1. Rapid Stimulation of L-Arginine Transport by D-Glucose
Involves p42/44mapk 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
Abstract—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 (Vmax 6.1 0.7 versus 4.4 0.1 pmol/ g
protein per minute) with no change in transport affinity (Km 105 9 versus 111 16 mol/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/p44mapk and Ser1177– endothelial NO
synthase phosphorylation, inhibited by NG-nitro-L-arginine methyl ester (L-NAME; NO synthesis inhibitor), gliben-
clamide (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 p42/44mapk. 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. (Circ Res. 2003;92:64-72.)
Key Words: humans endothelium glucose arginine nitric oxide
tivated protein (MAP) kinases (p42/44mapk).5,14,15 p42/44mapk
T he 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
activation may itself be dependent on PKC activation and NO
synthesis.5,14 However, the effect of short-term incubation
high-affinity (Km 100 to 400 mol/L) systems y /CAT-1 with elevated D-glucose on the endothelial L-arginine/NO
and y /CAT-2B (where CAT indicates cationic amino acid pathway has not been investigated.4,11,16,17
transporter) in human umbilical vein endothelial cells The present study shows that a 2-minute incubation with
(HUVECs).2,3 L-Arginine transport and NO synthesis (L- 25 mmol/L D-glucose increases L-arginine transport and NO
arginine/NO pathway) are increased in HUVECs from pa- synthesis in HUVECs. The underlying cellular mechanisms
tients with gestational diabetes.2 Interestingly, long-term involve phosphorylation of eNOS at Ser1177 via phosphatidyl-
incubation (24 hours) of HUVECs from normal pregnancies inositol 3-kinase (PI3-k) and activation of eNOS and p42/
with elevated D-glucose mimics the effect of gestational
p44mapk by D-glucose.18
diabetes on the L-arginine/NO pathway.4 In addition, elevated
D-glucose for 24 hours4,5 or 5 days6 increases eNOS gene
expression. A recent report shows that D-glucose infusion Materials and Methods
induces vasodilatation in humans,7 and in animal models, an
elevation of plasma D-glucose results in rapid (seconds to Cell Culture
minutes) vasodilatation.8 –10 Therefore, rapid fluctuations in Human umbilical vein endothelium was isolated (collagenase diges-
the D-glucose level are crucial in maintaining human fetal tion 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
endothelial function.2–5,11 serum, 10% fetal calf serum, 3.2 mmol/L L-glutamine, 100 mol/L
D-Glucose activates protein kinase C (PKC), an enzyme L-arginine, and 100 U/mL penicillin-streptomycin (primary culture
involved with long-term stimulation of the L-arginine/NO medium).2– 4 Before an experiment (24 hours), the incubation me-
pathway,5,12–14 and (within 1 hour) p42 and p44 mitogen-ac- dium was changed to serum-free medium 199.
Original received July 26, 2002; revision received October 31, 2002; accepted November 11, 2002.
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@udec.cl
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000048197.78764.D6
64

2. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 65
L-Arginine Transport Intracellular Ca2
L-Arginine transport (1 Ci/mL, 37°C, 1 minute) was determined in Cells on glass coverslips were loaded (30 minutes, 23°C) with the
cells preincubated (15 seconds to 5 minutes) with Krebs solution acetoxymethyl derivative of fluo 3 (5 mol/L). Coverslips were
(mmol/L: NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1, HEPES 20, transferred to an experimental bath with Krebs solution containing 5
CaCl2 2.5, and MgCl2 1 [pH 7.4, 37°C]) containing 5 or 25 mmol/L or 25 mmol/L D-glucose, and Ca2 was imaged using a Zeiss LSM
D-glucose, 25 mmol/L L-glucose, or 5 mmol/L D-glucose plus 410 confocal microscope.4
20 mmol/L D-mannitol (osmotic controls).2– 4 L-Arginine transport
was also determined in Krebs solution in which NaCl was replaced Western Blots
by equimolar concentrations of choline chloride2– 4 or in cells After pretreatment with 10 mol/L PD-98059 (30 minutes), 100
incubated (30 minutes) with KCl (5.5 to 131 mmol/L), with NaCl mol/L SNAP (2 to 5 minutes), or 30 nmol/L wortmannin (30
decreased equivalently, or with 131 mmol/L KCl for 2, 4, 10, 20, or minutes), the cells were incubated with 5 or 25 mmol/L D-glucose (2
30 minutes. In trans-stimulation experiments, cells were preincu- minutes). Cell protein extracts were probed with a primary poly-
bated (2 hours) with primary culture medium containing 10 mmol/L clonal mouse antiphosphorylated (1:1000) or nonphosphorylated
L-lysine. Cell-associated radioactivity and data analyses were per-
(1:1500) p44/p42mapk, rabbit anti-eNOS (1:2500) or anti-phosphory-
formed as described.2– 4 lated Ser1177– eNOS (1:2500) antibodies, and horseradish peroxidase–
conjugated goat secondary antibodies as described.3,5 Primary poly-
L-Arginine transport was assayed in cells preincubated (30 min-
clonal mouse anti-actin (1:2000) served as the internal control.
utes) with 100 mol/L NG-nitro-L-arginine methyl ester (L-NAME,
Proteins were detected by enhanced chemiluminescence and quan-
eNOS inhibitor),2,3 10 mol/L PD-98059 (MAP kinase kinase 1/2
tified by densitometry (Ultroscan XL enhanced laser densitometer,
[MEK1/2] inhibitor), 19 100 mol/L S-nitroso-N-acetyl- L , D - LKB Instruments).3,5
penicillamine (SNAP, NO donor), 1 mol/L KT-5823 (protein
kinase G [PKG] inhibitor),20 100 nmol/L calphostin C (PKC inhib-
Semiquantitative PCR
itor),21 10 mol/L glibenclamide (ATP-sensitive K [K ATP] channel
Extracted mRNA (Dynal) was reversed-transcribed into cDNA using
blocker),22 1 mol/L levcromakalim (K ATP activator),23 or 30
oligo(dT18) plus random hexamers (10-mer) and M-MLV reverse
nmol/L wortmannin (PI3-k inhibitor).24 transcriptase (Promega) for 1 hour at 37°C.3 Polymerase chain
reactions (PCRs) were performed in 20- L samples (2 L of 10
Membrane Potential PCR buffer, 0.8 L of 50 mmol/L Mg2 , 0.4 L dNTPs, 13.6 L
[3H]Tetraphenylphosphonium ([3H]TPP ) influx (46 nmol/L, 0.5 RNase-free H2O, 0.2 L Taq DNA polymerase, and 0.5 mol/L
Ci/mL, 15 to 120 seconds), a membrane potential–sensitive sequence-specific oligonucleotide primers for human CAT-1, CAT-
probe,2,3,25 was determined in Krebs solution containing 5 or 2A, or CAT-2B). Samples were incubated (4 minutes, 95°C),
25 mmol/L D-glucose, 100 mol/L L-arginine, and 5.5 or followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 57°C, 30
131 mmol/L KCl.2,3 Resting membrane potential (Em, whole-cell seconds at 72°C, and a final extension for 7 minutes at 72°C. -Actin
patch clamp) was recorded using an EPC-7 amplifier (List Medical) expression was used as a reference value. Reverse transcription
as described.2 Em was measured for at least 1 minute in the presence (RT)-PCR products were sequenced in both directions by Taq
of 5 or 25 mmol/L D-glucose. Only recordings with 0.1-mV dideoxy terminator cycle sequencing (automated DNA sequencer
variations were considered. Em was also determined in cells prein- 373A, Applied Biosystems).3
cubated for 2 hours with 10 mmol/L L-lysine. Oligonucleotide primers were as follows: hCAT-1 (sense)
5 -CCAGTACTTCCGACGAGTTAGA-3 , hCAT-1 (antisense)
5 -CATCCACACAGCAAACCGGACC-3 , hCAT-2A (sense) 5 -
PKC Activity TATCCCGATTTTTTTGCTGTGTGC-3 , hCAT-2A (antisense)
PKC activity (32P incorporation from [ -32P]ATP into a synthetic 5 -TGCAGTCAACGTGGCAGCAACT-3 , hCAT-2B (sense) 5 -
PKC substrate peptide analogue5) was determined in cells exposed to TCCCAATGCCTCGTGTAATCTA-3 , hCAT-2B (antisense)
5 or 25 mmol/L D-glucose (2 minutes) after pretreatment with 100 5 -GCATGCTGAAGCCCTGTCTCTGC-3 , -actin (sense) 5 -
nmol/L phorbol 12-myristate 13-acetate (5 minutes, PKC activator), AACCGCGAGAAGATGACCCAGATCATCTTT-3 , and -actin
100 nmol/L 4 -phorbol 12,13-didecanoate (5 minutes, less active (antisense) 5 -AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3 .
phorbol 12-myristate 13-acetate analogue), 100 nmol/L calphostin C Expected size products were as follows: hCAT-1, 450 bp; hCAT-2A,
(30 minutes), 10 mol/L PD-98059 (30 minutes), 100 mol/L 690 bp; hCAT-2B, 360 bp; and -actin, 350 bp.
SNAP (5 minutes), 100 mol/L L-NAME (30 minutes), or 30
nmol/L wortmannin (30 minutes). SOD Activity and -Tocopherol Experiments
Cells were homogenized in buffer containing 50 mmol/L Tris-(hy-
cGMP Determination droxymethyl)-aminomethane, 100 mmol/L potassium chloride,
Cells preincubated (30 minutes) in Krebs solution (37°C) containing 0.02% Triton X-100, 100 mmol/L sodium pyrophosphate, and
L -arginine (100 mol/L) and 3-isobutyl-1-methylxanthine 100 mmol/L sodium fluoride (pH 7.4), which was supplemented with
(0.5 mmol/L, phosphodiesterase inhibitor),2,3 in the absence or trypsin inhibitors (4 mg/mL aprotinin, 1 mg/mL benzamidine, 5
presence of L-NAME (100 mol/L), were exposed to 5 or g/mL leupeptin, and 200 mol/L sodium orthovanadate). Aliquots
25 mmol/L D-glucose for the last 2 minutes of the 30-minute (1 mg protein/mL) were incubated (25°C, 2 minutes) with potassium
phosphate buffer (50 mmol/L, pH 10.2) containing adrenochrome
incubation period with 3-isobutyl-1-methylxanthine. cGMP was
(200 mol/L) and epinephrine (10 mol/L), and absorbance was
determined in HCl-cell extracts by radioimmunoassay.2,3
measured at 480 nm. Superoxide dismutase (SOD) activity was
calculated from the inhibition curve for epinephrine auto-oxidation
L-Citrulline Assay versus protein concentration. Basal absorbance (100% activity) was
Cells were incubated with L-[3H]arginine (100 mol/L, 4 Ci/mL, the reaction in the absence of cell extracts.26 Cells were also
30 minutes, 37°C) in the absence or presence of L-NAME (100 preincubated (30 minutes) with -tocopherol (500 g/mL, 1%
mol/L). Cells were exposed to 5 or 25 mmol/L D-glucose for 30 ethanol).27
minutes or for the last 2, 4, 10, or 20 minutes of this incubation
period. Some experiments were performed in cells exposed only to Materials
L-[3H]arginine (4 Ci/mL) and 5 or 25 mmol/L D-glucose. Formic Sera, agarose, and buffers were from GIBCO Life Technologies.
acid– digested cells (200 L) were passed through a sodium ion form Collagenase type II (Clostridium histolyticum) was from Boehring-
of the cation ion-exchange resin Dowex 50W (50X8-200), and er-Mannheim, and Bradford protein reagent was from Bio-Rad
L-[3H]citrulline concentration was determined in the H2O eluate.3 Laboratories. L-NAME and SNAP were from Calbiochem. Ethidium

3. 66 Circulation Research January 10/24, 2003
bromide, Dowex (50WX8-400), and all other reagents were from
Sigma Chemical Co. L-[2,3-3H]Arginine (36.1 Ci/mmol), D-[1-
14
C]mannitol (49.3 mCi/mmol), [ -32P]ATP, and [3H]TPP (37
Ci/mmol) were from NEN. 3 ,5 -cGMP-TME was from ICN. Anti-
bodies 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
L-Arginine Transport
Elevated D-glucose (2 minutes), but not L-glucose or
D-mannitol, stimulated L-arginine transport (half-maximal
effect [K1/2] 13 2 mmol/L D-glucose) (Figure 1A). Basal
transport rates increased significantly after 30 seconds of
exposure to elevated D-glucose (K1/2 25 5 seconds), with
maximal rates achieved within 1 minute and sustained over 5
minutes (Figure 1B). Subsequent experiments were per-
formed using 25 mmol/L D-glucose for 2 minutes. D-Glu-
cose–stimulated L-arginine transport decreased to basal val-
ues 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).
Elevated D-glucose had no effect on the nonsaturable
component (KD) of overall L-arginine transport but increased
Vmax, with no change in apparent Km (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).
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
Figure 1. Effect of D-glucose on L-arginine transport and CAT
and changes in TPP influx and Em (Table 2). Levcromakalim expression. A, L-Arginine transport (100 mol/L, 1 minute, 37°C)
(K ATP channel activator) hyperpolarized the plasma mem- in HUVECs incubated (2 minutes) with increasing concentrations
brane and increased L-arginine transport and TPP influx only of D-glucose ( ), L-glucose ( ), or 5 mmol/L D-glucose plus 5 to
in 5 mmol/L D-glucose; effects were blocked by gliben- 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
clamide (Table 2). The effects of D-glucose were also blocked and D-glucose D-mannitol. B, Time course of effect of
by wortmannin (not shown). Preloading cells with L-lysine D-glucose on L-arginine transport (as in panel A) in cells incu-
did not alter Em ( 66.1 0.3 mV, P 0.05; n 29 cells) bated 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 (▫).
compared with control cells ( 67.5 0.5 mV, P 0.05; n 45 High D-glucose– containing Krebs solution was replaced (arrows)
cells). L-Arginine transport was inhibited by KCl with half- by 5 mmol/L D-glucose, and cells were incubated for 10 min-
maximal inhibition at 12 2 and 17 4 mmol/L KCl for 5 and utes. *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.
25 mmol/L D-glucose, respectively. The time needed to mRNA was reversed-transcribed into cDNA, and PCR was per-
induce half-maximal inhibition of transport with 131 mmol/L formed for human CAT-1 (lane 2, 449 bp), CAT-2A (lane 3, 690
KCl was similar in cells in 5 mmol/L D-glucose (6.5 0.6 bp), or CAT-2B (lane 4, 357 bp). Lane 5 is -actin (350 bp), and
minutes) compared with 25 mmol/L D-glucose (7.2 0.6 lane 1 is DNA ladder (100 to 2000 bp). Data are representative
of 14 cell cultures.
minutes).

4. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 67
lation at Ser1177, cGMP, and L-[3H]citrulline formation. Sim-
ilar 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). Intracel-
lular 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).
L-NAME also inhibited the effect of D-glucose on
L-arginine transport Vmax (Table 1), TPP influx, and Em
(Table 2); however, L-NAME did not alter TPP influx or Em
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).
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 inhibi-
tor) 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
Figure 2. Effect of D-glucose on kinetic parameters and trans- minutes) increased membrane PKC activity (not shown),
stimulation of L-arginine transport. A, Saturable L-arginine trans- confirming our previous observations in HUVECs.5 In con-
port (1 minute, 37°C) in HUVECs incubated (2 minutes) in trast, high D-glucose induced p42/p44mapk phosphorylation
5 mmol/L D-glucose ( ) or 25 mmol/L D-glucose ( ). B,
L-Arginine transport (100 mol/L, 1 minute, 37°C) in cells prein-
(Figure 6A), an effect blocked by PD-98059 and mimicked
cubated (2 hours) in medium 199 in the absence (control) or by brief exposure (2 minutes) to SNAP (Figure 6B). Inter-
presence of 10 mmol/L L-lysine. Transport assays were per- estingly, the effect of D-glucose on p42/44mapk phosphoryla-
formed in cells exposed to 5 mmol/L D-glucose (open bars) or tion was blocked by wortmannin (Figure 6C). PD-98059 also
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 blocked the stimulatory effects of elevated D-glucose and
(n 16). *P 0.04 vs all other values. SNAP on TPP influx, L-arginine transport, and Em (Table 2).
NO Involvement SOD Activity and -Tocopherol Effect
Elevated D-glucose increased eNOS phosphorylation at SOD activity in cells in 5 mmol/L D-glucose (5.5 0.6 U/mL)
Ser1177 (Figure 3A), L-[3H]citrulline (Figure 3B), and cGMP was not significantly altered (P 0.05, n 5) by 25 mmol/L
accumulation (Figure 3C). L-NAME inhibited the effect of D-glucose (6 1 U/mL). Extracellular SOD or -tocopherol
3
D-glucose on cGMP and L-[ H]citrulline formation but did not did not block (P 0.05, n 4 to 8) the effect of D-glucose on
alter eNOS-Ser1177 phosphorylation (not shown). However, L-arginine transport (5.6 0.3 and 5.8 0.6 pmol/ g protein
wortmannin inhibited D-glucose–induced eNOS phosphory- per minute, respectively), L-citrulline formation (3.1 0.2 and
TABLE 1. D-Glucose Effect on L-Arginine Transport in HUVECs
Vmax /Km, pmol/ g KD, pmol/ g
Vmax, pmol/ g Protein per Minute Protein per Minute
Km, mol/L Protein per Minute per mol/L per mol/L
5 mmol/L D-Glucose 105 9 4.4 0.1 0.044 0.011 0.002 0.0007
PD-98059 123 12 4.2 0.2 0.034 0.012 0.003 0.0012
L-NAME 97 16 3.8 0.3 0.039 0.016 0.003 0.0009
25 mmol/L D-Glucose 111 16 6.1 0.7* 0.055 0.012* 0.003 0.0011
PD-98059 133 22 3.7 0.3† 0.028 0.009† 0.004 0.0012
L-NAME 125 22 3.6 0.5† 0.029 0.007† 0.003 0.0008
L-Arginine transport (100 mol/L, 37°C) in cells preincubated (30 minutes) with 5 mmol/L D-glucose, in absence
or presence of 100 mol/L PD-98059 or 100 mol/L L-NAME, and exposed (2 minutes) to Krebs containing 5 or
25 mmol/L D-glucose. Values are mean SEM, n 6.
*P 0.05, †P 0.05 vs 5 and 25 mmol/L D-glucose, respectively.

5. 68 Circulation Research January 10/24, 2003
TABLE 2. D-Glucose Effect on L-Arginine Transport, [3H]TPP Influx, and Em in HUVECs
L-Arginine Transport, pmol/ g [3H]TPP Influx, pmol/mg
Protein per Minute Protein per Minute Em, mV
5 mmol/L D-Glucose
Control 1.8 0.3 2.1 0.3 67.2 0.5
KCl 0.2 0.1* 0.1 0.04* 8.1 0.5*
Glibenclamide 1.3 0.3 1.5 0.3 63.9 0.6
Levcromakalim 4.6 0.3* 5.6 0.3* 74.2 0.3*
Levcromakalim glibenclamide 1.6 0.2† 1.4 0.6† 64.5 0.2†
PD-98059 2.1 0.1 1.7 0.2 64.2 0.5
L-NAME 1.6 0.1 1.5 0.3 65.1 0.3
SNAP 5.1 0.3* 3.9 0.4* 76.2 1.0*
SNAP PD-98059 2.1 0.3† 1.9 0.5† 65.3 1.0†
KT-5823 1.9 0.5 1.9 0.3 66.8 0.9
25 mmol/L D-Glucose
Control 4.0 0.5* 3.7 0.3* 77.1 0.2*
KCl 0.3 0.2*‡ 0.3 0.03*‡ 6.2 0.5*‡
Glibenclamide 2.3 0.4‡ 2.4 0.4‡ 62.7 0.5‡
Levcromakalim 4.3 0.4* 4.7 0.2* 75.1 0.3*
Levcromakalim glibenclamide 2.2 0.3†‡ 1.8 0.3†‡ 61.9 0.3†‡
PD-98059 0.9 0.3‡ 1.7 0.1‡ 69.9 1.0‡
L-NAME 1.4 0.1‡ 1.8 0.2‡ 65.7 0.7‡
SNAP 4.1 0.4* 3.2 0.2* 78.2 1.0*
SNAP PD-98059 1.1 0.5†‡ 1.9 0.2†‡ 65.7 1.0†‡
KT-5823 1.9 0.5‡ 1.9 0.3‡ 65.3 0.5‡
L-Arginine transport (100 mol/L), 3H TPP influx (46 nmol/L), and Em (whole-cell patch clamp) in cells
preincubated (30 minutes) with 5 mmol/L D-glucose and 5.5 mmol/L (control) or 131 mmol/L KCl. Cells in 5.5 mmol/L
KCl were preincubated with 10 mol/L glibenclamide (5 minutes), 1 mol/L levcromakalim (5 minutes), 10 mol/L
PD-98059 (30 minutes), 100 mol/L L-NAME (30 minutes), 10 mol/L KT-5823 (30 minutes), or 100 mol/L SNAP
(2 minutes) and exposed (2 minutes) to 5 or 25 mmol/L D-glucose. Values are mean SEM, n 17.
*P 0.05 vs control in 5 mmol/L D-glucose; †P 0.05 vs corresponding values in SNAP or levcromakalim; ‡P 0.05
vs control in 25 mmol/L D-glucose.
2.8 0.4 pmol/ g protein per 30 minutes, respectively), TPP confirmed here, is Na independent and inhibited by mem-
influx (4.4 0.2 and 4.1 0.3 pmol/mg protein per minute, brane depolarization.2,3,32 Because of their similar kinetic
respectively), or changes in Em ( 76 0.3 and 78 0.3 mV, properties, CAT-1 and CAT-2B are hard to distinguish at the
respectively). functional level.31 Our results show that both high-affinity
hCAT-1 (Km 100 to 200 mol/L) and hCAT-2B (Km 200
Discussion to 400 mol/L), but not the low-affinity hCAT-2A trans-
The present study establishes that D-glucose induces a rapid porter (Km 2 to 5 mmol/L), are present in HUVECs,
concentration-dependent stimulation of L-arginine transport confirming previous reports.3,33 CAT-1 is more sensitive than
in HUVECs. This effect requires NO synthesis associated CAT-2B to trans-stimulation by cationic amino acids.3,31,34
with increased phosphorylation of eNOS at Ser1177 and acti- When we preloaded HUVECs with L-lysine, L-arginine trans-
vation of p42/p44mapk and PI3-k, and it is independent of PKC port was increased by 7-fold in 5 mmol/L D-glucose.
and intracellular Ca2 changes. These findings provide the However, the L-lysine trans-stimulatory effect was less ef-
first evidence that short-term hyperglycemia activates the fective ( 3-fold) in cells exposed for 2 minutes to 25 mmol/L
L -arginine/NO signaling pathway in human fetal D-glucose. Because L-arginine transport is trans-stimulated
endothelium. by 9.8-fold or 1.8-fold in Xenopus oocytes injected with
L-Arginine transport is mediated by systems y /CATs,2,3,12 hCAT-1 or hCAT-2B mRNA, respectively, 34 trans-
y L,29,30 and b0, 12 in HUVECs, with the first likely predom- stimulation in HUVECs in 5 mmol/L D-glucose may be
inating at the physiological concentration of extracellular preferentially mediated by hCAT-1. The reduced trans-
L-arginine. The cDNAs for four potential human y trans- stimulation of transport in high D-glucose may result from a
porters (hCAT-1, hCAT-2B, hCAT-2A, and hCAT-4) have state of maximal activity of L-arginine transporters already
been sequenced.31 L-Arginine transport in HUVECs occurs induced by L-lysine; therefore, high D-glucose could not
with relatively high affinity (Km 80 to 100 mol/L) and, as further increase L-arginine transport. In addition, the possi-

6. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 69
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 mol/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.
Vmax for L-arginine transport without altering the apparent Km.
As noted above, L-arginine transport is sensitive to changes in
extracellular K and Em. Because high D-glucose induced
membrane hyperpolarization, stimulation of L-arginine trans-
port could result from changes in Em. The stimulatory effect
Figure 3. Effect of D-glucose on eNOS activity. A, Immunoblot of D-glucose on TPP influx and L-arginine transport was
for total eNOS and phosphorylated eNOS at Ser1177 (eNOS P- blocked by glibenclamide, a K ATP channel blocker. In addi-
Ser1177) in HUVECs preincubated (30 minutes) with 5 mmol/L
D-glucose in the absence or presence of wortmannin and then tion, levcromakalim (K ATP activator)23 mimics D-glucose–
exposed (2 minutes) to 5 or 25 mmol/L D-glucose (see Materials induced changes in Em, L-arginine transport, and TPP influx.
and Methods). Top panel shows densitometry ratios. Data are K ATP channels are expressed in the endothelium35 and are
representative of 6 cell cultures. B, L-[3H]Citrulline formation
from L-[3H]arginine (4 Ci/mL, 37°C) in cells preincubated (30 activated by D-glucose36; thus, the effects of D-glucose may
minutes) with Krebs solution in the absence (control) or pres- involve changes in the activity of glibenclamide-sensitive
ence of L-NAME and exposed for the last 2 minutes to Krebs K ATP channels.
solution containing 5 mmol/L D-glucose (open bars) or
D-Glucose (24 hours) also increases eNOS expression4 and
25 mmol/L D-glucose (filled bars). C, Intracellular cGMP under
same experimental conditions as in panel B. Values are activity2 in HUVECs. In the present study, eNOS activity was
mean SEM (n 17). *P 0.05 vs all other values. increased in HUVECs exposed for 1 to 5 minutes to high
D-glucose, an effect associated with increased phosphoryla-
bility that L-lysine–stimulated L-arginine transport was due to tion of eNOS at Ser1177, a residue known to be associated with
membrane hyperpolarization is unlikely because Em was eNOS activation.37 The rapid eNOS stimulatory effect of
unaltered in L-lysine–preloaded cells. D-glucose was not further increased by longer incubations
Long-term incubation (24 hours) of HUVECs with high periods with D-glucose, which could be due to a maximal and
D-glucose increases Vmax for L-arginine transport.4 The pres- sustained activation of eNOS acutely induced by elevated
ent study shows that acute (2-minute) D-glucose increases D-glucose. Because D-glucose did not alter basal intracellular

7. 70 Circulation Research January 10/24, 2003
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 incu-
bated (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 mol/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.
Ca2 , it is likely that rapid eNOS activation by D-glucose is
Ca2 independent, supporting recent observations of Ca2 - Figure 6. NO and PI3-k involvement in effect of D-glucose on
independent eNOS activation in HUVECs.37,38 D-Glucose– p42/44mapk phosphorylation. A, Immunoblot for phosphorylated
p44mapk (p44 P) and p42mapk (p42 P) and nonphosphorylated
induced phosphorylation of eNOS at Ser1177, L-arginine trans- p44mapk (p44) or p42mapk (p42) in HUVECs preincubated (30 min-
port, and TPP influx were blocked by wortmannin, utes) with 5 mmol/L D-glucose in the absence or presence of
suggesting that the PI3-k pathway could be involved in the 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
effects of D-glucose. L-Arginine transport could be determi-
5 mmol/L D-glucose. C, Effect of wortmannin on p42/p42mapk in
nant for eNOS activity11; however, the possibility that the HUVECs (as in panel A). Data are representative of similar
D-glucose–induced increase of L-citrulline production was results in 17 cell cultures.
due to elevated L-arginine transport seems unlikely, inasmuch
as D-glucose–induced NO synthesis was unaltered in the synthase.2 NO causes membrane hyperpolarization in the
absence of extracellular L-arginine. endothelium,3,36 and NO (from SNAP) has been shown to
L-NAME blocked D-glucose–increased L-arginine trans- cause comparable increases in TPP influx and L-arginine
port and NO synthesis in HUVECs. This inhibitor does not transport and to cause membrane hyperpolarization to those
alter basal L-arginine transport in the endothelium2– 4,39; thus, caused by D-glucose, although SNAP treatment did not
NO most likely mediates changes in L-arginine transport, as further enhance the effects of high D-glucose, in HUVECs.
suggested in bovine aortic endothelium.40 This result is These findings support the hypothesis that NO acutely mod-
similar to that found in HUVECs from patients with gesta- ulates L-arginine transport by a mechanism that involves
tional diabetes; L-arginine transport in these cells is increased membrane hyperpolarization.
concomitantly with membrane hyperpolarization and NO NO-altered K channel activity may occur by both indirect
synthesis, and this increase is inhibited by blocking NO mechanisms via cGMP and direct NO action on channels.36

8. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 71
Our results show that the D-glucose increases in L-arginine fellowships. We thank the midwives of the Hospital Regional-
transport and TPP influx were mimicked by dbcGMP and Concepción (Chile) labor ward for the supply of umbilical cords and
Isabel Jara for secretarial assistance.
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
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