DOI 10.1007/s00424-004-1261-x
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
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
L-Arginine
transport
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
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].
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].
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
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
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)
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
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
Results
L-Arginine
L-arginine
transport
387
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
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
388
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
389
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
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
392
393
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