Published online before print June 21, 2001, doi: 10.1161/hh1301.092498
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Adrenomedullin Induces Endothelium-Dependent Vasorelaxation via the Phosphatidylinositol 3-Kinase/Akt–Dependent Pathway in Rat Aorta
From the Department of Urology (H.N., N.M., T.K.) and the Second Department of Internal Medicine (E.S., D.N., H.S., M.S., A.G., Y.H.), Faculty of Medicine, University of Tokyo, Japan; Division of Cardiovascular Research (K.W.), St. Elizabeth’s Medical Center of Boston, Boston, Mass; and National Cardiovascular Center Research Institute (K.K., H.M.), Fujishirodai, Japan.
Correspondence to Etsu Suzuki, MD, PhD, The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail suzuki-2im{at}h.u-tokyo.ac.jp
Abstract
Abstract—To study the mechanisms by which adrenomedullin (AM) induces endothelium-dependent vasorelaxation, we examined whether AM-induced endothelium-dependent vasodilation was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway in rat aorta, because it was recently reported that PI3K/Akt was implicated in the activation of endothelial NO synthase. AM-induced vasorelaxation in thoracic aorta with intact endothelium was inhibited by pretreatment with PI3K inhibitors to the same level as that in endothelium-denuded aorta. AM elicited Akt phosphorylation in a time- and dose-dependent manner. AM-induced Akt phosphorylation was inhibited by pretreatment with a calmodulin-dependent protein kinase inhibitor as well as with PI3K inhibitors. When an adenovirus construct expressing a dominant-negative Akt mutant (Ad/dnAkt) was injected into abdominal aortas so that the mutant was expressed predominantly in the endothelium layer, AM-induced vasodilation was diminished to the same level as that in endothelium-denuded aortas. Finally, AM-induced cGMP production, which was used as an indicator for NO production, was suppressed by PI3K inhibition or by Ad/dnAkt infection into the endothelium. These results suggested that AM induced Akt activation in the endothelium via the Ca2+/calmodulin-dependent pathway and that this was implicated in the production of NO, which in turn induced endothelium-dependent vasodilation in rat aorta.
Key Words: adrenomedullin • phosphatidylinositol 3-kinase • Akt • nitric oxide • gene transfer
Vascular endothelial cells (ECs) are critically implicated in the modulation of vascular tone by producing a variety of vasoactive substances. Among them, endothelin has a vasoconstrictive activity, whereas NO and prostacyclin possess a vasorelaxant activity. The modulation of vascular tone by ECs is known to be perturbed in pathophysiological states such as hypertension, diabetes mellitus, and hyperlipidemia.1
A novel peptide, which increases the level of cAMP in platelets, was recently isolated from human adrenal medulla and was dubbed adrenomedullin (AM).2 AM is a 52-amino acid peptide with a potent vasorelaxant activity and natriuretic activity.3 4 5 Although AM was originally isolated from human pheochromocytoma tissue, it is now well known that AM is also produced by vascular ECs, vascular smooth muscle cells, and macrophages,6 7 8 suggesting its role as a local mediator in normal and/or diseased vessels. Although little is known of the intracellular signaling pathways that are activated by AM, it is reported that AM increases intracellular cAMP and [Ca2+]i.9 AM also activates extracellular signal–regulated kinase (ERK), which appears to be involved in AM-induced cell proliferation,10 11 12 although AM seems to inhibit cell proliferation in some cells,13 depending on cell types. It was originally postulated that AM-induced vasorelaxation might be mediated by its effect on intracellular cAMP. However, we have recently shown that AM induced vasorelaxation, at least partly, in an endothelium-dependent manner and that endothelium-dependent vasorelaxation was mediated via the NO/cGMP-dependent pathway in rats.14 Furthermore, it has been reported that transgenic mice that were designed to express an excess amount of AM in vascular ECs showed lower blood pressure than wild-type mice and that the difference in blood pressure was abolished by administration of NG-monomethyl-L-arginine to those mice,15 suggesting the involvement of an NO/cGMP-dependent pathway for the AM-induced vasorelaxation, although the molecular mechanisms by which AM activated the NO/cGMP-dependent pathway remained to be elucidated.
It is well established that Ca2+/calmodulin (CaM) is a major activator of endothelial NO synthase (eNOS). When [Ca2+]i is increased, CaM binds to the CaM binding domain of eNOS, which in turn changes the conformation of eNOS by an "allosteric" effect and activates eNOS.16 17 18 It is therefore possible that AM activates eNOS via the Ca2+/CaM-dependent pathway. However, it has been reported that eNOS is activated by direct phosphorylation of serine 1179 (Ser1179) by Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K). When eNOS is phosphorylated on Ser1179, eNOS is activated under a very low concentration of Ca2+, which suggests that the affinity of eNOS to CaM increases by Ser1179 phosphorylation.19 20 Thus, it seemed to be of interest to examine whether AM induced activation of eNOS via the PI3K/Akt-dependent pathway.
In the present study, we examined the mechanisms by which AM induced endothelium-dependent vasorelaxation. We show that AM induces endothelium-dependent vasorelaxation and cGMP production via the PI3K/Akt-dependent pathway. We also show that AM induces phosphorylation of Akt and that AM-induced Akt phosphorylation depends on the Ca2+/CaM-mediated pathway.
Materials and Methods
Reagents
Phosphospecific anti-ERK1/2 antibody and
phosphospecific anti-Akt antibody that recognize catalytically active
ERK1/2 and Akt, respectively, were obtained from New England BioLabs.
Anti-Akt and -ERK1 antibodies were obtained from Santa Cruz
Biotechnology.
NG-nitro-L-arginine
methyl ester (L-NAME), forskolin, sodium nitroprusside (SNP), KN-93,
A23187, wortmannin, and LY294002 (LY) were purchased from Sigma,
and PD98059 (PD) was obtained from New England
Biolabs.
Ex Vivo Experiments
Effects of AM on the tension of rat aortic rings were
examined as previously
described.14 In brief,
thoracic aortas were excised from 12-week-old male Wistar rats, and
aortic rings with or without endothelium were mounted
in organ chambers filled with Krebs-Ringer bicarbonate solution at
37°C with constant bubbling of 95% oxygen/5% carbon dioxide.
Isometric tension was recorded with a force transducer. The
endothelium was denuded by gentle rubbing with a twist
of cotton, and this was confirmed by the lack of acetylcholine-induced
vasodilation. The aortic rings were precontracted with
L-norepinephrine,
and the effects of AM on vasodilation were studied. In some
experiments, several reagents such as L-NAME, wortmannin, LY, and PD
were added to the chambers before precontraction with
L-norepinephrine
to examine their effects on AM-induced vasorelaxation. To prepare
protein extracts, rat aortas were placed in tubes containing
oxygenated Krebs-Ringer bicarbonate solution at 37°C and
incubated with AM with or without pretreatment with several reagents
such as KN-93, wortmannin, LY, and PD.
Preparation of Protein Extracts
Rat aortas were homogenized on ice in a
Triton X-100 homogenization
buffer (in mmol/L, HEPES [pH 7.5] 50, NaCl 150, EDTA 1, and DTT
1, as well as 2% Triton X-100 and 10%
glycerol) containing 1 mmol/L PMSF, 2 µg/mL leupeptin, and 2
µg/mL aprotinin. After centrifugation for 20 minutes
at 4°C, the cleared supernatant was used for Western blot
analysis. Protein concentration was measured according to
Bradford’s method (Bio-Rad).
Western Blot Analysis
Western blot analysis was performed as
previously described.21
Antibodies that recognize catalytically active ERK1/2 and Akt were used
at a dilution of 1:500.
In Vivo Gene Transfer to Rat Abdominal
Aorta
Construction of an adenovirus expressing a
dominant-negative Akt mutant (Ad/dnAkt) has been described
previously.22 A recombinant
adenovirus that expresses green fluorescence protein (Ad/GFP)
was obtained from Qbiogene. Twelve-week-old male Wistar rats fed on a
normal diet were anesthetized by
intraperitoneal injection of pentobarbital (35
mg/kg) and placed on a thermoregulated surgical table. The abdominal
aorta was exposed by dissecting the mesenteries from intestines, and
the inferior mesenteric artery and lumbar vessels were
clamped with plastic forceps. The left femoral artery was then exposed
by a lower-quadrant incision. A polyethylene tube (PE-10) attached to a
microliter syringe was introduced into the artery and advanced up to
the proximal side of the bifurcation of the aorta into the common iliac
artery. The right common iliac artery was then clamped, and the left
one was ligated together with the PE-10 tube. The abdominal aorta was
clamped distal to the divergence of the renal arteries. After saline
was injected in the PE-10 tube to confirm that there was no leakage,
150 µL of Ad/dnAkt (1010 plaque-forming
units [PFU]/mL) or Ad/GFP (1010 PFU/mL)
was injected into the aorta and the vessel was incubated with the virus
suspension for 30 minutes. The aortas were excised 48 hours after
infection, and the tension of the aortic rings was examined. In some
experiments, the vessel was incubated with AM, and cGMP
production was measured.
Immunohistological
Analysis of Adenovirus-Infected Abdominal Aorta
Rats were anesthetized with pentobarbital 48
hours after the injection of Ad/GFP into the abdominal aorta. Abdominal
aortas were fixed by perfusion with 4%
paraformaldehyde in PBS for 5 minutes. They were then
excised and embedded in OCT (Sankyo-Miles Inc).
Sections of 10 µm each were prepared using a cryostat at -28°C
and then air dried and immersed in PBS. The sections were treated with
5% normal donkey serum (Jackson ImmunoResearch
Laboratories), followed by overnight incubation at 4°C with anti-GFP
antibody at a dilution of 1:200 (3E6 monoclonal antibody, Qbiogene).
After washing them with PBS, the sections were incubated for 1 hour
with 3.75 µg/mL of Cy-conjugated AffiniPure
Donkey Anti-Mouse IgG (H+L) (Jackson
ImmunoResearch Laboratories) and with To-PRO-3
iodide for nuclear staining at a dilution of 1:500
(Molecular Probes). Thereafter, the sections
were washed with PBS, mounted with antifade substance, and observed
under a confocal laser scanning microscope (MRC-1024, Japan
Bio-Rad Laboratories).
Measurement of cGMP
Rat aortas were homogenized in 4%
trichloroacetic acid (pH 4.0) on ice. After
centrifugation, the supernatant was extracted 4 times
with water-saturated ether and then evaporated. The pellets were
redissolved in a buffer included in the ELISA kit. ELISA was performed
according to the manufacturer’s recommendation
(Amersham).
Statistical Analyses
Values are mean±SEM. The statistical
analyses were performed using ANOVA followed by the
Student-Neumann-Keul test. Differences with a
P value of <0.05 were
considered statistically
significant.
Results
AM-Induced
Endothelium-Dependent Vasorelaxation Is Inhibited by
Pretreatment With PI3K Inhibitors
We first examined the effects of PI3K inhibition on
AM-induced endothelium-dependent vasorelaxation using
rat thoracic aorta
(Figure 1
). AM ranging from 10-9
to 10-7 mol/L induced vasorelaxation in
aortic rings with endothelium (E/+ aortic rings)
precontracted with norepinephrine in a dose-dependent
manner, whereas AM-induced vasorelaxation was diminished in aortic
rings when endothelium was denuded (E/- aortic
rings). In fact, AM-induced vasorelaxation was negligible in E/-
aortic rings when 10-9 mol/L AM was
administered, whereas 10-9 mol/L AM did
induce vasodilation in E/+ aortic rings. Although AM, at concentrations
of 10-8 and
10-7 mol/L, elicited slight vasorelaxation
in E/- aortic rings, the magnitude was 14.4% and 32.6%,
respectively, compared with its effects on E/+ aortic rings at each
concentration. Forskolin and SNP induced a potent vasodilation in E/-
aortic rings (data not shown). Thus, the reduction in AM-induced
vasorelaxation observed in E/- aortic rings did not seem to be due to
potential injuries to smooth muscle layers when
endothelium was denuded. Pretreatment of E/+ aortic
rings with 20 µmol/L LY or 100 nmol/L wortmannin reduced AM-induced
vasorelaxation almost to the same levels as AM-induced vasorelaxation
in E/- aortic rings. In contrast, pretreatment of E/+ aortic rings
with 25 µmol/L PD, an inhibitor of
mitogen-activated protein kinase kinase (MEK) 1/2, did not
inhibit AM-induced vasodilation. AM-induced vasodilation in E/+ aortic
rings was abolished by pretreatment with L-NAME. Furthermore,
pretreatment of E/+ aortic rings with LY or wortmannin did not suppress
forskolin- or SNP-induced vasodilation (data not shown). These results
indicated that AM elicited endothelium-dependent
vasodilation, at least in part, via the NO-dependent pathway in rat
aorta and that AM-induced endothelium-dependent
vasodilation was suppressed by PI3K inhibition but not by MEK
inhibition. The results also suggested that the suppression of
AM-induced endothelium-dependent vasodilation by PI3K
inhibitors did not seem to be a result of the cytotoxic
effects of LY and wortmannin on vascular smooth muscle cells, because
vasodilation induced by cAMP and NO donor was
intact.
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) endothelium were precontracted with 10-6 mol/L norepinephrine, and indicated doses of AM were administered to examine its vasorelaxant effects. In some experiments, aortic rings were pretreated with 20 µmol/L LY (
), 100 nmol/L wortmannin (
), 20 µmol/L PD (
), or 10-5 mol/L L-NAME (
) before precontraction with norepinephrine, and effects of AM on vasodilation were examined. *P<0.01 and #P<0.05 vs aortic rings with intact endothelium at 10-7 mol/L AM stimulation (n=4).
AM Induces Akt Phosphorylation
in Rat Aorta
We next examined whether AM induced
phosphorylation of Akt in rat aorta. AM induced Akt
phosphorylation in a time-dependent manner
(Figure 2A). The phosphorylation of Akt
peaked 
15 minutes after stimulation with
10-7 mol/L AM. AM also induced Akt
phosphorylation in a dose-dependent fashion
(Figure 2B). AM, even at a low concentration of
10-11 mol/L,
phosphorylated Akt and
10-7 mol/L AM increased Akt
phosphorylation by 5.3-fold (n=4,
P<0.01). We also examined the
extent of Akt phosphorylation in rat aorta in the
presence and absence of endothelium
(Figure 2C). The extent of Akt
phosphorylation in denuded aorta was 69.5% (n=3,
P<0.05) compared with that in
intact aorta. This finding indicated that 30% of total Akt
phosphorylation occurred in a monolayer of
endothelium in rat aorta, suggesting that enormous
amounts of Akt were phosphorylated by AM in vascular
endothelium.
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Previous reports showed that AM induced activation of
ERK. We, therefore, examined whether AM induced ERK
phosphorylation in rat aorta
(Figure 3). AM induced ERK phosphorylation in
a dose-dependent manner in our ex vivo system. AM at
10-7 mol/L increased ERK
phosphorylation by 2.4-fold (n=3,
P<0.01) 15 minutes after
stimulation, although the ERK-dependent pathway did not seem to be
involved in AM-induced endothelium-dependent
vasorelaxation.
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We next examined whether LY and wortmannin indeed inhibited
AM-induced Akt phosphorylation in our system. As shown
in
Figure 4, AM-induced Akt phosphorylation was
significantly inhibited by pretreatment of aortas with LY or
wortmannin, whereas pretreatment with PD did not significantly inhibit
AM-induced Akt phosphorylation. In fact, AM-induced Akt
phosphorylation was suppressed to a level lower than
the control (nonstimulated) level by pretreatment with LY or
wortmannin, suggesting a basal level of phosphorylation
of Akt in the vessels.
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AM-Induced Akt Phosphorylation
Is Mediated by the Ca2+/CaM-Dependent
Pathway
Previous reports showed that PI3K activation was
mediated by the Ca2+/CaM-dependent pathway
in some cells.23 It was also
reported that Akt was activated by the
Ca2+/CaM-dependent pathway in some
cells.24 We, therefore,
wondered whether AM-induced Akt phosphorylation was
mediated by the Ca2+/CaM-dependent pathway.
When the Ca2+ concentration of Krebs-Ringer
bicarbonate solution was reduced, AM-induced Akt
phosphorylation decreased significantly in a
dose-dependent manner
(Figure 5A). We also examined the effects of the
CaM-dependent protein kinase inhibitor KN93 on AM-induced
Akt phosphorylation. AM-induced Akt
phosphorylation was inhibited by pretreatment with KN93
in a dose-dependent fashion
(Figure 5B). Furthermore, A23187, a calcium ionophore,
increased Akt phosphorylation in a dose-dependent
fashion, and A23187-induced Akt phosphorylation was
abolished by pretreatment with PI3K inhibitors, suggesting
that Akt phosphorylation induced by
Ca2+/CaM was mediated by the PI3K-dependent
pathway in rat aorta (see online Figure 1
in data supplement available
at http://www.circresaha.org).
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AM-Induced
Endothelium-Dependent Vasodilation Is Diminished in Rat
Aortas Expressing a Dominant-Negative Akt Mutant in the
Endothelium
To examine more specifically the role of Akt expressed
in the endothelium on AM-induced
endothelium-dependent vasodilation, we infected
Ad/dnAkt in rat abdominal aorta and examined its effects. To confirm
that adenovirus particles injected into the aortas had predominantly
infected the endothelium, we injected Ad/GFP in
abdominal aortas and examined its localization by
immunofluorescence. Because rat aortas exhibited a
marked green autofluorescence (fluorescence emission
maxima
[Em]
509 nm), we used an anti-GFP antibody to examine the localization of
GFP expression in them. As shown in
Figure 6A, GFP was predominantly expressed in
endothelium (red fluorescence
[Em 570
nm], right panel), whereas its expression in the smooth muscle layer
was negligible. Red fluorescence corresponding to GFP
expression was not observed in the endothelium of
noninfected control aorta (left panel). Under the same conditions, AM
induced vasodilation in a dose-dependent manner in aortic rings
prepared from abdominal aortas infected with Ad/GFP
(Figure 6B). The extent of AM-induced vasodilation in
Ad/GFP-infected aortic rings was almost the same as that observed in
noninfected aortic rings with intact endothelium,
suggesting that adenovirus infection was not cytotoxic to
endothelium. In contrast, although AM elicited
vasodilation in aortic rings prepared from Ad/dnAkt-infected aortas in
a dose-dependent manner, AM-induced vasodilation was significantly
reduced compared with that in Ad/GFP-infected aortas. In fact, the
AM-induced vasodilation observed in Ad/dnAkt-infected aorta was reduced
almost to the same extent as that observed in
endothelium-denuded aorta, suggesting that Akt
expressed in endothelium played a major role in
AM-induced endothelium-dependent
vasodilation.
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AM-Induced cGMP Production Is
Suppressed by PI3K/Akt Inhibition
We originally tried to examine whether AM induced eNOS
phosphorylation and whether AM-induced eNOS
phosphorylation was mediated by the PI3K/Akt-dependent
pathway. We immunoprecipitated eNOS in protein extracts prepared from
rat aortas and examined its phosphorylation by
immunoblotting with antiphosphoserine antibody.
However, we did not detect a significant amount of eNOS
phosphorylation in aortas stimulated with AM (data not
shown), probably because the amount of phosphorylated
eNOS was under detectable levels. We therefore decided to use cGMP
production in aortas as an indicator for NO production
in endothelium. We examined whether AM induced cGMP
production in rat aortas and whether AM-induced cGMP
production was mediated by the PI3K/Akt-dependent pathway. AM
significantly increased cGMP production in rat thoracic aortas
in a dose-dependent manner
(Figure 7A). AM at 10-7 mol/L
increased the production of cGMP by 2.2-fold (n=3,
P<0.05). AM-induced increase
of cGMP production was significantly inhibited to basal levels
by pretreatment with LY. AM-induced increase of cGMP production
was also significantly inhibited to basal levels by chelating
extracellular Ca2+ with EGTA. Therefore, the
suppression of Akt phosphorylation by PI3K inhibition
or by inhibition of the Ca2+/CaM-dependent
pathway correlated with suppression of cGMP production by
inhibition of the PI3K- or
Ca2+/CaM-dependent pathway in rat aortas.
Furthermore, pretreatment with L-NAME also significantly inhibited
AM-induced cGMP production to basal levels, suggesting that
cGMP production could be an indicator of NO production
in rat aortas. Finally, we measured cGMP production in
abdominal aorta infected with Ad/GFP or Ad/dnAkt
(Figure 7B). Infection of the abdominal aorta with those
adenovirus constructs did not remarkably change the basal
production of cGMP. In contrast, AM-induced increase of cGMP
production was significantly inhibited in Ad/dnAkt-infected
aortas compared with that in Ad/GFP-infected and noninfected aortas,
indicating that Akt expressed in the endothelium was
involved in AM-induced NO/cGMP production.
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Additional results appear in an online data supplement available at http://www.circresaha.org.
Discussion
Although it was reported that AM had a potent
vasorelaxant activity,2 the
precise mechanisms were not clear. Because AM stimulates cAMP
production, it was originally thought that cAMP was a sole
second messenger for AM-induced vasodilation. On the other hand, we and
others reported that AM elicited endothelium-dependent
vasodilation via, at least partly, the NO/cGMP-mediated
pathway.14 15
Thus, it was possible that AM activated eNOS by stimulating the
direct binding of CaM to eNOS and a subsequent conformational change of
eNOS,16 17 18
because AM reportedly has the capacity to increase
[Ca2+]i in
endothelium.4 9
Here, we have shown that AM-induced
endothelium-dependent vasodilation and AM-induced
increase of cGMP production, which we used as an indicator for
NO production, depended on the PI3K/Akt-mediated pathway. To
our knowledge, this is the first report that shows that AM induces Akt
phosphorylation. Although we detected Akt
phosphorylation in whole vessels, several data
presented in this paper indicated that Akt
phosphorylation occurring in vascular
endothelium played a major role in AM-induced
endothelium-dependent vasodilation. First, pretreatment
with LY or wortmannin of rat aortas with intact
endothelium suppressed AM-induced vasodilation to the
same level as that in endothelium-denuded rat aortas.
LY and wortmannin did not seem to cause remarkable cytotoxic effects on
vascular smooth muscle cell layers, because forskolin and SNP potently
relaxed aortic rings pretreated with LY. Furthermore, pretreatment with
PD did not inhibit AM-induced vasodilation in aortas with intact
endothelium. Second, Akt
phosphorylation occurring in a single layer of vascular
endothelium was estimated to be 30% of the total
phosphorylation of Akt occurring in the whole aorta,
suggesting that a huge amount of Akt phosphorylation
occurred in vascular endothelium. Third, cGMP
production in rat aorta was significantly inhibited by
pretreatment with LY, and this inhibition correlated with the
suppression by LY of AM-induced endothelium-dependent
vasorelaxation. Fourth, a dnAkt mutant that expressed predominantly in
endothelium after infection with Ad/dnAkt inhibited
AM-induced vasodilation to the same level as that observed in
endothelium-denuded aortas. Finally, AM-induced
increase of cGMP production was significantly suppressed in
aortas that expressed dnAkt in the endothelium.
Recently, two studies have shown that Akt directly
phosphorylates eNOS on Ser1179 and that
phosphorylated eNOS is active at a low
Ca2+
concentration.19 20
Thus, eNOS phosphorylation on Ser1179 appears to be
critical for facilitating CaM binding to eNOS. Our results indicated
that AM-induced vasodilation was almost entirely
endothelium dependent at a low concentration of AM
(10-9 mol/L), at which concentration AM
could stimulate Akt phosphorylation and cGMP
production. In contrast, at high concentrations
(10-8 mol/L and
10-7 mol/L), AM-induced vasodilation was
mediated, partly, by an endothelium-independent
mechanism; this was probably a direct effect of AM on vascular smooth
muscle cells leading to an increase of intracellular cAMP and
vasodilation. Thus, although endothelium dependence of
AM-induced vasodilation reportedly relies on the species and vessels
used,5 25 26
it appears that, under physiological conditions,
AM-induced vasorelaxation is largely endothelium
dependent and mediated by Akt activation, at least, in rat
aorta.
Although it was reported that
Ca2+ was dispensable for shear
stress-induced Akt
phosphorylation,20
our results clearly showed that the
Ca2+/CaM-dependent pathway was necessary for
AM-induced Akt phosphorylation. Our results also
indicated that Ca2+/CaM-induced Akt
phosphorylation was mediated by the PI3K-dependent
pathway (see online Figure 1 in data supplement available at
http://www.circresaha.org). Thus, Ca2+/CaM
appeared to be necessary not only for direct activation of eNOS by
binding to the CaM binding domain in eNOS but also for activation of
eNOS via its phosphorylation by Akt. The involvement of
Ca2+/CaM in the activation of PI3K/Akt has
been suggested in several reports. CaM-dependent protein kinase kinase
directly phosphorylated Akt on threonine 308 and
activated Akt, which was required for antiapoptotic
effects induced by the Ca2+/CaM-dependent
pathway.24 Insulin-induced
activation of PI3K was inhibited in hepatocytes by
pretreatment with EGTA or the Ca2+ channel
inhibitors gadolinium and
nickel.23 Furthermore, the
p110 subunits of PI3K contained a highly conserved motif that bound to
CaM in a Ca2+-dependent
way.27 On the other hand, it
was reported that intracellular Ca2+ was not
required for epidermal growth factor– and platelet-derived growth
factor–induced Akt activation in BALB/c-3T3
fibroblasts.28 It was also
shown that the CaM-dependent protein kinase inhibitor KN-62
did not inhibit insulin-induced PI3K or Akt activity in rat skeletal
muscle.29 Thus, it appears
that the involvement of Ca2+/CaM in the
activation of PI3K/Akt differs depending on the mitogens and cell types
used.
The precise pathways leading to activation of Ca2+/CaM and PI3K/Akt by AM are not clear at present, partly because the cognate receptors for AM have not been sufficiently characterized.30 31 Although it was reported that AM increased [Ca2+]i via phospholipase C activation and inositol triphosphate formation,9 it is not clear how AM receptors activate phospholipase C. It also remains unclear whether AM receptors have a docking site for the p85 subunit of PI3K. Furthermore, there are no highly specific antagonists for AM receptors available at present. Thus, future studies are required to elucidate how AM receptors transmit signals to the Ca2+/CaM- and PI3K-dependent pathways.
Accumulated evidence has indicated that PI3K/Akt is implicated in a variety of biological processes including glycogen synthesis, antiapoptotic actions, modulation of cell proliferation and eNOS activation.19 20 32 33 34 Furthermore, it has been reported that PI3K/Akt is implicated in stimulation of angiogenesis.35 Thus, the PI3K/Akt-dependent pathway seems to play pivotal roles in vascular endothelium such as maintenance of vascular tone, protection against apoptotic cell death, repair of endothelium, and modulation of angiogenesis. Because AM has an activity to activate Akt and induce NO production, modulation of AM activity in the endothelium may be a useful strategy to inhibit progression of atherosclerosis.
Acknowledgments
This study was supported in part by Grants-in-Aid 09281206 and 10218202 from the Ministry of Education, Culture and Science of Japan (to Y.H.). We thank Reiko Sato, Etsuko Taira, and Marie Morita for technical assistance.
Footnotes
Original received January 24, 2001; revision received April 18, 2001; accepted May 1, 2001.
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