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(Circulation Research. 2003;92:64.)
© 2003 American Heart Association, Inc.

Cellular Biology

Rapid Stimulation of L-Arginine Transport by D-Glucose Involves p42/44^mapk and Nitric Oxide in Human Umbilical Vein Endothelium

Carlos Flores, Susana Rojas, Claudio Aguayo, Jorge Parodi, Giovanni Mann, Jeremy D. Pearson, Paola Casanello, Luis Sobrevia

From the Cellular and Molecular Physiology Laboratory (C.F., S.R., C.A., J.P., P.C., L.S.), Department of Physiology, Faculty of Biological Sciences, and the Department of Obstetrics and Gynaecology (P.C.), Faculty of Medicine, University of Concepción, Concepción, Chile, and King’s College London (G.M., J.D.P.), Guy’s Campus, London, UK.

Correspondence to Dr L. Sobrevia, Cellular and Molecular Physiology Laboratory (CMPL), Department of Physiology, Faculty of Biological Sciences, University of Concepción, PO Box 160-C, Concepción, Chile. E-mail lsobrev{at}udec.cl

	Abstract

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D-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High D-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of L-arginine transport (V_max 6.1±0.7 versus 4.4±0.1 pmol/µg protein per minute) with no change in transport affinity (K_m 105±9 versus 111±16 µmol/L). L-[³H]Citrulline formation and intracellular cGMP, but not intracellular Ca²⁺, were increased by high D-glucose. The effects of D-glucose were mimicked by levcromakalim (ATP-sensitive K⁺ channel blocker), paralleled by p42/p44^mapk and Ser¹¹⁷⁷–endothelial NO synthase phosphorylation, inhibited by N^G-nitro-L-arginine methyl ester (L-NAME; NO synthesis inhibitor), glibenclamide (ATP-sensitive K⁺ channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated D-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial L-arginine/NO signaling pathway is rapidly activated by elevated D-glucose via NO and p42/44^mapk. This could be determinant in pathologies in which rapid fluctuations of plasma D-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus.

Key Words: humans • endothelium • glucose • arginine • nitric oxide

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cationic amino acid L-arginine is the substrate for nitric oxide (NO) synthesis via endothelial NO synthase (eNOS)¹ and is taken up primarily by the Na⁺-independent high-affinity (K_m 100 to 400 µmol/L) systems y⁺/CAT-1 and y⁺/CAT-2B (where CAT indicates cationic amino acid transporter) in human umbilical vein endothelial cells (HUVECs).^2,3 L-Arginine transport and NO synthesis (L-arginine/NO pathway) are increased in HUVECs from patients with gestational diabetes.² Interestingly, long-term incubation (24 hours) of HUVECs from normal pregnancies with elevated D-glucose mimics the effect of gestational diabetes on the L-arginine/NO pathway.⁴ In addition, elevated D-glucose for 24 hours^4,5 or 5 days⁶ increases eNOS gene expression. A recent report shows that D-glucose infusion induces vasodilatation in humans,⁷ and in animal models, an elevation of plasma D-glucose results in rapid (seconds to minutes) vasodilatation.^8–10 Therefore, rapid fluctuations in the D-glucose level are crucial in maintaining human fetal endothelial function.^2–5,11

D-Glucose activates protein kinase C (PKC), an enzyme involved with long-term stimulation of the L-arginine/NO pathway,^5,12–14 and (within 1 hour) p42 and p44 mitogen-activated protein (MAP) kinases (p42/44^mapk).^5,14,15 p42/44^mapk activation may itself be dependent on PKC activation and NO synthesis.^5,14 However, the effect of short-term incubation with elevated D-glucose on the endothelial L-arginine/NO pathway has not been investigated.^4,11,16,17

The present study shows that a 2-minute incubation with 25 mmol/L D-glucose increases L-arginine transport and NO synthesis in HUVECs. The underlying cellular mechanisms involve phosphorylation of eNOS at Ser¹¹⁷⁷ via phosphatidylinositol 3-kinase (PI3-k) and activation of eNOS and p42/p44^mapk by D-glucose.¹⁸

	Materials and Methods

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Cell Culture
Human umbilical vein endothelium was isolated (collagenase digestion 0.25 mg/mL) and cultured (37°C, 5% CO₂, confluent passage 2) in medium 199 containing 5 mmol/L D-glucose, 10% newborn calf serum, 10% fetal calf serum, 3.2 mmol/L L-glutamine, 100 µmol/L L-arginine, and 100 U/mL penicillin-streptomycin (primary culture medium).^2–4 Before an experiment (24 hours), the incubation medium was changed to serum-free medium 199.

L-Arginine Transport
L-Arginine transport (1 µCi/mL, 37°C, 1 minute) was determined in cells preincubated (15 seconds to 5 minutes) with Krebs solution (mmol/L: NaCl 131, KCl 5.6, NaHCO₃ 25, NaH₂PO₄ 1, HEPES 20, CaCl₂ 2.5, and MgCl₂ 1 [pH 7.4, 37°C]) containing 5 or 25 mmol/L D-glucose, 25 mmol/L L-glucose, or 5 mmol/L D-glucose plus 20 mmol/L D-mannitol (osmotic controls).^2–4 L-Arginine transport was also determined in Krebs solution in which NaCl was replaced by equimolar concentrations of choline chloride^2–4 or in cells incubated (30 minutes) with KCl (5.5 to 131 mmol/L), with NaCl decreased equivalently, or with 131 mmol/L KCl for 2, 4, 10, 20, or 30 minutes. In trans-stimulation experiments, cells were preincubated (2 hours) with primary culture medium containing 10 mmol/L L-lysine. Cell-associated radioactivity and data analyses were performed as described.^2–4

L-Arginine transport was assayed in cells preincubated (30 minutes) with 100 µmol/L N^G-nitro-L-arginine methyl ester (L-NAME, eNOS inhibitor),^2,3 10 µmol/L PD-98059 (MAP kinase kinase 1/2 [MEK1/2] inhibitor),¹⁹ 100 µmol/L S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), 1 µmol/L KT-5823 (protein kinase G [PKG] inhibitor),²⁰ 100 nmol/L calphostin C (PKC inhibitor),²¹ 10 µmol/L glibenclamide (ATP-sensitive K⁺ [K⁺_ATP] channel blocker),²² 1 µmol/L levcromakalim (K⁺_ATP activator),²³ or 30 nmol/L wortmannin (PI3-k inhibitor).²⁴

Membrane Potential
[³H]Tetraphenylphosphonium ([³H]TPP⁺) influx (46 nmol/L, 0.5 µCi/mL, 15 to 120 seconds), a membrane potential–sensitive probe,^2,3,25 was determined in Krebs solution containing 5 or 25 mmol/L D-glucose, 100 µmol/L L-arginine, and 5.5 or 131 mmol/L KCl.^2,3 Resting membrane potential (E_m, whole-cell patch clamp) was recorded using an EPC-7 amplifier (List Medical) as described.² E_m was measured for at least 1 minute in the presence of 5 or 25 mmol/L D-glucose. Only recordings with <0.1-mV variations were considered. E_m was also determined in cells preincubated for 2 hours with 10 mmol/L L-lysine.

PKC Activity
PKC activity (³²P incorporation from [-³²P]ATP into a synthetic PKC substrate peptide analogue⁵) was determined in cells exposed to 5 or 25 mmol/L D-glucose (2 minutes) after pretreatment with 100 nmol/L phorbol 12-myristate 13-acetate (5 minutes, PKC activator), 100 nmol/L 4-phorbol 12,13-didecanoate (5 minutes, less active phorbol 12-myristate 13-acetate analogue), 100 nmol/L calphostin C (30 minutes), 10 µmol/L PD-98059 (30 minutes), 100 µmol/L SNAP (5 minutes), 100 µmol/L L-NAME (30 minutes), or 30 nmol/L wortmannin (30 minutes).

cGMP Determination
Cells preincubated (30 minutes) in Krebs solution (37°C) containing L-arginine (100 µmol/L) and 3-isobutyl-1-methylxanthine (0.5 mmol/L, phosphodiesterase inhibitor),^2,3 in the absence or presence of L-NAME (100 µmol/L), were exposed to 5 or 25 mmol/L D-glucose for the last 2 minutes of the 30-minute incubation period with 3-isobutyl-1-methylxanthine. cGMP was determined in HCl-cell extracts by radioimmunoassay.^2,3

L-Citrulline Assay
Cells were incubated with L-[³H]arginine (100 µmol/L, 4 µCi/mL, 30 minutes, 37°C) in the absence or presence of L-NAME (100 µmol/L). Cells were exposed to 5 or 25 mmol/L D-glucose for 30 minutes or for the last 2, 4, 10, or 20 minutes of this incubation period. Some experiments were performed in cells exposed only to L-[³H]arginine (4 µCi/mL) and 5 or 25 mmol/L D-glucose. Formic acid–digested cells (200 µL) were passed through a sodium ion form of the cation ion-exchange resin Dowex 50W (50X8-200), and L-[³H]citrulline concentration was determined in the H₂O eluate.³

Intracellular Ca²⁺
Cells on glass coverslips were loaded (30 minutes, 23°C) with the acetoxymethyl derivative of fluo 3 (5 µmol/L). Coverslips were transferred to an experimental bath with Krebs solution containing 5 or 25 mmol/L D-glucose, and Ca²⁺ was imaged using a Zeiss LSM 410 confocal microscope.⁴

Western Blots
After pretreatment with 10 µmol/L PD-98059 (30 minutes), 100 µmol/L SNAP (2 to 5 minutes), or 30 nmol/L wortmannin (30 minutes), the cells were incubated with 5 or 25 mmol/L D-glucose (2 minutes). Cell protein extracts were probed with a primary polyclonal mouse antiphosphorylated (1:1000) or nonphosphorylated (1:1500) p44/p42^mapk, rabbit anti-eNOS (1:2500) or anti-phosphorylated Ser¹¹⁷⁷–eNOS (1:2500) antibodies, and horseradish peroxidase–conjugated goat secondary antibodies as described.^3,5 Primary polyclonal mouse anti-actin (1:2000) served as the internal control. Proteins were detected by enhanced chemiluminescence and quantified by densitometry (Ultroscan XL enhanced laser densitometer, LKB Instruments).^3,5

Semiquantitative PCR
Extracted mRNA (Dynal) was reversed-transcribed into cDNA using oligo(dT₁₈) plus random hexamers (10-mer) and M-MLV reverse transcriptase (Promega) for 1 hour at 37°C.³ Polymerase chain reactions (PCRs) were performed in 20-µL samples (2 µL of 10x PCR buffer, 0.8 µL of 50 mmol/L Mg²⁺, 0.4 µL dNTPs, 13.6 µL RNase-free H₂O, 0.2 µL Taq DNA polymerase, and 0.5 µmol/L sequence-specific oligonucleotide primers for human CAT-1, CAT-2A, or CAT-2B). Samples were incubated (4 minutes, 95°C), followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 57°C, 30 seconds at 72°C, and a final extension for 7 minutes at 72°C. ß-Actin expression was used as a reference value. Reverse transcription (RT)-PCR products were sequenced in both directions by Taq dideoxy terminator cycle sequencing (automated DNA sequencer 373A, Applied Biosystems).³

Oligonucleotide primers were as follows: hCAT-1 (sense) 5'-CCAGTACTTCCGACGAGTTAGA-3', hCAT-1 (antisense) 5'-CATCCACACAGCAAACCGGACC-3', hCAT-2A (sense) 5'-TATCCCGATTTTTTTGCTGTGTGC-3', hCAT-2A (antisense) 5'-TGCAGTCAACGTGGCAGCAACT-3', hCAT-2B (sense) 5'-TCCCAATGCCTCGTGTAATCTA-3', hCAT-2B (antisense) 5'-GCATGCTGAAGCCCTGTCTCTGC-3', ß-actin (sense) 5'-AACCGCGAGAAGATGACCCAGATCATCTTT-3', and ß-actin (antisense) 5'-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3'. Expected size products were as follows: hCAT-1, 450 bp; hCAT-2A, 690 bp; hCAT-2B, 360 bp; and ß-actin, 350 bp.

SOD Activity and -Tocopherol Experiments
Cells were homogenized in buffer containing 50 mmol/L Tris-(hydroxymethyl)-aminomethane, 100 mmol/L potassium chloride, 0.02% Triton X-100, 100 mmol/L sodium pyrophosphate, and 100 mmol/L sodium fluoride (pH 7.4), which was supplemented with trypsin inhibitors (4 mg/mL aprotinin, 1 mg/mL benzamidine, 5 µg/mL leupeptin, and 200 µmol/L sodium orthovanadate). Aliquots (1 mg protein/mL) were incubated (25°C, 2 minutes) with potassium phosphate buffer (50 mmol/L, pH 10.2) containing adrenochrome (200 µmol/L) and epinephrine (10 µmol/L), and absorbance was measured at 480 nm. Superoxide dismutase (SOD) activity was calculated from the inhibition curve for epinephrine auto-oxidation versus protein concentration. Basal absorbance (100% activity) was the reaction in the absence of cell extracts.²⁶ Cells were also preincubated (30 minutes) with -tocopherol (500 µg/mL, 1% ethanol).²⁷

Materials
Sera, agarose, and buffers were from GIBCO Life Technologies. Collagenase type II (Clostridium histolyticum) was from Boehringer-Mannheim, and Bradford protein reagent was from Bio-Rad Laboratories. L-NAME and SNAP were from Calbiochem. Ethidium bromide, Dowex (50WX8-400), and all other reagents were from Sigma Chemical Co. L-[2,3-³H]Arginine (36.1 Ci/mmol), D-[1-¹⁴C]mannitol (49.3 mCi/mmol), [-³²P]ATP, and [³H]TPP⁺ (37 Ci/mmol) were from NEN. 3',5'-cGMP-TME was from ICN. Antibodies were from Cell Signaling, New England Biolabs.

Statistical Analysis
Values are mean±SEM, where n indicates the number of different cell cultures (4 to 8 replicates per experiment). Statistical analyses were carried out on raw data using the Peritz F multiple-means comparison test.²⁸ A Student t test was applied for unpaired data, and a value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
L-Arginine Transport
Elevated D-glucose (2 minutes), but not L-glucose or D-mannitol, stimulated L-arginine transport (half-maximal effect [K_1/2] 13±2 mmol/L D-glucose) (Figure 1A). Basal transport rates increased significantly after 30 seconds of exposure to elevated D-glucose (K_1/2 25±5 seconds), with maximal rates achieved within 1 minute and sustained over 5 minutes (Figure 1B). Subsequent experiments were performed using 25 mmol/L D-glucose for 2 minutes. D-Glucose–stimulated L-arginine transport decreased to basal values within 5 minutes after reexposure of cells to 5 mmol/L D-glucose (Figure 1B). RT-PCR analysis detected only hCAT-1 and hCAT-2B mRNA in HUVECs (Figure 1C).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of D-glucose on L-arginine transport and CAT expression. A, L-Arginine transport (100 µmol/L, 1 minute, 37°C) in HUVECs incubated (2 minutes) with increasing concentrations of D-glucose (), L-glucose (, or 5 mmol/L D-glucose plus 5 to 20 mmol/L D-mannitol (. *P<0.05 and **P<0.04 vs 5 or 7.5 mmol/L D-glucose and corresponding values in L-glucose and D-glucose+D-mannitol. B, Time course of effect of D-glucose on L-arginine transport (as in panel A) in cells incubated for 0 to 5 minutes in 5 mmol/L D-glucose (, 25 mmol/L D-glucose (), or 5 mmol/L D-glucose+20 mmol/L D-mannitol (. High D-glucose–containing Krebs solution was replaced (arrows) by 5 mmol/L D-glucose, and cells were incubated for 10 minutes. *P<0.04 vs all other values. Values are mean±SEM (n=13). C, RT-PCR for mRNA from cells in 5 mmol/L D-glucose. mRNA was reversed-transcribed into cDNA, and PCR was performed for human CAT-1 (lane 2, 449 bp), CAT-2A (lane 3, 690 bp), or CAT-2B (lane 4, 357 bp). Lane 5 is ß-actin (350 bp), and lane 1 is DNA ladder (100 to 2000 bp). Data are representative of 14 cell cultures.

Elevated D-glucose had no effect on the nonsaturable component (K_D) of overall L-arginine transport but increased V_max, with no change in apparent K_m (Figure 2A, Table 1). Cell incubation with L-lysine (10 mmol/L, 2 hours) increased 6.5-fold the L-arginine transport in 5 mmol/L D-glucose (Figure 2B). However, L-arginine transport was increased 2.9-fold in cells exposed to 25 mmol/L D-glucose (last 2 minutes of the 2-hour incubation with L-lysine) (Figure 2B). D-Glucose stimulation and trans-stimulation by L-lysine of L-arginine transport was unaltered in Na⁺-free Krebs solution (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of D-glucose on kinetic parameters and trans-stimulation of L-arginine transport. A, Saturable L-arginine transport (1 minute, 37°C) in HUVECs incubated (2 minutes) in 5 mmol/L D-glucose ( or 25 mmol/L D-glucose (). B, L-Arginine transport (100 µmol/L, 1 minute, 37°C) in cells preincubated (2 hours) in medium 199 in the absence (control) or presence of 10 mmol/L L-lysine. Transport assays were performed in cells exposed to 5 mmol/L D-glucose (open bars) or 25 mmol/L D-glucose (filled bars) for the last 2 minutes of the 2-hour incubation period with L-lysine. Values are mean±SEM (n=16). *P<0.04 vs all other values.

View this table: [in this window] [in a new window]   Table 1. D-Glucose Effect on L-Arginine Transport in HUVECs

TPP⁺ Influx and Membrane Potential
Elevated D-glucose increased TPP⁺ influx (1.8-fold) and caused membrane hyperpolarization (Table 2). TPP⁺ influx and L-arginine transport were inhibited by KCl-induced membrane depolarization. Glibenclamide (K⁺_ATP channel blocker) blocked D-glucose–increased L-arginine transport and changes in TPP⁺ influx and E_m (Table 2). Levcromakalim (K⁺_ATP channel activator) hyperpolarized the plasma membrane and increased L-arginine transport and TPP⁺ influx only in 5 mmol/L D-glucose; effects were blocked by glibenclamide (Table 2). The effects of D-glucose were also blocked by wortmannin (not shown). Preloading cells with L-lysine did not alter E_m (-66.1±0.3 mV, P>0.05; n=29 cells) compared with control cells (-67.5±0.5 mV, P>0.05; n=45 cells). L-Arginine transport was inhibited by KCl with half-maximal inhibition at 12±2 and 17±4 mmol/L KCl for 5 and 25 mmol/L D-glucose, respectively. The time needed to induce half-maximal inhibition of transport with 131 mmol/L KCl was similar in cells in 5 mmol/L D-glucose (6.5±0.6 minutes) compared with 25 mmol/L D-glucose (7.2±0.6 minutes).

View this table: [in this window] [in a new window]   Table 2. D-Glucose Effect on L-Arginine Transport, [3H]TPP+ Influx, and Em in HUVECs

NO Involvement
Elevated D-glucose increased eNOS phosphorylation at Ser¹¹⁷⁷ (Figure 3A), L-[³H]citrulline (Figure 3B), and cGMP accumulation (Figure 3C). L-NAME inhibited the effect of D-glucose on cGMP and L-[³H]citrulline formation but did not alter eNOS-Ser¹¹⁷⁷ phosphorylation (not shown). However, wortmannin inhibited D-glucose–induced eNOS phosphorylation at Ser¹¹⁷⁷, cGMP, and L-[³H]citrulline formation. Similar results were found in cells exposed to 25 mmol/L D-glucose for the last 4, 10, or 20 minutes of the 30-minute incubation period with L-[³H]arginine (not shown). Intracellular Ca²⁺ in cells incubated with 25 mmol/L D-glucose for 2 minutes (42±7 nmol/L) was not statistically different (P>0.05, n=125 cells) from values in cells incubated with 5 mmol/L D-glucose (35±5 nmol/L).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of D-glucose on eNOS activity. A, Immunoblot for total eNOS and phosphorylated eNOS at Ser1177 (eNOS~P-Ser1177) in HUVECs preincubated (30 minutes) with 5 mmol/L D-glucose in the absence or presence of wortmannin and then exposed (2 minutes) to 5 or 25 mmol/L D-glucose (see Materials and Methods). Top panel shows densitometry ratios. Data are representative of 6 cell cultures. B, L-[3H]Citrulline formation from L-[3H]arginine (4 µCi/mL, 37°C) in cells preincubated (30 minutes) with Krebs solution in the absence (control) or presence of L-NAME and exposed for the last 2 minutes to Krebs solution containing 5 mmol/L D-glucose (open bars) or 25 mmol/L D-glucose (filled bars). C, Intracellular cGMP under same experimental conditions as in panel B. Values are mean±SEM (n=17). *P<0.05 vs all other values.

L-NAME also inhibited the effect of D-glucose on L-arginine transport V_max (Table 1), TPP⁺ influx, and E_m (Table 2); however, L-NAME did not alter TPP⁺ influx or E_m in 5 mmol/L D-glucose. Other experiments show that SNAP (NO donor) induces TPP⁺ influx, L-arginine transport, and membrane hyperpolarization only in 5 mmol/L D-glucose (Table 2) and that dibutyryl cGMP (dbcGMP) increased L-arginine transport (Figure 4A) and TPP⁺ influx (Figure 4B). The effects of D-glucose and dbcGMP were blocked by KT-5823, a PKG inhibitor (Table 2).

View larger version (19K): [in this window] [in a new window]   Figure 4. cGMP involvement in the effect of D-glucose on L-arginine transport and [3H]TPP+ influx. HUVECs preincubated (30 minutes) with 5 mmol/L D-glucose in the absence (control) or presence of KT-5823 or dbcGMP were exposed (2 minutes) to 5 mmol/L D-glucose (open bars) or 25 mmol/L D-glucose (filled bars) in the presence of KT-5823 and/or dbcGMP, and L-arginine transport (100 µmol/L, 1 minute, 37°C) (A) and [3H]TPP+ influx (46 nmol/L, 1 minute, 37°C) (B) were determined. Values are mean±SEM (n=12). *P<0.05 vs all other values.

PKC and MAP Kinase Involvement
PKC activity was unaltered at up to 5 minutes of incubation with high D-glucose (Figure 5A) or after the addition of SNAP (not shown). Furthermore, calphostin C (PKC inhibitor) had no effect on D-glucose–increased L-arginine transport (Figure 5B), TPP⁺ influx, or NO synthesis (not shown). However, longer incubation with elevated D-glucose (>10 minutes) increased membrane PKC activity (not shown), confirming our previous observations in HUVECs.⁵ In contrast, high D-glucose induced p42/p44^mapk phosphorylation (Figure 6A), an effect blocked by PD-98059 and mimicked by brief exposure (2 minutes) to SNAP (Figure 6B). Interestingly, the effect of D-glucose on p42/44^mapk phosphorylation was blocked by wortmannin (Figure 6C). PD-98059 also blocked the stimulatory effects of elevated D-glucose and SNAP on TPP⁺ influx, L-arginine transport, and E_m (Table 2).

View larger version (18K): [in this window] [in a new window]   Figure 5. Lack of effect of calphostin C in the effect of D-glucose on L-arginine transport. A, PKC activity in cytosolic (open symbols) and membrane (filled symbols) fractions from HUVECs preincubated (30 minutes) with Krebs solution without (,) or with 100 nmol/L calphostin C (, ). Cells were incubated (30 seconds) in 25 mmol/L D-glucose in the absence or presence of calphostin C. B, Time-course effect of 25 mmol/L D-glucose on L-arginine transport (100 µmol/L, 1 minute, 37°C) (as in panel A) in Krebs solution without () or with 100 nmol/L calphostin C (). Values are mean±SEM (n=18). *P<0.05 vs all other values.

View larger version (30K): [in this window] [in a new window]   Figure 6. NO and PI3-k involvement in effect of D-glucose on p42/44mapk phosphorylation. A, Immunoblot for phosphorylated p44mapk (p44~P) and p42mapk (p42~P) and nonphosphorylated p44mapk (p44) or p42mapk (p42) in HUVECs preincubated (30 minutes) with 5 mmol/L D-glucose in the absence or presence of PD-98059 and then exposed (2 minutes) to 5 or 25 mmol/L D-glucose. B, Effect of SNAP on p42/p42mapk in HUVECs in 5 mmol/L D-glucose. C, Effect of wortmannin on p42/p42mapk in HUVECs (as in panel A). Data are representative of similar results in 17 cell cultures.

SOD Activity and -Tocopherol Effect
SOD activity in cells in 5 mmol/L D-glucose (5.5±0.6 U/mL) was not significantly altered (P>0.05, n=5) by 25 mmol/L D-glucose (6±1 U/mL). Extracellular SOD or -tocopherol did not block (P>0.05, n=4 to 8) the effect of D-glucose on L-arginine transport (5.6±0.3 and 5.8±0.6 pmol/µg protein per minute, respectively), L-citrulline formation (3.1±0.2 and 2.8±0.4 pmol/µg protein per 30 minutes, respectively), TPP⁺ influx (4.4±0.2 and 4.1±0.3 pmol/mg protein per minute, respectively), or changes in E_m (-76±0.3 and -78±0.3 mV, respectively).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study establishes that D-glucose induces a rapid concentration-dependent stimulation of L-arginine transport in HUVECs. This effect requires NO synthesis associated with increased phosphorylation of eNOS at Ser¹¹⁷⁷ and activation of p42/p44^mapk and PI3-k, and it is independent of PKC and intracellular Ca²⁺ changes. These findings provide the first evidence that short-term hyperglycemia activates the L-arginine/NO signaling pathway in human fetal endothelium.

L-Arginine transport is mediated by systems y⁺/CATs,^2,3,12 y⁺L,^29,30 and b^0,+12 in HUVECs, with the first likely predominating at the physiological concentration of extracellular L-arginine. The cDNAs for four potential human y⁺ transporters (hCAT-1, hCAT-2B, hCAT-2A, and hCAT-4) have been sequenced.³¹ L-Arginine transport in HUVECs occurs with relatively high affinity (K_m 80 to 100 µmol/L) and, as confirmed here, is Na⁺ independent and inhibited by membrane depolarization.^2,3,32 Because of their similar kinetic properties, CAT-1 and CAT-2B are hard to distinguish at the functional level.³¹ Our results show that both high-affinity hCAT-1 (K_m 100 to 200 µmol/L) and hCAT-2B (K_m 200 to 400 µmol/L), but not the low-affinity hCAT-2A transporter (K_m 2 to 5 mmol/L), are present in HUVECs, confirming previous reports.^3,33 CAT-1 is more sensitive than CAT-2B to trans-stimulation by cationic amino acids.^3,31,34 When we preloaded HUVECs with L-lysine, L-arginine transport was increased by 7-fold in 5 mmol/L D-glucose. However, the L-lysine trans-stimulatory effect was less effective (3-fold) in cells exposed for 2 minutes to 25 mmol/L D-glucose. Because L-arginine transport is trans-stimulated by 9.8-fold or 1.8-fold in Xenopus oocytes injected with hCAT-1 or hCAT-2B mRNA, respectively,³⁴ trans-stimulation in HUVECs in 5 mmol/L D-glucose may be preferentially mediated by hCAT-1. The reduced trans-stimulation of transport in high D-glucose may result from a state of maximal activity of L-arginine transporters already induced by L-lysine; therefore, high D-glucose could not further increase L-arginine transport. In addition, the possibility that L-lysine–stimulated L-arginine transport was due to membrane hyperpolarization is unlikely because E_m was unaltered in L-lysine–preloaded cells.

Long-term incubation (24 hours) of HUVECs with high D-glucose increases V_max for L-arginine transport.⁴ The present study shows that acute (2-minute) D-glucose increases V_max for L-arginine transport without altering the apparent K_m. As noted above, L-arginine transport is sensitive to changes in extracellular K⁺ and E_m. Because high D-glucose induced membrane hyperpolarization, stimulation of L-arginine transport could result from changes in E_m. The stimulatory effect of D-glucose on TPP⁺ influx and L-arginine transport was blocked by glibenclamide, a K⁺_ATP channel blocker. In addition, levcromakalim (K⁺_ATP activator)²³ mimics D-glucose–induced changes in E_m, L-arginine transport, and TPP⁺ influx. K⁺_ATP channels are expressed in the endothelium³⁵ and are activated by D-glucose³⁶; thus, the effects of D-glucose may involve changes in the activity of glibenclamide-sensitive K⁺_ATP channels.

D-Glucose (24 hours) also increases eNOS expression⁴ and activity² in HUVECs. In the present study, eNOS activity was increased in HUVECs exposed for 1 to 5 minutes to high D-glucose, an effect associated with increased phosphorylation of eNOS at Ser¹¹⁷⁷, a residue known to be associated with eNOS activation.³⁷ The rapid eNOS stimulatory effect of D-glucose was not further increased by longer incubations periods with D-glucose, which could be due to a maximal and sustained activation of eNOS acutely induced by elevated D-glucose. Because D-glucose did not alter basal intracellular Ca²⁺, it is likely that rapid eNOS activation by D-glucose is Ca²⁺ independent, supporting recent observations of Ca²⁺-independent eNOS activation in HUVECs.^37,38 D-Glucose–induced phosphorylation of eNOS at Ser¹¹⁷⁷, L-arginine transport, and TPP⁺ influx were blocked by wortmannin, suggesting that the PI3-k pathway could be involved in the effects of D-glucose. L-Arginine transport could be determinant for eNOS activity¹¹; however, the possibility that the D-glucose–induced increase of L-citrulline production was due to elevated L-arginine transport seems unlikely, inasmuch as D-glucose–induced NO synthesis was unaltered in the absence of extracellular L-arginine.

L-NAME blocked D-glucose–increased L-arginine transport and NO synthesis in HUVECs. This inhibitor does not alter basal L-arginine transport in the endothelium^2–4,39; thus, NO most likely mediates changes in L-arginine transport, as suggested in bovine aortic endothelium.⁴⁰ This result is similar to that found in HUVECs from patients with gestational diabetes; L-arginine transport in these cells is increased concomitantly with membrane hyperpolarization and NO synthesis, and this increase is inhibited by blocking NO synthase.² NO causes membrane hyperpolarization in the endothelium,^3,36 and NO (from SNAP) has been shown to cause comparable increases in TPP⁺ influx and L-arginine transport and to cause membrane hyperpolarization to those caused by D-glucose, although SNAP treatment did not further enhance the effects of high D-glucose, in HUVECs. These findings support the hypothesis that NO acutely modulates L-arginine transport by a mechanism that involves membrane hyperpolarization.

NO-altered K⁺ channel activity may occur by both indirect mechanisms via cGMP and direct NO action on channels.³⁶ Our results show that the D-glucose increases in L-arginine transport and TPP⁺ influx were mimicked by dbcGMP and blocked by the PKG inhibitor KT-5823. In addition, D-glucose–induced membrane hyperpolarization was also blocked by KT-5823. Thus, modulation of ion channel activity (and hence, L-arginine transport) could be due to the activation of PKG downstream from NO synthesis.

PKC activity is increased in subjects with diabetes mellitus or in endothelium chronically exposed to high D-glucose.^12,16,17 Activation of diacylglycerol/phorbol ester–sensitive PKC isozymes activates eNOS and the NO-dependent increased p42/p44^mapk phosphorylation in HUVECs exposed for 24 hours to high D-glucose.⁵ However, 25 mmol/L D-glucose for 1 to 5 minutes did not alter PKC activity in this cell type, suggesting that the rapid D-glucose effect on L-arginine transport was PKC independent. Because D-glucose induces a rapid (2-minute) p42/p44^mapk phosphorylation and because inhibition of the p42/p44^mapk phosphorylation by PD-98059 also inhibits the D-glucose increase in TPP⁺ influx and L-arginine transport, it is likely that p42/p44^mapk activation is involved in this pathway. Activation of p42/44^mapk requires PI3-k activity in HUVECs.⁴¹ Our results show that D-glucose–induced p42/44^mapk phosphorylation is blocked by wortmannin, suggesting that the D-glucose effect requires PI3-k activity in HUVECs. SNAP-increased p42/p44^mapk phosphorylation and L-arginine transport were blocked by PD-98059, complementing results showing that NO, via cGMP, causes rapid p42/p44^mapk phosphorylation in the endothelium.^4,42 Because D-glucose–induced and NO-induced membrane hyperpolarization are blocked by PD-98059, p42/p44^mapk activation could modulate ion channel activity and L-arginine transport in HUVECs.

Elevated D-glucose leads to overproduction of oxygen-derived free radicals in several cell types.^11,16,17 We found that SOD activity in HUVECs was unaltered by 25 mmol/L D-glucose and that SOD or -tocopherol did not block the effects of D-glucose, suggesting that short-term incubation with elevated D-glucose would not generate enough oxygen-derived free radicals to induce changes in the L-arginine/NO pathway in HUVECs.

The present study has established that high D-glucose rapidly activates the L-arginine/NO pathway in HUVECs. The effect of D-glucose involves PI3-k–dependent, but PKC-independent and intracellular Ca²⁺–independent, eNOS and p42/44^mapk activation. These results complement previous observations in animal models in which elevated plasma D-glucose results in a rapid (seconds to minutes) vasodilatation^8–10 and observations of D-glucose–induced vasodilatation in humans.⁷ Local NO synthesis could be one mechanism by which rapid alterations in plasma D-glucose result in vasodilatation and may have important implications in diabetic patients, in whom plasma D-glucose concentrations may change rapidly.^11,12,16,17

	Acknowledgments

 
This study was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT 1000354 and 7000354), Universidad de Concepción (DIUC 201.084.003-1) (Chile), and The Wellcome Trust (United Kingdom). P. Casanello, J. Parodi, and C. Aguayo hold Beca Docente Universidad de Concepción and CONICYT-PhD (Chile) fellowships. We thank the midwives of the Hospital Regional-Concepción (Chile) labor ward for the supply of umbilical cords and Isabel Jara for secretarial assistance.

Received July 26, 2002; revision received October 31, 2002; accepted November 11, 2002.

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