/- 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieve Vmax NO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mM EDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% of Vmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO from L-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.     

Uptake of agmatine into rat brain synaptosomes: possible role of cation channels

Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy- and temperature-dependent and saturable with a Km of 18.83 +/- 3.31 mM and a Vmax of 4.78 +/- 0.67 nmol/mg of protein/min. Treatment with ouabain (Na+,K+-ATPase inhibitor) or removal of extracellular Na+ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion-channel agents on agmatine uptake, only Ca2+-channel blockers inhibited the uptake, whereas a reduction in extracellular Ca2+ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na+-independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.