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(Stroke. 1995;26:2321-2327.)
© 1995 American Heart Association, Inc.
Articles |
Activity of Smooth Muscle Phosphatases 1 and 2A in Rabbit Basilar Artery in Vasospasm
From the Department of Neurosurgery, Hyogo (Japan) College of Medicine (M.F., E.T., I.Y., N.M.), and the Department of Physiology, Nagoya (Japan) University School of Medicine (A.T.).
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Abstract |
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Background and Purpose Subarachnoid hemorrhage frequently leads to a long-term cerebral artery narrowing called vasospasm. Recently, the involvement of myosin light chain kinase has been found in experimental vasospasm in our laboratory. We therefore measured the activity of serine/threonine protein phosphatases 1 and 2A in the rabbit basilar artery in vasospasm and in vasocontraction to study their role, particularly in regard to vasospasm compared with vasocontraction.
Methods Vasospasm was produced in the rabbit basilar artery by a two-hemorrhage method. Vasocontraction was induced by local application of KCl or serotonin to the rabbit basilar artery after a transclival exposure. The control animals were treated with saline instead of fresh blood. Serine/threonine protein phosphatase activity in the basilar artery was assayed with the use of [32P]phosphorylase-a as a substrate; protein phosphatase 1 activity was evaluated as protein phosphatase activity in the presence of 1 nmol/L okadaic acid, whereas protein phosphatase 2A activity was assessed as protein phosphatase activity inhibited by 1 nmol/L okadaic acid.
Results Values of mean activity of protein phosphatase 1 in myofibrillar extract were 3.58±0.26 nmol/min per milligram in the control group, 3.22±0.12 nmol/min per milligram in the spastic group on day 2, and 3.01±0.16 nmol/min per milligram in the spastic group on day 4 (a significant decrease in protein phosphatase 1 activity in the spastic group on days 2 and 4). In contrast, these values did not show any significant changes in the KCl and serotonin groups. Values of mean activity of protein phosphatase 2A in cytosolic extract were 0.90±0.07 nmol/min per milligram in the control group, 0.75±0.10 nmol/min per milligram in the spastic group on day 2, and 0.62±0.17 nmol/min per milligram in the spastic group on day 4 (a significant reduction in protein phosphatase 2A in the spastic group on days 2 and 4). There was no evidence of significant changes of protein phosphatase 2A in cytosolic extract in the KCl and serotonin groups.
Conclusions Protein phosphatase 1 in myofibrillar extract is reported to catalyze the dephosphorylation of myosin light chain and calponin, whereas protein phosphatase 2A in cytosolic extract catalyzes the dephosphorylation of calponin and caldesmon. In addition, the phosphorylation of calponin and caldesmon results in the loss of their ability to inhibit smooth muscle contraction. Therefore, the significant decrease in activity of protein phosphatases 1 and 2A in vasospasm may result in uninterrupted vascular smooth muscle contraction by the preservation of phosphorylation of not only myosin light chain but also calponin and caldesmon.
Key Words: muscle, smooth • potassium chloride • serotonin • vasospasm • rabbits
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The detailed mechanisms of cerebral vasospasm are as yet unknown. In experimental vasospasm, Ca2+-ATPase activity in the plasma membrane of vascular smooth muscle cells was significantly decreased,1 and µ-calpain in the cerebral artery was markedly activated.2 3 This suggests that Ca2+ homeostasis in vascular smooth muscle cells is persistently disturbed in vasospasm, because Ca2+-ATPase in the plasma membrane exports intracellular Ca2+ to extracellular space,4 and µ-calpain is activated by a rise of more than 1 mmol/L in intracellular Ca2+ levels.5 The elevation of intracellular Ca2+ levels may activate various protein kinases such as PKC and MLCK in the cerebral artery, but the mechanism of the involvement of PKC in vasospasm is still controversial at present. Membrane PKC activity was reported to increase with a reciprocal decrease in cytosolic PKC activity,6 namely, the activa-tion of PKC. Another report showed a decrease of 40% to 45% in cytosolic PKC activity without any significant changes in membrane PKC activity, and immunoreactive PKC
and PKC
but not immunoreactive
PKC
were decreased in spastic arteries.7 In addition,
there was a discrepancy between PKC activity and arterial
narrowing.8 A previous study in our laboratory
demonstrated that calphostin C, a specific PKC inhibitor
interacting with the regulatory domain, was unable to reverse
vasospasm, but the effect of calphostin C on the reversal of vasospasm
was greatly enhanced after a topical treatment with calpeptin, a
selective inhibitor of calpain,2 suggesting
that the catalytic domain of PKC is dissociated from the regulatory
domain by a limited proteolysis with calpain to result in the
activation of PKC. However, it may be also surmised that the limited
proteolysis of the PKC molecule by calpain, particularly by
µ-calpain, is related to the initiation of downregulation of the
enzyme.9 10 Generally, contraction of smooth muscle is triggered by the activation of Ca2+/calmodulin–dependent MLCK, which phosphorylates MLC, permitting the activation of myosin-ATPase by actin.11 Our recent study demonstrated that MLC in the canine basilar artery is phosphorylated by MLCK but not by PKC during experimental vasospasm, and a topical application of 1-(5-chloronaphthalene-sulfonyl)-1H-hexa-hydro-1,4-diazepine (ML-9), a selective MLCK inhibitor, reverses experimental cerebral vasospasm,12 supporting the involvement of MLCK in vasospasm. The major mechanism of relaxation in smooth muscle is dephosphorylation of MLC by smooth muscle serine/threonine PP1.13 The present study measures the activity of smooth muscle PP1 in myofibrillar extract and that of PP2A in cytosolic extract in the rabbit basilar artery in vasospasm and in KCl- or serotonin-induced vasocontraction and examines differences in involvement of the two PPs between vasospasm and voltage- or receptor-dependent vasocontraction.14 15 The results show a significant decrease in the activity of smooth muscle PP1 in myofibrillar extract and of PP2A in cytosolic extract only during vasospasm, which suggests uninterrupted vascular smooth muscle contraction induced by MLCK.
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Materials and Methods |
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Animal Preparation
The care of the animals in this study complied with the standards set by the US Public Health Service. Adult Japanese White rabbits (weight, 3.0 to 3.5 kg) were anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (20 mg/kg) and xylazine (5 mg/kg) and maintained with 70% nitrous oxide/30% oxygen. Muscle relaxation was ensured with an intravenous half-hourly injection of pancuronium bromide, and arterial CO2 tension was kept at 40±5 mm Hg by adjusting the respiratory pump or by adding CO2 to the inspired gas. Body temperature was kept at 37°C with a heating blanket, and mean arterial blood pressure and pulse rate were monitored continuously in the femoral artery, showing no changes during the procedure.
Spastic Group
Vertebral angiography via the femoral artery was performed as a
prespastic control, then cerebral vasospasm was produced by an
injection of 5 mL fresh autogenous nonheparinized arterial
blood into the cisterna magna, followed by another injection of 3 mL
fresh autogenous nonheparinized arterial blood 2 days
later. Vertebral angiography was repeated 2 days (day 2) and 4 days
(day 4) after the first injection of blood, and the caliber of the
basilar artery was measured at its narrowest point on the magnified
angiogram to confirm the angiographic vasospasm and expressed as
percentage of the prespastic caliber. Thus, the angiograms on day 2
were obtained in six animals after they received a single injection of
blood, whereas those on day 4 were obtained in six animals after two
injections of blood.
Control Group
In the control group, 5 mL saline was injected into the cisterna
magna of six rabbits instead of fresh blood. The angiographic caliber
of the basilar artery was examined 2 days after the injection.
KCl and Serotonin Groups
We exposed the normal basilar artery by gently removing the
clivus and carefully incising the dura and arachnoid under a surgical
microscope. The basilar artery was then contracted by a local
application of 40 mmol/L KCl or 0.1 mmol/L serotonin for 10
minutes ("KCl-10" and "serotonin-10" subgroups)
or 40 minutes ("KCl-40" and "serotonin-40"
subgroups). Each subgroup consisted of five rabbits. The concentration
of KCl or serotonin used in this study was the same as that
used in the production of KCl- or
serotonin-induced contraction of basilar artery in
rabbits.16 The KCl-induced contraction was done in the
presence of the -adrenergic blocker phentolamine (1
µmol/L) to minimize the effect of norepinephrine released
by high K+ depolarization.17 The reduced
caliber of the basilar artery was expressed as percentage of the
untreated control caliber.
Tissue Preparation
The animals were killed after perfusion at 75 mm Hg with 150 mL
of 4 mmol/L EDTA, 0.1% 2-mercaptoethanol, 1 mmol/L benzamidine, and
0.1 mmol/L PMSF, pH 7.0, and then the basilar artery was removed
together with the entire brain. In the spastic group, blood clot around
the spastic basilar artery and its branches was carefully removed on an
ice bath without any mechanical stimulation. The tissue was then
quickly frozen in dry ice–acetone after a brief washing with the
perfusion solution and transferred to liquid nitrogen.
The frozen basilar artery was pulverized in liquid nitrogen, transferred to the perfusion solution, and centrifuged at 15 000g for 10 minutes. The supernatant (first cytosolic extract) was removed. The myofibrillar pellet was reblended with 500 µL of the perfusion solution and recentrifuged as described above. The supernatant (second cytosolic extract) was again removed. The pellet was rehomogenized with 500 µL of 20 mmol/L triethanolamine/HCl (pH 7.5, 4°C), 0.1% 2-mercaptoethanol, 1 mmol/L benzamidine, and 0.1 mmol/L PMSF (solution B) containing 2 mmol/L EGTA, 0.5% Triton X-100, and 0.6 mol/L NaCl. After standing for 30 minutes, the homogenate was diluted with an equal volume of solution B and centrifuged at 15 000g for 10 minutes. The resulting supernatant (myofibrillar extract) as well as the above two cytosolic extracts were subjected to assays for PP1 and PP2A, respectively.
Preparation of
[32P]Phosphorylase-a
The GIBCO PP assay system (GIBCO BRL Life Technologies, Inc) was
used for preparation of
[32P]phosphorylase-a as the substrate for
the assay of PP activity. Isotope solution of
[
-32P]ATP (0.5 mCi/mL) was added to
phosphorylation reaction buffer (250 mmol/L Tris-HCl
buffer at pH 8.2/16.7 mmol/L MgCl2/1.67 mmol/L
ATP/0.83 mmol/L CaCl2/133 mmol/L
ß-glycerophosphate) and then added to kinase/substrate mixture
(110 µL of 100 mg/mL phosphorylase-b/10 µL of
phosphorylase kinase made up in a buffer [50 mmol/L
ß-glycerophosphate/2 mmol/L EDTA/0.1% 2-mercaptoethanol/50%
glycerol]) to start the kinase reaction. The kinase reaction was
allowed to proceed at 30°C for 60 minutes and then stopped by the
addition of 90% ammonium sulfate for 30 minutes on ice. Precipitated
protein was centrifuged at 12 000g for 10 minutes
at 4°C. The protein pellet was washed by 45% ice-cold ammonium
sulfate and centrifuged as above, and the washing of the
protein pellet was performed four times in this manner. The protein
pellet was dissolved in solubilization buffer (50 mmol/L Tris-HCl
buffer at pH 7.0/0.1 mmol/L EDTA/15 mmol/L caffeine/0.1%
2-mercaptoethanol), transferred to the upper reservoir of Centricon-30
concentrator (Amicon, Inc), and centrifuged at 5000g
for 20 minutes at 20°C. The centrifugation of the
concentrator was repeated after the addition of the solubilization
buffer. [32P]Phosphorylase-a solution was
removed from the upper reservoir of concentrator, which was then washed
with the solubilization buffer three times to solubilize any adhered
[32P]phosphorylase-a. The resulting
concentration of all [32P]phosphorylase-a
was approximately 3 mg/mL and was kept at 4°C.
Assay of Protein Phosphatase Activity
To start the PP reaction, 20 µL of
[32P]phosphorylase-a solution and 20 µL
of PP assay buffer (2 mmol/L EDTA/20 mg/mL bovine serum
albumin/400 mmol/L imidazole-HCl, pH 7.63/2%
2-mercaptoethanol) with or without 1 nmol/L okadaic acid were added to
20 µL of basilar artery extract diluted appropriately with PP assay
buffer and incubated for 10 minutes at 30°C. An appropriate dilution
with ice-cold PP assay buffer was selected to put PP activity on
the linear portion of the activity versus sample dilution curve; PP
activity should not exceed 0.02 nmol/min in the assay. The linearity of
the assay with respect to time was also kept during the assay. This
activity gave 30% dephosphorylation of
phosphorylase-a in 10 minutes. The reaction was stopped
by the addition of 180 µL of 20% ice-cold trichloroacetic acid
for 10 minutes. The reaction mixture was centrifuged at
12 000g for 3 minutes at 4°C. The clear supernatant was
placed in a scintillation vial containing scintillant to determine the
amount of radioactivity released in the assay.
[32P]Phosphorylase-a solution was
similarly counted to determine the total counts per minute added per
reaction for calculation. PP1 activity was evaluated as PP activity in
the presence of 1 nmol/L okadaic acid, whereas PP2A activity was
assessed as PP activity inhibited by 1 nmol/L okadaic acid. Protein
content in the sample was determined by the technique of
Bradford.18 The statistical significance of PP1 and PP2A
activity was examined by Student's t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Caliber of the Basilar Artery
No significant angiographic narrowing of the basilar artery was shown before the animals in the control group were killed. Representative angiograms of the basilar artery in the spastic group on days 2 and 4 are shown in Fig 1
, and
their mean percent calibers in Fig 2 indicate the
occurrence of vasospasm. The mean percent contractions of the basilar
artery in the animals in the KCl and serotonin groups
increased over a period of approximately 20 minutes after the treatment
and then stabilized (Fig 3).
Consequently, the specimens in the KCl-10 and serotonin-10
subgroups were taken in the early stage of increasing contraction,
whereas those in the KCl-40 and serotonin-40 subgroups were
obtained in the early stage of sustained contraction. The narrowing of
the basilar artery was more marked in the spastic group than in the KCl
or serotonin group.
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PP1 and PP2A Activity in the Basilar Artery
Mean PP1 activity in the cytosolic and myofibrillar extracts is
shown in Fig 4A and 4B, respectively.
Most of the PP1 activity was recovered in the myofibrillar extract in
the control group, at 3.58±0.26 nmol/min per milligram, and relatively
little PP1 activity was detected in the first and second cytosolic
extracts. PP1 activity in the myofibrillar extract was 3.22±0.12
nmol/min per milligram in the spastic group on day 2 and 3.01±0.16
nmol/min per milligram in the spastic group on day 4, which indicated
significant decreases in the spastic group on days 2 and 4 and a
significant reduction in the spastic group on day 4 compared with those
in the spastic group on day 2. PP1 activity did not change
significantly in the KCl-10, KCl-40, serotonin-10, and
serotonin-40 subgroups as well as between KCl-10 and KCl-40
subgroups and between the serotonin-10 and
serotonin-40 subgroups. Levels of PP1 activity in the
first, second, and total (first plus second) cytosolic extracts were
not changed significantly in any groups.
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Mean PP2A activity in the cytosolic and myofibrillar extracts is
demonstrated in Fig 5A and 5B,
respectively. Much of the PP2A activity was recovered in the second
cytosolic extract in the control group, at 0.67±0.07 nmol/min per
milligram, and some PP2A activity was found in the first cytosolic and
myofibrillar extracts. Levels of PP2A activity in the second cytosolic
extract in the spastic group were 0.49±0.09 nmol/min per milligram on
day 2 and 0.40±0.11 nmol/min per milligram on day 4, and those in the
total (first plus second) cytosolic extract were 0.90±0.07 nmol/min
per milligram in the control group, 0.75±0.10 nmol/min per milligram
in the spastic group on day 2, and 0.62±0.17 nmol/min per milligram in
the spastic group on day 4. Thus, levels of PP2A activity in the second
and total cytosolic extracts were significantly reduced in the spastic
group on days 2 and 4 without any significant difference between the
two, but those in the first cytosolic and myofibrillar extracts were
not changed significantly in any groups. In addition, significant
differences were found in PP2A activity in the second cytosolic extract
between the spastic group on day 2 and the serotonin-10
subgroup and between the spastic group on day 4 and the KCl-10, KCl-40,
or serotonin-10 subgroups. No significant changes were
shown in PP2A activity in any cytosolic extracts in the KCl-10, KCl-40,
serotonin-10, and serotonin-40 groups as well
as between the KCl-10 and KCl-40 subgroups and between the
serotonin-10 and serotonin-40 subgroups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein Phosphatases in Smooth Muscle
PPs are classified by Cohen19 into PP1 and PP2 on the basis of the substrate specificity toward phosphorylase kinase and the sensitivity to heat- and acid-stable PP inhibitors (I-1 and I-2). PP1 specifically dephosphorylates the ß-subunit of phosphorylase kinase and is inhibited by nanomolar concentrations of I-1 and I-2, whereas PP2 dephosphorylates the
-subunit of
phosphorylase kinase and is not inhibited by I-1 and I-2.
PP2 is further divided into PP2A (divalent cation–independent),
PP2B
(Ca2+/calmodulin–dependent),
and PP2C (Mg2+-dependent).19 20 21 Okadaic
acid is a specific inhibitor of serine/threonine
PPs22 and is used for the distinction of four
serine/threonine PPs, particularly PP1 and PP2A, because of the
differential inhibitory effects. The
Ki of okadaic acid is 150 nmol/L for PP1, 30
pmol/L for PP2A, 5 µmol/L for PP2B, and >>10 µmol/L for
PP2C.23 24 It is now well established that PP1 in cells is usually complexed to a variety of regulatory subunits that target it to particular locations, modify its specificity, and permit its regulation by extracellular signals.13 25 26 The myofibrils of smooth muscle contain smooth muscle PP1, which avidly dephosphorylates MLC and is a heterotrimer composed of a 37-kD PP1 catalytic subunit and a regulatory complex comprising a 130-kD catalytic subunit-binding component and a 20-kD protein of unknown function.13 In addition, the regulatory complex of smooth muscle PP1 enhances several-fold the rate at which the catalytic subunit dephosphorylates smooth muscle MLC but strongly diminishes both the activity toward phosphorylase, phosphorylase kinase, and glycogen synthase and the sensitivity to the cytosolic inhibitor proteins. Fernandez et al27 microinjected the purified PP1 and PP2A into the cytoplasm of mammalian fibroblasts, showing that the microinjected PP1 alone induced extensive dephosphorylation of MLC. Therefore, the present PP1 assay in myofibrillar extract measures the activity of smooth muscle PP1 released by Triton X-100 during the tissue preparation.
Protein Phosphatases in Vasospasm
The present tissue preparation for assay of PPs was performed
by the method used by Alessi et al13 for assay of
smooth muscle PP1 in chicken gizzard. They reported that 79.4±1.8% of
smooth muscle PP1 activity was present in the myofibrillar extract,
while only 11.8±1.5% was present in the total cytosolic extract.
The present levels of PP1 activity measured in the rabbit basilar
artery were 78.3±7.4% in the myofibrillar extract and 9.8±6.1% in
the total cytosolic extract, suggesting that the present assay
system is adequate for measuring PP1 activity.
When the effects of okadaic acid on protein phosphorylation in isolated cells are studied, higher concentrations may be needed in the incubation medium than those used in subcellular extract in vitro, because the intracellular concentrations of PP1 and PP2A often lie in the range 0.1 to 1.0 mmol/L.28 Okadaic acid has concentration-dependent dual effects in smooth muscle cells. At concentrations higher than 1 mmol/L, okadaic acid induces contraction29 30 by elevating MLC phosphorylation through interfering with the dephosphorylated step of smooth muscle PP1.31 32 33 In contrast, at lower concentrations okadaic acid inhibits contractions induced by high K+ depolarization and receptor agonists.34 35 36 37 The canine basilar artery is dilated by the administration of 1 and 10 nmol/L of okadaic acid into the cisterna magna.38 In addition, okadaic acid in concentrations of 10-7 and 10-6 mol/L also exerts a dose-dependent, long-lasting relaxation of isolated canine basilar artery in both the resting condition and precontracted with high K+ concentrations and receptor agonists.35 38 Abe and Karaki37 suggest that okadaic acid inhibits phosphatase antagonizing the cyclic AMP–dependent kinase to augment the phosphorylation due to cyclic AMP–dependent kinase in the rat aorta, resulting in relaxation. Okadaic acid in the present study is used for the distinction of PP1 and PP2A in the myofibrillar and cytosolic extracts in vitro but not in intact cells.
Our recent study demonstrated that MLC in the canine basilar artery is phosphorylated by MLCK but not by PKC in spastic, KCl, or serotonin groups.12 The present results demonstrate that smooth muscle PP1 activity is significantly reduced in vasospasm on days 2 and 4 and that this effect is stronger in vasospasm on day 4 than on day 2. The decreased activity of PP1 may not correlate with the degree of vasospasm, but rather the dephosphorylation of MLC may be more difficult to attain in vasospasm on day 4 than on day 2. On the contrary, smooth muscle PP1 activity is not significantly decreased after the treatment of the artery with KCl or serotonin. In vasospasm, the production of prostaglandins and leukotrienes in the canine basilar artery is usually increased,39 40 suggesting an active generation of arachidonic acid. Arachidonic acid stimulates phosphorylation of MLC and inhibits the dephosphorylation of MLC in vitro by releasing the catalytic subunit from the regulatory complex of smooth muscle PP1.41 In addition, the continuous elevation of intracellular Ca2+ in vasospasm may activate not only MLCK but also Ca2+/calmodulin–dependent kinase II, which phosphorylates MLCK to decrease its activity.42 Thus, the phosphorylation of MLC by MLCK in vasospasm may be regulated by two pathways: positively by the inhibition of smooth muscle PP1 activity, probably by arachidonic acid, and negatively by the decrease in activity of MLCK, possibly by Ca2+/calmodulin–dependent kinase II. However, it remains to be determined whether the action of Ca2+/calmodulin–dependent kinase II is involved in vasospasm as found in the stimulation of receptors by agonists.43
Calponin and caldesmon in the smooth muscle inhibit smooth muscle contraction by decreasing the actin-activated Mg-ATPase activity of smooth muscle myosin, and the phosphorylation of calponin and caldesmon by PKC or Ca2+/calmodulin–dependent kinase II results in loss of their ability to inhibit the actomyosin Mg-ATPase.44 45 46 47 48 49 PP2A in cytosolic fraction catalyzes the dephosphorylation of calponin and caldesmon,50 51 52 and calponin is a significantly better substrate of PP2A than caldesmon.51 Although the experimental procedures to prepare the first and second cytosolic extracts are similar, PP2A activity in the rabbit basilar artery is much more concentrated in the second cytosolic extract. The levels of PP2A activity in the second and total cytosolic extracts are decreased significantly in the spastic group on days 2 and 4 without any significant difference between the two. Smooth muscle PP1 also catalyzes the dephosphorylation of calponin.50 Although the phosphorylation of calponin and caldesmon has not yet been studied in vasospasm, the decreased activity of smooth muscle PP1 and PP2A in vasospasm might result in uninterrupted vascular smooth muscle contraction by the preservation of phosphorylation of not only MLC but also probably calponin and caldesmon.
In conclusion, the present study shows the difference in the
activity of PPs involved in basilar artery contraction between
vasospasm and voltage- or receptor-dependent vasocontraction:
significant decreases in PP1 and PP2A activity in vasospasm but not in
vasocontraction. In addition, µ-calpain is markedly
activated in vasospasm,2 3 and the activation of
µ-calpain is continuous in vasospasm but transient in
vasocontraction. The intracellular devices responsible for contraction
of the basilar artery, particularly talin, vinculin, and
-actinin, are degraded more severely in vasospasm than in
vasocontraction,53 probably by a proteolytic mechanism.
Those observations may suggest the difference in pathogenesis between
vasospasm and vasocontraction, and the understanding of that difference
may provide a new therapeutic direction.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by grant-in-aid No. 06454424 from the Ministry of Education, Science, and Culture, Japan.
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Footnotes |
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Reprint requests to Eiichi Tani, MD, Department of Neurosurgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan.
Received April 17, 1995; revision received July 18, 1995; accepted August 30, 1995.
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