(Stroke. 2000;31:2487.)
© 2000 American Heart Association, Inc.
Original Contributions |
Role of Phosphatidylinositol 3-Kinase in Acetylcholine-Induced Dilatation of Rat Basilar Artery
From the Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Correspondence to Jiro Kitayama, MD, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail kitayama{at}intmed2.med.kyushu-u.ac.jp
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Abstract |
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Background and Purpose—We tested the hypothesis that activation of phosphatidylinositol (PI) 3-kinase is involved in dilator responses of the basilar artery to acetylcholine in vivo.
Methods—Responses of the basilar artery were measured by the cranial window technique in anesthetized rats. To examine the role of PI 3-kinase in acetylcholine-induced calcium signaling, we measured intracellular free calcium concentration ([Ca2+]i) of cultured rat basilar arterial endothelial cells using a fluorescent calcium indicator, indo 1.
Results—Topical application of acetylcholine (10-6, 10-5.5, and 10-5 mol/L) increased the diameter of the basilar artery by 8±1%, 14±2%, and 24±3%, respectively. An inhibitor of PI 3-kinase, wortmannin (10-8 mol/L), did not change the baseline diameter of the artery. In the presence of wortmannin, acetylcholine (10-6, 10-5.5, and 10-5 mol/L) dilated the artery only by 3±2%, 6±2%, and 12±2%, respectively. Thus, wortmannin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05 versus control). Wortmannin had no effect on dilatation of the artery in response to a nitric oxide donor, sodium nitroprusside. LY294002, another inhibitor of PI 3-kinase, also inhibited dilator response of the basilar artery to acetylcholine. Acetylcholine produced an increase in [Ca2+]i of the endothelial cells. Genistein, an inhibitor of tyrosine kinase, markedly attenuated acetylcholine-induced calcium influx to the cells; however, wortmannin had no effect on acetylcholine-induced calcium changes.
Conclusions—These results suggest that acetylcholine-induced dilatation of the basilar artery is mediated, at least in part, by activation of PI 3-kinase in vivo. Acetylcholine-induced [Ca2+]i changes of the endothelial cells may not be mediated by activation of the kinase. PI 3-kinase as well as [Ca2+]i may play an important role in the acetylcholine-induced nitric oxide production of the basilar arterial endothelial cells.
Key Words: calcium • cells, cultured • nitric oxide • protein-tyrosine kinase • signal transduction
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Introduction |
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Endothelium-derived relaxing factor, which has been identified as nitric oxide (NO) or its related compound, appears to play a major role in regulation of cerebrovascular tone.1 It is reported that activation of endothelial nitric oxide synthase (eNOS) by various agonists, including acetylcholine and bradykinin, is mediated primarily by elevation in intracellular free calcium concentration ([Ca2+]i).2 3 4 On the other hand, several stimuli, such as insulin and shear stress, activate NO production in a Ca2+-independent manner.5 6 7 8 9 Thus, both Ca2+-dependent and -independent mechanisms may be involved in agonist-induced NO production of the endothelial cells (EC).
Phosphatidylinositol (PI) 3-kinase is a novel lipid kinase that phosphorylates the D-3 position of the inositol ring of inositol phospholipids and appears to play an important role in cell growth and oncogenesis.10 11 12 13 14 15 16 17 Very recently, it was reported that stimuli promoting NO release independent of elevation in [Ca2+]i, such as vascular endothelial growth factor (VEGF) and shear stress, activate PI 3-kinase and thereby increase the activity of eNOS through activation of protein kinase B (Akt).18 19 Thus, activation of PI 3-kinase plays a major role in NO production in response to these stimuli. It is still not known, however, whether the PI 3-kinase/Akt pathway is also involved in NO-dependent dilator responses of the cerebral blood vessels, especially in vivo.
We have shown recently that activation of tyrosine kinase is involved in acetylcholine-induced dilatation of rat basilar artery in vivo.20 Because the activity of PI 3-kinase appears to be closely related to protein tyrosine kinase,10 it may be possible that activation of PI 3-kinase is also involved in dilatation of the basilar artery in response to acetylcholine, whose receptor belongs to G protein–coupled receptors.21 Thus, the first goal of the present study was to test the hypothesis that acetylcholine-induced dilatation of the basilar artery is mediated by activation of PI 3-kinase in vivo.
It is reported that activation of tyrosine kinase is involved in agonist-induced changes in [Ca2+]i of cultured umbilical vein EC.22 23 24 On the other hand, the mechanism through the PI 3-kinase/Akt pathway may not be involved in agonist-induced calcium signaling, as previously discussed. Thus, it is important to determine whether activation of tyrosine kinase and PI 3-kinase is related to acetylcholine-induced changes in [Ca2+]i. The second goal of the present study was to investigate the role of both these protein kinases in acetylcholine-induced calcium signaling of basilar arterial EC.
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Materials and Methods |
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This study was performed under the control of the guidelines for animal experiments in the Graduate School of Medical Sciences, Kyushu University.
Cranial Window
Experiments were performed on male Sprague-Dawley rats
(mean±SEM weight, 354±8 g; age, 2 to 4 months; n=23),
anesthetized with amobarbital (50 mg/kg IP). Catheters were
placed in bilateral femoral arteries to measure systemic blood pressure
and to obtain arterial blood. The right femoral vein was
cannulated for infusion of supplemental anesthetic (20 to 25 mg/kg per
hour). The trachea was cannulated, and the animals were mechanically
ventilated with room air and supplemental oxygen. Arterial
blood gas monitored during the experiments had a pH of 7.42±0.00, a
PCO2 of 40±1 mm Hg, and a
PO2 of 135±6 mm Hg. Skeletal
muscle paralysis was produced with d-tubocurarine chloride
(2 mg/kg IV). Depth of anesthesia was evaluated by applying
pressure to a paw or the tail and observing changes in heart rate or
blood pressure. Additional anesthetic was administered when such
changes occurred. Body temperature was maintained with a heating pad
(36.6°C). A craniotomy was prepared over the ventral
brain stem as previously described in detail.25 26 27
In brief, after a part of the dura was opened, the cranial window was
suffused with artificial cerebrospinal fluid (aCSF)
(temperature=36.2°C; ionic composition [in mmol/L]: 132 NaCl,
2.95 KCl, 1.71 CaCl2, 0.65
MgCl2, 24.6 NaHCO3, 3.69
D-glucose) that was bubbled continuously with appropriate
gases. aCSF sampled from the cranial window had a pH of 7.40±0.01, a
PCO2 of 38±1 mm Hg, and a
PO2 of 117±3 mm Hg. After the
preparation was completed, the window was suffused with the aCSF for at
least 1 hour before the experiment.
Diameters of the blood vessels were measured with a microscope equipped with a television camera coupled to an autowidth analyzer (C3161, Hamamatsu Photonics). We examined responses of the basilar artery to topical application of acetylcholine (10-6, 10-5.5, and 10-5 mol/L), bradykinin (10-7 and 10-6 mol/L), and an NO donor, sodium nitroprusside (10-8, 10-7, and 10-6 mol/L). We decided the dose of these vasodilators according to prior reports in which cranial window technique was used to produce effective dilator responses of the basilar artery in Sprague-Dawley rats.20 28 29 The vasodilators were mixed in aCSF and suffused over the craniotomy for 5 minutes. Diameters of the basilar artery were measured immediately before and during the last minute of application of each agonist. After application of a specific agonist, vessel diameters returned to the baseline level within a few minutes before subsequent application of an agonist. The application sequence of agonists was randomized. We used 2 different inhibitors of PI 3-kinase, wortmannin (10-10, 10-9, and 10-8 mol/L) and LY294002 (2-[4-morpholinyl]-8-phenyl-1[4H]-benzopyran-4-one; 10-5 mol/L). The inhibitors were suffused 15 minutes before and during application of agonists.
Pretreatment of the basilar artery with NG-nitro-L-arginine (10-5 mol/L) almost abolished dilatation of the basilar artery in response to acetylcholine and bradykinin (data not shown). Intravenous administration of 10 mg/kg indomethacin, an inhibitor of cyclooxygenase, did not affect vasodilatation induced by acetylcholine and bradykinin (data not shown). Thus, vasodilator responses to these agonists were mediated primarily by NO.26
Wortmannin was dissolved in dimethyl sulfoxide and diluted with aCSF. The final concentration of dimethyl sulfoxide was <0.1%, and dimethyl sulfoxide at that concentration did not cause any significant change in diameter of the basilar artery (data not shown). Acetylcholine, sodium nitroprusside, LY294002, and bradykinin were dissolved in aCSF. Topical application of these agents did not cause any changes in systemic arterial pressure (data not shown).
Measurement of [Ca2+]i
EC were collected from the basilar artery of male Sprague-Dawley
rats aged 4 to 6 weeks. After the rats were anesthetized with
diethyl ether, they were decapitated, and the basilar artery was
quickly removed under sterile conditions. The arterial
segments were carefully cleaned of connective tissue, cut in small
fragments, and placed in culture dishes coated with collagen type 1.
The growth medium (Dulbecco’s modified Eagle’s medium) was
supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin
solution, 5 µg ·
mL-1 EC growth
supplements, and 10 U/mL heparin. The dishes were incubated at 37°C
in a humidified atmosphere of 95% air and 5%
CO2. After a few days, colonies of EC with
cobblestone pattern, which were typical for EC, proliferated from the
basilar arterial segments. After the arterial
segment was removed, the primary EC colonies were gently scraped off
and subcultured in other dishes. Final characterization of EC was
performed by demonstrating specific immunocytochemical staining for von
Willebrand factor.30 EC colonies were released
with 0.25% trypsin and subcultured for the measurement of on
coverslip-bottomed culture dishes (MatTek Corp) precoated with collagen
type 1. EC with passages 2 to 3 were used for measurements of
[Ca2+]i.
EC were washed with physiological salt solution (PSS) of the following composition (in mmol/L): 135 NaCl, 6.0 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 10 HEPES, 10 Tris, and 9.9 D-glucose and subsequently loaded with an acetoxymethyl ester form of indo 1 (indo 1-AM). The EC were incubated with PSS containing 5x10-6 mol/L of indo 1-AM for 30 minutes at room temperature, washed gently with PSS, and maintained for an additional 20 minutes to allow the cells to deesterify indo 1-AM. Monitoring of fluorescence images of EC was performed with an inverted confocal laser scanning microscope (LSM-GB200, Olympus). The excitation wavelength (351 nm) of indo 1 was applied by argon UV laser (Enterprise, Coherent) through a band-pass interference filter, and wavelength bands of emitted fluorescence were 380 to 455 nm ("405-nm channel") and 470 to 490 nm ("480-nm channel"), corresponding to the Ca2+-bound and Ca2+-free forms of the indicator, respectively. Emitted fluorescence was collected from a field of approximately 5 to 10 confluent cells, and the data were analyzed with software provided for the scanning system by Olympus. Autofluorescence of a comparable field of unloaded cells was subtracted from the emitted fluorescence image. The ratio of fluorescence at 405 and 480 nm was used as an index of [Ca2+]i.31 32 33 34 EC were exposed to wortmannin (5x10-8 mol/L) or genistein (2x10-5 mol/L), a tyrosine kinase inhibitor, for 20 minutes before application of acetylcholine. In the Ca2+-free buffer, extracellular Ca2+ was omitted and 1 mmol/L EGTA was added. Acetylcholine stock solution (10 µL) was added to the dish quietly (final concentration 10-5 mol/L), and the changes in the fluorescence ratio were measured approximately every 3 seconds for 3 minutes. We performed 8 independent experiments in each protocol. All experiments were performed at room temperature.
Materials
Acetylcholine, sodium nitroprusside, wortmannin, LY294002, and
genistein were all from Sigma. Bradykinin was from Wako. Cell culture
medium was also purchased from Sigma, except for fetal bovine serum,
which was from GIBCO. Collagen type 1, which was used to coat
coverslip-bottomed culture dishes, was from Iwaki. Indo 1-AM was from
Molecular Probes or Dojin.
Statistical Analysis
All values were expressed as mean±SEM. One-way
repeated-measures ANOVA was used to compare vasodilator responses under
control condition and in the presence of each inhibitor.
Post hoc analysis was made with a paired t test. A
value of P<0.05 was considered significant. Two-way
factorial ANOVA was used to compare each time course of
[Ca2+]i in response to
acetylcholine. In analyzing the statistical significance of each time
point, the Mann-Whitney U test was used.
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Results |
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Effects of PI 3-Kinase Inhibitors on Vasodilatation
Under control conditions, diameter of the basilar artery was 241±3 µm. Topical application of acetylcholine (10-6, 10-5.5, and 10-5 mol/L) produced dilatation of the basilar artery in a concentration-related manner (8±1%, 14±2%, and 24±3%, respectively; Figure 1a
). Acetylcholine-induced vasodilatation
was reproducible, since there was no significant attenuation of the
response during repeated application of acetylcholine (data not shown).
Wortmannin (10-8 mol/L)
had no effect on the baseline diameter of the basilar artery but
inhibited dilatation of the basilar artery in response to acetylcholine
(Figure 1a), with an IC50 value of
approximately 1.9x10-9
mol/L (Figure 1b). LY294002
(10-5 mol/L) did not
affect the baseline diameter of the basilar artery but inhibited
acetylcholine-induced vasodilatation significantly (Figure 2a).
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Application of sodium nitroprusside
(10-8,
10-7, and
10-6 mol/L) also produced
dilatation of the basilar artery in a concentration-related manner
(9±1%, 42±5%, and 67±4%, respectively; Figure 1c
). Neither
wortmannin (10-8 mol/L)
nor LY294002 (10-5 mol/L)
affected vasodilatation produced by sodium nitroprusside (Figures 1c and 2b).
Application of bradykinin
(10-7 and
10-6 mol/L) dilated the
basilar artery in a concentration-related manner (3.7±0.7% and
13.4±4.3%, respectively; Figure 3).
Wortmannin (10-8 mol/L)
also attenuated bradykinin-induced dilatation of the basilar artery
significantly (Figure 3).
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Effects of Tyrosine Kinase Inhibitor and PI 3-Kinase
Inhibitor on Acetylcholine-Induced
[Ca2+]i Changes of EC
Under control conditions, application of acetylcholine
(10-5 mol/L) caused
biphasic increase in
[Ca2+]i of cultured EC,
ie, an initial transient peak followed by a sustained increase (Figure 4). In the
Ca2+-free buffer, acetylcholine produced only an
initial transient peak but did not show the subsequent plateau phase.
Pretreatment of EC with genistein
(2x10-5 mol/L) did not
affect the initial transient peak but attenuated the sustained increase
significantly. Wortmannin
(5x10-8 nmol/L), however,
had no effect on acetylcholine-induced
[Ca2+]i changes of
EC.
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Discussion |
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There are 2 major new findings in the present study. First, acetylcholine-induced dilatation of rat basilar artery may be mediated, at least in part, by activation of PI 3-kinase in vivo. Because acetylcholine-induced dilatation of the artery appears to be mediated primarily by NO and activation of the kinase does not contribute to vasodilatation produced by an NO donor, activation of PI 3-kinase may have an important role in acetylcholine-induced NO production in the basilar arterial EC. The present study is the first thus far to show the role of PI 3-kinase in NO-dependent dilator responses of the cerebral artery in vivo. Second, activation of tyrosine kinase may contribute to acetylcholine-induced Ca2+ influx to the EC, but the activity of PI 3-kinase may not have any significant influence on acetylcholine-induced [Ca2+]i changes of the EC. Thus, activation of PI 3-kinase may have a role in acetylcholine-induced NO production of the basilar arterial EC in a calcium-independent manner.
Role of PI 3-Kinase in Acetylcholine-Induced
Vasodilatation
Several groups have shown recently that NO production of
EC stimulated by VEGF, insulin, and shear stress is mediated by
activation of PI 3-kinase.5 6 7 8 9 35 Because all these
stimuli do not increase
[Ca2+]i, PI 3-kinase
activate NO production in a calcium-independent manner.
We have shown recently that dilatation of the basilar artery in
response to acetylcholine, an agonist of G protein–coupled receptor,
is mediated, at least in part, by activation of tyrosine kinase in
vivo.20 Because PI 3-kinase has a Src homology 2 domain in
its regulatory subunit, which interacts with phosphotyrosine and is
closely related to the activity of tyrosine kinase, it may be possible
that PI 3-kinase is also involved in acetylcholine-induced dilator
responses of the basilar artery. There are no data, however, regarding
the role of PI 3-kinase in dilator responses of the cerebral blood
vessels to agonists of G protein–coupled receptors. In the present
study 2 different inhibitors of PI 3-kinase, wortmannin and
LY294002, produced marked inhibition of acetylcholine-induced
dilatation of the artery in vivo. Wortmannin also attenuated
vasodilatation induced by bradykinin, another G protein–dependent
agonist.36 Thus, PI 3-kinase is functionally active in the
basilar artery in vivo and may play an important role in vasodilator
responses to acetylcholine and bradykinin. Neither of these 2
inhibitors affected dilator responses of the basilar artery
to an NO donor, sodium nitroprusside. Thus, PI 3-kinase may not be
involved in NO actions on vascular muscle but may contribute to NO
production in the EC through activation of G protein–coupled
receptors.
The concern of this study may be specificity of the inhibitors of PI 3-kinase.37 38 39 In this study wortmannin inhibited vasodilator responses to acetylcholine in a concentration-dependent manner, and the IC50 (1.9x10-9 mol/L) was similar to those reported in recent studies (1.8 to 4.0x10-9 mol/L).39 Much higher concentrations of wortmannin are reported to be necessary for the inhibition of other kinases.37 39 LY294002, another inhibitor of PI 3-kinase, is a competitive inhibitor for the ATP binding site of PI 3-kinase and has no inhibitory effects on phosphatidylinositol 4-kinase and other ATP-requiring protein kinases and lipid kinases.40 Thus, the inhibitory action of the inhibitors on acetylcholine-induced vasodilatation is likely to be specific for PI 3-kinase. The findings that these 2 inhibitors did not affect vasodilatation produced by sodium nitroprusside may further support our interpretation.
Topical application of inhibitors of NO synthase (NOS) produces constriction of rat basilar artery in vivo.26 41 Thus, synthesis of NO influences the resting tone of the basilar artery. In the present study neither wortmannin nor LY294002 affected baseline diameter of the basilar artery. These findings may suggest that PI 3-kinase is not involved in basal production of NO in the basilar artery in vivo. The signal transduction pathway of basal production of NO may be different from that of agonist-induced NO production in the EC, although the precise molecular mechanisms are unclear. However, we cannot exclude the possibility that some compensatory mechanisms have counteracted the inhibitory actions of wortmannin and LY294002 on vasodilator responses under control conditions in vivo. For example, it is possible that activation of PI 3-kinase may also be involved in vasoconstrictor responses as well as vasodilator responses. Saward and Zahradka,42 using immunostaining, have shown the existence of PI 3-kinase in vascular muscle. Thus, inhibition of PI 3-kinase may have attenuated constrictor responses as well as dilator responses of the basilar artery and thereby masked inhibitory effects of PI 3-kinase inhibitors on vasodilator responses under control conditions. We have shown recently that both acetylcholine-induced dilatation20 and serotonin-induced constriction43 of the basilar artery were mediated by activation of tyrosine kinase and that the inhibitors of tyrosine kinase did not affect the baseline diameter of the basilar artery. Because the activity of PI 3-kinase appears to be closely related to tyrosine kinase,10 it may be possible that the inhibitors of PI 3-kinase also have actions on the basilar artery similar to those of the tyrosine kinase inhibitors.
Calcium Signaling and PI 3-Kinase
Several groups have reported that changes in
[Ca2+]i of cultured
vascular EC are dependent on activation of tyrosine
kinase.22 23 24 In the studies several different
inhibitors of tyrosine kinase attenuated agonist-induced
Ca2+ influx but not Ca2+
release from intracellular store sites in cultured EC. In the
present study we have also found that genistein inhibited the
sustained increase in
[Ca2+]i of cultured
basilar arterial EC, which reflects acetylcholine-induced
Ca2+ influx. These results suggest that
activation of tyrosine kinase may cause Ca2+
influx and thereby activate NOS in the basilar
arterial EC.
On the other hand, wortmannin did not affect acetylcholine-induced [Ca2+]i changes in the basilar arterial EC. Thus, activation of PI 3-kinase may not be involved in calcium signaling of the basilar arterial EC. PI 3-kinase may play an important role in acetylcholine-induced NO production of the basilar artery in a calcium-independent manner. Recent evidence has shown that G protein–coupled receptor-independent stimuli, such as VEGF and shear stress, induce activation of the PI 3-kinase/Akt pathway and thereby directly stimulate eNOS, which is independent of elevation in [Ca2+]i.18 19 The investigators discussed the possibility that Akt enhances the sensitivity of eNOS to calcium-calmodulin complex. Thus, it may be possible that acetylcholine activates PI 3-kinase, concomitant with the increase in [Ca2+]i, and thereby stimulates eNOS in a synergistic manner.
There are several possible mechanisms by which acetylcholine
activates PI 3-kinase. Acetylcholine receptors belong to a G
protein–coupled receptor family, and G proteins are reported to
activate PI 3-kinase directly in some cellular
types.21 44 Thus, it is possible that the ß
subunits
of the G protein coupled to acetylcholine receptors directly
activate both tyrosine kinase and PI 3-kinase. Another
possibility is that some phosphotyrosine produced by activation of
tyrosine kinase interacts with Src homology 2 domain of PI 3-kinase and
thereby activates PI 3-kinase in the EC.
In summary, activation of PI 3-kinase as well as tyrosine kinase may play an important role in acetylcholine-induced NO production of the basilar arterial EC and thereby contribute to acetylcholine-induced dilator responses of the artery in vivo. Tyrosine kinase may account for acetylcholine-induced Ca2+ influx to the EC, and PI 3-kinase may activate eNOS in a calcium-independent manner.
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Acknowledgments |
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This study was supported in part by a grant from Sankyo Foundation of Life Sciences, Japan, and by a research grant for cardiovascular diseases (11C-1) from the Ministry of Health and Welfare, Japan.
Received February 28, 2000; revision received May 22, 2000; accepted June 20, 2000.
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Editorial Comment
Department of Internal Medicine, Cardiovascular Division, University of Iowa College of Medicine, Iowa City, Iowa
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Studies from both experimental animals and humans have shown that NO plays a major role in regulation of cerebral vascular tone under basal conditions and in response to receptor-mediated agonists, including acetylcholine.R1 R2 R3 The goal of the present study was to better define the signal transduction pathway that mediates responses of the basilar artery to acetylcholine and bradykinin. Relaxation of this artery in response to these agonists is known to be endothelium dependent and mediated by the endothelial isoform of NOS (CC).R1 R2
The results of the present study suggest that dilatation of the basilar artery in response to acetylcholine and bradykinin is mediated, at least in part, by activation of PI 3-kinase. This conclusion is based on the finding that 2 different pharmacological inhibitors of PI 3-kinase selectively attenuated responses to these agonists. Because responses to an NO donor were not affected by the same inhibitors, the investigators suggest that PI 3-kinase activity was influencing production of NO. Additional studies with basilar artery endothelium and calcium imaging in vitro suggest that PI 3-kinase does not influence acetylcholine-induced calcium fluxes, raising the possibility that PI 3-kinase influences production of NO via a calcium-independent mechanism.
Another interesting finding from the study relates to the observation that inhibitors of PI 3-kinase attenuated responses to acetylcholine but had no effect on baseline diameter, even though production of NO appears to be relatively high in the basilar artery under basal conditions.R1 These findings raise the possibility that basal versus agonist-induced production of NO may be regulated in part through different mechanisms.
Received February 28, 2000; revision received May 22, 2000; accepted June 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Faraci FM. Role of nitric oxide in regulation of basilar artery tone in vivo. Am J Physiol.. 1990;259:H1216–H1221.
2. Faraci FM, DD Heistad. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev.. 1998;78:53–97.
3. Elhusseiny A, Hamel E. Muscarinic—but not nicotinic—acetylcholine receptors mediate a nitric oxide-dependent dilation in brain cortical arterioles: a possible role for the M5 receptor subtype. J Cereb Blood Flow Metab.. 2000;20:298–305.[Medline] [Order article via Infotrieve]
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