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Pflugers Arch - Eur J Physiol (2004) 448: 383394

DOI 10.1007/s00424-004-1261-x

CELL AN D MOL ECULA R PHY SIOLO GY

Marcelo Gonzlez . Carlos Flores . Jeremy D. Pearson .


Paola Casanello . Luis Sobrevia

Cell signalling-mediating insulin increase of mRNA expression


for cationic amino acid transporters-1 and -2 and membrane
hyperpolarization in human umbilical vein endothelial cells
Received: 20 December 2003 / Accepted: 24 February 2004 / Published online: 3 April 2004
# Springer-Verlag 2004

Abstract Insulin induces vasodilatation in human subjects and increases L-arginine transport and NO synthesis
in human umbilical vein endothelial cells (HUVEC). Cell
signalling events associated with insulin effects on activity
and mRNA expression of the human cationic amino acid
transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown.
L-Arginine transport and eNOS activity were determined
in HUVEC exposed to insulin. mRNA levels for hCAT-1,
hCAT-2B and eNOS were quantitated by real time RTPCR and endothelial NO synthase (eNOS) protein was
identified by Western blot analysis. Intracellular Ca2+, Larginine and L-citrulline levels, L-[3H]citrulline formation
from L-[3H]arginine, cGMP formation, nitrite level, ATP
release and membrane potential were determined. Insulin
increased L-arginine transport and the mRNA levels for
hCAT-1 and hCAT-2B and eNOS expression and activity.
Insulin also induced membrane hyperpolarization and
increased intracellular Ca2+, L-[3H]citrulline, cGMP and
nitrite formation. Insulin-mediated stimulation of the Larginine/NO pathway is thus associated with increased
M. Gonzlez . C. Flores . P. Casanello . L. Sobrevia (*)
Cellular and Molecular Physiology Laboratory (CMPL),
Department of Obstetrics and Gynaecology, Medical Research
Centre (CIM), School of Medicine, Faculty of Medicine,
Pontificia Universidad Catlica de Chile,
P.O. Box 114-D Santiago, Chile
e-mail: sobrevia@med.puc.cl
Fax: +56-2-6321924
J. D. Pearson
Centre for Cardiovascular Biology and Medicine, Kings
College London, Guys Campus,
London, SE1 1UL, UK
P. Casanello
Department of Pathophysiology, Program of Physiology,
Biomedical Sciences Institute (ICBM), Faculty of Medicine,
Universidad de Chile,
Santiago, Chile
Present address:
C. Flores
Universidad Austral de Chile,
Valdivia, Chile

hCAT-1 and hCAT-2B mRNA, and eNOS expression, via


mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have
characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human
subjects, and which could have implications in patients
in whom plasma insulin levels are altered, such as in
diabetes mellitus.
Keywords Human . Insulin . Endothelium . Glucose .
Umbilical . Vein . Arginine . Nitric oxide . Diabetes

Introduction
The biological effects of insulin, including endotheliumdependent modulation of vascular tone [4, 46], are
mediated by activation of phosphatidylinositol-3-kinase
(PI3-K), protein kinases C (PKC) and B (PKB/Akt) and
Ras/Raf/mitogen-activated protein kinase (MAPK) signalling pathways in several cell types [31, 46, 48]. Insulin
stimulates the synthesis and release of the potent vasodilator NO [39, 40, 45, 47], an effect involving activation of
PKB/Akt [13, 52], PI3-K [13, 51] and MAPK [27, 31]
pathways in human umbilical vein endothelial cells
(HUVEC). Insulin also stimulates membrane transport of
the cationic amino acid L-arginine, required for NO
synthesis in HUVEC [45]. NO is derived from L-arginine
metabolism catalysed by the Ca2+/calmodulin-sensitive
endothelial NO synthase (eNOS) [1] in this cell type [45].
In HUVEC, L-arginine is taken up primarily by the Na+independent, high-affinity (Km 100400 M) systems y+/
hCAT-1 and y+/hCAT-2B (human cationic amino acid
transporter) [3, 10] and, to a lesser extent, via system y+L
(Km 140 M) [2, 38, 42]. Insulin increases the activity of
system y+/CATs in HUVEC [45], rat pancreas [29], gastric
mucosa [6] and CAT-1 expression in rat liver cells [49] and
coronary myocytes [43]. However, there are no reports on
the cell signalling mechanisms involved in the modulation
of L-arginine transport and hCAT-1 and hCAT-2B expres-

384

sion in response to insulin in human endothelium [26, 46].


We therefore determined whether insulin-stimulation of Larginine transport is associated with changes in the hCAT1 and hCAT-2B mRNA expression, and examined the
signalling pathways involved in the modulation of mRNA
expression and activity of L-arginine transporters by
insulin in HUVEC.
Our results demonstrate that stimulation of L-arginine
transport by physiological concentrations of human insulin
is associated with higher hCAT-1 and hCAT-2B mRNA
expression in HUVEC. The underlying cellular mechanisms require activation of PI3-k, PKC, eNOS and p42/
p44mapk.

Materials and methods


Cell culture
Human umbilical vein endothelial cells were isolated by digestion
with collagenase (0.25 mg ml1, 8 min) and cultured (37 C, 5%
CO2, passage 2) in medium 199 (M199) containing 5 mM Dglucose, 10% new-born and 10% foetal calf serum, 3.2 mM Lglutamine, 100 M L-arginine and 100 i.u. ml1 penicillin-streptomycin (primary culture medium). Prior to an experiment (24 h) the
incubation medium was changed to 1% serum in M199 [3, 10, 37].

L-Arginine

transport

For transport assays endothelial cells were pre-incubated (010 h,


37 C) in M199 or M199 containing insulin (0.001100 nM). Cells
were then rinsed twice with Krebs solution (in mM: NaCl 131, KCl
5.6, NaHCO3 25, NaH2PO4 1, HEPES 20, CaCl2 2.5 and MgCl2 1;
pH 7.4, 37 C) supplemented with 100 M L-arginine. L-Arginine
transport (1 Ci ml1, 37 C, 1 min) was measured in Krebs
solution in absence or presence of insulin [45]. L-Arginine transport
was also measured in Krebs in which NaCl was replaced
(equimolarly) by choline chloride [3, 10], or in cells incubated
(30 min) with KCl (5.5131 mM), with NaCl decreased equivalently. Trans-stimulation experiments were performed in cells preincubated (8 h, 37 C) in M199 or M199 containing 0.1 nM insulin
and then exposed (2 h) to Krebs solution containing 10 mM Llysine, in the absence or presence of insulin [3, 10, 37, 45].
L-Arginine transport was assayed in cells co-incubated (8 h) with
30 nM wortmannin (PI3-K inhibitor) [16], 100 M NG-nitro-Larginine methylester (L-NAME, eNOS inhibitor) [3, 45], 10 M PD98059 (MAPK kinase 1/2 [MEK1/2] inhibitor) [20], 100 M Snitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), 100 nM
calphostin C (PKC inhibitor) [19], 10 M glibenclamide (ATPsensitive K+ [K+ATP] channel blocker) [14] or 1 M levcromakalim
(K+ATP channel activator) [18].

PKC activity
PKC activity was determined in membrane and cytosolic fractions
by measuring 32P incorporation from [-32P]ATP into a synthetic
PKC substrate peptide analogue, corresponding to a fragment of
glycogen synthase (GS) as described elsewhere [10, 37]. In brief, the
reaction mixture (100 l) consisted of 25 M GS peptide in 20 mM
TRIS-HCl, 10 mM MgCl2 and 50 M [-32P]ATP (specific activity
495 cpm pmol1), plus 0.5 mM EGTA or 0.5 mM CaCl2, 60 g ml1
phosphatidylserine and 3 g ml1 diolein (pH 7.4, 30 C, 10 min).
Reactions were started by addition of [-32P]ATP and stopped by
spotting a 40-l aliquot of the reaction mixture onto Whatman P-81
phosphocellulose filters (4 cm2) that were then soaked rapidly in
75 mM H3PO4. The filters were washed (3, 20 min) in the same
solution, dried and assayed for radioactivity in scintillation mixture.
PKC activity was calculated as the difference between 32P
incorporated into the GS substrate peptide in presence of CaCl2phosphatidylserine-diolein and that in the presence of EGTA. PKC
activity was determined in cells cultured (8 h) in absence or presence
of insulin (0.1 nM) and calphostin C (100 nM), PD-98059 (10 M),
L-NAME (100 M) or wortmannin (30 nM) [10, 37]. Cells were also
exposed to phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC
activator) or 4-phorbol 12,13-didecanoate (4-PDD, 100 nM, a
less-active PMA analogue) for the last 30-min of the 8-h incubation
period with insulin.

cGMP determination
Cells pre-incubated in Krebs (30 min, 37 C) containing L-arginine
(100 M) and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM, phosphodiesterase inhibitor), in the absence or presence of L-NAME
(100 M), were exposed (8 h) to insulin (0.1 nM). cGMP was
determined in HCl-cell extracts by radioimmunoassay as described
elsewhere [3, 10, 37, 45].

L-Citrulline

assay

Cells cultured in M199 containing 100 M L-arginine were exposed


to insulin (0.1 nM, 8 h) and incubated with L-[3H]arginine
(4 Ci ml1, 37 C, last 30 min of the 8-h incubation period with
insulin), in the absence or presence of L-NAME (100 M). L-[3H]
Citrulline was determined in H2O eluate in a cation ion-exchange
resin Dowex-50W (50X8-200, Na+ form) as described elsewhere [3,
10, 37].

Nitrate and nitrite determination


After nitrate conversion to nitrite in the presence of nitrate reductase
and cofactors, 100 l medium (collected at time 0 or after the 8-h
incubation period with insulin) was mixed with 100 l Griess
reagent (1% sulphanilamide plus 0.1% naphthylethylenediamine,
2% phosphoric acid) and the optical density determined at
540 nm [3].

Membrane potential
[3H]Tetraphenylphosphonium ([3H]TPP+) influx (46 nM,
0.5 Ci ml1, 15120 s, 37 C), was determined in cells incubated
(8 h) with 0.1 nM insulin and 5.5 or 131 mM KCl [39]. Resting
membrane potential (Em) was recorded using an EPC-7 amplifier
(List Medical, Darmstadt, Germany) as described elsewhere [3, 10,
45].

Intracellular Ca2+ determination


Cells on glass cover slips were exposed to insulin (0.1 nM, 8 h),
loaded (30 min, 37 C) with the acetoxymethyl derivative of fluo-3
(5 M) in Krebs containing insulin and Ca2+ and transferred to a
Zeiss LSM 410 confocal microscope [10, 37, 45].

385
avian Moloney murine leukaemia virus-reverse transcriptase (MMLV, Promega, Madison, Wisc., USA).

Western blots
Cells in M199 containing 100 M L-arginine were pre-incubated
(30 min) with PD-98059 (10 M), SNAP (100 M) or wortmannin
(30 nM) and then incubated with insulin (0.1 nM, 8 h) in the
continued presence of these molecules. Cell protein extracts were
probed with primary polyclonal mouse anti-phosphorylated
(1:1,000) or total p42/p44mapk (1:1,500), rabbit anti-eNOS
(1:2,500) or anti-phosphorylated serine1177-eNOS (1:2,500) antibodies and horseradish peroxidase-conjugated goat secondary
antibodies. Primary polyclonal mouse anti-actin (1:2,000) was
used as the internal control. Proteins were detected by enhanced
chemiluminescence and quantitated by densitometry (Ultroscan XL
enhanced laser densitometer, LKB, Bromma, Sweden) [3, 10, 37].

Quantitative real-time RT-PCR analysis

Cells were rinsed twice with Krebs solution and total RNA isolated
using a kit (RNeasy, Qiagen, Crawley, UK) according to
manufacturers instructions. RNA quality and integrity were insured
by gel visualization and spectrophotometric analysis (optical density
ratio OD260/280), quantitated at 260 nm and precipitated to obtain
4 g l1. Aliquots of 1 g total RNA were reversed transcribed into
cDNA using oligo (dT18) plus random hexamers (10-mers) and

Real-time PCR was performed using a LightCycler rapid thermal


cycler system (Roche Diagnostics, Lewes, UK). Reaction volume
was 10 l (0.5 M primers dNTPs, Taq DNA polymerase and
reaction buffer provided in the QuantiTect SYBR Green PCR
Master Mix, Qiagen). All real-time assays included a 95 C
denaturation step for 15 s followed by 20 s annealing at 54 C
(hCAT-1), 53 C (hCAT-2B), 54 C (eNOS) or 52 C (28S) and a
final extension at 72 C for variable times depending on product size
(hCAT-1, 10 s; hCAT-2B, 10 s; eNOS, 17 s; 28S, 10 s).The
fluorescent product was detected at the end of each cycle after an
additional 3-s step to 3 below the product melting temperature
(Tm). Product specificity was confirmed by agarose gel electrophoresis (1.5% v/v) and routinely by melting curve analysis. The
product Tm values were 79.17 C for hCAT-1, 77.84 C for hCAT2B, 86.43 C for eNOS and 82.88 C for 28S.
To prepare standards for real-time quantitative PCR, hCAT-1,
hCAT-2B, eNOS and 28S products were separated by agarose gel
electrophoresis (1.8% v/v) and visualized by staining with ethidium
bromide (0.5 g ml1). Each product was removed from the gel, spin

Table 1 Insulin effect on L-arginine transport, [3H]tetraphenylphosphonium ([3H]TPP+) influx and membrane potential (Em) in
human umbilical vein endothelial cells (HUVEC). L-Arginine
transport (100 M), [3H]TPP+influx (46 nM) and Em (whole-cell
patch clamp) in cells pre-incubated (30 min) in the absence or

presence of insulin (0.1 nM, 8 h), containing 5.5 (control) or


131 mM KCl (KCl). Cells in 5.5 mM KCl were co-incubated with
glibenclamide, levcromakalim, PD-98059, NG-nitro-L-arginine
methyl ester (L-NAME) or S-nitroso-N-acetyl-L,D-penicillamine
(SNAP). MeansSEM, n=17

Isolation of total RNA and reverse transcription

Without insulin
Control
KCl
Glibenclamide (10 M)
Levcromakalim (1 M)
Levcromakalim (1 M)
plus glibenclamide (10 M)
Wortmannin (30 nM)
L-NAME (100 M)
SNAP (100 M)
CalphostinC (100 nM)
PD-98059 (10 M)
With insulin
Control
KCl
Glibenclamide (10 M)
Levcromakalim (1 M)
Levcromakalim (1 M)
plus glibenclamide (10 M)
Wortmannin (30 nM)
L-NAME (100 M)
SNAP (100 M)
Calphostin C (100 nM)
PD-98059 (10 M)
a

L-Arginine

transport
(pmol/g protein per min)

[3H]TPP+ influx (pmol/mg


protein per min)

Em (mV)

2.10.2
0.30.2a
1.70.4
5.10.5a
1.80.3b

2.10.3
0.10.05a
1.90.4
6.00.8a
1.90.3b

65.50.4
11.50.7a
64.80.6
79.10.4a
64.60.4b

2.20.2
2.30.2
4.90.2a
1.90.4
2.50.4

1.70.4
2.50.5
5.60.7a
2.30.4
2.10.3

64.90.3
64.70.4
77.30.6a
65.30.5
65.20.3

12.20.9a
0.20.1a,c
2.50.7c
14.12.1a
2.60.6b,c

4.80.4a
0.20.01a,c
1.80.7c
4.50.6a
2.30.5b,c

82.30.4a
12.30.8a,c
64.90.3c
84.20.5a
65.90.6b,c

3.10.9c
2.60.4c
14.22.1a
2.70.6c
2.60.4c

1.90.5c
2.20.4c
6.21.1a
2.50.6c
1.90.3c

66.20.5c
64.90.6c
87.40.7a
64.70.7c
64.80.5c

P<0.05 vs. control in absence of insulin


P<0.05 vs. corresponding values in SNAP or levcromakalim
c
P<0.05 vs. corresponding values in control, SNAP or levcromakalim
b

386
Intracellular amino acid determination
Methanol (96%) cell extracts exposed to three cycles of freezethawing were centrifuged (1,500 rpm, 2 min). Aliquots of supernatant and standards (20 l) were injected onto a Hypersil
Ultratechsphere ODS-5 min reversed-phase HPLC column (Jones
Chromatography) in a Kontron 400 Series gradient HPLC system
(Kontron Instruments). L-Homoserine was used as internal standard [3, 45].

ATP determination
Culture medium (100 l) was mixed with 100 l luciferase reagent
(pH 7.7), and the reaction processed with the ATP bioluminescence
assay kit CLS II (Roche). Bioluminescence was monitored (562 nm,
10 s, 22 C) in a luminometer (Lumat LB 9501, Berthold). Detection
limit was 1 fmol ATP [36, 37].

Materials
Sera, agarose and buffers were from GIBCO Life Technologies,
Collagenase Type II (Clostridium histolyticum) from Boehringer
Mannheim (Mannheim, Germany) and Bradford protein reagent
(BioRad, Herts., UK). L-NAME and SNAP were from Calbiochem (La
Jolla, Calif., USA). All other reagents were from Sigma (St. Louis,
Mo., USA). L-[2,3,-3H]-Arginine (36.1 Ci mmol1), [-32P]ATP and
TPP bromide[phenyl-3H] (37 Ci mmol1) were from NEN (Dreieich,
Germany). 2-O-monosuccinyl guanosine-3,5-cyclic monophosphate tyrosyl methylester (GMP-TME, [tyrosine-125I]) was from
ICN (UK). Antibodies were from Cell Signalling, New England
Biolabs (UK).

Statistical analysis
Fig. 1A, B Effect of insulin on L-arginine transport. A L-Arginine
transport (100 M, 1 min, 37 C) in cultured human umbilical vein
endothelial cells (HUVEC) incubated (8 h) with M199 medium
(open circles) or M199 containing 0.1 nM insulin (solid circles).
The dotted line indicates replacement of M199 containing insulin by
insulin-free M199 (open squares). B L-Arginine transport (as in A)
in cells pre-incubated in M199 without (open bars) or with (solid
bars) 0.1 nM insulin (8 h, 37 C), and then exposed for a further 2 h
(37 C) to Krebs solution (control) or Krebs solution containing
10 mM L-lysine and 0.1 nM insulin. MeansSEM; n=12; *P<0.03
vs. all other values

Values are meansSEM, where n indicates the number of different


cell cultures (48 replicates). Statistical analyses were carried out on
raw data using the Peritz F-multiple means comparison test [12].
Students t-test was employed for unpaired data and P<0.05 was
considered significant.

column purified using a Qiaquick gel extraction kit (Qiagen) and


quantitated by densitometry with reference to the molecular weight
marker HpaII digest of pBluescript II 1 (SK+). Standards were
prepared by tenfold serial dilutions of each PCR product in tRNA to
obtain 101109 copies/2 l. Gene expression was quantitated using a
2-l sample of a tenfold dilution of HUVEC cDNA. All assays
included a tRNA or HUVEC RNA blank in addition to HUVEC
cDNA samples and standards.
Oligonucleotide primers were for hCAT-1 (sense) 5-GAGTTAGATCCAGCAGACCA-3, hCAT-1 (antisense) 5-TGTTCACAATTAGCCCAGAG-3, hCAT-2B (sense) 5-TCCCAATGCCTCGTGTAAACT-3, hCAT-2B (antisense) 5-GCACCCGATAAAGTAGCAA-3, eNOS (sense) 5-CCAGCTAGCCAAAGTCACCAT-3,
eNOS (antisense) 5-GTCTCGGAGCCATACAGGATT-3, 28S
(sense) 5-TTGAAAATCCGGGGGAGAG-3, 28S (antisense) 5ACATTGTTCCAACATGCCAG-3. Expected size products were
hCAT-1 148 bp, hCAT-2B 115 bp, eNOS 354 bp, and 28S 100 bp.

Complementing our previous results [7] insulin stimulated


transport with a concentration eliciting a halfmaximal effect (EC50) of 0.030.007 nM, and L-arginine
transport increased in response to 0.1 nM insulin (halfmaximal effect at 5.80.4 h) with a maximal rate achieved
within 8 h and sustained over 2 h (Fig. 1A). Subsequent
experiments were performed using 0.1 nM insulin for 8 h.
Insulin-stimulated L-arginine transport decreased to basal
values 68 h after removing insulin from the culture
medium (Fig. 1A). The effect of insulin on L-arginine
transport was blocked by wortmannin, calphostin C, LNAME or PD-98059. These inhibitors did not alter Larginine transport in absence of insulin (Table 1).
Insulin did not alter the non-saturable component (Kd)
of overall L-arginine transport, but increased Vmax with no
significant changes in the apparent Km for saturable
transport (Table 2). The Eadie-Hofstee analysis of

Results
L-Arginine

L-arginine

transport

387

saturable transport in presence or absence of insulin was


linear (not shown). Changes in Vmax for L-arginine
transport induced by insulin were blocked by wortmannin,
calphostin C, L-NAME or PD-98059. These inhibitors did
not alter kinetic transport parameters in absence of insulin.
Pre-incubation of cells with L-lysine (10 mM, 2 h)
increased L-arginine transport 6.9-fold in absence of
insulin (Fig. 1B). However, L-arginine transport was
unaltered (P<0.05) by L-lysine in cells pre-incubated
with insulin. L-Arginine transport stimulation by insulin or
+
L-lysine was unaltered in Na -free Krebs solution (not
shown).

Em. The K+ATP-channel activator levcromakalim induced


membrane hyperpolarization, increased L-arginine transport and TPP+ influx only in absence of insulin (Table 1).
The effect of levcromakalim was blocked by glibenclamide. Elevated extracellular KCl induced membrane
depolarization and inhibited TPP+ influx and L-arginine
transport as previously reported [3, 10]. In addition, the
effect of insulin on TPP+ influx and Em was blocked by
wortmannin, L-NAME, calphostin C and PD-98059
(Table 1).
NO involvement

hCAT-1 and hCAT-2B mRNA


Quantitative real time RT-PCR experiments showed that
HUVEC express hCAT-1 and hCAT-2B, confirming
previous reports in this cell type [3, 10, 15]. Insulin
(0.1 nM) increased hCAT-1 and hCAT-2B mRNA, but not
28S mRNA, in a time-dependent manner with a halfmaximal effect at 2.50.4 h for hCAT-1 and 3.10.5 h for
hCAT2-B (Fig. 2A), returning to basal levels after 8 h
incubation. In addition, the insulin effect (8 h) was
concentration-dependent (EC50 0.050.01 and 0.04
0.01 nM for hCAT-1 and hCAT-2B, respectively,
Fig. 2B). Insulin effects on hCAT-1 (Fig. 3A) and hCAT2B (Fig. 3B) mRNA were blocked by wortmannin, LNAME, PD-98059, or calphostin C. These inhibitors did not
alter hCAT-1 or hCAT-2B mRNA levels in absence of
insulin. None of the inhibitors altered basal expression of
28S mRNA (not shown).

Insulin increased L-[3H]citrulline, cGMP accumulation and


nitrite levels (Fig. 4AC, respectively). The effects of
insulin were blocked by L-NAME, wortmannin or calphostin
C. In the absence of insulin, L-[3H]citrulline synthesis,
cGMP accumulation and nitrite levels were inhibited only
by L-NAME. Insulin also increased total eNOS protein level
(2.1-fold) and eNOS phosphorylation at Ser1177 (5.5-fold,
Fig. 5A), and eNOS mRNA level (2.3-fold, Fig. 5B).
Insulin effects were inhibited by wortmannin or calphostin
C. In addition, intracellular L-arginine and L-citrulline
content were increased 3.4- and 2.1-fold respectively, by
insulin (Fig. 5C), an effect blocked by L-NAME, wortmannin or calphostin C. SNAP (an NO donor) induced TPP+
influx, L-arginine transport and membrane hyperpolarization in absence of insulin, but did not alter the insulinstimulatory effect on TPP+ influx, L-arginine transport and
Em (Table 1).
PKC and MAPK involvement

TPP+ influx and membrane potential


Insulin increased TPP+ influx (2.3-fold) and caused
membrane hyperpolarization (Table 1). The K+ATP-channel blocker glibenclamide blocked insulin-mediated increase in L-arginine transport, TPP+ influx and changes in

Insulin increased membrane-associated PKC activity, an


effect blocked by calphostin C and not further altered by
PMA (Fig. 6) or 4-PDD (not shown). The effect of
insulin on PKC activity was blocked by wortmannin, but
was unaltered by L-NAME. Insulin also induced p42/

Table 2 Insulin effect on kinetic parameters for L-arginine transport in HUVEC. L-Arginine transport (37 C, 1 min) in cells pre-incubated
(8 h) with 0.1 nM insulin, in the absence or presence of wortmannin, L-NAME, calphostin C or PD-98059 (see Methods). MeansSEM; n=8
Treatment
Control

Insulin

Wortmannin (30 nM)


L-NAME (100 M)
CalphostinC (100 nM)
PD-98059 (10 M)
(0.1 nM, 8 h)
Wortmannin (30 nM)
L-NAME (100 M)
Calphostin C (100 nM)
PD-98059 (10 M)

Km (M)

Vmax (pmol/g
protein per min)

Vmax/Km (pmol/g
protein per min per M)

Kd (pmol/g
protein per min per M)

8524
10316
8811
8717
9721
9320
9827
9312
12022
1049

3.90.3
4.10.3
3.90.5
4.40.4
3.70.5
9.91.2a
5.51.1b
4.70.9b
5.61.1b
5.90.7b

0.0460.011
0.0400.016
0.0440.014
0.0500.012
0.0380.015
0.1070.021a
0.0560.013b
0.0510.013b
0.0470.019b
0.0570.011b

0.00310.0022
0.00290.0011
0.00280.0009
0.00320.0021
0.00330.0007
0.00320.0009
0.00270.0010
0.00330.0016
0.00290.0012
0.00330.0013

P<0.05;bP<0.05 vs. values in absence or presence of insulin, respectively

388

Fig. 2AD Effect of insulin on expression of mRNA for human


cationic amino acid transporters-1 (hCAT-1) and -2B (hCAT-2B). A
Real time RT-PCR for hCAT-1 (148 bp, open bars) or hCAT-2B
(115 bp, solid bars) or 28S RNA (100 bp, hatched bars, internal
reference) obtained from HUVEC incubated (37 C) with 0.1 nM
insulin for the indicated periods. B Effect of incubation (8 h, 37 C)

with increasing concentrations of insulin (see Methods). Melting


temperatures for (C) hCAT-1 (79.17 C) or (D) hCAT-2B (77.84 C)
are shown. Bars show number of copies of hCAT-1, hCAT-2B or
28S mRNA. MeansSEM; n=1216; *P<0.03 vs. all other
corresponding values

p44mapk phosphorylation (Fig. 6C), an effect blocked by


PD-98059, wortmannin, L-NAME and calphostin C. PD98059 and calphostin C also blocked insulin-induced Larginine transport Vmax (Table 2), TPP+ influx and
membrane hyperpolarization (Table 1).

with higher eNOS mRNA and protein levels, and


increased eNOS phosphorylation at Ser1177. The effect of
insulin on hCAT-1 and hCAT-2B mRNA and L-arginine
transport requires NO synthesis and activation of PI3-K,
PKC and p42/p44mapk. Changes in transport activity and
hCAT-1 and hCAT-2B mRNA levels and eNOS expression
and activity induced by insulin are independent of
intracellular Ca2+. These findings provide the first direct
evidence that activation of L-arginine transport by insulin
at a physiological concentration is associated with changes
in hCAT-1 and hCAT-2B mRNA levels, membrane
hyperpolarization and requires eNOS expression and
activity in human endothelium.

Intracellular Ca2+ and ATP release


Basal intracellular [Ca2+] (403 nM, n=241 cells) was
increased (P<0.05) by insulin (37229 nM, n=241 cells).
The effect of insulin was unaltered (P<0.05, n=201329
cells) by L-NAME (35437 nM), wortmannin (32961 nM),
or calphostin C (32965 nM). These inhibitors did not
alter intracellular Ca2+ in absence of insulin (not shown).
Basal ATP release (6.10.2 nmol/106 cells) was unaltered
(P>0.05, n=612) by insulin (6.70.5 nmol/106 cells), LNAME
(5.60.5 nmol/106 cells), wortmannin (6.3
0.5 nmol/106 cells), or calphostin C (6.80.6 nmol/106
cells).

Discussion
This study demonstrates that human insulin increased
hCAT-1 and hCAT-2B mRNA levels in HUVEC. As has
been shown previously, insulin also increased L-arginine
transport [39] and NO synthesis [39, 45] in association

Effect of insulin on L-arginine transport


transport can be mediated by systems y+/
CATs [3, 10, 33, 39, 45], y+L [2, 38, 42] and b0,+ [33] in
HUVEC. Our results show that L-arginine transport occurs
with relatively high affinity (Km 85 M), is Na+-independent and inhibited by membrane depolarization, supporting previous reports showing that L-arginine transport in
these cells is mediated primarily by the high-affinity
membrane transporters hCAT-1 (Km100200 M) or
hCAT-2B (Km200400 M) [3, 10, 39, 45, 50]. Insulin
increases L-arginine transport in HUVEC [39, 45] and
activates Na+-independent L-lysine transport in rat panL-Arginine

389

Fig. 3A, B Effect of inhibitors on the effect of insulin on hCAT-1


and hCAT-2B mRNA. A Real time RT-PCR for hCAT-1 (148 bp) or
28S (100 bp, internal reference) obtained from HUVEC incubated
(8 h, 37 C) with M199 containing 0.1 nM insulin, in the absence
() or presence (+) of the indicated inhibitors. B Real time RT-PCR
for hCAT-2B (115 bp) as in A. Bars show number of copies of
hCAT-1 or hCAT-2B mRNA versus number of copies of 28S mRNA
(L-NAME NG-nitro-L-arginine methylester). MeansSEM; n=14;
*P<0.03 vs. all other values

creas [29] and L-arginine transport in gastric mucosa [6].


Our results show that the insulin-mediated increase in Larginine transport was associated with a higher Vmax with
no changes in the apparent Km. Thus, it is unlikely that the
insulin-mediated increase in L-arginine transport was due
to activation of a low-affinity transport system, such as
hCAT-2A (Km25 mM), or the very high affinity
transport system y+L [2, 3, 7, 26, 38, 42]. This is
supported by our results showing that hCAT-2A mRNA
was undetectable in HUVEC. Insulins stimulation of Larginine transport was not due to activation of Na+dependent transport since insulin-activated transport was
unaltered in absence of extracellular Na+.
Insulin-induced transport of cationic amino acids is
associated with a higher CAT-1 protein level in rat
hepatocytes [49] and CAT-1 mRNA in rat coronary
myocytes [43]. Similarly, insulin increased hCAT-1
mRNA levels in HUVEC, suggesting that one of the

Fig. 4AC Effect of insulin on endothelial NO synthase (eNOS)


activity. HUVEC monolayers were exposed (8 h) to M199 (open
bars) or M199 containing 0.1 nM insulin (solid bars), in the absence
or presence of L-NAME, wortmannin or calphostin C. A Activity of
eNOS monitored by measuring formation of L-[3H]citrulline from L[3H]arginine (4 Ci ml1, 37 C, 30 min). B Accumulation of
intracellular cGMP (5 min, 37 C). C Levels of nitrite in the culture
medium (see Methods). MeansSEM; n=14; *P<0.03 vs. all other
values

mechanisms by which insulin increased L-arginine transport is a higher expression of the transporter. It is well
documented that CAT-1 is more sensitive than CAT-2B to
trans-stimulation by cationic amino acids [3, 5, 7, 10, 26].
L-Lysine increased (~sevenfold) the L-arginine transport
only in cells cultured in absence of insulin, a value similar
to those reported in HUVEC [3, 10] and in Xenopus
oocytes injected with hCAT-1 mRNA [5]. These results
suggest that insulin-increased L-arginine transport could be
mediated preferentially by hCAT-1 in HUVEC, however
the involvement of hCAT-2B cannot be ruled out since
hCAT-2B mRNA level was also increased by insulin. LLysine did not trans-stimulate L-arginine transport in the
presence of insulin, possibly reflecting an already maximum L-arginine transporter activity induced by insulin. In

390
Fig. 5AC Effect of insulin on
eNOS expression and L-arginine
and L-citrulline content. A Immunoblot for eNOS and phosphorylated eNOS at Ser1177
(eNOS~P-Ser1177) in HUVEC
pre-incubated (8 h) with M199
containing 0.1 nM insulin, in the
absence () or presence (+) of
the indicated inhibitors (see
Methods). -Actin was the internal reference. B Real time
RT-PCR for eNOS (354 bp) or
28S RNA (100 bp, internal
reference) obtained from
HUVEC incubated (8 h, 37 C)
with M199 containing 0.1 nM
insulin as in A. Bars show the
ratio of the number of copies of
eNOS mRNA to the number of
copies of 28S mRNA. C Intracellular L-citrulline and L-arginine determined by HPLC under
same conditions as in A and B
(see Methods). In A data representative of eight cell cultures.
MeansSEM; n=1216;
*P<0.03 vs. all other values

addition, since Em was unaltered in L-lysine preloaded


cells, L-lysine-stimulated L-arginine transport was not due
to membrane hyperpolarization induced by L-lysine [3,
10].
Insulin-stimulated L-arginine transport is sensitive to
changes in extracellular K+ and Em. Since insulin induced
membrane hyperpolarization, stimulation of L-arginine
transport could result from changes in Em. Insulin
stimulation of TPP+ influx and L-arginine transport was
blocked by glibenclamide, an ATP-sensitive K+ (K+ATP)
channel blocker [14]. In addition, levcromakalim (K+ATP
activator) [10, 18] mimicked insulin-induced changes in
Em, TPP+ influx and L-arginine transport. K+ATP channels
are expressed in HUVEC [10, 50], porcine coronary

artery [32] and rat aorta and brain microvascular [17]


endothelium. Purinoceptor activation [23, 32, 50] or
elevated extracellular D-glucose [10, 45] induces membrane hyperpolarization associated with increased L-arginine transport and activation of K+ATP channels in
endothelial cells. Since insulin effects on Em and Larginine transport were blocked by glibenclamide, it is
likely that activity of glibenclamide-sensitive K+ATP
channels could be required for insulin effects in
HUVEC. The possibility that L-arginine transport resulted
from activation of P2Y purinoceptors, reported to be
expressed in this cell type [36], by ATP released from
HUVEC is unlikely since insulin did not alter basal ATP
release.

391
Fig. 6AC Effect of insulin on
activity of protein kinase C
(PKC) and p42/44mapk. A PKC
activity in membrane fractions
from HUVEC incubated (8 h) in
absence (open bars) or presence
(solid bars) of 0.1 nM insulin, in
the absence () or presence (+)
of calphostin C, wortmannin, LNAME, PD-98059 or phorbol 12myristate 13-acetate (PMA), as
indicated in Methods. B PKC
activity in cytosol fractions as in
A. C Immunoblot for phosphorylated mitogen-activated
protein kinases (MAPK)
p44mapk (p44~P) and p42mapk
(p42~P) and non-phosphorylated p44mapk (p44) or p42mapk
(p42) in the absence () or
presence (+) of insulin and
inhibitors (as in A and B). Data
representative of eight cell cultures. MeansSEM; n=812.
*P<0.05 vs. values in absence
of insulin or inhibitors,
**P<0.04 vs. values in presence
of insulin or insulin plus L-NAME
or PD-98059

Effect of insulin on NO synthesis


Local intra-arterial insulin in healthy subjects induces
vasodilatation via endothelium-derived NO [25, 40, 47].
Insulin also increases eNOS expression and activity in
HUVEC [27, 45, 52] human coronary artery (HCAEC) [9]
and bovine aortic [22] endothelial cells. In this study, the
insulin-mediated increase in eNOS activity was associated
with eNOS phosphorylation at Ser1177. These results are
similar to the reported Ser1177 phosphorylation in response
to 10 nM insulin in HCAEC [9] or 25 mM D-glucose in
HUVEC [19, 37], agreeing with reports showing that
Ser1177 phosphorylation is associated with eNOS activation [8]. In addition, insulin increased the formation of L[3H]citrulline, accumulation of cGMP, nitrite level and
intracellular Ca2+ and L-citrulline in HUVEC. Insulin also
increased eNOS protein and mRNA levels, suggesting that
insulin-increased NO synthesis results from a combination
of eNOS activation and increased eNOS expression in

HUVEC. L-NAME, which does not interfere with L-arginine


transport [3, 10, 45], blocked the increase in eNOS
activity, L-arginine transport and intracellular L-arginine
content induced by insulin, suggesting that NO most
probably mediates these effects of insulin as reported in
bovine aortic endothelium [30] and HUVEC [3, 10, 50].
NO induces membrane hyperpolarization in HUVEC [3,
10, 44, 50]. SNAP-derived NO caused comparable
increases in TPP+ influx and L-arginine transport, and
membrane hyperpolarization to that induced by insulin,
although SNAP treatment did not enhance insulin effects
further in HUVEC. Thus, insulin modulation of L-arginine
transport involves NO-induced membrane hyperpolarization. L-Arginine transport and TPP+ influx are increased by
exogenous dibutyryl cGMP in HUVEC [10, 37]. In this
study, cGMP level was increased by insulin, confirming
previous findings in this cell type [45] We therefore
hypothesize that NO-altered K+ channel activity may
occur by an indirect mechanism via cGMP in response to

392

insulin. Since NO also activates outwards K+ currents in


HUVEC [10, 44, 50] the possibility of a direct NO action
on channels cannot be excluded.
Involvement of PKC, MAPK and PI3-K in the effect
of insulin
PKC activity was increased by insulin, an effect blocked
by calphostin C. This inhibitor also blocked the insulinmediated increase in hCAT-1 and hCAT-2B mRNA levels
and L-arginine transport in HUVEC. Thus, insulin requires
calphostin C-sensitive PKC isozymes to modulate the
expression and activity of these isoforms of L-arginine
transporters in HUVEC. In addition, the insulin-stimulated
TPP+ influx, eNOS activation and eNOS expression were
blocked by calphostin C, supporting the possibility that
PKC could be a key modulator of insulin effects on Larginine/NO signalling pathway in human endothelium.
These results confirm previous observations in this cell
type [10, 33] and in Caco-2 cells [34], but contrast with
recent reports suggesting that PKC activation could lead to
inhibition of L-arginine transport via CAT-1 in the
endothelial cell line EA.hy926 [11] and in pig pulmonary
artery endothelium [21]. The discrepancies in the results
could be due to differences in the source of endothelial
cells used in these experiments, i.e. an endothelial cell line
or pig pulmonary endothelium on the one hand and freshly
isolated human umbilical vein endothelium on the other.

Fig. 7 Insulin modulation of L-arginine/NO endothelial signalling


pathway. Physiological concentrations of circulating insulin activate
receptors (IR) in the plasma membrane of HUVEC. Following the
activation of IR by insulin, phosphatidylinositol 3-kinase (PI3-K) is
activated and triggers series of reactions involving activation of
PKC, eNOS and the 44- and 42-kDa mitogen-activated protein
kinases (p42/44mapk). Activation of these molecules is required to
induce an increase (vertical arrows) of gene transcription for amino
acid transporter isoforms hCAT-1 and hCAT-2B and eNOS.
Increased hCAT-1 and hCAT-2B mRNA levels are associated with
increased Vmax for L-arginine transport. ? indicates the possibility

Insulin also increased p42/p44mapk phosphorylation, an


effect blocked by PD-98059 (a MAPK kinase 1/2
[MEK1/2] inhibitor) [20]. The effect of insulin on Larginine transport, hCAT-1 and hCAT-2B mRNA and Em
were also blocked by PD-98059, suggesting that each
requires MAPK activation. In addition, phosphorylation of
p42/p44mapk induced by insulin was blocked by calphostin
C and L-NAME, suggesting that it requires PKC and eNOS
activity in HUVEC. These results complement and
confirm previous observations in the same cell type
suggesting that phosphorylation of p42/p44mapk is an
event downstream from PKC and eNOS activation in
response to acute (minutes) [10] or chronic (hours) [28,
37] incubation with 25 mM D-glucose. Furthermore, it has
been reported that PI3-k activity is involved in p42/
p44mapk activation in HUVEC [10, 24, 41]. Our results
show that insulin-induced p42/p44mapk phosphorylation
was also blocked by wortmannin, suggesting that PI3-K is
required for the effects of insulin. Our results also show
that PI3-K is also involved in modulation of eNOS
expression and activity in response to insulin, since
wortmannin blocked insulin-increased eNOS activity and
expression in HUVEC. The insulin-mediated increases in
p42/p44mapk phosphorylation and L-arginine transport
were blocked by L-NAME, involving eNOS in the activation
of p42/p44mapk by insulin and agreeing with reports
suggesting that endogenous or exogenous NO induces
p42/p44mapk activation in endothelium [10, 35, 37]. Since
insulin-induced membrane hyperpolarization is blocked by
PD-98059, p42/p44mapk activation could also modulate

that hCAT-1 and/or hCAT-2B protein levels are also increased.


Increased eNOS mRNA is associated with increased eNOS protein
levels and activation of eNOS, leading to increased L-arginine
conversion to L-citrulline and nitric oxide (NO). The insulinmediated increase in L-arginine transport could also result, at least in
part, from membrane hyperpolarization due to activation of ATPsensitive K+ channels, possibly by NO (see [14, 15, 33, 45]). The
effect of insulin could be terminated by abnormally elevated levels
of NO that could inactivate eNOS or reduce eNOS expression and Larginine transport (see [23, 44, 50])

393

ion channel activity, and therefore L-arginine transport, in


response to insulin in HUVEC.
In summary, this study has confirmed that insulin
activation of L-arginine transport is associated with
increased hCAT-1 and hCAT-2B mRNA levels in human
foetal endothelium. As illustrated in Fig. 7 our results are
consistent with a model in which eNOS activity itself is
part of the cascade leading to enhanced L-arginine
transport in HUVEC via a mechanism that requires PI3K, PKC and p42/p44mapk activity and involves membrane
hyperpolarization. Modulation of mRNA expression and
activity of hCAT-1 and hCAT-2B by physiological
concentrations of insulin could be a mechanism by
which this hormone basally regulates the vascular tone,
having implications for type-II diabetic mellitus, in which
plasma insulin concentration, or tissue sensitivity to
insulin, are altered [4, 46].
Acknowledgements Supported by Fondo Nacional de Ciencia y
Tecnologa (FONDECYT 1030781, 1030607, 7030004, 7030109)
and The Wellcome Trust (UK). C.F. holds CONICYT-PhD (Chile)
fellowship. We thank Miss Alexandra Almeida for excellent
secretarial assistance and the midwives of Hospital Clnico of the
Pontificia Universidad Catlica de Chile labour ward for supply of
umbilical cords.

References
1. Alderton WK, Cooper CE, Knowles RG (2001) Nitric oxide
synthases: structure, function and inhibition. Biochem J
357:593615
2. Arancibia-Garavilla Y, Toledo F, Casanello P, Sobrevia L
(2003) Nitric oxide synthesis requires activity of the cationic
and neutral amino acid transport system y+L in human
umbilical vein endothelium. Exp Physiol 88:699710
3. Casanello P, Sobrevia L (2002) Intrauterine growth retardation
is associated with reduced activity and expression of the
cationic amino acid transport systems y+/hCAT-1 and y+/hCAT2B and lower activity of nitric oxide synthase in human
umbilical vein endothelial cells. Circ Res 91:127134
4. Cleland SJ, Petrie JR, Ueda S, Elliot HL, Connell JM (1998)
Insulin as a vascular hormone: implications for the pathophysiology of cardiovascular disease. Clin Exp Pharmacol Physiol
25:175184
5. Closs EI, Basha FZ, Habermeier A, Fstermann U (1997)
Interference of L-arginine analogues with L-arginine transport
mediated by the y+ carrier hCAT-2B. Nitric Oxide 1:6573
6. Contreras R, Fuentes O, Mann GE, Sobrevia L (1997) Diabetes
and insulin-induced stimulation of L-arginine transport and
nitric oxide synthesis in rabbit isolated gastric glands. J Physiol
(Lond) 498:787796
7. Devs R, Boyd CAR (1998) Transporters for cationic amino
acids in animal cells: Discovery, structure and function. Physiol
Rev 78:487545
8. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R,
Zeiher AM (1999) Activation of nitric oxide synthase in
endothelial cells by Akt-dependent phosphorylation. Nature
399:601605
9. Federici M, Menghini R, Mauriello A, Hribal ML, Ferrelli F,
Lauro D, Sbraccia P, Spagnoli LG, Sesti G, Lauro R (2002)
Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of
signalling proteins in human coronary endothelial cells. Circulation 106:466472

10. Flores C, Rojas S, Aguayo C, Parodi J, Mann G, Pearson JD,


Casanello P, Sobrevia L (2003) Rapid stimulation of L-arginine
transport by D-glucose involves p42/44mapk and nitric oxide in
human umbilical vein endothelium. Circ Res 92:6472
11. Graf P, Forstermann U, Closs EI (2001) The transport activity
of human cationic amino acid transporter hCAT-1 is downregulated by activation of protein kinase C. Br J Pharmacol
132:11931200
12. Harper JF (1984) Peritz F test: BASIC program of a robust
multiple comparison test for statistical analysis of all differences among group means. Comput Biol Med 14:437445
13. Hermann C, Assmus B, Urbich C, Zeiher AM, Dimmeler S
(2000) Insulin-mediated stimulation of protein kinase Akt: a
potent survival signalling cascade for endothelial cells.
Arterioscler Thromb Vasc Biol 20:402409
14. Holzmann S, Kukovetz WR, Braida C, Poch G (1992)
Pharmacological interaction experiments differentiate between
glibenclamide-sensitive K+ channels and cyclic GMP as
components of vasodilation by nicorandil. Eur J Pharmacol
29:17
15. Irie K, Tsukahara F, Fujii E, Uchida Y, Yoshioka T, He WR,
Shitashige M, Murota S, Muraki T (1997) Cationic amino acid
transporter-2 mRNA induction by tumor necrosis factor-alpha
in vascular endothelial cells. Eur J Pharmacol 339:289293
16. Isenovic E, Muniyappa R, Milivojevic N, Rao Y, Sowers JR
(2001) Role of PI3-kinase in isoproterenol and IGF-1 induced
ecNOS activity. Biochem Biophys Res Commun 285:954958
17. Janigro D, West GA, Gordon EL, Winn HR (1993) ATPsensitive K+ channels in rat aorta and brain microvascular
endothelial cells. Am J Physiol 265:C812C821
18. Katnik C, Adams DJ (1997) Characterization of ATP-sensitive
potassium channels in freshly dissociated rabbit aortic endothelial cells. Am J Physiol 272:H2507H2511
19. Kobayashi E, Ando K, Nakano H, Iida T, Ohno H, Morimoto
M, Tamaoki T (1989) Calphostins (UCN-1028), novel and
specific inhibitors of protein kinase C. I. Fermentation,
isolation, physico-chemical properties and biological activities.
J Antibiot (Tokyo) 42:14701474
20. Kolch W (2000) Meaningful relationships: the regulation of the
Ras/Raf/MEK/ERK pathway by protein interactions. Biochem
J 351:289305
21. Krotova KY, Zharikov SI, Block ER (2003) Classical isoform
of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells. Am J Physiol 284:L1037L1044
22. Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M,
Yamauchi T, Feener EP, Herbert TP, Rhodes CJ, King GL
(2000) Regulation of endothelial constitutive nitric oxide
synthase gene expression in endothelial cells and in vivo: a
specific vascular action of insulin. Circulation 101:676681
23. Kuo L, Chancellor JD (1995). Adenosine potentiates flowinduced dilatation of coronary arterioles by activating KATP
channels in endothelium. Am J Physiol 269:H541H549
24. Lal BK, Varma S, Pappas PJ, Hobson RW 2nd, Duran WN
(2001) VEGF increases permeability of the endothelial cell
monolayer by activation of PKB/akt, endothelial nitric-oxide
synthase, and MAP kinase pathways. Microvasc Res 62:252
262
25. Lind L, Fugmann A, Millgard J, Berne C, Lithell H (2002)
Insulin-mediates vasodilatation, but not glucose uptake or
endothelium-mediated vasodilatation, is enhanced in young
females compared with males. Clin Sci 102:241246
26. Mann GE, Yudilevich DL, Sobrevia L (2003) Regulation of
amino acid and glucose transporters in endothelial and smooth
muscle cells. Physiol Rev 83:183252
27. Montagnani M, Ravichandran LV, Chen H, Esposito DL, Quon
MJ (2002) Insulin receptor substrate-1 and phosphoinositidedependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells. Mol Endocrinol
16:19311942

394
28. Montecinos VP, Aguayo C, Flores C, Wyatt AW, Pearson JD,
Mann GE, Sobrevia L (2000) Regulation of adenosine transport
by D-glucose in human foetal endothelial cells: involvement of
nitric oxide, protein kinase C and mitogen-activated protein
kinase. J Physiol (Lond) 529:777790
29. Muoz M, Sweiry JH, Mann GE (1995) Insulin stimulates
cationic amino acid transport activity in the isolated perfused
rat pancreas. Exp Physiol 80:745753
30. Ogonowski AA, Kaesemeyer WH, Jin L, Ganapathy V, Leibach
FH, Caldwell RW (2000) Effects of NO donors and synthase
agonists on endothelial cell uptake of L-Arg and superoxide
production. Am J Physiol 278:C136C146
31. Okouchi M, Okayama N, Shimizu M, Omi H, Fukutomi T, Itoh
M (2002) High insulin exacerbates neutrophil-endothelial cell
adhesion through endothelial surface expression of intercellular
adhesion molecule-1 via activation of protein kinase C and
mitogen-activated protein kinase. Diabetologia 45:556559
32. Olanrewaju HA, Gafurov BS, Lieberman EM (2002) Involvement of K+ channels in adenosine A2A and A2B receptormediated hyperpolarization of porcine coronary artery endothelial cells. J Cardiovasc Pharmacol 40:4349
33. Pan M, Wasa M, Lind DS, Gertler J Abbott W, Souba WW
(1995) TNF-stimulated arginine transport by human vascular
endothelium requires activation of protein kinase C. Ann Surg
221:590601
34. Pan M, Meng QH, Wolfgang CL, Lin CM, Karinch AM, Vary
TC, Souba WW (2002) Activation of intestinal arginine
transport by protein kinase C is mediated by mitogen-activated
protein kinases. J Gastrointest Surg 6:876882
35. Parenti A, Morbidelli L, Cui XL, Douglas JG, Hood JD,
Granger HJ, Ledda F, Ziche M (1998) Nitric oxide is an
upstream signal of vascular endothelial growth factor-induced
extracellular signal-regulated kinase 1/2 activation in postcapillary endothelium. J Biol Chem 273:42204226
36. Parodi J, Flores C, Aguayo C, Rudolph MI, Casanello P,
Sobrevia L (2002) Inhibition of nitrobenzylthioinosine-sensitive adenosine transport by elevated D-glucose involves activation of P2Y2 purinoceptors in human umbilical vein endothelial
cells. Circ Res 90:570577
37. Rojas S, Rojas R, Lamperti L, Casanello P, Sobrevia L (2003)
Hyperglycaemia inhibits thymidine incorporation and cell
growth via protein kinase C, mitogen-activated protein kinases
and nitric oxide in human umbilical vein endothelium. Exp
Physiol 88:209219
38. Sala R, Rotoli BM, Colla E, Visigalli R, Parolari A, Bussolati
O, Gazzola GC, DallAsta V (2002) Two-way arginine
transport in human endothelial cells: TNF-alpha stimulation is
restricted to system y+. Am J Physiol 282:C134C143

39. Salt IP, Morrow VA, Brandie FM, Connel JMC, Petrie JR
(2003) High glucose inhibits insulin-stimulated nitric oxide
production without reducing endothelial nitric-oxide synthase
Ser1177 phosphorylation in human aortic endothelial cells. J
Biol Chem 278:1879118797
40. Scherrer U, Randin D, Vollenweider P, Vollenweider L, Nicod
P (1994) Nitric oxide release accounts for insulins vascular
effects in humans. J Clin Invest 94:25112515
41. Shaul PW (2002) Regulation of endothelial nitric oxide
synthase: location, location, location. Annu Rev Physiol
64:749774
42. Signorello MG, Pascale R, Leoncini G (2003) Transport of Larginine and nitric oxide formation in human platelets. Eur J
Biochem 270:20052012
43. Simmons WW, Closs EI, Cunningham JM, Smith TW, Kelly
RA (1996) Cytokines and insulin induces cationic amino acid
transporter (CAT) expression in cardiac myocytes. Regulation
ofL-arginine transport and NO production by CAT-1, CAT-2A,
and CAT-2B. J Biol Chem 271:1169411702
44. Sobey CG (2001) Potassium channel function in vascular
disease. Arterioscler Thromb Vasc Biol 21:2838
45. Sobrevia L, Nadal A, Yudilevich DL, Mann GE (1996)
Activation ofL-arginine transport (system y+) and nitric oxide
synthase by elevated glucose and insulin in human endothelial
cells. J Physiol (Lond) 490:775781
46. Steinberg HO, Baron AD (2002) Vascular function, insulin
resistance and fatty acids. Diabetologia 45:623634
47. Steinberg HO, Brechtel G, Johnson A, Fineberg N, Baron AD
(1994) Insulin-mediated skeletal muscle vasodilation is nitric
oxide dependent. A novel action of insulin to increase nitric
oxide release. J Clin Invest 94:11721179
48. Taha C, Klip A (1999) The insulin signalling pathway. J Membr
Biol 169:112
49. Wu JY, Robinson D, Kung HJ, Hatzoglou M (1994) Hormonal
regulation of the gene for the type C ecotropic retrovirus
receptor in rat liver cells. J Virol 68:16151623
50. Wyatt AW, Steinert JR, Wheeler-Jones CP, Morgan AJ, Sudgen
D, Pearson JD, Sobrevia L, Mann GE (2002) Early activation
of the p42/44MAPK pathway mediates adenosine-induced nitric
oxide production in human endothelial cells: a novel calciuminsensitive mechanism. FASEB J 16:15841594
51. Zeng G, Quon MJ (1996) Insulin-stimulated production of
nitric oxide is inhibited by wortmannin. Direct measurement in
vascular endothelial cells. J Clin Invest 98:894898
52. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M,
Mostowski H, Quon MJ (2000) Roles for insulin receptor, PI3kinase, and Akt in insulin-signalling pathways related to
production of nitric oxide in human vascular endothelial
cells. Circulation 101:15391545