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ARTICLE |
CORRESPONDENCE Emilio Hirsch: Emilio.Hirsch{at}unito.it OR Giuseppe Lembo: lembo{at}neuromed.it
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E. Hirsch and G. Lembo contributed equally to this work.
Angiotensin II is the primary effector peptide of the renin-angiotensin system and acts as a hormonal and local factor. It plays a key role in blood pressure homeostasis; high plasma levels of the peptide are a main trait of renovascular hypertension. In addition, angiotensin II overactivity has been involved in other widely diffused cardiovascular diseases, such as atherosclerosis and congestive heart failure.
The effects of angiotensin II are exerted on several target organs; however, especially the vascular action explains its impact on blood pressure. Angiotensin II increases vascular tone by activating calcium-flux, oxidative stress, and cell growth in vascular smooth muscle and, concomitantly, by promoting an inflammatory reaction in the vessel wall.
Several pharmacological interventions have been developed to attenuate angiotensin II vascular effects. In particular, inhibition of angiotensin II synthesis and, subsequently, blocking of its high affinity subtype-1 (AT1) have allowed the targeting of angiotensin II-dependent negative effects.
Recent evidence suggests that the vasculotoxic effects of angiotensin II can be mediated via PI3K signaling pathways (1). PI3Ks are a family of lipid and protein kinases that are responsible for the phosphorylation of PtdIns at the position D3 of the inositol ring. These molecules act as secondary messengers and influence a variety of cellular responses, including proliferation, survival, and cytoskeletal remodeling (2). In vivo, PI3Ks of the class I subfamily produce PtdIns(3,4,5)P3 that serves as a docking site for the pleckstrin homology domain that is present in numerous proteins that act as PI3K downstream effectors. Class I PI3Ks are divided in two subgroups depending on their biochemical properties. The class IA group consists of PI3Ks thatwith the exception of PI3Kß that also can respond to GPCRsare activated mainly by tyrosine kinase receptors (3). Conversely, the unique member of class IB, PI3K (p110
), is activated exclusively by GPCRs; it binds directly to the ß
subunits of heterotrimeric G proteins (4) but its activity also can be modulated by interaction with an adaptor protein, p101 (5). Deletion of the PI3K
gene in mice is compatible with life and causes a protection from leukocyte recruitment by inflammatory stimuli (6, 7). A growing set of evidence indicates that PI3K
also is expressed in the cardiovascular system where it negatively controls cardiomyocyte contractility (810).
The specific PI3K isoform that is involved in angiotensin II signaling is still controversial. Using pharmacological inhibitors that block PI3K function without distinguishing between isoforms, it has been found that, in vascular smooth muscle cells, angiotensin II requires a PI3K activity to stimulate calcium channels and induce the calcium influx that governs the vascular contractile response (11). Although in porcine coronary artery smooth muscles, tyrosine phosphorylation and class IA PI3Ks may be involved (12), in rat portal vein myocytes, the free ß13 dimers that are generated by the activation of the G13-coupled AT1A receptor directly stimulate PI3K activity; this indicates a crucial role for the class IB enzyme, PI3K
(13). Recent evidence indicates that PI3K
and PI3K
, but not PI3Kß, are expressed by myocytes freshly isolated from rat portal veins (14). Although classes IA and B PI3K isoforms are present in rat portal vein myocytes, injection of antibodies that recognize different PI3K isoforms into these cells indicates that the angiotensin II-dependent activation of L-type Ca2+ current is inhibited by blocking PI3K
but not PI3K
; this suggests a crucial role for PI3K
in angiotensin II signal transduction (15).
Despite the finding that smooth muscle cells require PI3K for the angiotensin IImediated intracellular Ca2+ concentration increase, in vivo studies that address the role of PI3K
in vascular responses to angiotensin II are missing. We examined the vascular responses to angiotensin II stimulation in mice lacking PI3K
and found that PI3K
/ vessels show reduced contractile responses to angiotensin II, a markedly decreased angiotensin IImediated ROS production, and intracellular Ca2+ mobilization. As a consequence of these effects, mice lacking PI3K
are protected strongly from the hypertension that is induced by administration of angiotensin II in vivo.
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RESULTS |
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Impaired angiotensin II-dependent Akt phosphorylation in PI3K-deficient aortas
To test a possible involvement of PI3K in angiotensin IImediated signal transduction and in the vascular responses that lead to hypertension, primary smooth muscle cells were isolated from aortas of wild-type and PI3K
null mice. The cells were stained positive for smooth muscle actin and, when derived from wild-type animals, presented the mRNA for PI3K
(Fig. 4 A). Similarly, only wild-type cells showed the expression of the PI3K
protein, albeit at very low levels (Fig. 4 B, top). In PI3K
/ samples, no changes in expression of other PI3Ks (e.g., PI3Kß) were detected in mutant samples (Fig. 4 B, bottom). Because PI3K
could play a role in GPCR-mediated signaling, cultures were expanded for no more than five passages and cells were stimulated with angiotensin II. In apparent contrast to a putative role of PI3K
in angiotensin II signaling, analysis of the PI3K-dependent phosphorylation of protein kinase B (PKB)/Akt at Ser473 did not show any difference between wild-type and mutant cells (Fig. 4 C). However, in aortic smooth muscle cells (ASMCs) of both genotypes, the angiotensin II activation of PKB/Akt was blocked by the EGFR kinase blocker, AG1478 (Fig. 4 C). These results are in agreement with the previous finding that in vascular smooth muscle cells, angiotensin IImediated signaling triggers EGFR transactivation (17), and thus, bypasses PI3K
function by an EGFR-dependent activation of class IA PI3Ks.
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PI3K/ mice are protected from angiotensin IIevoked vascular ROS generation
In hypertension, the increase of vascular ROS contributes to endothelial dysfunction and vessel contraction. Because angiotensin II induces ROS production in vascular smooth muscle cells, the involvement of PI3K in this process was evaluated next. To test the effects of angiotensin IImediated ROS production, vascular contractility was measured after the combined administration of angiotensin II and tiron, a potent ROS scavenger. As expected, in wild-type vessels, tiron significantly reduced the angiotensin IIdependent enhancement of vascular wall tension. In contrast, in mutant vessels, the addition of tiron did not modify further the blunted response to angiotensin II; this suggests that angiotensin II-evoked ROS generation requires the activation of PI3K
(Fig. 6 A). To prove further that PI3K
was involved in the angiotensin IImediated ROS production, the generation of ROS was measured directly in wild-type and mutant vessels after angiotensin II stimulation. The levels of ROS production in tissues of the two genotypes were comparable in basal conditions (Fig. 6 B). After administration of angiotensin II, wild-type vessels responded with a significant increase of ROS generation over the basal level (370% ± 70% over control, P < 0.01); this effect was blocked by preincubation with wortmannin (Fig. 6 B). In contrast, stimulated PI3K
-deficient aortas showed a response that was reduced by 70% over that of similarly treated wild-type samples (P < 0.05); wortmannin did not affect this response further. PI3K
KD/KD mice showed a similarly impaired response (unpublished data); this confirms that the catalytic activity of PI3K
is a key event that is necessary for the angiotensin IIdependent generation of ROS. This effect was mediated by AT1 receptors because it was inhibited by candesartan, but not by PD123319 (unpublished data). Angiotensin II-mediated ROS production was inhibited significantly by pretreatment with PTX; this further suggested an involvement of a G
i-coupled angiotensin II receptor in vessel (Fig. 6 B). In addition, vascular ROS production that was evoked by phenylephrine, an agonist of a G
q-coupled receptor, was very weak and seemed to be similar in both mouse strains (Fig. 6 B).
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In the vasculature, superoxide can react with other free radicals, such as nitric oxide, and generate peroxynitrites that lead to nitrotyrosine production. In wild-type samples, tyrosine nitration was undetectable in basal conditions but became apparent after stimulation with angiotensin II (Fig. 6 D, left). In contrast, the level of nitrotyrosines in aortas that were derived from PI3K/ mice was undetectable in basal conditions and after angiotensin II stimulation (Fig. 6 D, right).
PI3K is required for angiotensin IImediated extracellular Ca2+ entry through activation of PKB/Akt
Although in wild-type vessels, contractility in response to angiotensin II was reduced by tiron, the impact of the antioxidant was smaller than that caused by the lack of PI3K (Fig. 6 A). This observation suggested that in the absence of PI3K
, the impaired angiotensin IImediated vasoconstriction was due not only to reduced ROS production. During vessel contraction, a crucial role is played by the increase of intracellular Ca2+, which stimulates the contractile machinery. To test whether the reduced contractile response to angiotensin II in PI3K
/ vessels was due to a defective induction of intracellular calcium increase, the ability of angiotensin II to enhance [Ca2+]i was evaluated in wild-type and mutant mesenteric artery preparations. In resting conditions, vessels of wild-type and mutant mice showed similarly low fluorescence levels (Fig. 7 A). The addition of angiotensin II caused a time-dependent increase in fluorescence intensity with a maximal peak at 70 s after agonist exposure (Fig. 7 A). However, the peak fluorescence that was detected in PI3K
-deficient vessels was 73% weaker than that measured in wild-type controls (Fig. 7 B, n = 7). In contrast, phenylephrine elicited a comparable fluorescence response in wild-type and mutant samples (Fig. 7 B).
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L-type Ca2+ channels can be regulated by the levels of the second messenger, cAMP. Our previous results indicated that in the heart, PI3K helps to reduce cAMP concentration independently of its kinase activity (10). PI3K
/ mice, where PI3K
is absent, show increased cAMP in basal conditions but no effect can be detected in PI3K
KD/KD mice that express a catalytically inactive PI3K
. As increased cAMP levels could influence L-type Ca2+ channels of smooth muscle negatively, cAMP concentration was determined in wild-type and PI3K
/ aortas. In agreement with the finding that in PI3K
/ and in PI3K
KD/KD vessels angiotensin IIdependent vasoconstriction is affected equally, measurements in resting conditions and after stimulation with the ß-adrenergic agonist, isoproterenol (1 µM), never revealed differences between wild-type and PI3K
-deficient aortas (basal [cAMP]: 0.081 ± 0.019 nmol/g and 0.062 ± 0.022 nmol/g, respectively; isoproterenol-induced [cAMP]: 0.391 ± 0.140 nmol/g and 0.280 ± 0.174 nmol/g, respectively; n = 6 for each genotype). The finding that angiotensin II-dependent vasoconstriction requires the kinase activity of PI3K
demonstrates the involvement of signaling pathways that are linked to PtdIns(3,4,5)P3 production. Because the enzymatic activity of PI3K
is required for PKB/Akt phosphorylation, PKB/Akt activation might mechanistically couple PI3K
to extracellular Ca2+ entry. To explore this hypothesis, PKB/Akt was blocked by transfecting a DN-Akt in isolated vessels. A reduction of angiotensin IIevoked vasoconstriction was detected only in DN-Akttransfected wild-type samples that were leveled to the response of PI3K
/ vessels (24 ± 6% and 19 ± 8% increase over basal in wild-type and PI3K
/ samples, respectively; not significant; n = 4 for each genotype). This result was supported fully by data on calcium fluctuations which showed that DN-Akt expression blunted angiotensin II effects only in wild-type vessels (Fig. 7 E). In contrast, expression of DN-Akt did not affect the PKB/Akt-independent vascular response and calcium mobilization that are induced by potassium (unpublished data). These data indicate that angiotensin II acts on L-type Ca2+ channels through a PI3K
/Akt pathway.
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DISCUSSION |
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It is well known that angiotensin II represents one of the major mediators that is involved in the development of hypertension. The protection from blood pressure increase after angiotensin II administration that was seen in PI3K/ mice has to be ascribed mainly to the vascular phenotype and not to the previously reported enhancement of cardiac contractility (8, 10), that should, in principle, increase cardiac output and blood pressure. In agreement with this view, several studies demonstrated that in the pathogenesis of angiotensin IImediated hypertension, a critical role is played by ROS production (21) as well as imbalanced homeostasis of calcium (22, 23). Therefore, it is possible that the protection from angiotensin IIinduced hypertension that is observed in the absence of PI3K
could be due to the beneficial reduction of oxidative stress and intracellular calcium concentration. However, PI3K
-deficient vessels also were protected from the chronic vascular remodeling that occurred as a consequence of hypertensive vascular insult. Although a minor involvement of inflammatory response, evoked particularly by vascular ROS production, could not be excluded, our data point to a crucial role of PI3K
in vascular smooth muscle, rather than in the onset of vascular inflammation.
Although a previous report suggests that PI3K is not present in rat aortas (24), our finding of PI3K
expression in aortic smooth muscle is in agreement with the established notion of its existence in rat portal vein myocytes (14, 15). Nevertheless, PI3K
was expressed at low levels and it is possible that the different nature of the antibodies that were used in the two studies might account for this discrepancy. Our data further located p110
expression in cultured ASMCs; however, analysis of angiotensin II stimulation revealed that mutant cultured cells responded equally as well as wild-type controls. In contrast, PI3K
/ intact vessels showed a clearly impaired response to angiotensin II. As an explanation for these divergent results, and in agreement with previous studies (17) in ASMCs cultured in vitro, our results suggest that the angiotensin IIdependent transactivation of the EGFR induces class IA PI3K-dependent activation and bypasses the requirement for PI3K
function. Our data suggest that in intact aortas, the pathway that is dependent on EGFR transactivation is less critical, and that, in vivo, PI3K
is used preferentially for angiotensin IImediated PKB/Akt activation. Thus, our apparently contrasting findings indicate that for the analysis of vascular signal transduction events, in vivo experiments in genetically altered organisms are required; in vitro studies with primary ASMCs might be limited by the adaptation of cells to culture conditions. A similar situation was described in rat portal vein myocytes that, when freshly isolated, express only p110
, -
, and -
, but start to express p110ß after a few days of culture (14). Because angiotensin II receptors can relay equally to PI3Kß and -
(14), the presence of PI3Kß in cultured cells might compensate for the absence of PI3K
. The limited amount of low-passage primary ASMCs that is obtainable from mice and the lack of specific antibodies for immunohistochemistry prevented us from investigating whether PI3Kß is absent from ASMCs.
Nonetheless, our analysis of in vivo stimulated aortas clearly indicated that multiple angiotensin IImediated vascular responses depend on PI3K. So far, several mechanisms have been proposed to explain the activation of PI3K and the subsequent phosphorylation of PKB/Akt that is induced by angiotensin II. Although some studies include a role for tyrosine kinaseactivated class IA PI3Ks (1, 12, 17), others reported that activation of Gq-coupled angiotensin II receptors can mediate PKB/Akt phosphorylation (25). Despite these indications, we showed by genetic means that angiotensin II signals through PI3K
in intact aortas and that this process is mediated by the activation of a Gi-coupled receptor. In general, signaling by the angiotensin II AT1 receptors mainly is dependent on Gq-containing heterotrimeric G proteins (26); however, other studies also point to an involvement of Gicoupled angiotensin II AT1 receptors, particularly in the angiotensin IIdependent inhibition of adenylate cyclase (27). In addition, an involvement of Gi and PI3K is crucial for the signal transduction events that lead to Raf-1 activation in response to angiotensin II (28). In further agreement with a role of Gi in the angiotensin IImediated signal transduction in smooth muscle cells, we and others (29) found that treatment with PTX inhibits the angiotensin IIdependent ROS generation. Our data indicate that the Gi-dependent branch of angiotensin II signaling might have considerable importance in the vascular responses that lead to vasoconstriction and hypertension.
The finding that the addition of wortmannin, a PI3K inhibitor with no isoform selectivity, in wild-type tissues blunted the angiotensin IIdependent vascular response to the level that was observed in mutant arteries, indicated that in intact vessels, PI3K probably is the unique PI3K isoform that is involved in the process. In vivo blockade of EGFR-dependent signaling did not exert significant effects on angiotensin IImediated PKB/Akt phosphorylation. Because other agonists, such as phenylephrine or acetylcholine, exerted identical effects in the two genotypes, the absence of PI3K
did not induce a generalized impairment of vascular function; this demonstrates a specific involvement of PI3K
in the angiotensin IIdependent vascular contractile response. In agreement with previous reports that suggested a role of ROS in vasoconstriction (30), scavenging of angiotensin IImediated ROS production reduced vasoconstriction in wild-type preparations but was unable to exert a further inhibitory effect in PI3K
/ samples. Consistently, we found a marked reduction of ROS production in response to angiotensin II in PI3K
-null vessels. As an explanation for this defective ROS generation, PI3K
seemed to be crucial for angiotensin II activation of Rac, a key event that is required for NAD(P)H oxidase assembly and ROS generation (17, 31). Our finding is in agreement with the PtdIns (3,4,5)P3-dependent activation of GTP exchange factors triggering Rac. The involvement of a similar mechanism in the activation of NAD(P)H oxidase recently was outlined by the cloning of P-Rex, a GTP exchange factor for Rac, that, in neutrophils, mediates the respiratory burst response in a GPCR- and PI3K-dependent way (32).
The mechanism by which free radicals increase vascular tone has been attributed to a direct smooth muscle effect and a reduced nitric oxide bioavailability (33). The finding of increased nitration of tyrosines in wild-type, but not mutant, vessels that were stimulated with angiotensin II, further supports this view and suggests that PI3K contributes to the angiotensin IIdependent depletion of vascular nitric oxide. Although antioxidant agents reduced the angiotensin IIdependent vasoconstriction in wild-type control samples, the overall effect that was caused by the lack of PI3K
was significantly stronger. This fact suggested that mechanisms other than decreased ROS production could be triggered concomitantly by PI3K
signaling and contribute to the impact on angiotensin II vascular response. In addition to through oxidative stress, angiotensin II can control vascular tone by increasing intracellular calcium concentration in smooth muscle cells (34). It is known that voltage-gated L-type Ca2+ channels represent the major pathway for calcium entry and play an important role in excitation-contraction coupling (35). Previous reports indicated that PI3K
may act as a key mediator of angiotensin IIdependent voltage gated L-type Ca2+ channel activation in rat portal vein myocytes (13, 14). In these cells, stimulation of angiotensin II receptors frees the ß
dimer of the G13 protein and activates PI3K
, which, in turn, causes an increase in intracellular Ca2+ concentration. Furthermore, intracellular infusion of an anti-PI3K
antibody causes reduced PtdIns(3,4,5)P3 generation, and the inhibition of angiotensin II elicited stimulation of Ca2+ current (15). In agreement with these data, our results clearly showed that the angiotensin IImediated elevation of intracellular Ca2+ concentration was defective in PI3K
/ vessels. Our results with pharmacological inhibitors further clarify that PI3K
is involved in the mobilization of extracellular Ca2+ through L-type channels, and does not influence Ca2+ release from intracellular stores. Thus, the PI3K emerging signaling that was described to be involved in the regulation of Ca2+ release from intracellular stores in cardiac cells through membrane anchoring of Tec and subsequent phospholipase C activation seems not to be recruited by angiotensin II at the vascular level (36). Recently, PI3K
was implicated, in this case in the heart, in a kinase-independent activation of PDE3B, an enzyme that hydrolyzes cAMP, a secondary messenger that controls L-type Ca2+ channels function (10). Despite this fact, cAMP concentration in aortas was not affected by the absence of PI3K
; in addition, although cardiac contractility was different in mice lacking PI3K
(PI3K
/) or expressing a kinase-dead mutant (PI3K
KD/KD) (10), the angiotensin IImediated vasoconstriction was equally blunted in the two PI3K
/ and PI3K
KD/KD genotypes. This clearly excludes a cAMP-related kinase-independent function of PI3K
in the angiotensin IImediated modulation of L-type Ca2+ channel activity at the vascular level. Conversely, in agreement with an involvement of the catalytic activity of PI3K
, PI3Ks were found to enhance native voltage-dependent L-type Ca2+ currents through the activation of PKB/Akt, which causes rapid plasma membrane relocalization of channel subunits (37). Similarly, the expression of a constitutively active PKB/Akt mutant in the murine heart leads to increased L-type Ca2+ channel activation and enhanced contractility (38). ROS production can sustain PKB/Akt phosphorylation by the oxidative stressdependent inactivation of the PtdIns(3,4,5)P3 3-phosphatase, phosphatase with tensin homology (39). In this way, it could be hypothesized that the concurrent production of PtdIns(3,4,5)P3 and ROS, induced by angiotensin II, cooperate to keep PKB/Akt in its phosphorylated state, and consequently, increase L-type Ca2+ channel activity. Our results, which were obtained with transfection of vessels with a dominant-negative form of PKB/Akt blocked angiotensin IIevoked [Ca2+]i increase, clearly indicate that a PI3K
/Akt signaling pathway crucially regulates angiotensin IImediated vascular contractility.
In light of these results, PI3K represents a crucial intracellular signaling molecule which drives multiple mechanisms that are responsible for the angiotensin IIdependent vasculotoxic and hypertensive effects; thus, targeting this enzyme with specific inhibitors could be exploited to expand the therapeutic strategy that is aimed at treating hypertension.
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MATERIALS AND METHODS |
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Evaluation of blood pressure in conscious mice.
Radio-telemetric analysis of blood pressure and echocardiography were assessed as described (40). Angiotensin II (0.5 mg/kg/d in 0.9% NaCl), phenylephrine (0.15 mg/kg/d in 0.2% ascorbic acid), or vehicle were infused for 21 d through osmotic minipumps. Because blood pressure response in the first 3 d of chronic infusion was masked by the stress response to surgical procedures, acute blood pressure response to angiotensin II was evaluated by arterial catheterization as described (41).
Evaluation of vascular reactivity.
Vascular reactivity and structure were assessed in mesenteric arteries as described previously (42). Increasing doses of angiotensin II (109 to 106 M) were tested alone and in the presence of the PI3K inhibitor, wortmannin (107 M, 30 min); the antioxidant agent, tiron (103 M, 10 min); the AT1 antagonist, candesartan (106 M, 15 min); or the AT2 antagonist, PD123319 (106 M, 15 min). Moreover, phenylephrine (109 to 105 M) and acetylcholine (109 to 105 M) vascular responses were tested in all vessels. For selected experiments, mechanical removal of the endothelial layer was demonstrated by the absence of acetylcholine-mediated vasorelaxation.
Isolation of ASMCs.
ASMCs were isolated from male wild-type and PI3K/ mouse aortas using published procedures (43). All experiments were performed using cells at the fourth/fifth passage. Cell lysates were prepared from ASMCs at 80% confluence, starved for 24 h, and then stimulated with the agonists. For RT-PCR analysis, total RNA was extracted using RNAeasy columns (QIAGEN). Primers used were described previously (6).
In vivo aortic tissue stimulation.
For in vivo angiotensin II stimulation, mice were anesthetized with i.p. thiopental (50 mg/kg). Angiotensin II was infused by i.p. injection. For PTX inhibition experiments, mice were injected i.p. with PTX (150 mg/kg) 24 h before angiotensin II stimulation. Effectiveness of the PTX treatment was assayed by the detection of full inhibition of muscarinic chronotropic response. At the end of angiotensin II stimulation, thoracic aortas were removed and proteins were extracted.
Three distinct methods were used to evaluate oxidative stress: chemiluminescence with lucigenin (16), histochemistry with dihydroethidium, and immunohistochemistry with anti-nitrotyrosine antibodies (44). Vascular Rac activity was measured using a commercially available kit (Upstate Biotechnology).
Antibodies.
Mouse monoclonal and rabbit polyclonal antibodies against PI3K were provided by R. Wetzker (University Hospital, Jena, Germany). Immunohistochemistry was performed on paraffin sections of mouse aorta by the ABC peroxidase method (Strepta ABComplex/HRP; DakoCytomation). Antibodies against murine F4/80 antigen (CI:A3-1) and CD18 (YTS 213.1) were from BMA Biomedicals. Rabbit polyclonal antibodies against phospho-Ser473-Akt, Akt, and phospho-ERK1/2 were obtained from New England BioLabs, Inc., antibodies against ERK1 (C16) were obtained from Santa Cruz Biotechnology, Inc.
Evaluation of vascular angiotensin IIdependent Ca2+ flux.
Mesenteric arteries were placed at 37°C, in Krebs' buffer (mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4 x 7 H2O, 1.2 KH2PO4, 25 NaHCO3, 5.6 glucose), in a Mulvany micromyograph (Danish Myo Technology), and stretched to the appropriate tension. The myograph was placed on the stage of an inverted confocal microscope (Nikon). After the equilibration period, the vascular responsiveness was tested with 80 mM KCl three times. To view intracellular Ca2+ oscillations, vessels were incubated for 2.5 h with Fluo4-AM (60 µM) plus 0.2% pluronic acid. Vessels were washed three times with Krebs' buffer and stimulated with angiotensin II (1 µM) or phenylephrine (1 µM). Confocal images and [Ca2+]i measurements were acquired as described previously (45).
Mesenteric artery transfection.
Vessels of PI3K/ and wild-type mice were transfected as described previously (46). Vessels were placed in a Mulvany pressure system with DMEM/F12 medium, containing pCMV6 Vector (Origene Technologies) carrying a HA-tagged dominant negative mutation of PKB/Akt (K179
M179) at the concentration of 3 µg/ml. An empty plasmid was used as a negative control. The vessels were perfused at 100 mm Hg of pressure for 1 h, and, subsequently, at 60 mm Hg for 5 h. Transfection efficacy was tested by immunofluorescence with anti-HA monoclonal antibodies (BabCo) in transfected and control sections of mesenteric artery. The vascular contractility of transfected vessels, perfused at constant flow, was assessed by pressure changes that were induced by angiotensin II (1 µM) or KCl (80 mM).
Statistical analysis.
Data are expressed as the mean ± SEM. Comparisons used were unpaired Student's t test for differences between wild-type and PI3K-deficient mice, or repeated measures two-way ANOVA followed by Bonferroni post hoc test. p-value of <0.05 was assigned statistical significance.
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Acknowledgments |
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This work was supported by Human Frontier Science Project and the European Union Fifth Framework Programme QLG1-2001-02171 (to E. Hirsch and M.P. Wymann), by the Murst Cofin 2002 (to E. Hirsch), by a grant from Ministero della Salute (to G. Lembo and E. Hirsch), and by an FIRB grant (to E. Hirsch, F. Altruda, G. Lembo, and G. Tarone).
The authors have no conflicting financial interests.
Submitted: 19 May 2004
Accepted: 1 February 2005
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References |
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