(Stroke. 2001;32:154.)
© 2001 American Heart Association, Inc.
Original Contributions |
Relaxant Effect of U0126 in Hemolysate-, Oxyhemoglobin-, and Bloody Cerebrospinal Fluid–Induced Contraction in Rabbit Basilar Artery
From the Department of Neurosurgery, University of Mississippi Medical Center, Jackson.
Correspondence to John H. Zhang, MD, PhD, Department of Neurosurgery, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216-4505. E-mail jzhang{at}neurosurgery.umsmed.edu
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Background and Purpose—It has been suggested that mitogen-activated protein kinase (MAPK) is involved in cerebral vasospasm after subarachnoid hemorrhage. The present study was undertaken to explore the inhibitory effect of U0126, a novel MAPK inhibitor, in the contraction of the rabbit basilar artery by 3 spasmogens: hemolysate, oxyhemoglobin, and bloody cerebrospinal fluid (CSF) from patients with vasospasm.
Methods—The contraction and relaxation of rabbit basilar arteries were measured by isometric tension. MAPK immunoprecipitation was assessed by Western blot analysis.
Results—(1) Pretreatment of the rabbit basilar arteries with U0126 reduced contractions to hemolysate, oxyhemoglobin, or bloody CSF applied subsequently. (2) In the absence of endothelial cells, U0126 produced an inhibitory effect similar to the contractions induced by hemolysate, oxyhemoglobin, or bloody CSF. (3) U0126 relaxed the sustained contraction induced by hemolysate, oxyhemoglobin, or bloody CSF. (4) Hemolysate, oxyhemoglobin, and bloody CSF enhanced MAPK immunoprecipitation. (5) U0126 reduced MAPK immunoprecipitation induced by hemolysate, oxyhemoglobin, and bloody CSF. (6) Hemolysate, oxyhemoglobin, and bloody CSF significantly increased MAPK activity in the rabbit basilar artery. (7) U0126 abolished the effect of hemolysate, oxyhemoglobin, or bloody CSF on MAPK activation.
Conclusions—This study demonstrated a role of MAPK in the contraction of rabbit basilar arteries by hemolysate, oxyhemoglobin, and bloody CSF. MAPK inhibitor U0126 may be useful in the treatment of cerebral vasospasm.
Key Words: cerebrospinal fluid • hemolysis • oxyhemoglobins • protein kinases • vasospasm • rabbits
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Introduction |
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It has been suggested that tyrosine kinases are involved in the Ca2+ elevation,1 smooth muscle contraction,2 3 and fibroblast compaction4 induced by hemolysate or other spasmogens and may be involved in cerebral vasospasm. Mitogen-activated protein kinase (MAPK), a tyrosine kinase substrate, was activated by oxyhemoglobin5 and hemolysate6 and contributed to cerebral arterial contraction. MAPK immunoprecipitation was enhanced in the cerebral arteries in a canine double-hemorrhage model of subarachnoid hemorrhage, and a tyrosine kinase inhibitor, genistein, transiently reversed vasospasm.7 MAPK inhibitor PD98059 reduced the contraction of a rabbit basilar artery by hemolysate6 and endothelin-1.8
U0126 is a novel and potent MAPK inhibitor. U0126 has been shown to be more effective in the inhibition of MAPK activity than PD98059.9 In a rabbit basilar artery, U0126 seemed to be more potent in the inhibition of the endothelin-1–induced contraction, a causative agent for cerebral vasospasm.8 Although the etiologic factors for vasospasm are still debatable, hemolysate and its components, such as oxyhemoglobin, are believed to be the most likely candidates for vasospasm.10 In this study 3 spasmogens—hemolysate, oxyhemoglobin, and cerebrospinal fluid (CSF)—were used in the contraction and MAPK immunoprecipitation, and the inhibitory effect of U0126 was tested.
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Materials and Methods |
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Materials
U0126 was purchased from Promega. Anti-MAPK (extracellular signal–regulated kinase [ERK]1/ERK2) antibodies were purchased from Zymed Laboratories. Other chemicals were purchased from Sigma.
Hemolysate Preparation
Hemolysate was prepared as previously
described.11 Briefly,
heparinized dog arterial blood was centrifuged at
2500g for 15 minutes at 4°C,
and the supernatant was aspirated. The erythrocyte-rich precipitate was
washed 3 times with a sterile saline (saline/erythrocyte fraction
ratio, 1:3) and lysed by ultrasonic waves. The particulate material was
centrifuged at 15 000g
for 90 minutes at 4°C, and the supernatant (erythrocyte lysate) was
collected and stored at -80°C. The concentration of oxyhemoglobin
in the preparation of 100% hemolysate was 12.2±0.9 mmol/L (n=5).
The hemolysate was used in vitro, and therefore no immunologic
cross-reaction from the rabbit basilar artery was
expected.
Preparation of Oxyhemoglobin
Oxyhemoglobin was prepared as previously
described.12 Briefly, human
hemoglobin was reduced to oxyhemoglobin with 10-fold molar excess of
sodium dithionite. The sodium dithionite was later removed by dialysis
against 200 volumes of normal saline for 18 hours at 4°C. The normal
saline was replaced every 6 hours. The concentration of oxyhemoglobin
was determined spectrophotometrically. Oxyhemoglobin was stored at
-80°C before use.
Cerebrospinal Fluid
CSF samples were collected from 3 patients with
aneurysmal ruptures and cerebral vasospasm. Severe cerebral
vasospasm was confirmed in these patients by angiogram,
transcranial Doppler ultrasonography, and clinical
diagnosis. The peak of vasospasm was detected by
transcranial Doppler ultrasonography, and on that day
samples were collected and used in this study. CSF was prepared as
previously described.13 In
brief, CSF samples were obtained from ventricular drainage
and were immediately centrifuged for 5 minutes at
2500g at 4°C. The supernatant
was collected and kept at -80°C before use. The concentration of
the oxyhemoglobin was 6.0±3 µmol/L (n=3).
Isometric Tension
New Zealand White rabbits (n=40), of either sex and
weighing 5 to 6 pounds, were anesthetized with an
intravenous injection of thiopental (20 mg/kg) and
euthanatized by an overdose of phenobarbital (120 mg/kg). All
procedures were approved by the Animal Care and Use Committee at the
University of Mississippi Medical Center.
The basilar arteries were removed and cut into 3-mm rings in a dissecting chamber filled with modified Krebs-Henseleit bicarbonate solution that was bubbled with 95% O2 and 5% CO2. No attempt was made to remove the endothelial cells. The modified Krebs-Henseleit solution contained the following (mmol/L): NaCl 120, KCl 4.5, MgSO4 1, NaHCO3 27, KH2PO4 1, CaCl2 2.5, and dextrose 10.
The rings were suspended at 500 mg resting tension (Radnoti transducer, Radnoti Glass) between the stainless steel hooks in 10 mL water-jacketed tissue baths (Radnoti Glass). The tissue bath was filled with modified Krebs-Henseleit buffer and bubbled with 95% O2/5% CO2 at 37°C. The rings were equilibrated for 90 minutes, and the bath solution was changed every 20 minutes. After equilibration, the tissues were incubated with KCl (90 mmol/L) 2 times at 30-minute intervals to obtain stable contractions. Only data with recovery of 90% to 110% of the initial contraction by KCl (90 mmol/L) were included. The tension was continuously recorded with a force-displacement transducer as described previously.6
The arteries were divided into 2 groups. In the first group, samples were preincubated with U0126 (30 µmol/L) for 30 minutes, and then dose-dependent responses to hemolysate (0.1% to 10%), oxyhemoglobin (0.1 to 100 µmol/L), or CSF (30%) were studied. In the second group, the arterial samples were contracted with hemolysate (10%), oxyhemoglobin (10 µmol/L), or CSF (30%), and then a dose-dependent relaxation was initiated with U0126 (1 to 100 µmol/L). Each ring was used with only 1 agonist to avoid cross-reaction.
Another series of studies was performed to evaluate the contribution of endothelium. The arteries were divided into 2 groups. In one group no attempt was made to remove endothelial cells; in another the endothelial cells were removed with the stainless rod. The endothelial removal was confirmed by the absence of relaxation effect of acetylcholine (1 µmol/L) on serotonin-induced contraction (30 µmol/L). Experiments similar to the aforementioned experiments were performed in the absence of endothelial cells.
Western Blot Analysis
The basilar arteries were removed from the base of
the brain stem and incubated with hemolysate (10%), oxyhemoglobin (10
µmol/L), or 30% CSF. In another group the vessels were preincubated
with U0126 (30 µmol/L) for 30 minutes and then incubated with
hemolysate (10%), oxyhemoglobin (10 µmol/L), or bloody CSF (30%).
After the treatment, the arteries were immediately frozen in liquid
nitrogen. The arteries were homogenized for 20 minutes at
4°C in the following (mmol/L): Tris-HCl (pH 7.5) 50, NaCl 100, EDTA
5, phenylmethylsulfonyl fluoride 1, and IGEPAL CA-630 100 µL.
The insoluble materials were removed by centrifugation
(13 000g, for 10 minutes, at
4°C). The samples (30 µg of protein) were applied to 12.5%
SDS-PAGE. After the electrophoretic transfer of the separated
polypeptides to the nitrocellulose membrane, the membranes were blotted
with the use of 8% nonfat milk in Tris-buffered PBS (TBS) for 1 hour.
The membranes were washed with TBS and incubated at 4°C overnight in
a 1:5000 dilution of mouse anti-MAPK antibodies (ERK1/ERK2, monoclonal
mouse antibody, Zymed Laboratories). These antibodies recognize both
phosphorylated and nonphosphorylated
MAPK. The nitrocellulose membranes were later washed with TBS and
incubated with a 1:1000 dilution of a goat anti-mouse IgG antibody,
which was linked with horseradish peroxidase. The enhanced
chemiluminescence system (Amersham) was used for visualization of the
protein bands. The results were quantified by Quantity One software
(Biorad).
MAPK Activity Assay
The basilar arteries were removed from the brain and
were exposed to hemolysate (10%), oxyhemoglobin
(10-4 mol/L),
or bloody CSF (30%) for 5 minutes. In some samples, the basilar
arteries were pretreated with U0126 (30 µmol/L) for 30 minutes and
then were exposed to hemolysate (10%), oxyhemoglobin
(10-4 mol/L),
or bloody CSF (30%) for 5 minutes. After the treatment, the arteries
were immediately frozen in liquid nitrogen. The MAPK activity was
studied according to the method described in the MAPK assay manual (New
England Biolabs, Inc). In brief, arteries were sonicated in lysis
buffer provided in the kit, and the lysate was incubated with
immobilized phospho-p44/42 MAPK monoclonal antibodies
overnight at 4°C. After the incubation, the samples were spun down,
and the pellets were incubated with Elk-1 fusion protein for 30 minutes
at 30°C. The samples (30 µL) were applied to 12.5% SDS-PAGE and
then transferred to the nitrocellulose membrane. The membrane was
incubated with phospho-Elk-1 antibodies overnight and then for 1 hour
with horseradish peroxidase–conjugated anti-rabbit secondary
antibodies. The phospho-Elk-1 protein bands were visualized with
LumiGLO. The density of the bands was quantified with Quantity One
software (Biorad).
Data Analysis
Data are expressed as mean±SEM. Statistical
differences between the control and other groups were compared with a
1-way ANOVA and then the Tukey-Kramer multiple comparison procedure, if
significant variance was found. A
P value of <0.05 was
considered statistically
significant.
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Results |
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Contractions Induced by Hemolysate, Oxyhemoglobin, and Bloody CSF
The initial significant contraction to hemolysate was obtained with 0.1% hemolysate (Figure 1A
), and further contraction was achieved by 1% to
10% hemolysate. The initial contraction to oxyhemoglobin occurred at
0.1 µmol/L, with maximal contraction obtained at 10 µmol/L of
oxyhemoglobin
(Figure 1B). Thirty percent of bloody CSF
(Figure 1C) was used in this study because a lower level
(10%) failed to produce a consistent contraction (data not
shown).
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Effects of U0126 on Arterial
Contraction
Preincubation of the basilar artery rings with U0126
(30 µmol/L) for 30 minutes reduced significantly the contractions to
hemolysate, oxyhemoglobin, and bloody CSF
(Figure 1A
through 1C).
Figures 2 through 4 summarize the results from the rings of
at least 5 rabbits for each agonist. U0126 did not change resting
tension in any of the rings
(Figure 1). At a higher concentration, U0126 (100 µmol/L)
completely abolished the contraction induced by hemolysate
(Figure 2A; P<0.05
to P<0.01, ANOVA) and
significantly reduced contractions to oxyhemoglobin
(Figure 3A; P<0.05
to P<0.001, ANOVA).
Contractions induced by bloody CSF were significantly reduced by 1
µmol/L of U0126 and completely abolished by 30 µmol/L of U0126.
(Figure 4A; P<0.05,
ANOVA). The higher concentration of U0126 was not used against the
contractions induced by bloody CSF since U0126 at 30 µmol/L
essentially abolished the contraction to bloody CSF
(Figure 4A).
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In the absence of endothelial cells, U0126
(30 µmol/L) achieved an inhibitory response to the
contractions induced by hemolysate
(Figure 2B), oxyhemoglobin
(Figure 3B), and bloody CSF
(Figure 4B) that was similar to the inhibitory
effects obtained in the presence of endothelial cells,
as mentioned above.
In another series of studies, arterial rings
were precontracted with hemolysate (10%), oxyhemoglobin (10 µmol/L),
or CSF (30%), and once a stable contraction was obtained, a
dose-dependent relaxation was induced with U0126 (1 to 100 µmol/L).
Although U0126 induced a partial relaxation at 60 µmol/L, the high
dose of U0126 (100 µmol/L) was required to produce significant
relaxation
(Figure 5).
Figure 6 summarizes the results of relaxation induced by
U0126. U0126 completely reversed
(P<0.05) the contraction to
CSF (30%), while it relaxed only approximately 50%
(P<0.05) of the initial
contraction induced by hemolysate and
oxyhemoglobin.
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Effects of U0126 on MAPK
Immunoprecipitation
Rabbit basilar arteries were treated with hemolysate,
oxyhemoglobin, and bloody CSF for 5 minutes, and an enhanced MAPK
immunoprecipitation was observed for all the agonists
(Figure 5). We chose 5 minutes of incubation time
because hemolysate induced a peak MAPK immunoprecipitation in the
rabbit basilar artery in a previous
study.6 Preincubation of the
rabbit basilar arteries with U0126 (30 µmol/L) for 30 minutes
markedly reduced the effect of hemolysate, oxyhemoglobin, and bloody
CSF on MAPK immunoprecipitation
(Figure 7A).
Figure 7B summarizes the results of hemolysate-,
oxyhemoglobin-, and bloody CSF–induced MAPK immunoprecipitation
(P<0.001) and the
inhibitory effect of U0126
(P<0.05 to
P<0.001).
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Effects of U0126 on MAPK Activity
Hemolysate, oxyhemoglobin, and bloody CSF significantly
increased MAPK activity
(P<0.05 to
P<0.001, ANOVA)
(Figure 8A). Pretreatment of the basilar arteries with U0126
(30 µmol/L) for 30 minutes completely abolished this increase of MAPK
activity caused by studied agonists. U0126 slightly reduced the MAPK
activity in control vessels without statistical significance
(P>0.05)
(Figure 8B).
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Discussion |
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Several findings were presented in this report. (1) U0126 significantly inhibited the contractions of rabbit basilar arteries to hemolysate, oxyhemoglobin, or bloody CSF. (2) In the absence of endothelial cells, U0126 achieved similar inhibitory effects. (3) U0126 significantly relaxed all rings precontracted with hemolysate, oxyhemoglobin, or bloody CSF. (4) Hemolysate, oxyhemoglobin, or bloody CSF significantly enhanced MAPK immunoprecipitation. (5) U0126 significantly reduced MAPK immunoprecipitation induced by hemolysate, oxyhemoglobin, and bloody CSF. (6) Hemolysate, oxyhemoglobin, and bloody CSF significantly increased MAPK activity in the rabbit basilar artery. (7) U0126 abolished the effect of hemolysate, oxyhemoglobin, or bloody CSF on MAPK activation.
Role of MAPK in Prolonged Arterial
Contraction
MAPK is the last protein phosphorylated
in the processing of the signal from the receptor to the nucleus. There
are at least 2 major pathways for the activation of MAPK: through
tyrosine kinase receptors or through G-protein–coupled receptors. The
adapter protein Grb2 links the tyrosine-phosphorylated
receptor to SOS, which acts as a guanine nucleotide
exchange factor for p21ras, and the active
GTP-bound p21ras stimulates Raf-1 kinase
activity toward mitogen-activated protein kinase kinase (MEK).
The MEK activation leads to MAPK phosphorylation on
both threonine and tyrosine residues and subsequently to
phosphorylation of transcription factors
c-myc,
c-jun, and
c-fos. The most studied MAPK is
the ERK cascade, which is an established pathway responding to most
vasoactive agents, such as endothelin-1 and angiotensin II,
as well as growth factors, such as epidermal growth factor and
platelet-derive growth
factor.14 15 The
protein tyrosine kinase seems to be important in MAPK activation
because the tyrosine kinase inhibitor genistein
significantly reduced MAPK phosphorylation by
serotonin in bovine carotid
arteries.16 Another pathway
for the activation of MAPK is by the activation of G-protein–coupled
receptors. For example, angiotensin II–induced activation
of MAPK in vascular smooth muscle cells is mainly mediated by a
Ca2+/calmodulin-dependent
tyrosine kinase through phosphoinositide-specific
phospholipase C–mediated Ca2+
release coupled with
Gq.17
In swine carotid arteries, both 42- and 44-kDa isoforms of MAPK
(ERK1/ERK2) were activated during agonist-dependent or membrane
depolarization–dependent
contraction.18 The mechanism
of MAPK activation and smooth muscle contraction may involve caldesmon.
Identification of the phosphorylation sites on
caldesmon as p44/42 MAPK
sites19 supports a role for
p44/42 MAPK in this signaling. In resting smooth muscle cells from
swine carotid arteries, caldesmon inhibits crossbridge
interactions.20 The
phosphorylation of caldesmon produces de-inhibition of
the actin-myosin coupling, forming a force-bearing noncycling
crossbridge,21 which leads
to a prolonged contraction. In addition, the
phosphorylated caldesmon is involved in the regulation
of the microtubule structure in smooth muscle
cells,22 restructuring
smooth muscle cytoskeleton and thus supporting a prolonged
contraction.
MAPK and Cerebral Vasospasm
Intercepting intracellular signals induced by
spasmogens has been used as a new therapy to prevent or reverse
cerebral vasospasm. First, protein kinase C was identified as a protein
that was activated in cerebral
vasospasm.22 23
Inhibitors of protein kinase C were proven to have an
effect on the development of
vasospasm,24 and the protein
kinase C inhibitor fasudil hydrochloride had a partial and
transient relaxant effect in
humans.25 Later, several
studies demonstrated the involvement of the protein tyrosine kinase in
vasospasm.1 2 3
All protein tyrosine kinase antagonists are relatively
nonspecific to the type of tyrosine kinase, and in vivo animal studies
are needed to confirm the role of tyrosine kinases in
vasospasm.
The activation of MAPK, a substrate of tyrosine kinase, was demonstrated in a dog double-hemorrhage model.7 MAPK was activated within 2 days and stayed above baseline up to 7 days after subarachnoid hemorrhage. The involvement of MAPK in the contraction of the cerebral arteries was demonstrated in the isometric tension studies.6 8 In our previous studies we demonstrated that hemolysate and endothelin-1 were able to activate MAPK and that specific MAPK antagonist PD98059 significantly reduced MAPK activation. PD98059 was also effective in the reduction of oxyhemoglobin-induced contraction of canine basilar arteries.5
In the recent past, a novel specific MAPK inhibitor, U0126, became available. The data from our laboratory demonstrated that U0126 was equal to, if not more potent than, PD98059 in the inhibition of endothelin-1–induced contraction of rabbit basilar arteries.8 In this study we used U0126 with known pathogens of cerebral vasospasm. U0126 appeared to be an effective inhibitor of the contractions induced by hemolysate, oxyhemoglobin, and bloody CSF in rabbit basilar artery. Indeed, a high dose of U0126 (100 µmol/L) completely abolished hemolysate-induced contractions, while lower doses (30 µmol/L) were enough to completely abolish bloody CSF–induced contractions.
Mechanism of MAPK Activation by
Spasmogens
Oxyhemoglobin is an established causative agent for
vasospasm.10 Oxyhemoglobin
produced contraction in cerebral arteries in multiple species and
origins, including cerebral, coronary, mesenteric, renal, and
femoral arteries from dogs and monkeys. Studies showed that cerebral
arteries are more sensitive to oxyhemoglobin than the
peripheral arteries. The initial contraction induced by a
low concentration of oxyhemoglobin (0.1 µmol/L) was obtained in
cerebral arteries from dogs and monkeys and, in the present study,
in rabbit basilar arteries.
The mechanism for oxyhemoglobin-induced contraction remains unresolved despite intensive studies in the past. Oxidation of oxyhemoglobin to methemoglobin generates free radicals, such as reactive ferryl radicals and hydroxyl radicals, that can interfere with the membrane lipids and initiate lipid peroxidation and activation of phospholipase A2, thus releasing the products of the arachidonic acid cascade.26 27 28 Most of the eicosanoids are able to activate phospholipase C, leading to the formation of inositol 1,4,5-triphosphate, which will subsequently release Ca2+ from internal stores. The elevation of intracellular Ca2+ has been observed in smooth muscle cells exposed to oxyhemoglobin.29 Free radicals are involved in the activation of protein tyrosine kinase and MAPK in rat aortic smooth muscle cells30 and in canine cerebral smooth muscle cells.5 However, more evidence is needed to clarify the mechanism of oxyhemoglobin-induced MAPK activation.
The mechanism of bloody CSF–induced MAPK activation is difficult to identify. Bloody CSF may contain many spasmogens, including oxyhemoglobin and other factors released either from a blood clot or from the vessel wall. Bloody CSF induced elevation of intracellular Ca2+ in cerebral smooth muscle and endothelial cells31 32 and produced a contraction of cerebral arteries.33 Although bloody CSF was more potent than normal CSF in producing a contraction of canine basilar arteries, the contractile potency of the bloody CSF was significantly variable, and the degree of contraction did not correlate with the severity of vasospasm in patients.34 The level of oxyhemoglobin in bloody CSF may influence contractility, and the bloody CSF with higher oxyhemoglobin concentration was more potent in inducing contraction.33 Indeed, the concentration of oxyhemoglobin in the bloody CSF varied from 2 to 11 µmol/L in the present study from 3 patients, and the bloody CSF with higher oxyhemoglobin content caused more potent contraction (data not shown).
Factors other than oxyhemoglobin may contribute to the
contraction induced by bloody CSF. As shown in
Figure 4, 30% CSF induced a degree of contraction similar
to that of hemolysate and oxyhemoglobin
(Figures 2 and 3), even though bloody CSF contains only 1.8
µmol/L oxyhemoglobin (30% of 6 µmol/L), and oxyhemoglobin and
hemolysate solutions contain 10 to 100 µmol/L and 1.2 mmol/L
oxyhemoglobin, respectively. Thus, other factors in bloody CSF may
produce additional contraction or enhance the effect of
oxyhemoglobin.
Hemolysate is also a mixture of different substances from
the lysed erythrocytes. Some small molecules of hemolysate produced an
elevation of intracellular Ca2+ in cerebral
smooth muscle
cells.32 35 One
of the factors in hemolysate is ATP, which may be responsible for the
effect of hemolysate in the contraction of cerebral
arteries35 and vasospasm in
animal models.36 In addition
to its contractility properties, ATP binds with
P2 receptors in endothelial
cells and produces relaxation. The relaxant effect of ATP may explain
why hemolysate that contains 10 times more oxyhemoglobin produced a
contraction similar to that of oxyhemoglobin or bloody CSF
(Figures 2 through 4). Nevertheless, ATP in hemolysate may
activate MAPK by the activation of P2
receptors (a G-protein–coupled receptor). Other unknown factors in
hemolysate may also be involved in the activation of
MAPK.
Conclusions
The present study demonstrated the involvement of
MAPK in the prolonged contraction of rabbit basilar arteries induced by
hemolysate, oxyhemoglobin, and bloody CSF. MAPK may be a final common
pathway for these spasmogens and may be involved in cerebral vasospasm
after subarachnoid hemorrhage. The mechanisms of
activation of MAPK by these spasmogens require further
investigation.
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Acknowledgments |
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This study was supported in part by a Grant-in-Aid from the American Heart Association (Dr Zhang).
Received March 15, 2000; revision received August 30, 2000; accepted September 22, 2000.
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