I. INTRODUCTION

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The endothelial lining of blood vessels provides a barrier for the exchange of nutrients and is itself actively involved in the local control of vascular homeostasis. Blood-borne and tissue-derived mediators act on endothelial cells, stimulating the synthesis and release of soluble vasoactive factors and the expression of surface adhesion molecules for circulating leukocytes (reviewed in Refs. 124, 223, 464), while the actin- and myosin-based contractile cytoskeleton in endothelium regulates responses to changes in blood flow and shear stress (see Ref. 514). Endothelium-dependent vascular relaxation is markedly impaired in diseases such as diabetes mellitus, atherosclerosis, hypertension, and preeclampsia. Because disease-induced alterations in plasma levels of L-arginine and related amino acids, glucose, and insulin modulate vascular relaxation, it is surprising that only recent studies have examined regulation of transport and metabolism of amino acids and glucose in vascular endothelial and smooth muscle cells. During the last two decades, the L-arginine-nitric oxide signaling pathway has emerged as one of the key second messenger systems involved in the regulation of vascular tone and permeability (reviewed in Refs. 285, 326, 412). The discovery that L-arginine is the physiological precursor for nitric oxide (NO) biosynthesis precipitated research into the role of circulating and intracellular arginine in the regulation of vascular function in health and disease. In 1998 the Nobel Prize in Physiology and Medicine was awarded to Robert Furchgott, Louis Ignarro, and Ferid Murad for their contribution to the discovery of NO as a key signaling molecule (for commentaries, see Refs. 274, 643).

This review aims to highlight the mechanisms regulating amino acid and glucose transporters in endothelial cells derived from peripheral vascular beds and the blood-brain and blood-retinal barriers. Although nutrient transport and metabolism by the brain endothelium (and to a lesser extent retinal and corneal endothelium) have been reviewed previously (see Refs. 75, 76, 455, 544), the recent advances in our understanding of transport processes in endothelial cells derived from peripheral vascular beds provide a basis for comparing the specificity, kinetics, and regulation of amino acid and glucose transport. We have focused principally on transport processes in endothelial cells but have also reviewed the available literature for vascular smooth muscle cells, in view of the modulation of smooth muscle tone by endothelium-derived mediators and proinflammatory cytokines. Because there are excellent reviews on the molecular biology of amino acid (51, 120, 125, 128, 130, 131, 162, 163, 186, 256, 301, 371, 375, 449, 450, 625) and glucose (22, 23, 95, 106, 294, 324, 414, 445, 516, 594) transporters, we have chosen to focus this review on the regulation of nutrient transport in vascular endothelial and smooth muscle cells.

Endothelial cell metabolism and the general characteristics of amino acid and glucose transport systems (and recently cloned transporters, see Table 1) expressed in mammalian cells are intended only as a brief overview. These sections, however, provide the basis for a detailed comparison of the selectivity, ionic dependence, and kinetic properties of amino acid and glucose transporters in endothelial cells derived from the blood-brain barrier, blood-retinal barrier, and peripheral vascular beds, such as fetal umbilical vein, placenta, aorta, lung, heart, and adrenal gland. Regulation of endothelial cell amino acid and glucose transport by vasoactive agonists, NO, insulin, hypoxia, substrate deprivation, and development is reviewed subsequently, highlighting where possible differences in the responses of endothelial cells derived from cerebral and peripheral circulations. In view of the importance of NO as a modulator of vascular tone in inflammation, we have critically evaluated the evidence that transport of amino acids and glucose in endothelial and smooth muscle cells is modulated by bacterial endotoxin, proinflammatory cytokines, and oxidatively modified low-density lipoproteins. This review aims to provide a comprehensive overview of the mechanisms regulating the activity and expression of amino acid and glucose transporters in vascular cells. With the increasing advances in the molecular identification and understanding of the function of amino acid and glucose transporters in other mammalian tissues, we believe that vascular physiology merits similar research initiatives.

	II. ENDOTHELIAL AND SMOOTH MUSCLE CELL METABOLISM

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Microvascular endothelial cells are able to use certain amino acids as fuels for oxidative phosphorylation (332). In rat coronary microvascular endothelial cells deprived of glucose, L-glutamate and L-glutamine are rapidly oxidized, whereas oxidation rates for L-asparagine, L-alanine, L-isoleucine, and L-arginine are intermediate. The negligible oxidation of L-valine and L-tyrosine suggests that coronary endothelial cells may lack specific enzymes required for the degradation of these amino acids. Amino acids with the highest rates of oxidation (glutamate, glutamine, alanine, asparagine) are degraded by no more than three intermediate steps before entering the Krebs cycle, and catabolism of these substrates has been characterized in bovine pulmonary artery endothelial cells (344). Bovine coronary venular endothelial cells metabolize L-glutamine to ammonia, L-glutamate, and L-aspartate (398), and the high activity of glutaminase in bovine pulmonary artery endothelial cells (~20-fold higher than in lymphocytes) indicates that L-glutamine provides an important respiratory fuel (395, 556). However, as cultured endothelial cells may lack a fully functional urea cycle, it seems unlikely that L-glutamine can be metabolized via L-ornithine to L-citrulline and L-arginine (see Fig. 2 in Ref. 654), confirming the lack of an effect of extracellular L-glutamine on intracellular L-arginine levels (398).

A comparative study of L-glutamine metabolism in bovine coronary venular, bovine aortic, human mesenteric, and human umbilical vein endothelial cells has confirmed that CO₂ is the major metabolic product of glutamine-derived L-glutamate (648), highlighting the importance of glutamine as an energy substrate in different endothelial cell types. Although formation of L-glutamate and ammonia from L-glutamine are similar in micro- and macrovascular endothelial cells, Wu et al. (648) concluded that glutamate dehydrogenase was inhibited by ammonia generated from L-glutamine by mitochondrial phosphate-dependent glutaminase. Their study also identified a novel pathway for L-ornithine synthesis from L-glutamine via pyrroline-5-carboxylate synthase, but unlike intestinal epithelial cells, glutamine-derived L-ornithine was not converted into L-citrulline and L-arginine due to the absence of carbamoylphosphate synthase-1 (see Fig. 2 in Ref. 654).

NO is a labile vasodilator synthesized in endothelial cells from the semiessential cationic amino acid L-arginine (reviewed in Refs. 285, 412). The metabolism of L-arginine by mammalian cells has been reviewed in detail (see Refs. 30, 654), and in endothelial cells a constitutive, Ca²⁺/calmodulin (CaM)-sensitive NO synthase (eNOS) metabolizes L-arginine to NO and the neutral amino acid L-citrulline (451). eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobioptherin (BH₄), NADPH, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) as additional cofactors for its activity (see Refs. 326, 521). Vasoactive agonists normally elevate intracellular Ca²⁺ in endothelial cells (292, reviewed in Ref. 403) with binding of Ca²⁺/CaM to eNOS stimulating NO production, while fluid shear stress leads to phosphorylation of the serine/threonine protein kinase Akt (protein kinase B) in a phosphatidylinositol (PI) 3-kinase-dependent manner and activation of eNOS at basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) levels (see Refs. 144, 166, 202, 209).

Figure 1 depicts the described transport systems mediating cationic amino acid influx in vascular endothelial (and those known for smooth muscle) cells and further illustrates the effects of agonist-induced increases in intracellular Ca²⁺ and NO on cross-talk between endothelial and smooth muscle cells. We envisage system y⁺ as the primary carrier mediating facilitated transport of L-arginine, in which the negative membrane potential leads to an accumulation of cationic amino acids within the cell. Intracellular L-arginine concentrations in cultured endothelial cells range between 0.1 and 0.8 mM, although concentrations up to ~2-4 mM have been measured in freshly isolated endothelial cells (42, 43, 227, 398). Figure 2 compares amino acid concentrations in bovine aortic and human umbilical vein endothelial cells in culture and highlights the differential effect of L-arginine deprivation on intracellular concentrations of L-arginine, L-lysine, L-ornithine, and L-citrulline (see sect. VIIA).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Schematic representation of cationic amino acid transport systems in endothelial cells and the role of arginine in nitric oxide (NO) biosynthesis. In endothelial cells, transport of L-arginine and other cationic amino acids is mediated principally via the Na+-independent cationic amino acid transport systems y+ and y+L, with negligible entry mediated by Na+-dependent or Na+-independent transport systems Bo,+ or bo,+, respectively, or passive diffusion (see sect. IIIA). Arginine is metabolized by constitutive NO synthase (eNOS) to generate basal and agonist-stimulated NO, which rapidly diffuses to underlying smooth muscle cells to activate soluble guanylyl cyclase (sGC) resulting in smooth muscle relaxation (see Refs. 285, 412). Vascular smooth muscle cells also express system y+ and x transport activity, and both cell types express other amino acid (and glucose) transport systems (see Tables 2, 3, and 7). Vasoactive agonists such as bradykinin, ATP, and histamine elevate intracellular Ca2+ and activate cGMP-dependent Ca2+-activated K+ channels, with the ensuing membrane hyperpolarization increasing the driving force for arginine entry via system y+ (see Refs. 66, 97, 307, 547). Inset: acute exposure of endothelial cells to NO donors activates system y+, whereas prolonged exposure to NO inhibits L-arginine transport and stimulates L-cystine transport via the anionic transport system x (349, 440, 461).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Comparison of the effects of arginine deprivation on intracellular amino acid concentrations in bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC). Confluent endothelial cell monolayers (~2 × 105 cells) were cultured in Dulbecco's modified Eagle's medium (BAEC, top panel) or medium 199 (HUVEC, bottom panel) containing L-arginine (~300 µM) and 20% serum. Cell monolayers were then washed and cultured for 1-24 h in L-arginine-free medium supplemented with 20% dialyzed serum. Cell pellets were lysed with 90% methanol, and the deproteinized supernatant was analyzed by reverse-phase HPLC following precolumn derivatization with o-phthaldialdehyde at pH 9. Amino acid concentrations were determined with reference to the internal standard L-homoserine and the measured intracellular water space of 1 pL. Deprivation of L-arginine rapidly reduces intracellular concentrations of cationic amino acids and the neutral amino acid L-citrulline in BAEC, whereas intracellular amino acid concentrations are maintained close to basal levels in HUVEC. [Data replotted from Baydoun et al. (42, 43).]

Within endothelial cells, recycling of L-citrulline to L-arginine occurs at a rate of ~0.7-1.9 nmol L-arginine·10⁶ cells¹·h¹, with L-citrulline converted into L-arginine via argininosuccinate synthase in the presence of aspartate and Mg-ATP and argininosuccinate lyase (255, 406, 522, 652; see Fig. 3). Although Sessa et al. (522) reported that L-glutamine (0.2 mM) inhibited recycling of L-citrulline to L-arginine in L-arginine-depleted bovine aortic endothelial cells, Wu and Meininger (652) noted that L-glutamine only reduced L-arginine synthesis from L-citrulline in human mesenteric, but not bovine aortic or coronary venular endothelial cells. These discrepancies have not been resolved, and differences in cell culture conditions and/or experimental protocols (e.g., microcarrier cultures vs. static monolayers) seem the most likely explanation.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Metabolism of L-arginine in endothelial cells. An important step in L-arginine metabolism in endothelial cells is the formation of the labile gas nitric oxide (NO) via constitutive endothelial nitric oxide synthase (eNOS) (see Refs. 255, 285, 326, 412, 521). eNOS utilizes L-arginine (EC50 ~6 µM), molecular oxygen, and reduced nicotinamide adenine dinucleotide (NADH) as cosubstrates and tetrahydrobioptherin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and protoporphyrin IX haem as cofactors. L-Arginine is mono-oxygenated and converted to N-hydroxyarginine by the enzyme arginine N-hydroxylase (HDX) in the presence of BH4, nicotinamide adenine dinucleotide phosphate (NADPH), H+, and O2. N-hydroxyarginine is then oxidized by the action of N-hydroxyarginine mono-oxygenase (HAMO) to form L-citrulline and NO in the presence of NADPH, H+, and O2 (see Refs. 330, 410). N-hydroxyarginine, but not L-arginine, can potentiate NO synthesis in endothelial cells (reviewed in Ref. 521), suggesting that the L-arginine mono-oxidation step (probably synthesis of BH4) may be rate limiting the overall reaction. L-Citrulline can be recycled into L-arginine via argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASSL). ASSL catalyzes the conversion of argininosuccinate into L-arginine and fumarate, which is recycled through the Krebs cycle into L-aspartic acid. Hecker et al. (255) have also described conversion of L-citrulline into L-arginine via a transamination reaction. Recycling of L-citrulline into L-arginine is dependent on availability of exogenous L-citrulline and is sensitive to inhibition by L-glutamine (Ki ~50-100 µM), with L-glutamine (L-Gln) reported to decrease L-arginine synthesis from L-citrulline by inhibiting membrane transport of L-citrulline (see Refs. 522, 652, 654). More recent studies in bovine venular endothelial cells have shown that L-glutamine and its metabolite glucosamine can reduce intracellular NADPH levels and thereby inhibit NO synthesis (see Ref. 649). L-NMMA, N-monomethyl-L-arginine; ADMA, asymmetrical dimethyl-L-arginine.

Agonist-stimulated NO production is also sensitive to inhibition by L-glutamine [inhibitory constant (K_i) ~50-100 µM] (15, 255, 522), and the initial studies by Hecker et al. (255) attributed this inhibition to a direct action of L-glutamine on the citrulline-arginine cycle in endothelial cells. Arnal et al. (15) subsequently reported that the effect of L-glutamine on NO release from bovine aortic endothelial cells was dependent on the stimulus used, e.g., L-glutamine inhibited NO release in cells challenged with bradykinin but slightly enhanced NO release in the presence of the calcium ionophore A23187. In contrast, another study in bovine coronary venular and aortic endothelial cells reported that L-glutamine inhibits A23187-stimulated NO release (398). Although there are no immediate explanations for the discrepancy in the effects of L-glutamine on A23187-stimulated NO production in bovine aortic endothelial cells, it is worth emphasizing that L-glutamine does not inhibit L-arginine synthesis from either L-citrulline or argininosuccinate in endothelial cell lysates, whereas inhibition of L-citrulline transport by L-glutamine is associated with inhibition of the citrulline-arginine cycle (398, 652). This same group reported that L-citrulline transport was inhibited competitively by L-glutamine (0.2-1 mM) but unaffected by 0.5 mM L-arginine, L-alanine, L-glutamate, or L-lysine. Our studies in J774 murine macrophages established that L-citrulline was transported via a saturable [Michaelis constant (K_m) = 0.16 mM, maximum binding velocity (V_max) = 32 pmol·µg protein¹·min¹], pH insensitive, neutral amino acid carrier insensitive to inhibition by L-arginine or the cationic NOS inhibitor N-monomethyl-L-arginine (L-NMMA) (41, 45). We further reported that, unlike transport of L-arginine and L-NMMA, kinetics of L-citrulline transport were not altered by bacterial lipopolyassachride (LPS), and recycling of L-citrulline to L-arginine could only sustain limited NO production (41). In view of the fact that responses to endothelium-dependent vasodilators in vivo are often less than those observed in vitro, Arnal et al. (15) suggested that studies with isolated arterial rings or cultured endothelial cells should consider supplementing incubation media with L-glutamine.

More recent studies in bovine coronary venular endothelial cells have established a role for glutamine:fructose-6-phosphate amidotransferase (GFAT, EC 2.6.1.16), the rate-limiting enzyme in the synthesis of hexosamine from glutamine and fructose-6-phosphate, in the modulation of the L-arginine-NO pathway by L-glutamine (649, 650). Increased glucose flux through the hexosamine biosynthetic pathway results in the generation of glucosamine-6-phosphate from fructose-6-phosphate by GFAT. The inhibition of NO generation by L-glutamine or its metabolite glucosamine was attributed to an inhibition of the pentose cycle and decreased availability of cellular NADPH. Neither L-glutamine nor glucosamine had any effect on radiolabeled L-arginine uptake or intracellular concentrations of L-arginine, BH₄, and Ca²⁺. A note of caution in the interpretation of the above findings is that GFAT activity is markedly elevated in cultured endothelial cells compared with freshly isolated cells, suggesting that cell differentiation or in vitro culture conditions may upregulate GFAT expression (650). This provides a plausible explanation for the notable discrepancy between the significant expression of GFAT in human cultured mesenteric microvascular and umbilical vein endothelial cells (650) and the lack of immunohistochemical staining for GFAT in the endothelium of human blood vessels (428). In summary, inhibition of L-citrulline transport by L-glutamine and inhibitory actions of L-glutamine (and its metabolite glucosamine) on the pentose cycle can modulate the NO generation in cultured endothelial cells. Due to the unusually high activity of GFAT in cultured endothelial cells, future studies should determine whether L-glutamine effectively inhibits the L-arginine-NO pathway in different vascular beds in vivo.

Methylated arginines are excreted into the urine and accumulate in the plasma of patients with renal insufficiency or hypercholesterolemia, with asymmetric dimethylarginine (ADMA) significantly attenuating endothelium-dependent relaxation (see Refs. 136, 290, 367, 368, 604). ADMA and L-NMMA can be metabolized via dimethylarginine dimethylaminohydrolase (DDHA) to L-citrulline (439), with inhibition of DDHA activity in vascular disease leading to an accumulation of ADMA, normally extensively metabolized in vivo. In bovine aortic endothelial cells, L-NMMA, but not N-nitro-L-arginine (L-NNA), is rapidly metabolized to L-citrulline and subsequently L-arginine (255). Experiments in human umbilical and saphenous vein endothelial cells further confirmed that L-[¹⁴C]NMMA can be metabolized to L-[¹⁴C]citrulline via an enzyme with properties similar to DDHA (367). Treatment of a transformed human cell line ECV304 (which may have limited value as a human endothelial cell model) with oxidized low-density lipoprotein or tumor necrosis factor (TNF)- causes a time-dependent decrease in the activity of DDHA with maximal ADMA concentrations of ~4 µM measured in the culture medium (290). Although there is an ongoing debate concerning the validity of the ECV304 cell line as a model for endothelium (82, 569), the identical genotype of ECV304 and T24/83 bladder carcinoma cell lines argues against the use of ECV304 cells for the study of endothelial cell biology.

Arginases are responsible for the metabolism of L-arginine into L-ornithine and urea, and at least two isoforms have been identified: arginase I, a cytosolic enzyme expressed highly in the liver, and arginase II, a mitochondrial enzyme expressed at lower levels in extrahepatic tissues (reviewed in Ref. 654). Arginase I preferentially directs L-ornithine to polyamine biosynthesis via ornithine decarboxylase with arginase II preferentially directing L-ornithine to L-proline and L-glutamate synthesis via ornithine aminotransferase. Arginase isoforms are expressed in activated macrophages and vascular smooth muscle cells (113, 179, 633), and unstimulated rat aortic and porcine coronary arteriolar endothelial cells express arginase I constitutively (94, 666). Activation of rat aortic endothelial cells with bacterial LPS induces arginase II and the inducible Ca²⁺/CaM-insensitive isoform of NOS (iNOS) (for review, see Ref. 326). Under these conditions, urea production appears to be inhibited as a result of intracellular N-hydroxyl-L-arginine accumulation following N-hydroxylation of L-arginine with insertion of one oxygen atom from dioxygen and consumption of two electrons from NADPH (409, 410, see Fig. 3). The inhibition of arginase by N-hydroxyl-L-arginine in endothelial cells expressing iNOS was implicated as a mechanism for sustaining intracellular L-arginine concentrations during sustained production of NO (94).

Recent studies with bovine coronary venular endothelial cells transfected with rat arginase I cDNA and murine arginase II cDNA have confirmed that overexpression of arginases attenuates NO production even though intracellular concentrations of L-arginine were reduced by only 11-25% (348). These authors speculated that a distinct intracellular pool of L-arginine available for eNOS may have been depleted in cells overexpressing arginase and that close association of arginase I with eNOS might have reduced L-arginine levels near the site of NO production. Conversely, inhibition of arginase with difluoromethylornithine (DFMO) enhances endothelium-dependent relaxation of porcine coronary arterial rings challenged with adenosine or serotonin, implying that inhibition of arginase may specifically increase the availability of L-arginine for NO synthesis in coronary arterial endothelium (666). Interestingly, inhibition of NO synthesis in renal mesangial cells is not associated with enhanced arginase activity (623), suggesting that increased availability of L-arginine is not necessarily diverted to arginase.

Due to the much higher L-arginine concentrations in tissue-culture media (~400 µM), it is possible that rates of N-hydroxyl-L-arginine production by cultured endothelial cells are greater than in vivo, and thus more effective in inhibiting arginase. Because the K_m of arginase for L-arginine is high (1-3 mM) whereas the K_m of NOS isoforms for L-arginine is relatively low (~3-10 µM) (see Refs. 94, 470, 653), arginase may not necessarily limit the availability of intracellular L-arginine for NO production in all endothelial cell types. Intracellular L-arginine levels in endothelial cells are maintained within the range of the K_m for arginase, and this together with efficient recycling of L-citrulline to L-arginine and the prevailing membrane potential may explain the high intracellular L-arginine levels (42, 43, 227, 398).

Accumulating evidence now suggests that supply of L-arginine for NO synthesis may be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool (132, 244, 396, 647), e.g., near invaginations of the plasma membrane referred to a caveolae or "lipid rafts" (reviewed in Refs. 210, 536). Colocalization of eNOS and the cationic amino acid transport system y⁺ in caveolae (see sect. X) may explain the "arginine paradox," concerning the discrepancy in the sensitivity of eNOS to extracellular L-arginine in cell-free systems (K_m of NOS for L-arginine in the low micromolar range, see Ref. 470) and studies in vivo where L-arginine supply seems to be rate-limiting for NO synthesis in hypercholesterolemia (148, 224, see also Ref. 136) despite high intracellular and circulating levels of L-arginine. To our knowledge there is no evidence that arginase and eNOS are colocalized in plasmalemmal caveolae, although a recent study has identified eNOS and argininosuccinate synthase in the caveolar fraction of bovine aortic endothelial cells (see Ref. 201, discussed in sect. X).

Glucose is actively metabolized in endothelial cells (219) and sustains anaerobic and aerobic metabolism (i.e., ~20-50 nmol ATP·mg protein¹·min¹, see Refs. 332, 402). In the presence of 5 mM D-glucose, catabolism of amino acids, palmitate, and lactate is reduced significantly, with oxidation rates for L-glutamine, L-alanine, and L-arginine decreased significantly (332). In rat coronary microvascular endothelial cells, >98% of incorporated glucose is metabolized to lactate (332). At physiological concentrations of glucose, the contribution of the hexose monophosphate pathway accounts for ~1.2% of glucose metabolism and the Krebs cycle for only ~0.04%, suggesting that in microvascular endothelial cells almost all of the energy obtained from catabolism of glucose is generated glycolytically. At lower glucose concentrations (~1 mM), oxidation of glucose via the Krebs cycle is higher. Thus oxidative metabolism in endothelial cells is inhibited at physiological concentrations of glucose, demonstrating that endothelial cells express the Crabtree effect (i.e., an inhibitory effect of glucose on mitochondrial respiration, Ref. 332).

Endothelial cells synthesize ATP primarily via glycolysis with a relatively low O₂ consumption (152, 167, 402). Studies employing calorimetry and ³¹P nuclear magnetic resonance have shown that porcine aortic endothelial cells deprived of glucose for 2 h exhibit a marked loss of nucleoside triphosphates and inhibition of protein synthesis, yet are capable of metabolizing endogenous triglycerides for de novo purine synthesis, recovering most of their adenine nucleotides following readministration of glucose (151). Thus endothelial cells are able to withstand prolonged periods of substrate deprivation and can adapt to hypoxia (see sect. VIIB) due to their low energy demand and high glycolytic activity (150, 151, 402). Recent evidence in human umbilical vein endothelial cells suggests that fatty acids can also serve as an energy fuel (154). Stimulation of AMP-activated protein kinase (AMPK) in human umbilical vein endothelial cells by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR, 2 mM) results in a decrease in malonyl coenzyme (CoA) levels and the activity of acetyl CoA carboxylase, increased oxidation of palmitate, and decreased glucose uptake and glycolysis (154). Despite a predicted decrease in the rate of ATP production, ATP levels increased by ~35%, reflecting potentially increased ATP generation from fatty acid oxidation. However, oxidation of fatty acids in umbilical vein endothelial cells only accounts for ~25% of the calculated ATP production in cells incubated with 5 mM glucose. As discussed by Dagher et al. (154), further studies are required to determine whether this reflects an underestimate for ATP generation from fatty acid oxidation and/or and actual decrease in cellular ATP utilization.

The effects of elevated glucose on endothelial cell function are often cell specific (see Refs. 298, 333, 334, 376). The cytosol of endothelial cells is reduced by accumulation of NADH and transformation of pyruvic acid to lactate, as described in microvascular endothelial cells from bovine corpus cavernosum (167) and brain microvessels (263). In some, but not all, endothelial cell types, the polyol pathway can reduce glucose to sorbitol via aldose reductase (AR), which has an extremely low affinity for glucose (K_m ~100 mM) but is activated by glucose itself or glucose-6-phosphate (reviewed in Refs. 133, 590). Conversion of glucose to sorbitol by aldose reductase forms NADP⁺ and may compete with other NADPH-requiring reactions such as conversion of oxidized glutathione (GSSG) to reduced (GSH) glutathione (17, 306). Kashiwagi et al. (306) emphasized that glucose-induced activation of the polyol pathway in endothelial cells may not be directly responsible for the associated decrease in NADPH content, but rather that activation of the pentose phosphate pathway and NADP supply to the GSH redox cycle is impaired by H₂O₂ generated in cells exposed to high glucose. There is lack of consensus concerning the importance of the polyol pathway in glucose-mediated endothelial dysfunction, e.g., sorbitol fails to accumulate in canine retinal capillary endothelial cells exposed to 30 mM glucose (511) while advanced glycation end products increase AR mRNA and protein in human dermal microvascular cells (420).

Elevated glucose also increases the generation of superoxide anions known to react with NO to form peroxynitrite, which upon decomposition generates a strong oxidant with reactivity similar to hydroxyl radicals (47). Human endothelial cells exposed to hyperglycemia in established diabetes mellitus (see sect. VIIC) are more sensitive to reactive oxygen species, since intracellular levels of glutathione, vitamin E, superoxide dismutase, catalase, and ascorbic acid are reduced significantly (reviewed in Refs. 173, 240, 644).

Vascular smooth muscle cells have a high rate of glycolysis, relying to a large extent on glycolytically generated ATP to sustain a variety of cell functions. Vascular smooth muscle metabolism and the influence of contraction on the metabolic fate of glucose and fatty acids have been studied extensively (see Refs. 6, 34-37, 242, 243). The glycogen content of vascular smooth muscle ranges from 1 to 13.9 µmol/g, and the rapid depletion of glycogen reserves in the absence of additional subtrates raises doubts whether glycogen is an important oxidative substrate for vascular smooth muscle. Allen and Hardin (6), using pig carotid arteries, concluded that glycogen contributed minimally (~10%) to substrate oxidation in vascular smooth muscle whilst oxidation of glucose comprised ~40-50% of the total substrates entering the tricarboxylic acid cycle. Other than glucose, vascular smooth muscle cells utilize several different substrates including short- or medium-chain fatty acids such as acetate and octanoate. Supply of mitochondrial substrates is thought to inhibit phosphofructokinase via elevated citrate levels, resulting in an inhibition of carbohydrate metabolism. During sustained isometric contraction of arterial smooth muscle induced by KCl, oxidation of fatty acid substrates increases whilst glucose metabolism declines progressively (34). Acetate, unlike octanoate, is not a major substrate in resting arterial muscle, yet KCl-induced contractions increase oxidation of both acetate and octanoate. Interestingly, norepinephrine-induced contraction is associated with a decrease in glucose uptake by vascular smooth muscle cells (34).

Adenosine modulates oxidative metabolism in cardiac and vascular smooth muscle by increasing O₂ consumption and the concentration of high-energy phosphate and adenine nucleotides (31). In contrast to the stimulatory effects of adenosine on glucose uptake in cardiac muscle, adenosine has no effect on glucose uptake or oxidation of glucose and octanoate in porcine carotid artery smooth muscle (35). Vascular smooth muscle isolated from porcine cerebral microvessels can simultaneously utilize fructose (a glycolytic intermediate) for gluconeogenesis and glucose for glycolysis, suggesting that exogenous fructose does not mix with fructose derived from glucose metabolism (356). These authors hypothesized that, because intermediates of glycolysis and gluconeogenesis appear not to mix freely within the cytoplasm of cerebral vascular smooth muscle cells, specific membrane microdomains containing glucose and dicarboxylate transporters may account for metabolite channeling, where "intermediates are transferred from one enzyme to another without complete equilibration with the surrounding medium" (448). Thus localization of glucose transporters and glycolytic enzymes to plasmalemmal caveolae could allow direct entry of exogenous glucose to the glycolytic pathway (see Fig. 7 in Ref. 356).

Recent studies in rat aortic smooth muscle cells have established that metabolism of L-arginine is modulated by physiologically relevant cyclic stretch (179). Exposure of aortic smooth muscle cells to cyclic stretch (~10% at 1 Hz for 72 h) resulted in a stimulation of L-arginine transport and metabolism via the induction of the CAT-2 transporter and arginase I. Cyclic stretch increased L-arginine metabolism to L-proline by concomitantly inhibiting ornithine decarboxylase (ODC) activity and polyamine biosynthesis. The implication of these studies is that hemodynamic stretch stimulates collagen synthesis in vascular smooth muscle cells by regulating transport and metabolism of cationic amino acids, leading potentially to a stabilization of vascular lesions in disease.

	III. GENERAL CHARACTERISTICS OF MAMMALIAN AMINO ACID AND GLUCOSE TRANSPORT

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As reviewed by Christensen (123), multiple transport systems mediate the influx of cationic, neutral, sulfonic, and anionic amino acids across the plasma membrane of mammalian cells. Molecular cloning approaches have led to the identification of Na⁺-dependent and Na⁺-independent amino acid transporters, and Table 1 summarizes that nomenclature (see citations) and the classical nomenclature used to designate these different amino acid transport systems (reviewed in Refs. 123, 662). The ionic dependency and K_m corresponding to the different transport systems were compiled from the cited publications and do not necessarily reflect transport properties of all cell types.

The different carrier proteins mediating transport of cationic amino acids include the Na⁺-independent systems y⁺, y⁺L, b^o,+, b⁺, and Na⁺-dependent system B^o,+. System b⁺, originally described in mouse blastocysts, is highly specific for cationic amino acids (610), whereas the other systems can also transport neutral amino acids. System y⁺ is the principal cationic amino acid transport system expressed in NO producing cells (41, 67, 130, 131, 177, 429, 486) and thus most likely plays a key role in regulating L-arginine supply for NOS. Although there is limited information on ASCT, PROT, GLYT, TAUT, and EAAT associated amino acid transport in vascular cells (see below and Table 1), recent evidence indicates that bovine aortic endothelial cells express a taurine transporter sharing a high degree of sequence homology with that of the mTAUT cDNA isolated from brain (see Ref. 477).

Cationic amino acid transport activity was initially assigned to the classical Na⁺-independent amino acid transport system y⁺ (639-641). Cationic amino acid transporter (CAT) proteins were among the first amino acid carriers identified in mammalian cells and are classified as members of the solute carrier family 7 (SLC7). In the early 1990s, expression of the ecotropic MuLV receptor in Xenopus oocytes demonstrated that this receptor mediated Na⁺-independent transport of cationic amino acids (318, 632). The receptor was renamed mCAT-1 for mouse cationic amino acid transporter and is an integral membrane protein with 14 putative transmembrane domains and intracellular NH₂ and COOH termini. Transport of L-arginine, L-lysine, and L-ornithine via CAT-1 (system y⁺) is pH independent, sensitive to trans-stimulation, and saturable at circulating plasma concentrations (~0.1-0.2 mM). Because CAT-1 is sensitive to changes in membrane potential (97, 307, 547), hyperpolarization induced by vasoactive agonists increases the driving force for cationic amino acid transport in endothelial and other cell types (see sects. IVB and VIA). The voltage dependence of other human CAT transporter isoforms has recently been investigated in Xenopus oocytes and confirmed that L-arginine-induced currents were usually larger in CAT-2A compared with CAT-2B (splice variants of CAT-2, see below) expressing oocytes (425). With the exception of the liver, system y⁺ transport activity is expressed ubiquitously, and the majority of studies in endothelial and smooth muscle cells have established that transport of L-arginine and cationic L-arginine analogs is mediated predominantly by a Na⁺-independent system with characteristics resembling system y⁺ (see sects. IV and VI-IX).

Four additional related cationic transport proteins, designated CAT-2A, CAT-2B, CAT-3, and CAT-4, have now been identified in different mammalian species (see Refs. 128-131, 162, 307, 370, 450, 555). CAT-1, -2A, and -2B are glycosylated, suggesting that these carriers are located in the plasma membrane, with CAT-2A and -2B splice variants differing only in a stretch of 42 amino acids (128, 129, 371). CAT-2A is predominantly expressed in liver, whereas CAT-2B is usually induced under inflammatory conditions in a variety of cells including T cells, macrophages, lung, and testis (see Table 6 and Refs. 46, 178, 429, 486, 524). CAT-2A is a low-affinity carrier for cationic amino acids and, unlike CAT-1, is relatively insensitive to trans-stimulation. CAT-3 isolated from mouse and rat brain mediates Na⁺-independent transport of cationic amino acids, although it is worth noting that 1) the substrate specificity differs from that for other CAT isoforms and 2) the K_m for CAT-3-mediated transport in oocytes is ~100-fold lower than that for CAT-1 (270, 291). In mouse and rat, CAT-3-mediated L-arginine transport is inhibited by other cationic amino acids, as well as L-citrulline, L-methionine, L-cysteine, L-aspartate, and L-glutamate, but not homoserine, and interestingly the recognition of neutral amino acids by CAT-3 is Na⁺ independent (270, 291). Closs and colleagues (612) recently succeeded in cloning a cDNA encoding human CAT-3, which was found to be glycosylated and targeted principally to the plasma membrane in human cells and oocytes. Unlike mouse and rat brain CAT-3 (270, 291), human CAT-3 is not neuron specific, exhibits a high selectivity for cationic amino acids, and does not transport L-citrulline, L-methionine, L-cysteine, or L-glutamate (612). The discrepancies in the specificity of the CAT-3 transporters remain to be resolved. Although Sperandeo et al. (555) identified a cDNA (designated CAT-4) in human placenta with 41-42% sequence identity to members of the CAT family, recent evidence indicates a lack of cationic amino acid transport activity in Xenopus oocytes or glioblastoma cells (U373 MG cell line) overexpressing CAT-4 (645). Whether CAT-3 and CAT-4 play a functional role in endothelial and/or smooth muscle cell cationic amino acid transport remains to be investigated.

Heterodimeric amino acid transporters, a subfamily of SLC7, are comprised of two subunits, a heavy chain (rBAT or 4F2hc) and an associated light chain linked by a disulfide bridge (126, 163, 387, 614, 625). The heavy subunit may be necessary for trafficking of the complex to the membrane, whereas the light chain may catalyze transport (186, 419, 467). To date, seven different light chain cDNAs have been identified, namely, LAT1, LAT2 (encoding system L), y⁺LAT-1, and y⁺LAT-2 (system y⁺L), xCT (system x), Asc-1 (system asc), and b^o,+AT (system b^o,+), with all but the latter associating with 4F2hc (reviewed in Refs. 163, 450, 614, 625).

In 1992 Devés et al. (164) were the first to describe system y⁺L activity in erythrocytes. System y⁺L-like activity has subsequently been described in intestine, placenta, lymphocytes, and platelets, and it is worth noting that this carrier exhibits a much a higher affinity for cationic amino acids (K_m for lysine ~10 µM) than any other cationic amino acid transport system (reviewed in Ref. 163). System y⁺L is stereoselective, electroneutral, and sensitive to trans-stimulation and mediates high-affinity, Na⁺-independent cationic amino acid transport, whereas the affinity of this carrier for neutral amino acids decreases significantly following substitution of Na⁺ by K⁺ (164; reviewed in Ref. 163). The affinity of system y⁺L for neutral amino acids differs, with L-leucine, L-methionine, L-isoleucine, and L-glutamine exhibiting higher affinities than L-alanine, L-serine, or L-cysteine. Moreover, in human erythrocytes, N-ethylmaleimide (200 µM) appears to be a relatively selective inhibitor of y⁺ activity, permitting the resolution of cationic amino acid fluxes via the low-capacity system y⁺L and higher capacity system y⁺ (164, 401).

Expression of the ubiquitous transmembrane protein 4F2hc (also named CD98) in Xenopus occytes induces amino acid transport activity resembling system y⁺L (50, 52, 186, 636). These studies further demonstrated that association of 4F2hc with a membrane oocyte protein was required for the expression of system y⁺L transport activity, providing evidence for a heterodimeric structure of an amino acid carrier (reviewed in Refs. 120, 163, 625). More recent studies have established that 4F2hc associates with y⁺LAT-1 and y⁺LAT-2 (~56 kDa) to induce system y⁺L transport activity (see Table 1). The apparent affinity of L-arginine in the presence of Na⁺ for y⁺LAT-1 is approximately twofold lower than that reported by Devés and colleagues for human erythrocytes, suggesting that y⁺LAT-2 rather than y⁺LAT-1 is related to the red cell system y⁺L (467). Both y⁺LAT-1 and y⁺LAT-2 mediate transport of dibasic amino acids in the absence of Na⁺ and neutral amino acids in the presence of Na⁺ (see Ref. 625). These transporters are likely to be involved in interorgan and intracellular transfer of amino acids. It has been suggested that y⁺LAT-2 is a glutamine (leucine)/arginine exchanger, which could play a role in the Na⁺-dependent uptake of L-leucine and L-glutamine into neurons, as well as in the supply of L-arginine for certain brain cells (86). As discussed in section IVB2, y⁺LAT-1 and y⁺LAT-2 are expressed in human umbilical vein endothelium (503).

The classical Na⁺-independent transport system L is most reactive with branched chain and aromatic neutral amino acids and is often characterized using the selective nonmetabolizable analog 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH; see Refs. 123, 531). Transport via system L is trans-stimulated by intracellular substrates of this carrier and in some cases may be increased by lowered extracellular pH. Studies in rat glioma cells, primary astroglial cells, and lymphocytes suggested that 4F2hc serves as a necessary component for expression of system L-like transport activity (87). This same group reported that system y⁺L was not involved in L-isoleucine or L-arginine transport in rat glioma cells and that overexpression of 4F2hc in Chinese hamster ovary cells was associated with increased Na⁺-independent transport of L-isoleucine (88). More recently, cDNAs encoding system L-like transporters have been isolated (see Table 1). Coexpression of 4F2hc and LAT-1 or LAT-2 in oocytes induces Na⁺-independent transport with a broad specificity for small and large zwitterionic amino acids and sensitivity to trans-stimulation (305, 469, 518). Interestingly, the expression of a tumor variant of LAT-1, tumor-associated gene-1/L amino acid transporter-1 (TA1/LAT-1) is dramatically upregulated in hepatocytes deprived of L-arginine, suggesting that it could act as a sensor of amino acid deprivation (100).

A Na⁺-independent anionic amino acid transport system designated x has been described in a variety of cell types, including vascular endothelial and smooth muscle cells (24-26, 39, 158-160, 539, 576, see Table 1). System x is insensitive to inhibition by dibasic and neutral amino acids and mediates exchange of L-cystine for intracellular L-glutamate with a 1:1 stoichiometry. Cysteine autoxidizes to L-cystine in the extracellular fluid, and within cells L-cystine is rapidly reduced to L-cysteine, the rate-limiting precursor for GSH synthesis (see Refs. 24, 26). Transport of L-cystine via system x is cis-inhibited by L-glutamate and homocysteate and markedly induced in cells exposed to oxidative stress and/or electrophilic compounds (26). Bannai and colleagues (509) recently isolated a cDNA encoding the system x transporter in activated murine macrophages. Expression of system x activity in Xenopus oocytes required two cDNA transcripts, with one identical to 4F2hc and the other a novel protein of 502 amino acids with 12 putative transmembrane domains. The designated protein xCT is a new member of a family of amino acid transporters known to form a heteromultimeric complex with 4F2hc (reviewed in Refs. 120, 162, 163, 450, 625), and human cDNAs for system x have now been isolated (39, 78, 319, 510, 529). mRNA levels and system x activity are increased in macrophages exposed to LPS, electrophilic agents such as diethylmaleate (DEM), and increased oxygen, and neither AP-1 nor NF-B appears to be involved in the transcription of xCT mRNA by LPS (508). It is known that the transcription factor Nrf2 binds to the antioxidant/electrophile response element (ARE/EpRE) in the 5'-flanking region of stress response genes, and interestingly, system x activity is not induced in Nrf2-deficient macrophages (288). Recent studies have confirmed that gene expression and activity of the xCT transporter is enhanced after treatment of mouse brain endothelial cells (272) and rat retinal capillary endothelial cells (595) with DEM. As in umbilical vein endothelial (407) and artery smooth muscle cells (539), induction of L-cystine transport activity was paralleled by a concomitant increase in intracellular glutathione levels.

In several cell types, L-aspartate and L-glutamate are accumulated by a high-affinity Na⁺- and K⁺-dependent system X (213), with one report in human umbilical vein endothelial cells also describing a Na⁺-independent anionic transporter (designated x) markedly inhibited by L-aspartate and L-glutamate (454).

This Na⁺-independent, high-affinity transport system mediates entry of small neutral amino acids such as L-alanine, L-serine, L-cysteine, glycine, L-threonine, and 2-aminoisobutyric acid (AIB), with transport activity not inhibited by the system A analog MeAIB (see Refs. 208, 603). Recent molecular strategies have successfully isolated cDNAs from mouse and human brain encoding asc-type amino acid transport (208, 423). The encoded proteins were designated Asc-1 and hAsc-1 and are structurally related to the family of amino acid transporters linked via disulfide bonds to the type II membrane glycoproteins such as 4F2hc and rBAT. Functional expression of these Asc-1 transporters required 4F2hc, was not dependent on Na⁺ or Cl, and mediated uptake of D-serine required activation of the glutamate N-methyl-D-aspartate receptor (see Refs. 208, 423, 625). The characteristics of another asc-type amino acid transporter (Asc-2, 32% identify with Asc-1) have recently been described (110). Coexpression of Asc-2 with either 4F2hc or rBAT in oocytes or COS-7 cells did not induce transport activity, whereas a fusion protein of the COOH terminus of Asc-2 with the NH₂ terminus of either 4F2hc or rBAT mediated L-serine uptake. This same group recently identified another transporter AGT1, structurally related to Asc-2, which as a fusion protein with either rBAT or 4F2hc exhibits a high affinity for Na⁺-independent transport of L-aspartate and L-glutamate (388). These authors speculated that Asc-2 and AGT1 represent a new subgroup of the heterodimeric amino acid transporter family, whose members associate not with rBAT or 4F2hc but with as yet unknown heavy chains.

Detection of system b^o,+ in the intestine and renal tubules has generated considerable interest, since a defect in system b^o,+ in the human kidney causes inherited hyperaminoaciduria cystinuria (reviewed in Refs. 120, 162, 450, 625). Unlike system b^o,+ in blastocysts (610), human b^o,+ transports L-cystine as well as neutral and dibasic amino acids. The affinity of diabasic and L-cystine is severalfold higher than for neutral amino acids, with a negative membrane potential stimulating inward flux of diabasic amino acids in exchange for neutral amino acids (50). The rapid reduction of cystine to cysteine intracellularly provides the chemical gradient for L-cystine transport. Expression cloning identified rBAT (also referred to as NBAT or D2) as a potential subunit of system b^o,+ (50, 52, 636), which has a predicted molecular mass of ~54 kDa and has also been detected in heart, liver, placenta, and lung (see Ref. 625). There is limited evidence that overexpression of 4F2hc leads to an interaction with the b^o,+AT light chain in mammalian cells and oocytes (85, 483), although Wagner et al. (625) have questioned the physiological relevance of these findings since 4F2hc is localized predominantly on basolateral membranes whereas b^o,+AT is targeted to apical membranes. Limited system b^o,+ transport activity has been described in vascular endothelial cells (see Table 3).

System B^o,+ represents a Na⁺-dependent transport system identified for cationic and neutral amino acids (609). Although the substrate specificity of systems b^o,+ and B^o,+ is similar, the latter also accepts L-alanine, L-serine, and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (reviewed in Refs. 162, 450). A cDNA encoding a Na⁺/Cl-dependent carrier (designated ATB^o,+) has been shown to transport cationic and neutral amino acids (540). This transporter has a sequence homology with neurotransmitter transporters and exhibits a broad specificity for neutral amino acids and a high affinity for cationic and neutral amino acids. Because cationic amino acid transport in endothelial and smooth muscle cells is predominantly Na⁺ independent, the ATB^o,+ transporter may play a negligible role in mediating L-arginine transport for NO synthase.

The classical Na⁺-dependent system A is expressed ubiquitously and is a key target for hormonal regulation, with transport acitivity also upregulated in response to amino acid deprivation or hypertonic stress (reviewed in Refs. 123, 236, 531, see sects. VII and IX). Neutral amino acids with short, polar, linear, and N-methylated side chains (e.g., MeAIB) are most reactive with system A, providing a useful tool for discriminating transport from other systems. System A transport activity is reduced markedly at lowered extracellular pH and subject to trans-inhibition by intracellular substrates for this carrier. The molecular identification of the system A was elusive during the 1990s, and cDNAs encoding system A have only been isolated within the past 2 years (3, 249, 284, 351, 570, 571, 658, see Table 1). Albers et al. (3) have briefly reviewed the nomenclature for the three reported variants of system A (ATA1, ATA2, ATA3), clarifying that ATA isoforms have also been designated as SAT1/2/3 or SA1/2/3 (see Table 1). Notably, a neuronal glutamine transporter (GlnT) was one of the first members of the system A family of transporters to be cloned (608), and this same group subsequently renamed the transporter SAT1 (658). ATA1 and ATA2 have similar functional characteristics, with transport in ooctyes saturable, voltage and Na⁺ dependent, pH sensitive, and inhibitable by MeAIB. Tissue expression of ATA isoforms varies, with ATA1 expressed principally in placenta and brain (631), ATA2 expressed ubiquitously in mammalian tissues including endothelial cells (4, 571, 658), and ATA3 virtually restricted to the liver (249, 571). Although expression of human ATA3 in retinal pigment epithelial cells evokes Na⁺-coupled neutral amino acid transport (lower affinity for MeAIB than ATA1 or ATA2), ATA3 exhibits a greater affinity for cationic compared with neutral amino acids, which the authors suggested may provide an important mechanism for L-arginine transport in hepatocytes (249). In summary, the ubiquitous expression of ATA2 in mammalian tissues suggests that ATA2 may encode the classical, Na⁺-dependent system A. In addition to adaptive increases in ATA2-mediated transport in response to amino acid deprivation and hypertonic stress (4, 351), steady-state levels of ATA2 are elevated by increased intracellular cAMP (250), a characteristic of system A in hepatocytes (reviewed in Ref. 531).

System ASC is a ubiquitously expressed Na⁺-dependent system that prefers small neutral amino acids such as L-alanine, L-serine, and L-cysteine. Transport exhibits a marked stereoselectivity and sensitivity to trans-stimulation and is unaffected by amino acid starvation (123, 316, 375). As extracellular pH is lowered, anionic amino acids such as L-glutamate may become more effective substrates and inhibitors of system ASC (602). A cDNA (ASCT1) encoding system ASC has been isolated from human hippocampal libraries, sharing ~40% sequence identity with the excitatory amino acid transport (EAAT) family of glutamate transporters (see Refs. 16, 524, 635). A second identified cDNA (ASCT2) encoding system ASC is believed to play an important role in facilitating the efflux of L-glutamine from glial cells (601). The ASCT transporters primarily mediate exchange of neutral amino acids rather than net uptake, and transport is associated with a substrate-dependent anion conductance (665).

Classical studies in hepatocytes demonstrated that system N exhibits a preference for L-glutamine, L-asparagine, and L-histidine, an intolerance for N-methylated substrates, an insensitivity to regulation by glucagon and insulin, and a reduced rate of transport at lowered extracellular pH (see Refs. 123, 315, 531). Unlike systems A and ASC, amino acid transport via system N tolerates substitution of Li⁺ for Na⁺ as the cotransported ion. Subtypes of system N with different functional characteristics and tissue distribution patterns have been described. The subtype of system N in skeletal muscle was designated system N^m and exhibits a much weaker Li⁺ tolerance and pH sensitivity than the hepatic system N (2, 278). Two further subtypes of system N have been described in the brain, with characteristics of transport in astrocytes (418) similar to hepatocytes and the system in neurons (421) distinct from systems N and N^m. The neuronal system N^b exhibits a similar Li⁺ tolerance and pH sensitivity to system N but is inhibited by L-glutamate.

A system N subtype was recently isolated from a rat brain cDNA library and designated SN1, with expression highest in liver and much lower in kidney, heart, and brain (115). Heterologously expressed rat or human SN1 mediates Na⁺-coupled transport of L-glutamine and other neutral amino acids, with efflux of H⁺ through the transporter resulting in intracellular alkalinization. Transport mediated by rat and human SN1 appears to be electrogenic, with inward transport of two Na⁺ and an amino acid coupled to efflux of one H⁺ (193). SN1 mediated L-glutamine transport is modulated by changes in membrane potential, with depolarization resulting in a switch from uptake to efflux, as well as by transmembrane L-glutamine gradients. A new member of this gene family (designated SN2) was cloned from rat brain and a human liver cell line, with broader expression including lung and stomach (421, 422). Gu et al. (234) subsequently identified another member of system N (SN3) with sequence homology to both system A and N transporters and expression in liver, muscle, kidney, and pancreas. For an overview of the molecular advances in our understanding of L-glutamine transport, we refer readers to a review by Bode (65).

Classical mammalian facilitative glucose transporters (GLUTs) belong to a supergene family, and four of them were initially identified as glucose transporters (GLUT1, GLUT2, GLUT3, GLUT4) and one as a fructose transporter (GLUT5). These transporters differ in their kinetic properties, sugar specificity, tissue localization, and regulation (see Refs. 22, 294, 415, 594). Three further glucose transporters have been cloned, namely, GLUT6 (352), previously referred to as GLUT9 by Doege et al. (169) and Phay et al. (468), GLUT8 (103, 171), and GLUT11 (detected exclusively in human heart and skeletal muscle and with sequence similarity to GLUT5, see Ref. 170). The distribution of GLUT transporters in mammalian cells is widespread, including endothelial cells from peripheral blood vessels and the blood-brain barrier. GLUT1, GLUT3, and GLUT4 have a higher affinity for glucose, with K_m values around ~2 mM, whereas GLUT2 has a lower affinity for glucose (K_m ~20 mM).

Glucose transport has been a major interest in blood-brain barrier research, and the high capacity of glucose transport into brain microvessels occurs primarily via GLUT1. The human erythrocyte glucose transporter GLUT1 can carry glucose, galactose, and mannose and is expressed in fetal tissues (239, 459). GLUT1 is also widely expressed in adult tissues but is most abundant in fibroblasts, erythrocytes, and brain endothelial cells compared with muscle, liver, and adipose tissue, suggesting that GLUT1 transport activity may be associated with developmental adaptation. In this context, children with glucose transporter protein syndrome (GLUT1 deficiency) exhibit impaired glucose transport across the blood-brain barrier, associated with infantile seizures and developmental delay (323). GLUT1 transporters in adipocytes have been shown to translocate to the plasma membrane in response to insulin (99), suggesting a regulatory mechanism and/or intracellular signaling pathway similar to GLUT4 in insulin-sensitive tissues. Interestingly, GLUT1 appears to be absent in human iris and corneal capillaries in diabetes mellitus (334).

The low-affinity glucose transporter GLUT2 has been identified in cells near the abluminal surface of liver cells, small intestine, and kidney and may facilitate glucose uptake or efflux from tissues depending on their nutritional status (reviewed in Ref. 294). To our knowledge, there are no reports of GLUT2 expression in endothelial or smooth muscle cells. Neuronal tissues have a high expression of GLUT3, which may be the main isoform involved in moving glucose into nerves and brain (417). GLUT3 seems to be more abundant in human brain tumor cells (433), and thus, as for GLUT1, its expression may be modulated by disease. In situ hybridization has detected GLUT3 mRNA and protein in the endothelium of human intraplacental microvessels, where it was proposed to play a potential role with GLUT1 in sustaining glucose supply to the developing fetus (254). However, a physiological role for GLUT3 in the placental vasculature remains questionable, since an earlier study failed to detect GLUT3 in the human syncytiotrophoblast (38).

The insulin-sensitive glucose transporter isoform GLUT4 is expressed mainly in adult skeletal muscle, cardiac muscle, and adipose tissue (reviewed in Refs. 294, 594) and has only been detected in very low abundance in the rat forebrain microvasculature using high-stringency hybridization of poly(A)⁺ RNA (394). To our knowledge, there is no functional evidence for GLUT4 activity in the cerebral or retinal vasculature. Regulation of GLUT4 by insulin has been studied extensively in adipocytes, where it is situated in perinuclear membranes and is translocated to the plasma membrane following stimulation with insulin (see Refs. 415, 438). Gene regulation of GLUT4 expression is now recognized as an essential process in the modulation of glucose transport, particularly in diabetes and hypoxia (438).

GLUT5 has been isolated from cDNA libraries for human, rat, and rabbit intestinal epithelial cells (see Ref. 294), with mRNA detected in human kidney, small intestine, skeletal muscle, adipocytes, and microglial cells. GLUT5 is constitutively expressed in the plasma membrane of muscle and adipocytes, and its distribution is not affected by insulin treatment. Although GLUT5 has been detected in the brain microvasculature (382, 383, 584), there is no convincing evidence implicating a transport role for GLUT5 in the blood-brain or blood-retinal barriers.

GLUT6 and GLUT8 exhibit glucose transport activity and form a separate branch of the GLUT family, with marked differences from GLUT1-5. GLUT6 mRNA has been detected in brain, spleen, and leukocytes (169) and more recently in adipose tissue (352). Initial reports from Doege et al. (169) show that GLUT6 can be recognized as a sugar anion transporter with a high K_m, based on cytochalasin binding studies. Expression of GLUT6 and GLUT8 has been achieved in transiently transfected primary rat adipose cells, and translocation is not responsive to phorbol ester, hyperosmolarity, or insulin (352), even though this isoform exhibits similarities to the insulin-responsive GLUT4. The putative GLUT6 and GLUT8 proteins have 44 and 31% sequence identity to GLUT5 and GLUT3, respectively, which could be one of the reasons why GLUT6 or GLUT8 expressed in adipocytes do not respond to translocation induced by insulin (352). GLUT11 is only expressed in human heart and skeletal muscle, with overexpression of GLUT11 cDNA in COS-7 cells resulting in a two- to threefold stimulation of glucose transport, inhibitable by an excess of fructose (170). The sequence similarity of GLUT11 and GLUT5 and sensitivity of GLUT11 transport activity to inhibition by fructose suggests that GLUT11 may be a fructose transporter. There is no evidence that GLUT6, GLUT8, or GLUT11 is expressed or has a functional role in vascular endothelial and smooth muscle cells.

	IV. AMINO ACID TRANSPORTERS IN ENDOTHELIAL CELLS

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