While transport processes for amino acids and glucose have long been known to be expressed in the luminal and abluminal membranes of the endothelium comprising the blood-brain and blood-retinal barriers, it is only within the last decades that endothelial and smooth muscle cells derived from peripheral vascular beds have been recognized to rapidly transport and metabolize these nutrients. This review focuses principally on the mechanisms regulating amino acid and glucose transporters in vascular endothelial cells, although we also summarize recent advances in the understanding of the mechanisms controlling membrane transport activity and expression in vascular smooth muscle cells. We compare the specificity, ionic dependence, and kinetic properties of amino acid and glucose transport systems identified in endothelial cells derived from cerebral, retinal, and peripheral vascular beds and review the regulation of transport by vasoactive agonists, nitric oxide (NO), substrate deprivation, hypoxia, hyperglycemia, diabetes, insulin, steroid hormones, and development. In view of the importance of NO as a modulator of vascular tone under basal conditions and in disease and chronic inflammation, we critically review the evidence that transport of L-arginine and glucose in endothelial and smooth muscle cells is modulated by bacterial endotoxin, proinflammatory cytokines, and atherogenic lipids. The recent colocalization of the cationic amino acid transporter CAT-1 (system y(+)), nitric oxide synthase (eNOS), and caveolin-1 in endothelial plasmalemmal caveolae provides a novel mechanism for the regulation of NO production by L-arginine delivery and circulating hormones such insulin and 17beta-estradiol.

1. Modulation of L-arginine transport (system y+) and release of nitric oxide (NO) and prostacyclin (PGI2) by elevated glucose and insulin were investigated in human cultured umbilical vein endothelial cells. 2. Elevated glucose induced a time-(6-12 h) and concentration-dependent stimulation of L-arginine transport, which was reversible and associated with a 3-fold increase in intracellular cGMP accumulation (index of NO synthesis) and 75 % decrease in PGI2 production. 3. Elevated glucose had no effect on the initial transport rates for L-serine, L-citrulline, L-leucine, L-cystine or 2-deoxyglucose. 4. Resting membrane potential was unaffected by elevated glucose whereas basal intracellular [Ca2P] increased from 65 + 5 nm to 136 + 16 nM.5. Insulin induced a protein synthesis-dependent stimulation of L-arginine transport and increased NO and PGI2 production in cells exposed to 5 mm glucose. 6. In cells exposed to high glucose, insulin downregulated elevated rates of L-arginine transport and cGMP accumulation but had no effect on the depressed PGI2 production. 7. Our findings suggest that insulin's normal stimulatory action on human endothelial cell vasodilator pathways may be impaired under conditions of sustained hyperglycaemia.Endothelium-dependent relaxation is impaired in longlasting diabetes mellitus (reviewed by Cohen, 1993;Poston & Taylor, 1995), yet in the early stages of the disease blood flow is actually increased in various organs (reviewed by Tooke, 1989). The generalized vasodilatation in diabetic patients appears to be influenced by the degree of hyperglycaemia and is attenuated upon lowering blood glucose levels (see Tooke, 1989). As endothelium-derived nitric oxide (NO) plays a key role in the regulation of vascular tone (Moncada, Palmer & Higgs, 1991), it seems likely that prolonged hyperglycaemia may modulate the L-arginine-NO signalling pathway. We have recently shown that gestational diabetes induces phenotypic changes in human fetal endothelial cells, which are associated with a membrane hyperpolarization, activation of the L-arginine transporter (system y+), elevation of basal NO synthesis and decreased basal and histamine-stimulated PGI2 release (Sobrevia, Cesare, Yudilevich & Mann, 1995). Recent evidence also implicates insulin as a regulator of the L-arginine-NO vasodilator pathway in man (see Baron, 1994). Moreover, insulin has been reported to enhance the synthesis and expression of a high-affinity cationic amino acid transporter (system y+/ murine cationic amino acid transporter-1 (MCAT-1): Kim, Closs, Albritton & Cunningham, 1991) in rat liver cells (Wu, Robinson, Kung & Hatzoglou, 1994 b).In the present study we have investigated the effects of elevated glucose and human insulin on L-arginine transport and generation of NO and PGI2 in human umbilical vein endothelial cells isolated from normal pregnancies. In addition, we examined whether elevated glucose altered the resting membrane potential and intracellular Ca2+ levels. Our findings establish that both hyperglycaemia and human ...

1. The activity of the human endothelial cell L-arginine transporter (system y+) has been correlated with cGMP production (index of nitric oxide) and prostacyclin (PGJ2) release in umbilical vein endothelial cells cultured from normal or gestational diabetic pregnancies. 2. In non-diabetic and diabetic cells, transport of L-arginine was Na+ and pH independent, inhibited by other cationic L-arginine analogues and unaffected by neutral amino acids. 3. Diabetes was associated with an increased Vmax for saturable L-arginine transport (4-6 + 0-13 vs. 9-9 + 0.5 pmol (ug protein)-' min-', P< 0X01), but had no effect on initial rates of transport for L-serine, L-citrulline, L-leucine or 2-deoxyglucose.4. In non-diabetic and diabetic cells, elevated K+ resulted in a concentration-dependent inhibition in the initial rates of transport for L-arginine and the membrane potentialsensitive probe tetra[3H]phenylphosphonium (TPP+).5. When resting membrane potential was measured using the whole-cell patch voltage clamp technique, diabetic cells were hyperpolarized (-78 + 0 3 mV) compared with non-diabetic cells (-70 + 0 04 mV, P< 0 04). Accumulation of [3H]TPP+ was also increased in diabetic compared with non-diabetic cells. 6. Basal intracellular cGMP levels were elevated 2-5-fold in diabetic cells, and L-NAME (100 uM), an inhibitor of nitric oxide synthase, abolished basal cGMP accumulation in nondiabetic and diabetic cells.7. Histamine (10 juM) had no effect on L-arginine transport but evoked significant increases in cGMP in non-diabetic and diabetic cells, which were completely inhibited by L-NAME but unaffected by superoxide dismutase. 8. 9.Basal and histamine-stimulated PGI2 release was decreased markedly in diabetic cells. Our findings demonstrate that gestational diabetes is associated with phenotypic changes in fetal endothelial cells, which result in a membrane hyperpolarization, activation of the human endothelial cell L-arginine transporter (system y+), elevation of basal nitric oxide synthesis and decreased PGI2 production.Abnormalities in vascular endothelial cell function occur early in the pathogenesis of insulin-dependent diabetes mellitus (reviewed by Bar, 1992;Cohen, 1993;Poston & Taylor, 1995). Nitric oxide (NO) has emerged as an important regulator of vascular tone and mediates vasodilatation by elevating cGMP levels in smooth muscle cells (reviewed by Knowles & Moncada, 1994 Tilton et al. 1993;Smulders et al. 1994). Of the few studies that havTe reported an enhanced production of NO in diabetes (Langenstroer & Pieper, 1992;Tilton et al. 1993;Smulders et al. 1994), some are based on the observation that aminoguanidine (an inhibitor of NO synthase and advanced glycosylation end products) reverses endothelial cell dysfunction in diabetes (Bucala, Tracey & Cerami, 1991; see review by Poston & Taylor, 1995). Most studies itt vitro have not added L-arginine to the organ bath, which could lead to substrate depletion, if basal NO synthesis and L-arginine transport were enhanced. In addition to altered endothelial c...