Journal of Cerebral Blood Flow and Metabolism 12:784-793 © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York
Global Forebrain Ischemia Results in Decreased Immunoreactivity of Calcium/Calmodulin-Dependent Protein Kinase II
*Severn B. Churn, t Amy Yaghmai, tJohn Povlishock, * Azhar Rafiq, and *:j:Robert J. DeLorenzo Departments of*Neurology, tAnatomy, and tPharmacology, Medical College of Virginia, Richmond, Virginia, U.S.A.
Summary: Previous studies utilizing crude brain homoge nates have shown that forebrain ischemia results in inhi bition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (lC33D6) directed against the 13- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia induced inhibition of CaM kinase II. Immunohistochemi cal investigations showed that the ischemia-induced de creased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia sensitive cells such as pyramidal cells of the hippocam pus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells
of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, au tophosphorylation of CaM kinase II in the presence of low (7 f-lM) or high (500 f-lM) ATP did not alter immuno reactivity of the enzyme with IC3-3D6. The data demon strate the production of a monoclonal antibody that rec ognizes the l3-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. To gether with previous studies, the data support the hypoth esis that rapid, ischemia-induced inhibition of CaM ki nase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke. Key Words: Antigen-Autophosphorylation-Cell loss Immunohistochemistry-Monoclonal antibody-Stroke.
Cerebral ischemia produces morphological and functional modifications in neurons that can result in delayed neuronal death (Pulsinelli and Brierly, 1979; Kirino, 1982; Churn et aI., 1990a,b). Alter ation of calcium homeostasis and calcium-depen dent mechanisms may play a critical role in the isch emia-induced cascade of events resulting in delayed neuronal cell death. The involvement of calcium dependent protein kinase systems in the regulation
of cell function has been intensely investigated in recent years. Calcium-dependent protein phospho rylation modulates many neuronal processes in cluding neurotransmitter synthesis and release (De Lorenzo and Freedman, 1978; Albert et aI., 1984; Vulliet et aI., 1984; Nose et aI., 1985; Griffith and Schulman, 1988; Nichols et aI., 1990), synaptic ves icle mobilization (DeLorenzo et aI., 1979; Llinas et aI., 1985; Hemmings et aI., 1989), postsynaptic events such as cytoskeletal dynamics (Jameson et aI., 1980; Burke and DeLorenzo, 1982; Yamamoto et aI., 1985), and regulation of membrane conduc tances (Sakakibara et aI., 1986). Therefore, alter ation of neuronal calcium-dependent protein kinase systems by ischemia may play a role in mediating some of the effects of calcium in ischemia-induced cell death. Calcium-dependent protein phosphorylation has been shown to be very sensitive to the effects of
Received November 11, 1991; final revision received February IS, 1992; accepted February 21, 1992. Address correspondence and reprint requests to Dr. S. B. Chum at Department of Neurology, Medical College of Virginia, Box 599 MCV Station, Richmond, VA 2329S, U. S. A. Abbreviations used: CaM kinase II, calcium/calmodulin dependent protein kinase II; ELISA, enzyme-linked immunosor bent assay; NHS, normal horse serum; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, so dium dodecyl sulfate; TPBS, PBS containing TWEEN-20.
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CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
ischemia (Goldenring et aI., 1983; Wasterlain and Powell, 1986; Taft et al., 1988; Churn et al., 1990a,b, Zivin et aI., 1990). Thus, inhibition of pro tein phosphorylation may represent a pathway lead ing to alteration of neuronal function and possibly neuronal cell death. To investigate the effects of ischemia on protein phosphorylation, our labora tory has concentrated on studying the ischemia-in duced inhibition of the multifunctional calcium/ calmodulin-dependent protein kinase II (CaM ki nase lI). CaM kinase II activity is inhibited immedi ately after 5 min of ischemia, and the inhibition remains until significant neuronal necrosis is ob served (Taft et aI., 1988; Churn et aI., 1990a). In addition, the decrease in CaM kinase II activity is not likely to be due to large-scale proteolysis. No significant differences are observed in CaM kinase II subunit levels or calmodulin binding properties in forebrain homogenates (Taft et aI., 1988, Churn et aI., 1990a) or purified CaM kinase II enzyme frac tions (Churn et aI., 1991) isolated from control and ischemic animals. In this report, we characterize a monoclonal IgG3 antibody directed against CaM ki nase II and describe the ischemia-induced decrease in immunoreactivity with CaM kinase II both in vitro and in situ. The data indicate that ischemia produces a posttranslational modification of CaM kinase II that alters the ability of the antibody to recognize the f3-subunit of the enzyme. Character ization of this monoclonal antibody may provide a powerful resource for investigating the role of CaM kinase II in cerebral ischemia. METHODS AND MATERIALS Materials
All materials are reagent grade unless otherwise spec ified. [-y_32p]ATP was purchased from New England Nu clear (Wilmington, DE, U.S.A.). Nitrocellulose mem brane (0.45 fl.m) was purchased from Bio-Rad (Richmond, CA, U.S.A.). Purified calmodulin was purchased from Pharmacia (Piscataway, NJ, U.S.A.). Gerbil ischemia model
Bilateral carotid occlusion was performed as previ ously described (Churn et a!., 1990a,b). Briefly, male Mongolian gerbils (Meriones unquiculatus) weighing 5070 g were anesthetized under 2.5% halothane/60% nitrous oxide/40% oxygen and placed on a heated surgical table. The ventral neck area was soaked with Operanol (Redi Products) and shaved using a safety razor. An incision was made by lifting the skin above the sternum and mak ing two to three cuts directed rostrally up the neck. Chest muscles were separated from the trachea by a stripping motion parallel to the trachea to expose the carotid arter ies. To induce ischemia, the carotid arteries were raised and Heifitz aneurysm clips were attached. After 5 min of occlusion, the aneurysm clips were removed and recircu-
785
lation was confirmed visually. The skin was folded over and the wound sutured with 5-0 silk. Surgery, ischemia, and wound closure required 16-20 min. Temperature was monitored continuously during surgery via a lubricated rectal probe (YSI). CaM kinase II reactions
CaM kinase II was purified by standard column chro matography (Goldenring et a!., 1983). Purified Sephacryl fractions (>1,OOO-fold enrichment) or highly enriched cal modulin-Sepharose fractions (>400-fold enriched) were used in the assays. For autophosphorylation studies, CaM kinase II was reacted under standard conditions, except where noted (Goldenring et aI., 1983; Churn et a!., 1990a). Standard phosphorylation reaction solutions contained 10 mM pi perazine-N-N' -bis[2-ethanesulfonate] (pH 7.4), 10 mM MgCI2, and 7 fl.M [-y_32p]ATP. For maximal CaM kinase II activity, 5 mM CaCl2 and 1 fl.g calmodulin were in cluded. Reactions were initiated with the addition of cal cium, continued for 1 min, and then terminated by the addition of 5% sodium dodecyl sulfate (SDS) stop solu tion. Proteins were resolved by SDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) and either sub jected to Western analysis or stained for protein resolu tion with Coomassie Brilliant Blue or the more sensitive silver stain as previously described (Goldenring et aI., 1983; Churn et a!., 1990a). Stained gels were dried and exposed to x-ray film (XRP-l; Kodak, Rochester, NY, U.S.A.) for autoradiography. Development of monoclonal antibody
Mice (Mus musculus, BALB-c; Charles River) were immunized with two intraperitoneal injections (100 fl.g/ injection, 21 days apart) of purified CaM kinase II. Thirty days following the second injection, the mice were killed by cervical dislocation, and a spleen cell suspension was made. The splenocytes were fused with a nonsecreting mouse myeloma cell line (P3X63-AG8.653). Monoclonal producing cells were obtained from antibody-secreting polyclonal hybridomas by limited dilution methods (Ken net, 1979). Subtyping of antibody directed against CaM kinase II was performed by enzyme-linked immunoassay (ELISA) (Kennet, 1979). Specificity of lC3-3D6
The specificity of the monoclonal antibody was tested by Western analysis. Samples of crude homogenate or purified CaM kinase II were resolved on one-dimensional 8.5% SDS-PAGE or two-dimensional isoelectric focusing gels combined with SDS-PAGE and electroblotted onto nitrocellulose (Goldenring et aI., 1982; Churn et aI., 1990a). The nitrocellulose was immersed in phosphate buffered saline (PBS; IO mM NaP04/0.9% NaCI, pH 7.5), containing Tween-20 (TPBS; Sigma, St. Louis, MO, U.S.A.). The strip was incubated in antibody-containing TPBS for I h and washed by three changes of TPBS. The blot was incubated in a solution of biotinylated secondary antibody in TPBS. Excess secondary antibody was washed by three changes of PBS and labeled proteins were reacted with an alkaline phosphatase-staining kit (Vector). In addition to Western analysis, ELISA was performed to observe linearity of immunoreactivity with respect to CaM kinase II concentration or antibody concentration. ELISA plates were coated with varying amounts of CaM =
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kinase II for 24 h. The coated plates were washed with TPBS. Background was decreased by incubating coated plates in blocking solution (5 g Carnation Milk/loo ml PBS). Blocked plates were incubated with antibody for 2 h and then washed four times with TPBS. The washed plates were incubated with peroxidase-conjugated anti mouse IgG for 2 h. Staining was performed by reacting plates with substrate solution (Vector) and wells were read at OD 405 nm on an EIA reader (Biotech, Winooski, VT, U.S.A.).
Homogenate
CaM KII
80
Immunohistochemistry and histology
For histological slides, gerbils were subjected to isch emia or sham operation as described above. At the spec ified times postischemia, the animals were killed and per fused and the brains processed for histological examina tion as described previously (Churn et aI., 1990a). Coronal sections of brain were dehydrated in a series of ethanol and xylene baths prior to paraffin embedding. Serial sections 7 J..lm thick containing the dorsal hippo campus were cut using an American Optical AL micro tome (Buffalo, NY, U.S.A.), mounted on slides, and stained with cresyl violet. For immunocytochemical studies, animals were anes thetized with an injection of sodium pentobarbital (50 mg/ kg i.p.). Gerbils were then transcardially perfused with saline followed by fixative composed of 4% paraformal dehyde/O.l M phosphate buffer. After fixation, the brains were removed and placed in fresh fixative for an addi tional hour. They were stored in phosphate buffer at 4°C until sectioned. For sectioning, the harvested brain ma terial was blocked in a sagittal plane and 40-j.tm semiserial sections were cut with a vibratome. The sections were washed in PBS (pH 7.2), incubated for 1 h in 10% normal horse serum (NHS)/0.3% Triton-X, and then incubated in a solution of lC3-3D6 diluted 1: 1, 000 into 1% NHS in PBS for 18 h at 4°C. Following incubation in the primary antibody, the sections were washed 10 min in 1% NHS in PBS and then incubated for 60 min in biotinylated anti mouse IgG (Vector) diluted into 1% NHS/PBS. The sec tions were washed 10 min in PBS and then immersed for 1 h in avidin-biotin-horseradish peroxidase complex (Vector). Incubated sections were washed 10 min in PBS and transferred to a development chamber. The sections were reacted in 0.05% diaminobenzidene and 0.01% hy drogen peroxide for 5 min. Following diaminobenzidene reaction, the sections were mounted onto glass slides, dehydrated, cleared, and covered with a coverslip.
RESULTS Specificity of monoclonal IgG3 anti-CaM kinase II
The selectivity of lC3-3D6 for the (3-subunit of CaM kinase II is shown in Fig. 1. In crude homoge nate fractions, lC3-3D6 reacted with a band of �60 kDa molecular mass. The monoclonal lC3-3D6 did not react with any other protein within the crude homogenate fraction. Highly purified CaM kinase II was subjected to Western analysis and reacted with lC3-3D6 as described in Materials and Methods. A similar band of 60 kDa molecular mass was ob served in the purified fraction as seen in the crude protein fraction. The 60-kDa band identified by J Cereb Blood Flow Me/ab, Vol. 12, No. 5, 1992
-
-
60 50
27
17
Protein
Ab
Ab
FIG. 1. Specificity of 1C3-306 for the l3-subunit of calcium! calmodulin-dependent protein kinase II (CaM kinase II). A monoclonal antibody (Ab) directed against CaM kinase II was generated in our laboratory (see Materials and Methods). The specificity of 1C3-306 was tested against crude homogenate protein and purified (>1,OOO-fold) CaM kinase II subjected to Western analysis. Monoclonal 1C3-306 reacted with a major band of -60 kOa in crude homogenate fraction (lanes 1-2), which was shown to be the l3-subunit of CaM kinase II (lane 3). When analyzed in high concentration, a minor band of -58 kOa was also observed in both crude and purified frac tions. The minor band co-migrates with the 13'-subunit of CaM kinase II (Bennett and Kennedy, 1987).
lC3-3D6 co-migrated with the (3- (60-kDa) subunit (Goldenring et aI., 1983; Bennett and Kennedy, 1987) of highly purified CaM kinase II (Fig. I). A minor band of molecular mass 58 kDa was also identified when high concentrations of protein were studied in both the crude homogenate and the puri fied fractions of CaM kinase II (Fig. 1). This band co-migrated with the 13' -subunit of CaM kinase II identified by Bennett and Kennedy (1987). Immunochemical analysis of CaM kinase II sub jected to two-dimensional SDS-PAGE was per formed to further investigate the remote possibility that a 60-kDa contaminating protein was the immu noreactive agent observed in Fig. 1. Western anal ysis was performed on highly purified CaM kinase II, which was subjected to two-dimensional isoelec tric focusing and SDS-PAGE. The monoclonal lC33D6 reacted with a band of 60-kDa molecular mass
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
and with an isoelectric point of �6.9 (data not shown). The immunoreactive peptide co-migrated 3 with the 2P-Iabeled and protein-stained l3-subunit of CaM kinase II identified in the same gel. Thus, lC3-3D6 reacted with a peptide of 60 kDa molecular mass that co-purified with CaM kinase II and has the same isoelectric point as the l3-subunit of CaM kinase II. The data indicate that lC3-3D6 selec tively recognized the 13- and 13' -subunits of CaM kinase II. Linearity range of immunoreactivity with lC3-3D6
A standard curve for immunoreactivity of CaM kinase II with lC3-3D6 was performed utilizing ELISA. The immunoassay with purified CaM ki nase II was linear over a concentration range from 30 to 760 ng (�6-150 ng l3-subunit, correlation co efficient 0.972, p < 0.001, different from 0, two tailed Student t test, n 18; Fig. 2A). A CaM ki nase II concentration of 380 ng, which was in the middle of the linear portion of the curve, was used =
=
A 1.00 0.75 8 ....
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c:i c:i 0.50
0.00 '--"'--'---'---,--..o..-.J o .1 2 .3 .4 .5 .6 .7 .8 CaM kinase II ('9) 1.5 r----,
1.0 c o
to perform a standard titration curve for the con centration of lC3-3D6 (Fig. 2B). A linear curve was generated with an antibody range of 0.027 j.1g to the highest concentration tested, 0.54 j.1g (correlation coefficient 0.967, p < 0.001, different from 0, two-tailed Student t test, n 18). This represented a dilution curve of 1:2,000 to 1:1. Therefore, for immunohistochemical studies, a dilution of 1:1,000 (0.054 j.1g lC3-3D6/j.11 reaction volume) was used to reduce background, yet remain in the linear portion of immunoreactivity. =
=
Decreased immunoreactivity of CaM kinase II resulting from ischemia
In the gerbil, 5 min of global forebrain ischemia causes a decrease in CaM kinase II activity without changing total protein levels or calmodulin binding properties of the enzyme (Taft et aI., 1988; Churn et aI., 1990a,b). Ischemia produced a decrease in im munoreactivity of CaM kinase II demonstrated by Western analysis both in starting homogenate frac tions and in highly enriched calmodulin-Sepharose eluant (Fig. 3). The decreased immunoreactivity was in agreement with activity observed in purified (>1,OOO-fold) fractions (Churn et aI., 1990b, 1991). Immunoreactivity of CaM kinase II was reduced 57.6 ± 10.5% in a fraction exhibiting 55% inhibition of CaM kinase II activity (Fig. 3C; p < 0.01, Stu dent t test, n 4). The decreased immunoreactivity occurred in fractions where equivalent protein staining and calmodulin binding of the (X- and l3-sub units were observed (Taft et aI., 1988; Churn et aI., 1990a,b) (Fig. 3). The data suggest that global fore brain ischemia in the gerbil induced a posttransla tional modification of CaM kinase II that did not affect calmodulin binding or total protein levels sig nificantly but reduced antibody recognition of the enzyme. =
025
B.
787
Effect of autophosphorylation on immunoreactivity
0.5
of CaM kinase II
•
0.0 '--'---'--'---' .6 .5 .4 .3 2 .1 o lC3-306 Concentration ('9) FIG. 2. Linear regression curves of immunoreactivity under various total concentrations of calcium/calmodulin dependent protein kinase II (CaM kinase II) or various con centrations of 1C3-3D6. Enzyme-linked immunosorbent as say was performed on vessels containing various concentra tions of antigen or antibody. A: CaM kinase II concentration was varied between 18 and 760 ng. A standard curve was generated with a correlation coefficient of 0.972 (p < 0.001, different from 0, two-tailed Student t test, n 18). B: Con centration of 1C3-3D6 was varied and reacted with 380 ng of CaM kinase II. A standard curve was generated with a corre lation coefficient of 0.967 (p < 0.001, different from 0, two tailed Student t test, n 18). Each point represents average ± SEM of three replications. =
=
The extent of autophosphorylation has been shown to alter the immunoreactivity of CaM kinase II with some monoclonal antibodies (Hendry and Kennedy, 1986). Therefore, we investigated whether autophosphorylation alters immunoreac tivity with 1C3-3D6 (Fig. 4). CaM kinase II was reacted under standard conditions (7 j.1M ATP), re solved on SDS-PAGE, and subjected to Western analysis with >80% transfer efficiency. In addition to standard reactions, reactions where the level of ATP was increased to 500 j.1M were performed (Lou et aI., 1986). Autophosphorylation of CaM kinase II at either standard (7 j.1M) or high (500 j.1M) levels of ATP did not alter immunoreactivity with 1C3-3D6. J Cereb Blood Flow Metab, Vol. 12, No. 5, 1992
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A.
HOMOGENATE FRACTION ANTIBODY
CALMODULIN
...... -.... . .� .. --
C
C
CaM KINASE II FRACTION
B.
ANTIBODY
CALMODULIN
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C
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'0 :::
100
t: 0
() '0 2ft: 0 '';::::;
75
50
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Ischemic
Control
FIG. 3. Ischemia results in decreased immunoreactivity of calcium/calmodulin-dependent protein kinase II (CaM kinase II) with 1C3-306. Protein samples were obtained from gerbils subjected to sham operation or 5 min of bilateral carotid occlusion. Decreased immunoreactivity with 1C3-3D6 was observed in samples isolated from ischemic animals. The de creased immunoreactivity was observed in crude homoge nate fractions (A) and in enriched CaM kinase II fractions (B). The decreased immunoreactivity occurred under conditions where equivalent calmodulin binding is observed between control (C) and ischemic (I) fractions. Also shown is quanti fication of ischemia-induced inhibition of immunoreactivity of CaM kinase II with 1C3-3D6 (e). CaM kinase II was isolated from control and ischemic animals and subjected to Western analysis. CaM kinase II isolated from ischemic animals dis played 42.4 ± 10.4% of control immunoreactivity. Ab, anti body. "p < 0.01, Student's t test, n 4. =
Therefore, changes in immunoreactivity of CaM ki nase II with lC3-3D6 are not secondary to auto phosphorylation of the enzyme. Decreased immunohistochemical reactivity with lC3-3D6 resulting from ischemia
Delayed neuronal loss after 5 min of ischemia is observed in pyramidal cells of the CAl and CA3 J Cereb Blood Flow Metab, Vol. 12, No.5, 1992
subregions of the hippocampus (Pulsinelli and Bri erly, 1979; Kirino, 1982; Churn et aI., 1990a,b) (Fig. 5). Histological loss of pyramidal neurons is not ob served until 3-4 days following 5 min of global fore brain ischemia and fully developed after only 7 days (Pulsinelli and Brierly, 1979; Kirino, 1982; Churn et aI., 1990a,b) (Fig. 5C-F). To further study the effects of ischemia on the immunoreactivity of CaM kinase II with lC3-3D6, immunohistochemistry was performed on brains harvested from naive and ischemic animals. Local ization of forebrain immunoreactivity observed with lC3-3D6 is similar to that observed or a mono clonal antibody directed against the a- subunit of CaM kinase II (Ouimet et a!., 1984a,b; Erondu and Kennedy, 1985; Odonera et aI., 1990). CaM kinase II is concentrated in neuronal cells and is especially prominent in the pyramidal cells of the hippocam pus, cortical neurons, and striatal neurons. In addi tion to the forebrain, significant immunoreactivity toward lC3-3D6 was observed in the cerebellum (data not shown). Loss of CaM kinase II activity is observed in the hippocampus immediately following 5 min of global forebrain ischemia in the gerbil (Taft et aI., 1988; Churn et aI., 1990b). Loss of immunoreactivity oc curs within 30 min in hippocampal cells, which show significant delayed neuronal loss following 5 min of global forebrain ischemia (Fig. 6). In addi tion, the loss of immunoreactivity is observed at both 2 and 24 h of reperfusion (data not shown). This loss is observed before the histological damage induced by 5 min of global forebrain ischemia (Figs. 5 and 6). Thus, the ischemia-induced loss of immu noreactivity with lC3-3D6 occurs only in neurons that show significant histological cell loss after tran sient global forebrain ischemia. In addition, loss of immunoreactivity with lC3-3D6 significantly pre cedes histological observation of ischemia-induced neuronal necrosis. DISCUSSION
CaM kinase II is an important neuronal enzyme that is significantly inhibited in several models of ischemia, including the gerbil global forebrain isch emia model (Taft et aI., 1988; Churn et aI., 1990a,b), the rabbit spinal chord ischemia model (Zivin et aI., 1990), and decapitation ischemia in the rat (Golden ring et aI., 1983; Wasterlain and Powell, 1986). Isch emia has also been shown to alter immunoreactivity of CaM kinase II with monoclonal antibodies di rected against the enzyme (Wasterlain and Powell, 1986; Churn et aI., 1991). CaM kinase II has been purified from control and ischemic gerbils, and
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
789
Low ATP Autophosphorylation
-
Immunoreactivity
-
-
-
FIG. 4. Autophosphorylation of cal
cium/calmodulin-dependent protein kinase II (CaM kinase II) does not alter immunoreactivity with 1C3-3D6. CaM kinase II was reacted for various amounts of time under standard con ditions (7 fLM ATP) or standard condi tions with high (500 fLM) ATP. Lett: In creasing the time of reaction resulted in increased 32p incorporation into the a- (50-kDa) and 13- (60-kDa) subunits of CaM kinase II. Right: Increasing the extent of autophosphorylation did not alter immunoreactivity of CaM kinase II with 1C3-3D6 at either standard (7 fLM) or high (500 fLM) ATP. Data shown are representative of four experi ments.
Time
.5
5
10
20
.5
5
10
20
High ATP
Autophosphorylation
-
-
Time
Immunoreactivity
.5
when balanced for protein staining and calmodulin binding, the ischemia-induced inhibition of the en zyme remained. The previous observations support the hypothesis that ischemia induced a posttransla tional modification of CaM kinase II (Churn et al., 1990a,b, 1991). In this report, we characterized a monoclonal antibody directed against the l3-subunit of CaM kinase II. It was demonstrated that isch emia induced an alteration of CaM kinase II that resulted in decreased immunoreactivity of the en zyme. The decreased immunoreactivity was not due to autophosphorylation of the enzyme and oc curred under conditions that did not alter the con centration of protein staining or calmodulin binding of the l3-subunit of the kinase. The monoclonal IgG3 antibody developed in this study was highly selective for the l3-subunit of CaM kinase II. Immunoreactivity of 1C3-3D6 in a crude
5
10
20
-
-
.5
5
10
20
homogenate fraction was specific to a 60-kDa band on SDS-PAGE. This band was shown to be the l3-subunit of CaM kinase II by Western blot analysis of purified enzyme with lC3-3D6 on one- and two dimensional PAGE systems. The monoclonal lC33D6 antibody reacted with a peptide displaying the same molecular mass and the same isoelectric point of the l3-subunit of CaM kinase II (Goldenring et aI., 1983). Ischemia resulted in decreased immunoreac tivity of CaM kinase II when studied by Western analysis. The decreased immunoreactivity was ob served in both crude homogenate and highly en riched fractions. This decreased immunoreactivity agrees with data shown in other models of ischemia such as the Levine preparation (Wasterlain and Powell, 1986; Odonera et aI., 1990). The mecha nisms whereby ischemia produces alterations in CaM kinase II immunoreactivity are not presently
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790
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r
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"
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FIG. 5. Transient ischemia results in delayed, selective neuronal cell loss after 7 days. Histological studies of hippocampal pyramidal cells were performed on sham-operated gerbils and gerbils subjected to 5 min of global forebrain ischemia at 39°C. A: Photomicrograph ( x35 original magnification) of sham-operated gerbil, showing clearly defined pyramidal cell layer. B: High-magnification (original magnification x475) photomicrograph of CA1 pyramidal cell region from sham-operated gerbil, showing clearly defined cells with dark-staining nuclei. C: Photomicrograph ( x35 original magnification) of ischemic gerbil allowed to recover for 2 h postischemia, showing no significant loss of pyramidal neurons. 0: High magnification (original magnification x475) of CA1 pyramidal cells from ischemic gerbil in C, showing clearly defined neurons with dark-staining nuclei. E: Photomicrograph ( x35 original magnification) of ischemic gerbil allowed to recover for 7 days postischemia. Note selective cell loss in the CA1-3 subregion of the hippocampus with remarkable sparing of granule cells of the dentate gyrus. F: High magnification (original magnification x475) of CA1 pyramidal cell region taken from gerbil in E. Pyramidal cells are fragmented, dense, with no discernible nuclei.
known. To investigate the ischemia-induced alter ation(s) of CaM kinase II, mapping of the 1C3-3D6 recognition site will be performed. Autophosphorylation of CaM II kinase has been shown to alter both enzymatic activity (Lai et aI.,
J Cereb Blood Flow Metab. Vol. 12. No.5. 1992
1986; Lou et aI., 1986; Nichols et aI., 1990) and immunoreactivity (Hendry and Kennedy, 1986). Therefore, we studied whether autophosphoryla tion of CaM kinase II resulted in decreased immu noreactivity of the enzyme with lC3-3D6. Under
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
791
FIG. 6. Global forebrain ischemia results in loss of calcium/calmodulin-dependent protein kinase II (CaM kinase II) immunore activity in selectively vulnerable cells prior to observation of histological damage. A: Photomicrograph (x40 original magnifi cation) of 1C3-3D6 immunoreactivity in hippocampus of sham-operated gerbil, showing enriched immunoreactivity in the soma and dendritic areas of granule and pyramidal cell layers. B: Photomicrograph (x40 original magnification) of 1C3-3D6 immu noreactivity in gerbil subjected to 5 min of global forebrain ischemia (39°C) and immediately perfused (see Materials and Methods). Note loss of immunoreactivity in CA1-3 pyramidal cells with no significant loss of immunoreactivity in granule cells of the dentate gyrus. C: High magnification (original magnification x475) of CA1 pyramidal cell layer taken from gerbil in A. Note enriched staining of pyramidal soma area. 0: High magnification (original magnification x475) of CA1 pyramidal cell layer taken from ischemic gerbil in B. Note significant loss of 1C3-3D6 immunoreactivity in pyramidal soma layer.
standard conditions, ATP has been shown to mod ulate the kinetics of CaM kinase II autophosphory lation (Lou and Schulman, 1986). Low ATP con centrations, such as those around the Kd for ATP (7 fLM), resulted in autophosphorylation that leads to inhibition of the enzyme. However, in the presence of high ATP levels (500 fLM), which are closer to physiological ATP concentrations, autophosphory lation does not lead to inhibition of the enzyme. Since the level of ATP modulates the extent of autophosphorylation, CaM kinase II was reacted under standard conditions in the presence of both low (7 fLM) and high (500 fLM) ATP. Immunoreac tivity of CaM kinase II with lC3-3D6 was not al tered after extensi'/e autophosphorylation of the en zyme at either ATP level. Therefore, the ischemia induced decrease in immunoreactivity of CaM
kinase II was not due to autophosphorylation of the enzyme. Previous studies provide evidence that the isch emia-induced inhibition of CaM kinase II activity may be involved in the cascade of events resulting in delayed neuronal cell death. First, CaM kinase II activity is decreased at an early time point following global ischemia in the gerbil and remains inhibited until significant cell loss is observed (Taft et aI., 1988; Churn et aI., 1990b). In addition, parameters that modulate the extent of neuronal cell loss, such as intraischemic temperature, modulate the extent of CaM kinase II inhibition in a parallel manner (Churn et aI. , 1990a,b). To further investigate whether CaM kinase II inhibition might be involved with delayed neuronal cell loss, immunohistochem istry was performed on brain sections from isch-
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emic gerbils. Loss of CaM kinase II immunoreac tivity was noted in the CAl_3 subregions of the hip pocampus. Under the conditions of ischemia, these are the cell populations that are most sensitive to the effects of ischemia (Pulsinelli and Brierly, 1979; Kirino, 1982; Churn et al., 1990a). No significant loss of immunoreactivity or neuronal necrosis was observed in ischemia-resistant cells such as in the dentate gyrus. What is significant about the alter ation in immunoreactivity of CaM kinase II is that it occurs rapidly, long before cell loss is noted. Loss of CaM kinase II activity following ischemia is ob served after 10 s and persists until significant cell loss occurs (Taft et al., 1988; Churn et al., 1990b). The decreased immunoreactivity of CaM kinase II with lC3-3D6 occurs within 30 min following the ischemic insult and remains after 24 h of reperfu sion. Thus, decreased immunoreactivity of CaM ki nase II is an early event following ischemia and occurs in cells that are sensitive to the effects of ischemia. Ischemia results in decreased immunore activity that temporally parallels the ischemia induced inhibition of CaM kinase enzymatic activ ity and precedes ischemia-induced neuronal cell loss. Since the loss of immunoreactivity of CaM kinase II parallels the histological loss of neuronal cells, the immunohistochemistry studies provide further evidence that alteration of CaM kinase II activity may be involved in the cascade of events that ultimately result in neuronal death. The anti body may also be a useful early marker of cells that have sustained significant ischemic injury and will ultimately die. CaM kinase II has been successfully isolated from ischemic gerbils (Churn et all., 1990b, 1991). Ischemic CaM kinase II displays equivalent cal modulin and substrate recognition and reaction ki netics when compared with control enzyme (Churn et al., 1991). In this report, it was shown that isch emia alters CaM kinase II, which results in de creased immunoreactivity of the enzyme. This de creased immunoreactivity is not due to autophos phorylation of the enzyme, nor is it due to significant proteolysis. The data from this study, in combination with previously described results (Taft et al., 1988; Churn et al., 1990a,b) suggest that isch emia induces a posttranslational alteration of CaM kinase II, which results in decreased immunoreac tivity of the enzyme. Since the decreased immuno reactivity occurs in cells that are sensitive to the effects of ischemia, alteration of CaM kinase II may be important in the cascade of events that result in ischemia-induced delayed neuronal cell death. The use of lC3-3D6 as a marker of the ischemia-induced
J Cereb Blood Flow Metab. Vol. 12. No. 5. 1992
postranslational modification of CaM kinase II will provide a powerful tool to study the role of this major enzyme system in stroke. Acknowledgments: This work was supported by an NINDS Jacob Javits Award (RO I-NS23350) and the Ep ilepsy Program Project (PO I-NS25630) to R.J.D. and an NIH postdoctoral grant (HL07537) to S.B.C. The au thors would like to thank Pam Schwartz (Hybridoma Laboratory, Medical College of Virginia) for her expert assistance.
REFERENCES Albert KA, Hellmer-Matyjek E, Nairn AC, Muller TH, Haycock JW, Greene LA, Goldstein M, Greengard P (1984) Calcium/ phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase. Proc Natl Acad Sci USA 81:7713-7717 Bennett MK, Kennedy M (1987) Deduced primary structure of the beta subunit of brain type II calcium/calmodulin dependent protein kinase determined by molecular cloning. Proc Natl Acad Sci USA 84:1794-1798 Burke BE, DeLorenzo Rl (1982) Calcium and calmodulin dependent phosphorylation of endogenous synaptic vesicle tubulin by a vesicle-bound calmodulin kinase system. J Neu rochem 38:1205-1218 Chum SB, Taft WC, Billingsley ML, Blair RE, DeLorenzo Rl (1990a) Temperature modulation of ischemia-induced neu ronal cell death and ischemia-induced inhibition of calcium! calmodulin-dependent protein kinase II in gerbils. Stroke 21: 1715-1721 Chum SB, Taft WC, DeLorenzo Rl (1990b) Effects of ischemia on multifunctional calcium/calmodulin-dependent protein ki nase II in the gerbil. Stroke 21 (suppl 3):112-116 Chum SB, Taft WC, Billingsley ML, Sankaran B, DeLorenzo RJ (1992) Global forebrain ischemia induces a posttranslational modification of calcium- and calmodulin-dependent kinase II. J Neurochem 59: 1221-1232 DeLorenzo RJ, Freedman SD (1978) Calcium dependent neuro transmitter release and protein phosphorylation in synaptic vesicles. Biochem Biophys Res Commun 80:183-192 DeLorenzo Rl, Freedman SD, Yohi WB, Maurer SC (1979) Stimulation of Ca2 + -dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by cal modulin and a calmodulin-like protein isolated from synaptic vesicles. Proc Natl Acad Sci USA 76:1838-1842 Erondu NE, Kennedy MB (1985) Regional distribution of type II calcium!calmodulin-dependent protein kinase in rat brain. J Neurosci 5:3270-3277 Goldenring JR, Gonzalez B, Mcguire IS Jr, DeLorenzo RJ (1983) Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated protein. J Bioi Chem 268:1263212640 Griffith LC, Schulman H (1988) The multifunctional calcium! calmodulin-dependent protein kinase mediates calcium dependent phosphorylation of tyrosine hydroxylase. J Bioi Chem 263:9542-9549 Hemmings HC Jr, Nairn AC, McGuinness TL, Huganir RL, Greengard P (1989) Role of protein phosphorylation in neu ronal signal transduction. FASEB J 3:1583-1592 Hendry SHC, Kennedy MB (1986) Immunoreactivity for a cal modulin-dependent protein kinase is selectively increased in Macaque striate cortex after monocular deprivation. Proc Natl Acad Sci USA 83:1536-1540 Jameson L, Frey T, Zeeberg B, Da1dorf F, Caplow M (1980) Inhibition of microtubule assembly by phosphorylation of mi crotubule-associated proteins. Biochemistry 19:24472-2479
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
Kennet RG (1979) Monoclonal antibodies. Hybrid myelomas-a revolution in serology and immunogenetics. Am J Hum Genet 31:539-547 Kirino T (1982) Delayed neuronal death in the gerbil hippocam pus following ischemia. Brain Res 239:57-69 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 Lai Y, Nairn AC, Greengard P (1986) Autophosphorylation re versibly regulates the calcium/calmodulin-dependence of calcium/calmodulin-dependent protein kinase II. Proc Natl Acad Sci USA 83:4253-4257 Llinas R, McGuinness TL, Leonard CS, Sugimori M, Greengard P (1985) Intraterminal injection of synapsin I or calcium and calmodulin-dependent kinase II alters neurotransmitter re lease at the squid giant synapse. Proc Natl Acad Sci USA 82:3035-3039 Lou LL, Lloyd SJ, Schulman H (1986) Activation of the multi functional calcium/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an au tonomous enzyme. Proc Natl Acad Sci USA 83:9497-9501 Miller SG, Kennedy MB (1986) Regulation of brain type II cal cium/calmodulin-dependent protein kinase by autophosphor ylation: a calcium-triggered molecular switch. Cell 44: 861-870 Nichols RA, Sihra TS, Czernik AJ, Nairn AC, Greengard P (1990) Calcium and calmodulin-dependent protein kinase II increases glutamate and norepinephrine release from synap tosomes. Nature 343:647-650 Nose PS, Griffith LC, Schulman H (1985) Calcium-dependent protein phosphorylation of tyrosine hydroxylase in PC12 cells. J Cell Bioi 101:1182-1190 Odonera H, Hara H, Kogure K, Fukunaga K, Ohta Y, Miyamoto E (1990) Ca2 + /calmodulin-dependent protein kinase II im munoreactivity in the rat hippocampus after forebrain isch emia. Neurosci Lett 113:134--138
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Ouimet CC, McGuiness TL, Greengard P (1984a) Immunocyto chemical localization of calcium/calmodulin-dependent ki nase II in rat brain. Proc Natl Acad Sci USA 81:5604--5608 Ouimet CC, McGuiness TL, Greengard P (1984b) Immunocyto chemical localization of calcium/calmodulin-dependent ki nase II in rat brain. Proc Natl Acad Sci USA 81:5604--5608 Pulsinelli W, Brierly JBA (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10:267272 Sakakibara M, Alkon DL, DeLorenzo RJ, Goldenring JR, Neary JT, Heldman E (1986) Modulation of calcium-mediated in activation of ionic currents by calcium/calmodulin-depen dent protein kinase II. Biophys J 50:319-327 Shields SM, Vernon PJ, Kelly PT (1984) Autophosphorylation of calmodulin-kinase II in synaptic junctions modulates endog enous kinase activity. J Neurochem 43:1599-1609 Taft WC, Tennes-Rees KA, Blair RE, Clifton GL, DeLorenzo RJ (1988) Cerebral ischemia decreases endogenous calcium dependent protein phosphorylation in gerbil brain. Brain Res 447:159-163 Vuliet PR, Woodgett JR, Cohen P (1984) Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase. J Bioi Chem 259:13680-13683 Wasterlain CG, Powell CL (1986) Calcium-dependent protein phosphorylation in cerebral ischemia. In Acute Brain Isch emia: Medical and Surgical Therapy (Battistini N, Fiorani P, Ciurbein R, Plum F, Fieschi C, eds), New York, Raven Press, pp 67-72 Yamamoto H, Fukunaga K, Goto S, Tanaka E, Miyamoto E (1985) Calcium, calmodulin-dependent regulation of micro tubule formation via phosphorylation of microtubule associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation. J Neuro chem 44:759-768 Zivin JA, Kochhar A, Saitoh T (1990) Protein phosphorylation during ischemia. Stroke 21 (Suppl 3):117-124
J Cereb Blood Flow Metab, Vol. 12, No.5, 1992
Global Forebrain Ischemia Results in Decreased Immunoreactivity of Calcium/Calmodulin-Dependent Protein Kinase II
*Severn B. Churn, t Amy Yaghmai, tJohn Povlishock, * Azhar Rafiq, and *:j:Robert J. DeLorenzo Departments of*Neurology, tAnatomy, and tPharmacology, Medical College of Virginia, Richmond, Virginia, U.S.A.
Summary: Previous studies utilizing crude brain homoge nates have shown that forebrain ischemia results in inhi bition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (lC33D6) directed against the 13- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia induced inhibition of CaM kinase II. Immunohistochemi cal investigations showed that the ischemia-induced de creased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia sensitive cells such as pyramidal cells of the hippocam pus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells
of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, au tophosphorylation of CaM kinase II in the presence of low (7 f-lM) or high (500 f-lM) ATP did not alter immuno reactivity of the enzyme with IC3-3D6. The data demon strate the production of a monoclonal antibody that rec ognizes the l3-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. To gether with previous studies, the data support the hypoth esis that rapid, ischemia-induced inhibition of CaM ki nase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke. Key Words: Antigen-Autophosphorylation-Cell loss Immunohistochemistry-Monoclonal antibody-Stroke.
Cerebral ischemia produces morphological and functional modifications in neurons that can result in delayed neuronal death (Pulsinelli and Brierly, 1979; Kirino, 1982; Churn et aI., 1990a,b). Alter ation of calcium homeostasis and calcium-depen dent mechanisms may play a critical role in the isch emia-induced cascade of events resulting in delayed neuronal cell death. The involvement of calcium dependent protein kinase systems in the regulation
of cell function has been intensely investigated in recent years. Calcium-dependent protein phospho rylation modulates many neuronal processes in cluding neurotransmitter synthesis and release (De Lorenzo and Freedman, 1978; Albert et aI., 1984; Vulliet et aI., 1984; Nose et aI., 1985; Griffith and Schulman, 1988; Nichols et aI., 1990), synaptic ves icle mobilization (DeLorenzo et aI., 1979; Llinas et aI., 1985; Hemmings et aI., 1989), postsynaptic events such as cytoskeletal dynamics (Jameson et aI., 1980; Burke and DeLorenzo, 1982; Yamamoto et aI., 1985), and regulation of membrane conduc tances (Sakakibara et aI., 1986). Therefore, alter ation of neuronal calcium-dependent protein kinase systems by ischemia may play a role in mediating some of the effects of calcium in ischemia-induced cell death. Calcium-dependent protein phosphorylation has been shown to be very sensitive to the effects of
Received November 11, 1991; final revision received February IS, 1992; accepted February 21, 1992. Address correspondence and reprint requests to Dr. S. B. Chum at Department of Neurology, Medical College of Virginia, Box 599 MCV Station, Richmond, VA 2329S, U. S. A. Abbreviations used: CaM kinase II, calcium/calmodulin dependent protein kinase II; ELISA, enzyme-linked immunosor bent assay; NHS, normal horse serum; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, so dium dodecyl sulfate; TPBS, PBS containing TWEEN-20.
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CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
ischemia (Goldenring et aI., 1983; Wasterlain and Powell, 1986; Taft et al., 1988; Churn et al., 1990a,b, Zivin et aI., 1990). Thus, inhibition of pro tein phosphorylation may represent a pathway lead ing to alteration of neuronal function and possibly neuronal cell death. To investigate the effects of ischemia on protein phosphorylation, our labora tory has concentrated on studying the ischemia-in duced inhibition of the multifunctional calcium/ calmodulin-dependent protein kinase II (CaM ki nase lI). CaM kinase II activity is inhibited immedi ately after 5 min of ischemia, and the inhibition remains until significant neuronal necrosis is ob served (Taft et aI., 1988; Churn et aI., 1990a). In addition, the decrease in CaM kinase II activity is not likely to be due to large-scale proteolysis. No significant differences are observed in CaM kinase II subunit levels or calmodulin binding properties in forebrain homogenates (Taft et aI., 1988, Churn et aI., 1990a) or purified CaM kinase II enzyme frac tions (Churn et aI., 1991) isolated from control and ischemic animals. In this report, we characterize a monoclonal IgG3 antibody directed against CaM ki nase II and describe the ischemia-induced decrease in immunoreactivity with CaM kinase II both in vitro and in situ. The data indicate that ischemia produces a posttranslational modification of CaM kinase II that alters the ability of the antibody to recognize the f3-subunit of the enzyme. Character ization of this monoclonal antibody may provide a powerful resource for investigating the role of CaM kinase II in cerebral ischemia. METHODS AND MATERIALS Materials
All materials are reagent grade unless otherwise spec ified. [-y_32p]ATP was purchased from New England Nu clear (Wilmington, DE, U.S.A.). Nitrocellulose mem brane (0.45 fl.m) was purchased from Bio-Rad (Richmond, CA, U.S.A.). Purified calmodulin was purchased from Pharmacia (Piscataway, NJ, U.S.A.). Gerbil ischemia model
Bilateral carotid occlusion was performed as previ ously described (Churn et a!., 1990a,b). Briefly, male Mongolian gerbils (Meriones unquiculatus) weighing 5070 g were anesthetized under 2.5% halothane/60% nitrous oxide/40% oxygen and placed on a heated surgical table. The ventral neck area was soaked with Operanol (Redi Products) and shaved using a safety razor. An incision was made by lifting the skin above the sternum and mak ing two to three cuts directed rostrally up the neck. Chest muscles were separated from the trachea by a stripping motion parallel to the trachea to expose the carotid arter ies. To induce ischemia, the carotid arteries were raised and Heifitz aneurysm clips were attached. After 5 min of occlusion, the aneurysm clips were removed and recircu-
785
lation was confirmed visually. The skin was folded over and the wound sutured with 5-0 silk. Surgery, ischemia, and wound closure required 16-20 min. Temperature was monitored continuously during surgery via a lubricated rectal probe (YSI). CaM kinase II reactions
CaM kinase II was purified by standard column chro matography (Goldenring et a!., 1983). Purified Sephacryl fractions (>1,OOO-fold enrichment) or highly enriched cal modulin-Sepharose fractions (>400-fold enriched) were used in the assays. For autophosphorylation studies, CaM kinase II was reacted under standard conditions, except where noted (Goldenring et aI., 1983; Churn et a!., 1990a). Standard phosphorylation reaction solutions contained 10 mM pi perazine-N-N' -bis[2-ethanesulfonate] (pH 7.4), 10 mM MgCI2, and 7 fl.M [-y_32p]ATP. For maximal CaM kinase II activity, 5 mM CaCl2 and 1 fl.g calmodulin were in cluded. Reactions were initiated with the addition of cal cium, continued for 1 min, and then terminated by the addition of 5% sodium dodecyl sulfate (SDS) stop solu tion. Proteins were resolved by SDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) and either sub jected to Western analysis or stained for protein resolu tion with Coomassie Brilliant Blue or the more sensitive silver stain as previously described (Goldenring et aI., 1983; Churn et a!., 1990a). Stained gels were dried and exposed to x-ray film (XRP-l; Kodak, Rochester, NY, U.S.A.) for autoradiography. Development of monoclonal antibody
Mice (Mus musculus, BALB-c; Charles River) were immunized with two intraperitoneal injections (100 fl.g/ injection, 21 days apart) of purified CaM kinase II. Thirty days following the second injection, the mice were killed by cervical dislocation, and a spleen cell suspension was made. The splenocytes were fused with a nonsecreting mouse myeloma cell line (P3X63-AG8.653). Monoclonal producing cells were obtained from antibody-secreting polyclonal hybridomas by limited dilution methods (Ken net, 1979). Subtyping of antibody directed against CaM kinase II was performed by enzyme-linked immunoassay (ELISA) (Kennet, 1979). Specificity of lC3-3D6
The specificity of the monoclonal antibody was tested by Western analysis. Samples of crude homogenate or purified CaM kinase II were resolved on one-dimensional 8.5% SDS-PAGE or two-dimensional isoelectric focusing gels combined with SDS-PAGE and electroblotted onto nitrocellulose (Goldenring et aI., 1982; Churn et aI., 1990a). The nitrocellulose was immersed in phosphate buffered saline (PBS; IO mM NaP04/0.9% NaCI, pH 7.5), containing Tween-20 (TPBS; Sigma, St. Louis, MO, U.S.A.). The strip was incubated in antibody-containing TPBS for I h and washed by three changes of TPBS. The blot was incubated in a solution of biotinylated secondary antibody in TPBS. Excess secondary antibody was washed by three changes of PBS and labeled proteins were reacted with an alkaline phosphatase-staining kit (Vector). In addition to Western analysis, ELISA was performed to observe linearity of immunoreactivity with respect to CaM kinase II concentration or antibody concentration. ELISA plates were coated with varying amounts of CaM =
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S. B. CHURN ET AL.
kinase II for 24 h. The coated plates were washed with TPBS. Background was decreased by incubating coated plates in blocking solution (5 g Carnation Milk/loo ml PBS). Blocked plates were incubated with antibody for 2 h and then washed four times with TPBS. The washed plates were incubated with peroxidase-conjugated anti mouse IgG for 2 h. Staining was performed by reacting plates with substrate solution (Vector) and wells were read at OD 405 nm on an EIA reader (Biotech, Winooski, VT, U.S.A.).
Homogenate
CaM KII
80
Immunohistochemistry and histology
For histological slides, gerbils were subjected to isch emia or sham operation as described above. At the spec ified times postischemia, the animals were killed and per fused and the brains processed for histological examina tion as described previously (Churn et aI., 1990a). Coronal sections of brain were dehydrated in a series of ethanol and xylene baths prior to paraffin embedding. Serial sections 7 J..lm thick containing the dorsal hippo campus were cut using an American Optical AL micro tome (Buffalo, NY, U.S.A.), mounted on slides, and stained with cresyl violet. For immunocytochemical studies, animals were anes thetized with an injection of sodium pentobarbital (50 mg/ kg i.p.). Gerbils were then transcardially perfused with saline followed by fixative composed of 4% paraformal dehyde/O.l M phosphate buffer. After fixation, the brains were removed and placed in fresh fixative for an addi tional hour. They were stored in phosphate buffer at 4°C until sectioned. For sectioning, the harvested brain ma terial was blocked in a sagittal plane and 40-j.tm semiserial sections were cut with a vibratome. The sections were washed in PBS (pH 7.2), incubated for 1 h in 10% normal horse serum (NHS)/0.3% Triton-X, and then incubated in a solution of lC3-3D6 diluted 1: 1, 000 into 1% NHS in PBS for 18 h at 4°C. Following incubation in the primary antibody, the sections were washed 10 min in 1% NHS in PBS and then incubated for 60 min in biotinylated anti mouse IgG (Vector) diluted into 1% NHS/PBS. The sec tions were washed 10 min in PBS and then immersed for 1 h in avidin-biotin-horseradish peroxidase complex (Vector). Incubated sections were washed 10 min in PBS and transferred to a development chamber. The sections were reacted in 0.05% diaminobenzidene and 0.01% hy drogen peroxide for 5 min. Following diaminobenzidene reaction, the sections were mounted onto glass slides, dehydrated, cleared, and covered with a coverslip.
RESULTS Specificity of monoclonal IgG3 anti-CaM kinase II
The selectivity of lC3-3D6 for the (3-subunit of CaM kinase II is shown in Fig. 1. In crude homoge nate fractions, lC3-3D6 reacted with a band of �60 kDa molecular mass. The monoclonal lC3-3D6 did not react with any other protein within the crude homogenate fraction. Highly purified CaM kinase II was subjected to Western analysis and reacted with lC3-3D6 as described in Materials and Methods. A similar band of 60 kDa molecular mass was ob served in the purified fraction as seen in the crude protein fraction. The 60-kDa band identified by J Cereb Blood Flow Me/ab, Vol. 12, No. 5, 1992
-
-
60 50
27
17
Protein
Ab
Ab
FIG. 1. Specificity of 1C3-306 for the l3-subunit of calcium! calmodulin-dependent protein kinase II (CaM kinase II). A monoclonal antibody (Ab) directed against CaM kinase II was generated in our laboratory (see Materials and Methods). The specificity of 1C3-306 was tested against crude homogenate protein and purified (>1,OOO-fold) CaM kinase II subjected to Western analysis. Monoclonal 1C3-306 reacted with a major band of -60 kOa in crude homogenate fraction (lanes 1-2), which was shown to be the l3-subunit of CaM kinase II (lane 3). When analyzed in high concentration, a minor band of -58 kOa was also observed in both crude and purified frac tions. The minor band co-migrates with the 13'-subunit of CaM kinase II (Bennett and Kennedy, 1987).
lC3-3D6 co-migrated with the (3- (60-kDa) subunit (Goldenring et aI., 1983; Bennett and Kennedy, 1987) of highly purified CaM kinase II (Fig. I). A minor band of molecular mass 58 kDa was also identified when high concentrations of protein were studied in both the crude homogenate and the puri fied fractions of CaM kinase II (Fig. 1). This band co-migrated with the 13' -subunit of CaM kinase II identified by Bennett and Kennedy (1987). Immunochemical analysis of CaM kinase II sub jected to two-dimensional SDS-PAGE was per formed to further investigate the remote possibility that a 60-kDa contaminating protein was the immu noreactive agent observed in Fig. 1. Western anal ysis was performed on highly purified CaM kinase II, which was subjected to two-dimensional isoelec tric focusing and SDS-PAGE. The monoclonal lC33D6 reacted with a band of 60-kDa molecular mass
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
and with an isoelectric point of �6.9 (data not shown). The immunoreactive peptide co-migrated 3 with the 2P-Iabeled and protein-stained l3-subunit of CaM kinase II identified in the same gel. Thus, lC3-3D6 reacted with a peptide of 60 kDa molecular mass that co-purified with CaM kinase II and has the same isoelectric point as the l3-subunit of CaM kinase II. The data indicate that lC3-3D6 selec tively recognized the 13- and 13' -subunits of CaM kinase II. Linearity range of immunoreactivity with lC3-3D6
A standard curve for immunoreactivity of CaM kinase II with lC3-3D6 was performed utilizing ELISA. The immunoassay with purified CaM ki nase II was linear over a concentration range from 30 to 760 ng (�6-150 ng l3-subunit, correlation co efficient 0.972, p < 0.001, different from 0, two tailed Student t test, n 18; Fig. 2A). A CaM ki nase II concentration of 380 ng, which was in the middle of the linear portion of the curve, was used =
=
A 1.00 0.75 8 ....
•
c:i c:i 0.50
0.00 '--"'--'---'---,--..o..-.J o .1 2 .3 .4 .5 .6 .7 .8 CaM kinase II ('9) 1.5 r----,
1.0 c o
to perform a standard titration curve for the con centration of lC3-3D6 (Fig. 2B). A linear curve was generated with an antibody range of 0.027 j.1g to the highest concentration tested, 0.54 j.1g (correlation coefficient 0.967, p < 0.001, different from 0, two-tailed Student t test, n 18). This represented a dilution curve of 1:2,000 to 1:1. Therefore, for immunohistochemical studies, a dilution of 1:1,000 (0.054 j.1g lC3-3D6/j.11 reaction volume) was used to reduce background, yet remain in the linear portion of immunoreactivity. =
=
Decreased immunoreactivity of CaM kinase II resulting from ischemia
In the gerbil, 5 min of global forebrain ischemia causes a decrease in CaM kinase II activity without changing total protein levels or calmodulin binding properties of the enzyme (Taft et aI., 1988; Churn et aI., 1990a,b). Ischemia produced a decrease in im munoreactivity of CaM kinase II demonstrated by Western analysis both in starting homogenate frac tions and in highly enriched calmodulin-Sepharose eluant (Fig. 3). The decreased immunoreactivity was in agreement with activity observed in purified (>1,OOO-fold) fractions (Churn et aI., 1990b, 1991). Immunoreactivity of CaM kinase II was reduced 57.6 ± 10.5% in a fraction exhibiting 55% inhibition of CaM kinase II activity (Fig. 3C; p < 0.01, Stu dent t test, n 4). The decreased immunoreactivity occurred in fractions where equivalent protein staining and calmodulin binding of the (X- and l3-sub units were observed (Taft et aI., 1988; Churn et aI., 1990a,b) (Fig. 3). The data suggest that global fore brain ischemia in the gerbil induced a posttransla tional modification of CaM kinase II that did not affect calmodulin binding or total protein levels sig nificantly but reduced antibody recognition of the enzyme. =
025
B.
787
Effect of autophosphorylation on immunoreactivity
0.5
of CaM kinase II
•
0.0 '--'---'--'---' .6 .5 .4 .3 2 .1 o lC3-306 Concentration ('9) FIG. 2. Linear regression curves of immunoreactivity under various total concentrations of calcium/calmodulin dependent protein kinase II (CaM kinase II) or various con centrations of 1C3-3D6. Enzyme-linked immunosorbent as say was performed on vessels containing various concentra tions of antigen or antibody. A: CaM kinase II concentration was varied between 18 and 760 ng. A standard curve was generated with a correlation coefficient of 0.972 (p < 0.001, different from 0, two-tailed Student t test, n 18). B: Con centration of 1C3-3D6 was varied and reacted with 380 ng of CaM kinase II. A standard curve was generated with a corre lation coefficient of 0.967 (p < 0.001, different from 0, two tailed Student t test, n 18). Each point represents average ± SEM of three replications. =
=
The extent of autophosphorylation has been shown to alter the immunoreactivity of CaM kinase II with some monoclonal antibodies (Hendry and Kennedy, 1986). Therefore, we investigated whether autophosphorylation alters immunoreac tivity with 1C3-3D6 (Fig. 4). CaM kinase II was reacted under standard conditions (7 j.1M ATP), re solved on SDS-PAGE, and subjected to Western analysis with >80% transfer efficiency. In addition to standard reactions, reactions where the level of ATP was increased to 500 j.1M were performed (Lou et aI., 1986). Autophosphorylation of CaM kinase II at either standard (7 j.1M) or high (500 j.1M) levels of ATP did not alter immunoreactivity with 1C3-3D6. J Cereb Blood Flow Metab, Vol. 12, No. 5, 1992
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A.
HOMOGENATE FRACTION ANTIBODY
CALMODULIN
...... -.... . .� .. --
C
C
CaM KINASE II FRACTION
B.
ANTIBODY
CALMODULIN
C
C
c. �
'0 :::
100
t: 0
() '0 2ft: 0 '';::::;
75
50
'c
Cl 0 U Q)
a::
25
.c
< o
Ischemic
Control
FIG. 3. Ischemia results in decreased immunoreactivity of calcium/calmodulin-dependent protein kinase II (CaM kinase II) with 1C3-306. Protein samples were obtained from gerbils subjected to sham operation or 5 min of bilateral carotid occlusion. Decreased immunoreactivity with 1C3-3D6 was observed in samples isolated from ischemic animals. The de creased immunoreactivity was observed in crude homoge nate fractions (A) and in enriched CaM kinase II fractions (B). The decreased immunoreactivity occurred under conditions where equivalent calmodulin binding is observed between control (C) and ischemic (I) fractions. Also shown is quanti fication of ischemia-induced inhibition of immunoreactivity of CaM kinase II with 1C3-3D6 (e). CaM kinase II was isolated from control and ischemic animals and subjected to Western analysis. CaM kinase II isolated from ischemic animals dis played 42.4 ± 10.4% of control immunoreactivity. Ab, anti body. "p < 0.01, Student's t test, n 4. =
Therefore, changes in immunoreactivity of CaM ki nase II with lC3-3D6 are not secondary to auto phosphorylation of the enzyme. Decreased immunohistochemical reactivity with lC3-3D6 resulting from ischemia
Delayed neuronal loss after 5 min of ischemia is observed in pyramidal cells of the CAl and CA3 J Cereb Blood Flow Metab, Vol. 12, No.5, 1992
subregions of the hippocampus (Pulsinelli and Bri erly, 1979; Kirino, 1982; Churn et aI., 1990a,b) (Fig. 5). Histological loss of pyramidal neurons is not ob served until 3-4 days following 5 min of global fore brain ischemia and fully developed after only 7 days (Pulsinelli and Brierly, 1979; Kirino, 1982; Churn et aI., 1990a,b) (Fig. 5C-F). To further study the effects of ischemia on the immunoreactivity of CaM kinase II with lC3-3D6, immunohistochemistry was performed on brains harvested from naive and ischemic animals. Local ization of forebrain immunoreactivity observed with lC3-3D6 is similar to that observed or a mono clonal antibody directed against the a- subunit of CaM kinase II (Ouimet et a!., 1984a,b; Erondu and Kennedy, 1985; Odonera et aI., 1990). CaM kinase II is concentrated in neuronal cells and is especially prominent in the pyramidal cells of the hippocam pus, cortical neurons, and striatal neurons. In addi tion to the forebrain, significant immunoreactivity toward lC3-3D6 was observed in the cerebellum (data not shown). Loss of CaM kinase II activity is observed in the hippocampus immediately following 5 min of global forebrain ischemia in the gerbil (Taft et aI., 1988; Churn et aI., 1990b). Loss of immunoreactivity oc curs within 30 min in hippocampal cells, which show significant delayed neuronal loss following 5 min of global forebrain ischemia (Fig. 6). In addi tion, the loss of immunoreactivity is observed at both 2 and 24 h of reperfusion (data not shown). This loss is observed before the histological damage induced by 5 min of global forebrain ischemia (Figs. 5 and 6). Thus, the ischemia-induced loss of immu noreactivity with lC3-3D6 occurs only in neurons that show significant histological cell loss after tran sient global forebrain ischemia. In addition, loss of immunoreactivity with lC3-3D6 significantly pre cedes histological observation of ischemia-induced neuronal necrosis. DISCUSSION
CaM kinase II is an important neuronal enzyme that is significantly inhibited in several models of ischemia, including the gerbil global forebrain isch emia model (Taft et aI., 1988; Churn et aI., 1990a,b), the rabbit spinal chord ischemia model (Zivin et aI., 1990), and decapitation ischemia in the rat (Golden ring et aI., 1983; Wasterlain and Powell, 1986). Isch emia has also been shown to alter immunoreactivity of CaM kinase II with monoclonal antibodies di rected against the enzyme (Wasterlain and Powell, 1986; Churn et aI., 1991). CaM kinase II has been purified from control and ischemic gerbils, and
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
789
Low ATP Autophosphorylation
-
Immunoreactivity
-
-
-
FIG. 4. Autophosphorylation of cal
cium/calmodulin-dependent protein kinase II (CaM kinase II) does not alter immunoreactivity with 1C3-3D6. CaM kinase II was reacted for various amounts of time under standard con ditions (7 fLM ATP) or standard condi tions with high (500 fLM) ATP. Lett: In creasing the time of reaction resulted in increased 32p incorporation into the a- (50-kDa) and 13- (60-kDa) subunits of CaM kinase II. Right: Increasing the extent of autophosphorylation did not alter immunoreactivity of CaM kinase II with 1C3-3D6 at either standard (7 fLM) or high (500 fLM) ATP. Data shown are representative of four experi ments.
Time
.5
5
10
20
.5
5
10
20
High ATP
Autophosphorylation
-
-
Time
Immunoreactivity
.5
when balanced for protein staining and calmodulin binding, the ischemia-induced inhibition of the en zyme remained. The previous observations support the hypothesis that ischemia induced a posttransla tional modification of CaM kinase II (Churn et al., 1990a,b, 1991). In this report, we characterized a monoclonal antibody directed against the l3-subunit of CaM kinase II. It was demonstrated that isch emia induced an alteration of CaM kinase II that resulted in decreased immunoreactivity of the en zyme. The decreased immunoreactivity was not due to autophosphorylation of the enzyme and oc curred under conditions that did not alter the con centration of protein staining or calmodulin binding of the l3-subunit of the kinase. The monoclonal IgG3 antibody developed in this study was highly selective for the l3-subunit of CaM kinase II. Immunoreactivity of 1C3-3D6 in a crude
5
10
20
-
-
.5
5
10
20
homogenate fraction was specific to a 60-kDa band on SDS-PAGE. This band was shown to be the l3-subunit of CaM kinase II by Western blot analysis of purified enzyme with lC3-3D6 on one- and two dimensional PAGE systems. The monoclonal lC33D6 antibody reacted with a peptide displaying the same molecular mass and the same isoelectric point of the l3-subunit of CaM kinase II (Goldenring et aI., 1983). Ischemia resulted in decreased immunoreac tivity of CaM kinase II when studied by Western analysis. The decreased immunoreactivity was ob served in both crude homogenate and highly en riched fractions. This decreased immunoreactivity agrees with data shown in other models of ischemia such as the Levine preparation (Wasterlain and Powell, 1986; Odonera et aI., 1990). The mecha nisms whereby ischemia produces alterations in CaM kinase II immunoreactivity are not presently
J Cereb Blood Flow Metab, Vol. 12, No.5, 1992
S. B. CHURN ET AL.
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FIG. 5. Transient ischemia results in delayed, selective neuronal cell loss after 7 days. Histological studies of hippocampal pyramidal cells were performed on sham-operated gerbils and gerbils subjected to 5 min of global forebrain ischemia at 39°C. A: Photomicrograph ( x35 original magnification) of sham-operated gerbil, showing clearly defined pyramidal cell layer. B: High-magnification (original magnification x475) photomicrograph of CA1 pyramidal cell region from sham-operated gerbil, showing clearly defined cells with dark-staining nuclei. C: Photomicrograph ( x35 original magnification) of ischemic gerbil allowed to recover for 2 h postischemia, showing no significant loss of pyramidal neurons. 0: High magnification (original magnification x475) of CA1 pyramidal cells from ischemic gerbil in C, showing clearly defined neurons with dark-staining nuclei. E: Photomicrograph ( x35 original magnification) of ischemic gerbil allowed to recover for 7 days postischemia. Note selective cell loss in the CA1-3 subregion of the hippocampus with remarkable sparing of granule cells of the dentate gyrus. F: High magnification (original magnification x475) of CA1 pyramidal cell region taken from gerbil in E. Pyramidal cells are fragmented, dense, with no discernible nuclei.
known. To investigate the ischemia-induced alter ation(s) of CaM kinase II, mapping of the 1C3-3D6 recognition site will be performed. Autophosphorylation of CaM II kinase has been shown to alter both enzymatic activity (Lai et aI.,
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1986; Lou et aI., 1986; Nichols et aI., 1990) and immunoreactivity (Hendry and Kennedy, 1986). Therefore, we studied whether autophosphoryla tion of CaM kinase II resulted in decreased immu noreactivity of the enzyme with lC3-3D6. Under
CaM KINASE II IMMUNOREACTIVITY IN ISCHEMIA
791
FIG. 6. Global forebrain ischemia results in loss of calcium/calmodulin-dependent protein kinase II (CaM kinase II) immunore activity in selectively vulnerable cells prior to observation of histological damage. A: Photomicrograph (x40 original magnifi cation) of 1C3-3D6 immunoreactivity in hippocampus of sham-operated gerbil, showing enriched immunoreactivity in the soma and dendritic areas of granule and pyramidal cell layers. B: Photomicrograph (x40 original magnification) of 1C3-3D6 immu noreactivity in gerbil subjected to 5 min of global forebrain ischemia (39°C) and immediately perfused (see Materials and Methods). Note loss of immunoreactivity in CA1-3 pyramidal cells with no significant loss of immunoreactivity in granule cells of the dentate gyrus. C: High magnification (original magnification x475) of CA1 pyramidal cell layer taken from gerbil in A. Note enriched staining of pyramidal soma area. 0: High magnification (original magnification x475) of CA1 pyramidal cell layer taken from ischemic gerbil in B. Note significant loss of 1C3-3D6 immunoreactivity in pyramidal soma layer.
standard conditions, ATP has been shown to mod ulate the kinetics of CaM kinase II autophosphory lation (Lou and Schulman, 1986). Low ATP con centrations, such as those around the Kd for ATP (7 fLM), resulted in autophosphorylation that leads to inhibition of the enzyme. However, in the presence of high ATP levels (500 fLM), which are closer to physiological ATP concentrations, autophosphory lation does not lead to inhibition of the enzyme. Since the level of ATP modulates the extent of autophosphorylation, CaM kinase II was reacted under standard conditions in the presence of both low (7 fLM) and high (500 fLM) ATP. Immunoreac tivity of CaM kinase II with lC3-3D6 was not al tered after extensi'/e autophosphorylation of the en zyme at either ATP level. Therefore, the ischemia induced decrease in immunoreactivity of CaM
kinase II was not due to autophosphorylation of the enzyme. Previous studies provide evidence that the isch emia-induced inhibition of CaM kinase II activity may be involved in the cascade of events resulting in delayed neuronal cell death. First, CaM kinase II activity is decreased at an early time point following global ischemia in the gerbil and remains inhibited until significant cell loss is observed (Taft et aI., 1988; Churn et aI., 1990b). In addition, parameters that modulate the extent of neuronal cell loss, such as intraischemic temperature, modulate the extent of CaM kinase II inhibition in a parallel manner (Churn et aI. , 1990a,b). To further investigate whether CaM kinase II inhibition might be involved with delayed neuronal cell loss, immunohistochem istry was performed on brain sections from isch-
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S. B. CHURN ET AL.
emic gerbils. Loss of CaM kinase II immunoreac tivity was noted in the CAl_3 subregions of the hip pocampus. Under the conditions of ischemia, these are the cell populations that are most sensitive to the effects of ischemia (Pulsinelli and Brierly, 1979; Kirino, 1982; Churn et al., 1990a). No significant loss of immunoreactivity or neuronal necrosis was observed in ischemia-resistant cells such as in the dentate gyrus. What is significant about the alter ation in immunoreactivity of CaM kinase II is that it occurs rapidly, long before cell loss is noted. Loss of CaM kinase II activity following ischemia is ob served after 10 s and persists until significant cell loss occurs (Taft et al., 1988; Churn et al., 1990b). The decreased immunoreactivity of CaM kinase II with lC3-3D6 occurs within 30 min following the ischemic insult and remains after 24 h of reperfu sion. Thus, decreased immunoreactivity of CaM ki nase II is an early event following ischemia and occurs in cells that are sensitive to the effects of ischemia. Ischemia results in decreased immunore activity that temporally parallels the ischemia induced inhibition of CaM kinase enzymatic activ ity and precedes ischemia-induced neuronal cell loss. Since the loss of immunoreactivity of CaM kinase II parallels the histological loss of neuronal cells, the immunohistochemistry studies provide further evidence that alteration of CaM kinase II activity may be involved in the cascade of events that ultimately result in neuronal death. The anti body may also be a useful early marker of cells that have sustained significant ischemic injury and will ultimately die. CaM kinase II has been successfully isolated from ischemic gerbils (Churn et all., 1990b, 1991). Ischemic CaM kinase II displays equivalent cal modulin and substrate recognition and reaction ki netics when compared with control enzyme (Churn et al., 1991). In this report, it was shown that isch emia alters CaM kinase II, which results in de creased immunoreactivity of the enzyme. This de creased immunoreactivity is not due to autophos phorylation of the enzyme, nor is it due to significant proteolysis. The data from this study, in combination with previously described results (Taft et al., 1988; Churn et al., 1990a,b) suggest that isch emia induces a posttranslational alteration of CaM kinase II, which results in decreased immunoreac tivity of the enzyme. Since the decreased immuno reactivity occurs in cells that are sensitive to the effects of ischemia, alteration of CaM kinase II may be important in the cascade of events that result in ischemia-induced delayed neuronal cell death. The use of lC3-3D6 as a marker of the ischemia-induced
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postranslational modification of CaM kinase II will provide a powerful tool to study the role of this major enzyme system in stroke. Acknowledgments: This work was supported by an NINDS Jacob Javits Award (RO I-NS23350) and the Ep ilepsy Program Project (PO I-NS25630) to R.J.D. and an NIH postdoctoral grant (HL07537) to S.B.C. The au thors would like to thank Pam Schwartz (Hybridoma Laboratory, Medical College of Virginia) for her expert assistance.
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Ouimet CC, McGuiness TL, Greengard P (1984a) Immunocyto chemical localization of calcium/calmodulin-dependent ki nase II in rat brain. Proc Natl Acad Sci USA 81:5604--5608 Ouimet CC, McGuiness TL, Greengard P (1984b) Immunocyto chemical localization of calcium/calmodulin-dependent ki nase II in rat brain. Proc Natl Acad Sci USA 81:5604--5608 Pulsinelli W, Brierly JBA (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10:267272 Sakakibara M, Alkon DL, DeLorenzo RJ, Goldenring JR, Neary JT, Heldman E (1986) Modulation of calcium-mediated in activation of ionic currents by calcium/calmodulin-depen dent protein kinase II. Biophys J 50:319-327 Shields SM, Vernon PJ, Kelly PT (1984) Autophosphorylation of calmodulin-kinase II in synaptic junctions modulates endog enous kinase activity. J Neurochem 43:1599-1609 Taft WC, Tennes-Rees KA, Blair RE, Clifton GL, DeLorenzo RJ (1988) Cerebral ischemia decreases endogenous calcium dependent protein phosphorylation in gerbil brain. Brain Res 447:159-163 Vuliet PR, Woodgett JR, Cohen P (1984) Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase. J Bioi Chem 259:13680-13683 Wasterlain CG, Powell CL (1986) Calcium-dependent protein phosphorylation in cerebral ischemia. In Acute Brain Isch emia: Medical and Surgical Therapy (Battistini N, Fiorani P, Ciurbein R, Plum F, Fieschi C, eds), New York, Raven Press, pp 67-72 Yamamoto H, Fukunaga K, Goto S, Tanaka E, Miyamoto E (1985) Calcium, calmodulin-dependent regulation of micro tubule formation via phosphorylation of microtubule associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation. J Neuro chem 44:759-768 Zivin JA, Kochhar A, Saitoh T (1990) Protein phosphorylation during ischemia. Stroke 21 (Suppl 3):117-124
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