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NEUROPHARMACOLOGY
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (F.H., K.F.); New Product Research Laboratories II, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan (Y.S.); and Tohoku University 21st Century Center of Excellence Program, Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation (CRESCENDO), Sendai, Japan (F.H., K.F.)
Received August 30, 2005; accepted February 6, 2006.
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Abstract |
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). ME-induced ischemic injury includes many pathological events, such as release of excitatory amino acids and deprivation of oxygen/glucose (Smith et al., 1990; Takeo et al., 1992). These events then trigger calcium influx into neurons, thereby leading to activation of Ca2+-dependent signaling, such as Ca2+/CaM-dependent enzymes and Ca2+-dependent protease calpains (Hayashi et al., 1998; Takagi et al., 2001). Brain ischemia/reperfusion injury is associated with calpain-induced proteolysis of the cytoskeleton, including fodrin/spectrin (Fokuda et al., 1998; Sato et al., 1999; Yakota et al., 2003). We also documented nitric oxide production through activation of Ca2+/CaM-dependent nitric-oxide synthase (NOS) in ME-induced ischemia, thereby producing protein tyrosine nitration (Shirakura et al., 2005). The novel CaM inhibitor DY-9760e rescues neurons from ME-induced neuronal damage with concomitant inhibition of both NO production and protein tyrosine nitration (Hashiguchi et al., 2003). Interestingly, the neuroprotective effect of DY-9760e is also highly correlated with inhibition of calpain-induced breakdown of fodrin, a cytoskeletal and CaM-binding protein (Sato et al., 1999). Therefore, we hypothesize that inhibition of fodrin breakdown by calpain is partly involved in the neuroprotective action of DY-9760e.
In addition to calpain activation, caspase-3 plays pivotal roles in apoptotic cell death by inducing Bid and poly(ADP-ribose) polymerase activation (Affar et al., 2001; Degli Esposti et al., 2003). Inappropriate imbalances between calpain and calpastatin activation may play critical roles in the delayed neuronal death including apoptosis (Rami et al., 2003). Inhibition of nitric oxide production and fodrin breakdown by DY-9760e may suppress apoptotic signals such as caspase-3 activation through maintaining membrane integrity and suppressing mitochondrial damage. Indeed, cross-talk between calpain and caspase-3 has been documented in brain ischemia. For example, calpain-mediated N-terminal truncation of caspase-3 to a p30 polypeptide enhanced caspase-3 activation in one study (Blomgren et al., 2001) and inhibited its activation in another (McGinnis et al., 1999). Calpain-mediated N-terminal truncation of caspase-9 results in the loss of ability to activate caspase-3 (Chua et al., 2000; Lankiewicz et al., 2000). Calpastatin, an endogenous calpain inhibitor, is cleaved by caspase-3, thereby leading to loss of its inhibitory effect on calpain (Rami et al., 2003). Calpastatin also inhibits translocation of calpain to the plasma membrane, thereby inhibiting its proteolytic activity toward membranous cytoskeletal proteins such as fodrin (Melloni et al., 1996; Kosower et al., 2000). In addition, calpastatin is a CaM-binding protein; therefore, it leads to the hypothesis that DY-9760e may affect proteolysis of calpastatin, which is mediated by caspase-3.
Little is known about the cross-talk between calpain and caspase-3 in brain ischemia-induced neuronal death in vivo. Therefore, we asked whether cross-talk of calpain/caspase-3 participates in ME-induced neuronal damage through calpastatin. We also determined that DY-9760e has an inhibitory effect on calpastatin degradation during ischemia and showed that inhibition of cross-talk between calpain and caspase-3 by DY-9760e probably underlies its neuroprotective action in brain ischemia. Therefore, we propose a novel neuroprotective mechanism of DY-9760e.
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Materials and Methods |
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). Neurologic scores were determined 0.5, 24, 48, and 72 h after microsphere injection. Scoring was as follows: grade 0, no deficit; grade 1, right side forelimb weakness and torso turning to the left side when held by tail; grade 2, circling to left side; grade 3, circling to left side and inability to bear weight on the right side; and grade 4, no spontaneous locomotor activity. Rats with grade 0 and grade 4 were not used for subsequent experiments. Rats were decapitated at 2, 6, 12, 24, 48, and 72 h after injection of microspheres, and the striatum and cortical regions of the ipsilateral hemisphere were dissected for immunoblotting analyses. Drug Treatments. DY-9760e was dissolved with 100% DMSO and diluted in a 5% glucose solution to a final concentration of 1% DMSO. The microsphere-induced cerebral ischemic model was prepared as described above. After microsphere injection, rats were treated with DY-9760e at 12.5 or 25 mg/kg intraperitoneally. In each case, DY-9760e was injected twice 30 min and 6 h after microsphere injection. Vehicle-treated animals were administered the same volume of 1% DMSO in 5% glucose 30 min and 6 h after microsphere injection, and sham groups received the same experimental procedures without microsphere injection. The dose of DY-9760e used did not affect the mean of arterial blood pressure monitored 30 min after DY-9760e administration (data not shown).
Western Blotting Analysis. After decapitation, brains were removed and rinsed once with ice-cold 0.32 M sucrose. Coronal sections of 2 mm were prepared by a hand brain slicer, and ipsilateral and contralateral hemispheres including cortex and striatum were dissected and stored at –80°C. Frozen brain tissues were homogenized in buffer containing 50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 4 mM EGTA, 10 mM EDTA, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 50 mM NaF, 100 nM calyculin A, 50 µg/ml leupeptin, 25 µg/ml pepstatin A, 50 µg/ml trypsin inhibitor, and 1 mM dithiothreitol (DTT). Insoluble material was removed by a 20-min centrifugation at 15,000g. After determining protein concentration in each fraction using Bradford's solution, samples containing equivalent amounts of protein were applied to 10 to 15% acrylamide-denaturing gel (SDS-PAGE) (Laemmli, 1970). Proteins were then transferred to an Immobilon polyvinylidene difluoride transfer membrane for 2 h at 70 V. Membranes were blocked in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 containing 5% fat-free milk powder for 1 h at room temperature and incubated with fodrin (rabbit polyclonal antibody, 1:2000) (Sato et al., 1999), cleaved caspase-3 (rabbit polyclonal antibody, 1:1000; Cell Signaling Technology, Beverly, MA), calpastatin (goat polyclonal antibody, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), and 
-tubulin (mouse monoclonal antibody, 1:10,000; Sigma, St. Louis, MO) antibodies overnight at 4°C. After washing, membranes were incubated 60 min at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody diluted in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 solution for 60 min at room temperature. Immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Amersham Life Science, Little Chalfont, Buckinghamshire, UK). Images were scanned and analyzed semiquantitatively using the Image Gauge Software (Fuji Film, Tokyo, Japan).
Immunohistochemical Studies. Rats were anesthetized and transcardially perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) as described previously (Kawano et al., 2002). Whole brains were immediately removed and postfixed overnight at 4°C in the same fixative. Then, coronal brain sections at the coordinates of 1 mm posterior to bregma (35-µm thick) were prepared using a vibratome. Sections were incubated at room temperature with 0.01% Triton X-100 in PBS for 30 min and for another hour in 3% bovine serum albumin in PBS. For immunolabeling, slices were probed with anticalpastatin (goat polyclonal antibody, 1:200; Santa Cruz Biotechnology), anticleaved caspase-3 (rabbit polyclonal antibody, 1:500; Cell Signaling Technology), and anti-NeuN (mouse monoclonal antibody, 1:500; Chemicon International, Temecula, CA) overnight at 4°C. After washing steps, sections were incubated with biotinylated anti-goat or rabbit IgG (1:5000) in TNB buffer [0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.5% blocking reagent (supplied in TSA-Direct kit; NEN Life Science Products, Boston, MA)] for 1 h, followed by both streptavidin-horseradish peroxidase (1:5000) and Alexa 594 anti-mouse IgG (Molecular Probes, Eugene, OR) in TNB buffer (1:400) and labeled for 2 h. Sections were then stained with tetramethylrhodamine tyramide for 10 min using the TSA-Direct kit. Immunofluorescent images were taken with a confocal laser scanning microscope (TCS SP; Leica Microsystems). To normalize immunoreactivity with anticalpastatin antibody, we analyzed cortical slices without changing the confocal laser intensity and laser aperture. Furthermore, sections were double-stained with anti-NeuN antibody as a neuronal marker. We confirmed that the intensity of immunoreactivity with anti-NeuN antibody was the same between control and ischemic cortical slices. We further quantified the NeuN-positive neurons in six consecutive sections of the ipsilateral cortex under confocal laser microscope at 72 h after ME to evaluate neuroprotective action of DY-9760e. The photographed regions are borders of the infarction, in which neither obvious morphological nor immunohistochemical changes with anti-NeuN antibody were observed in neurons compared with sham-operated animals.
Capase-3 Activity Assay. Caspase-3 activity was measured by cleaving N-acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin) (BioMol, Plymouth Meeting, PA), a selective substrate for caspase-3. The ipsilateral hemisphere containing both striatum and cortex was obtained 24 h after ischemia from both nonischemic and ischemic groups. Tissues were homogenized in a homogenizing buffer containing 15 mM Tris-HCl, pH 7.4, 320 mM sucrose, 1 mM EGTA, 2 mM EDTA, 50 mM NaF, 1 mM MgCl2, 1 mM Na3VO4, and 30 mM sodium pyrophosphate plus protease inhibitors and centrifuged at 14,000g for 60 min at 4°C. Protein concentration of supernatants was measured by Bradford's method, and equal amounts of proteins (100 µg) were incubated in a total volume of 400 µl comprised of 20 mM HEPES, pH 7.4, 10% glycerol, and 2 mM DTT. The reaction was started by addition of caspase-3 fluorogenic substrate Ac-DEVD-AMC. After incubation for 60 min at 37°C, cleavage of the fluorogenic substrate was detected using a PerkinElmer LS50B luminescence spectrometer (PerkinElmer Life and Analytical Sciences, Boston, MA) with excitation and emission wavelengths of 380 and 460 nm, respectively. Activities of caspase-3 were expressed as changes in DEVDase activity.
Preparation of Brain Extracts and Incubation with Exogenous Caspase-3 Enzymes. Dissected brain tissue including cortex and striatum was homogenized in buffer containing 50 mM Tris-HCl, pH 7.4, 320 mM sucrose, 50 µg/ml leupeptin, 25 µg/ml pepstatin A, 50 µg/ml trypsin inhibitor, and 1 mM DTT. After centrifugation at 15,000g for 20 min, supernatants were incubated for 15 min in 60°C to denature endogenous protease activities. Brain extracts (20 µg of protein) were incubated for 30 min with 0.2 µg of purified caspase-3 at 30°C in a 50 µl-reaction medium containing 50 mM HEPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, and 10% glycerol. Under the same condition, heat-treated brain extracts were incubated with caspase-3 in the presence of 2.5, 5, or 20 µM DY-9760e with or without Ca2+/CaM. After incubation with purified caspase-3, brain extracts were subjected to SDS-PAGE followed by immunoblotting with anticalpastatin and anticaspase-3 antibodies as described above.
Statistical Analysis. Data were represented as means ± S.D. and obtained from at least four independent animals. Significance differences between treated and control animals at each time point were assessed by Student's t test. Multiple comparisons between experimental groups were performed by one-way analysis of variance, followed by Dunnett's test. P < 0.05 was considered significant.
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-tubulin showed an equal amount of loaded protein in each lane (Fig. 1A). To determine whether changes in expression of calpastatin, an endogenous calpain inhibitor, underlie further activation of calpain, calpastatin levels were assessed by immunoblotting using anticalpastatin antibody. In sham-operated animals, a 110-kDa immunoreactive protein was detected by SDS-PAGE (Fig. 1B). Interestingly, levels of calpastatin were significantly reduced 6 to 24 h after ME induction. Such decreased levels in calpastatin were correlated with further increases in FDBP generation seen after 12 h, as shown in Fig. 1A.
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Effects of DY-9760e on the Neurological Deficits. Neurological scores were determined to verify the outcome of neurological functions at 0.5, 24, 48, and 72 h after microsphere injection. There was no significant difference in scores between vehicle (2.90 ± 0.22) and DY-9760e-treated groups (3.10 ± 0.42) at 0.5 h after ME. However, the neurological scores in DY-9760e-treated rats significantly improved at 24, 48, and 72 h compared with vehicle-treated rats (Fig. 3).
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DY-9760e Treatment Blocked Activation of Caspase-3 after ME. We next asked whether DY-9760e suppresses caspase-3 activation after ME. As shown in Fig. 5, the level of 17-kDa caspase-3 active fragment significantly increased 24 h after ME and after treatment with DY-9760e (25 and 50 mg/kg) inhibited increased active caspase-3 production in a dose-dependent manner (Fig. 5A). In accord with this, caspase-3 activity also increased at 24 h after ME, as production of 17-kDa active fragment and increased activity was reduced by DY-9760e treatment (Fig. 5B).
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Immunohistochemical Calpastatin Expression Is Reduced in Neurons after ME-Induced Ischemia. To confirm that decreased calpastatin and caspase-3 activation occurs in neurons after ME, we performed immunohistochemical studies using anticalpastatin and active caspase-3 antibodies. When cortical sections were double-stained with anti-NeuN antibody, calpastatin immunoreactivity colocalized with NeuN immunoreactivity in both the cytosol and nucleus of cortical neurons (Fig. 6). Both immunoreactivities against calpastatin and NeuN markedly decreased 24 h after ME in cortical neurons compared with those seen in sham-operated animals. Immunoreactivity with calpastatin was markedly reduced, even in surviving NeuN-positive neurons. Finally, treatment with DY-9760e partially restored immunostaining patterns against both calpastatin and NeuN. Like cortical neurons, decreased calpastatin immunoreactivity was also evident in striatal neurons 24 h after ME (data not shown). In contrast with decreased calpastatin immunoreactivity, dramatic increases in active caspase-3 immunoreactivity were observed in cortical neurons. The appearance of active caspase-3 in cortical neurons was largely abolished by treatment with DY-9760e as expected. We further confirmed that DY-9760e treatment led to a significantly increase in the number of survival neurons (825 ± 225 per mm2; P < 0.05) compared with vehicle group (542 ± 199 per mm2) in the ipsilateral cortex 72 h after ME.
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Ca2+/CaM-Induced Proteolysis of Calpastatin by Caspase-3 and Its Inhibition by DY-970e Treatment. Finally, we confirmed that caspase-3 induced proteolysis of calpastatin in a Ca2+/CaM-dependent manner in vitro using purified caspase-3 and calpastatin in brain extracts. After inactivation of endogenous caspases and proteases by heating, brain extracts (20 µg) were incubated with or without purified caspase-3 in the presence or absence of Ca2+/CaM, and amounts of calpastatin were determined by immunoblotting. As shown in Fig. 7, the addition of caspase-3 to the incubation medium resulted in proteolysis of 110-kDa calpastatin in a Ca2+/CaM-dependent manner. Ca2+/CaM-dependent proteolysis of calpastatin was inhibited by DY-9760e in a dose-dependent manner. To verify whether DY-9760e directly inhibits caspase-3 activity in vitro, we tested its effect on caspase-3 autolysis without substrate. The treatment with DY-9760e did not affect autolysis of caspase-3 up to 20 µM (data not shown).
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Discussion |
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; Hashiguchi et al., 2003). In the previous study, we defined two possible neuroprotective mechanisms of DY-9760e using brain ischemia/reperfusion models. DY-9760e is a potent CaM inhibitor that primarily inhibits Ca2+/CaM-dependent NOS (Hashiguchi et al., 2003) and fodrin breakdown (Sato et al., 1999) after ischemia/reperfusion in rat brain. Generation of NO after brain ischemia/reperfusion has detrimental effects on neurons by producing peroxynitrite (Hashiguchi et al., 2003). The fodrin breakdown probably mediates loss of membrane integrity, thereby leading to necrosis in neurons (Sato et al., 1999). Here, we found a novel mechanism of DY-9760e-induced neuroprotection, in which cross-talk between caspase-3 and calpain through calpastatin underlies its neuroprotective action. A key observation in the present study is that proteolysis of calpastatin by caspase-3 is dependent on Ca2+/CaM in vivo. We also confirmed Ca2+/CaM-dependent proteolysis of calpastatin in vitro by purified caspase-3 using brain extracts. We also found that DY-9760e, a novel neuroprotective agent, could rescue neurons in part by inhibiting cross-talk between caspase-3 and calpain after brain ischemia. Because cross-talk between caspase-3 and calpain plays crucial roles in apoptotic neuronal death after not only brain ischemia but also neurodegenerative disorders (Zhao et al., 2000; Blomgren et al., 2001; Pike et al., 2004), our results showing inhibition of this activity by a novel CaM antagonist is potentially important for development of novel neuroprotective drugs for these conditions.
Calpains are expressed as two isoforms (I or µ-calpain and II or m-calpain), which have different requirements for Ca2+ in the micromolar and millimolar ranges, respectively. µ-Calpain is known to function in both apoptotic and necrotic neuronal death after brain ischemia (Rami, 2000). In the case of apoptotic signaling, calpain-mediated cleavage of caspase-3, -7, -8, -9, and -12 is apparently involved in apoptotic signaling (Chua et al., 2000; Lankiewicz et al., 2000; Nakagawa et al., 2000; Blomgren et al., 2001). However, the functional relevance of cleavage of caspases by calpain is unclear. Calpain-mediated cleavage directly activated procaspase-7 and -12 (Ruiz-Vela et al., 1999; Nakagawa et al., 2000). In addition to the calpain-mediated caspases, caspase-mediated cleavage of calpastatin has been documented in cell culture models of apoptosis (Porn-Ares et al., 1998; Wang et al., 1998; Kato et al., 2000; Neumar et al., 2003). Caspase-mediated cleavage of calpastatin also results in activation of calpain. Therefore, the relevance of cross-talk between calpains and caspases should be tested in vivo ischemia models. We here show that caspase-mediated cleavage of calpastatin accounts for activation of calpain in an in vivo ischemia model. Furthermore, cleavage of calpastatin by caspase-3 occurred in a Ca2+/CaM-dependent manner in the brain. Because calpastatin is ubiquitously expressed and is a potent inhibitor of µ- and m-calpain, the balance of calpain-calpastatin levels possibly controls pathological Ca2+-induced events in mammalian tissues. Overexpression of calpastatin by gene transfer ameliorates contractile dysfunction in rat hearts subjected to ischemia/reperfusion (Maekawa et al., 2003). In rabbit hippocampus regions, µ-calpain is highly expressed in pyramidal neurons of both the CA1 and CA3 regions, and studies of loss of calpastatin in CA1 pyramidal neurons suggest vulnerability of CA1 neurons to ischemic injury (Fukuda et al., 1990). A previous report also showed that calpastatin is proteolyzed by calpain in the rat hippocampus after ischemia/reperfusion (Saido et al., 1997). In the present study, both calpastatin and caspase-3 activation after ME was almost completely abolished by treatment with DY-9760e (50 mg/kg), whereas fodrin breakdown by calpain was partly inhibited DY-9760e treatment. Taken together, DY-9760e could preferentially inhibited Ca2+/CaM-dependent calpastatin breakdown compared with fodrin breakdown. Because the membrane-permeable calpain inhibitor MDL28,170 blocked brain injury in a rat model of focal stroke (Markraf et al., 1998), pathological activation of calpain probably leads to necrosis and apoptosis of neurons. On the other hand, physiological and transient activation of calpain in the limited compartment of the synapse is also essential for neuronal plasticity such as long-term potentiation (Jourdi et al., 2005). In our studies, caspase-3-mediated calpastatin degradation was closely associated with delayed increases in calpain activity, which were associated with irreversible neuronal death including apoptosis. Moreover, significant calpastatin degradation was observed 12 h after ME induction. The delayed calpastatin degradation is particularly important concerning the wide therapeutic time window for treatment of neuroprotective drugs.
Another interesting feature of the present study is finding that downstream targets of calpain and caspase-3, such as fodrin and calpastatin, respectively, are CaM binding proteins. Binding of Ca2+/CaM to fodrin, which acts as a scaffold protein between ion channels and actin filaments, promotes its degradation, thereby causing loss of membrane integrity and down-regulation of ion channel functions. The Ca2+/CaM-mediated susceptibility of fodrin to calpain is reduced by DY-9760e, which has potent neuroprotective action (Sato et al., 1999; Takagi et al., 2001; Hashiguchi et al., 2003). We believe that ME-induced ischemia causes early and mild activation of calpain by Ca2+ influx through glutamate receptors such as the N-methyl-D-aspartate receptor (Fig. 8). The early calpain activation was evident 2 h after ME induction and probably leads to cleavage of caspase-3. Thereafter, Ca2+/CaM accelerates calpastatin breakdown by the activated caspase-3. Thus, the inhibition of calpastatin proteolysis by DY-9760e treatment possibly accounts for suppression of further activation of calpain and caspase-3. Thus, we assumed that calpastatin is the key player in cross-talk between calpain and caspase-3, at least in rat ME-induced brain damage. In the rat transient forebrain ischemia model, calpain is predominantly activated 36 to 72 h after ischemia without significant elevation of caspase-3 activity (Zhang et al., 2002). By contrast, we show here that ME-induced permanent ischemia causes marked increases in both calpain and caspase-3 activities 12 h after ME induction. In particular, fodrin degradation by calpain was seen immediately after ME. Therefore, ME ischemic insults may induce aberrant and prolonged intracellular Ca2+ elevation compared with transient forebrain ischemia, resulting in prolonged enhancement of the Ca2+/CaM-dependent fodrin breakdown. In ischemic brain damage, caspase-3 is activated not only by calpain but also by activation of apoptotic signal through extracellular regulated signals such as Fas ligand and tumor necrosis factor 
. Delayed calpain activation through caspase-3 mediated calpastatin degradation possibly turns on a switch leading to irreversible apoptotic or necrotic neuronal death.
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In summary, our results provide evidence that caspase-3-mediated calpastatin proteolysis accounts for delayed and pronounced activation of calpain, thereby leading to irreversible neuronal death. Like transient forebrain ischemia, DY-9760e has a potent neuroprotective effect against ME-induced ischemic brain damage. In addition to inhibition of NOS activity by DY-9760e as described previously (Fukunaga et al., 2000; Hashiguchi et al., 2003), DY-9760e-mediated inhibition of fodrin and calpastatin degradation possibly contributes its neuroprotective action.
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Footnotes |
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ABBREVIATIONS: ME, microsphere embolism; NOS, nitric-oxide synthase; DY-9760e, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]-ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydro-chloride 3.5 hydrate; CaM, calmodulin; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; AMC, 7-amino-4-methylcoumarin; FBDP, fodrin breakdown product; MDL28,170, carbobenzoxy-valinyl-phenylalaninal.
Address correspondence to: Dr. Kohji Fukunaga, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki-Aoba Aoba-ku, Sendai 980-8578, Japan. E-mail: fukunaga{at}mail.pharm.tohoku.ac.jp
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