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(Circulation Research. 2001;89:63.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Adrenomedullin Induces Endothelium-Dependent Vasorelaxation via the Phosphatidylinositol 3-Kinase/Akt–Dependent Pathway in Rat Aorta

Hiroaki Nishimatsu, Etsu Suzuki, Daisuke Nagata, Nobuo Moriyama, Hiroshi Satonaka, Kenneth Walsh, Masataka Sata, Kenji Kangawa, Hisayuki Matsuo, Atsuo Goto, Tadaichi Kitamura, Yasunobu Hirata

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 N^G-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. N^G-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.

View larger version (17K): [in this window] [in a new window]   Figure 1. AM-induced endothelium-dependent vasorelaxation is suppressed by PI3K inhibition. Rat aortic rings with (•) or without () 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.

View larger version (40K): [in this window] [in a new window]   Figure 2. AM induces Akt phosphorylation. A, AM phosphorylates Akt in a time-dependent manner. Rat aortas were incubated with 10-7 mol/L AM for indicated periods. Fifty micrograms of each protein extract was immunoblotted with a phosphospecific anti-Akt antibody (*pAkt), which recognizes catalytically active Akt, or anti-Akt antibody (Total Akt), which recognizes total Akt1/2, regardless of whether Akt is phosphorylated or not. B, AM phosphorylates Akt in a dose-dependent manner. Rat aortas were incubated with AM at concentrations ranging from 10-11 to 10-7 mol/L for 15 minutes. Immunoblot analyses were performed in the same way as in panel A. Histogram shows relative intensity of Akt phosphorylation by 10-7 mol/L AM stimulation compared with nonstimulated control. *P<0.01 vs control. C, Effects of endothelium denudation on AM-induced Akt phosphorylation. Rat aortas with or without endothelium, E(+) or E(-), respectively, were incubated with 10-7 mol/L AM for 15 minutes. Immunoblot analyses were performed in the same way as in panel A. Histogram shows relative intensity of Akt phosphorylation in rat aorta with or without endothelium. #P<0.05 vs E(-) (n=4).

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.

View larger version (31K): [in this window] [in a new window]   Figure 3. AM stimulates ERK phosphorylation. A, Rat aortas were incubated with AM at concentrations ranging from 10-11 to 10-7 mol/L for 15 minutes. Fifty micrograms of each protein extract was immunoblotted with a phosphospecific anti-ERK1/2 antibody (*pERK), which recognizes catalytically active ERK, or anti-ERK antibody (Total ERK), which recognizes total ERK1/2, regardless of whether ERK is phosphorylated or not. B, Histogram showing relative intensity of ERK phosphorylation by 10-7 mol/L AM stimulation compared with nonstimulated control. #P<0.01 vs control (n=3).

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.

View larger version (44K): [in this window] [in a new window]   Figure 4. AM-induced Akt phosphorylation is inhibited by LY and wortmannin. A, Rat aortas were pretreated with 20 µmol/L LY, 100 nmol/L wortmannin (WT), or 20 µmol/L PD for 30 minutes and stimulated with 10-7 mol/L AM for 15 minutes. Immunoblot analyses were performed as described in Figure 2 legend. B, Histogram showing relative intensity of phosphorylated Akt. *P<0.001 vs 10-7 mol/L AM stimulation (n=4).

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).

View larger version (44K): [in this window] [in a new window]   Figure 5. AM-induced Akt phosphorylation depends on the Ca2+/CaM-mediated pathway. A, Rat aortas were incubated in Krebs-Ringer bicarbonate solution with decreasing concentrations of Ca2+. Ca2+-free solution was obtained by adding 1 mmol/L EGTA to the solution. Rat aortas were then stimulated with 10-7 mol/L AM for 15 minutes. Immunoblot analyses were performed as described in Figure 2 legend. Histogram shows relative intensity of phosphorylated Akt. *P<0.01 and #P<0.05 vs 2.5 mmol/L Ca2+ (n=4). B, Rat aortas were incubated together with increasing doses of CaM-dependent protein kinase inhibitor KN93. Rat aortas were then stimulated with 10-7 mol/L AM for 15 minutes. Immunoblot analyses were performed as described in Figure 2 legend. Relative intensity of phosphorylated Akt is also shown.

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 [E_m] 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 [E_m 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.

View larger version (54K): [in this window] [in a new window]   Figure 6. AM-induced vasodilation is suppressed in rat aorta expressing a dominant-negative Akt mutant in endothelium. A, Adenovirus particles injected into rat abdominal aorta are predominantly expressed in the endothelium layer. Ad/GFP (109 PFU) was injected into rat abdominal aortas, and aortas were fixed 48 hours after infection. Noninfected rat abdominal aortas were used as negative control. GFP-positive cells were stained with Cy (red fluorescence). Nuclei in aortas were stained with To-PRO-3 iodide (blue fluorescence). Areas showing green fluorescence represent autofluorescence in vessels. Note that nuclei in the endothelium that were double-stained with Cy and To-PRO-3 iodide show pink fluorescence. Arrowheads indicate GFP-positive nuclei in the endothelium. Bars=10 µm. B, Ad/dnAkt () or Ad/GFP (•) was infected in the endothelium of rat abdominal aorta, and AM-induced vasodilation was examined 48 hours after adenovirus infection. Noninfected abdominal aortas with () or without () endothelium were used as the control. #P<0.05 vs Ad/GFP infection at each concentration of AM stimulation (n=4).

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.

View larger version (33K): [in this window] [in a new window]   Figure 7. Effects of PI3K/Akt inhibition and low Ca2+ on AM-induced cGMP production in rat aorta. A, Rat thoracic aortas were pretreated with 20 µmol/L LY or L-NAME or incubated in Ca2+-free Krebs-Ringer bicarbonate solution with 1 mmol/L EGTA. Rat aortas were then stimulated with 10-7 mol/L AM for 5 minutes, and cGMP extracted from aortas was measured by ELISA. *P<0.05 vs 10-7 mol/L AM stimulation (n=5); #P<0.05 vs nonstimulated control (n=5). B, Ad/dnAkt (109 PFU) or Ad/GFP (109 PFU) was injected into rat abdominal aortas, and experiments were performed 48 hours after adenovirus infection in the same way as in panel A. **P<0.001 and ##P<0.01, compared with cGMP level in Ad/dnAkt-infected aorta stimulated with AM (n=3).

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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