HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 8, 6382-6390, February 22, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/8/6382    most recent M108081200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Takagi, N. Articles by Takeo, S. Search for Related Content PubMed PubMed Citation Articles by Takagi, N. Articles by Takeo, S. Social Bookmarking                      What's this?

Altered Extracellular Signal-regulated Kinase Signal Transduction by the Muscarinic Acetylcholine and Metabotropic Glutamate Receptors after Cerebral Ischemia^*

Norio Takagi, Keiko Miyake-Takagi, Kaori Takagi, Hiroshi Tamura^§, and Satoshi Takeo

From the Faculty of Pharmaceutical Sciences, Department of Pharmacology and the ^§ Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received for publication, August 22, 2001, and in revised form, November 8, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To determine whether muscarinic acetylcholine receptors (mAChR) in the post-ischemic hippocampus may be involved in altered extracellular signal-regulated kinases (ERK) signal transduction, we have investigated changes in the activity of ERK1/2 induced by a muscarinic agonist, carbachol. Cerebral ischemia was produced in the rat by injecting 900 microspheres (48 µm in diameter) into the right internal carotid artery. Applying carbachol to the contralateral hippocampal slices from ischemic rats increased the phosphorylation of ERK1/2 but did not increase phosphorylation in the ipsilateral hippocampus. Analysis of M₁ mAChR binding showed that there was no significant difference in the number and K_d values between the hippocampi from naïve and ischemic rats. Immunoblotting analysis showed no significant difference in the amount of M₁ mAChR in both hemispheres. In contrast to carbachol stimulation, the protein kinase C activator induced an activation of ERK1/2 in the ipsilateral hippocampus. This increase was shown to occur in neurons by immunofluorescence colocalization study. Carbachol-stimulated tyrosine phosphorylation of the G_q/11, inositol 1,4,5-trisphosphate formation, and association of G_q/11 with phospholipase C1 were attenuated in the ipsilateral hippocampus. We also found that stimulation of group I metabotropic glutamate receptors, which are linked to G_q/11, failed to increase in phosphorylation of ERK1/2 in the ipsilateral hippocampus. These results suggest that failure in receptor-mediated tyrosine phosphorylation of the G_q/11 subunit and a defect in receptor-G_q/11-effector coupling in the ischemic hippocampus may be involved in alterations of ERK signal transduction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Accumulating evidence indicates that muscarinic acetylcholine receptors (mAChR)¹ play a pivotal role in the regulation of numerous physiological responses to neurotransmitter acetylcholine in the central nervous system. The mAChR is composed of five heterogeneous subtypes (M₁-M₅), which couple to heterotrimeric guanine nucleotide-binding (G) proteins and initiate intracellular signal transduction cascades (1). The M₂ and M₄ receptors couple preferentially to inhibit adenylyl cyclase activity via the G_i protein family, although in some cell types, these receptors can also activate phospholipase C (PLC) (2, 3) and mitogen-activated protein (MAP) kinase by the subunit of G protein (4). The M₁, M₃, and M₅ receptors preferentially couple via G_q/11 protein to activate PLC, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP₃) (5). Diacylglycerol is a physiological activator of protein kinase C (PKC), whereas IP₃ elicits calcium release from intracellular stores (5-7). It has been shown that direct activation of PKC by phorbol esters can induce Ras-dependent or Ras-independent MAP kinase activation (8, 9), suggesting that G_q/11-coupled receptor activation of MAP kinase may be PKC-dependent (9). Among G_q-coupled mAChR subtypes, the M₁ receptor represents the majority of muscarinic receptors in the rat hippocampus (10, 11) and has been suggested to play an important role in the hippocampus-based memory and learning and neuronal plasticity (12). In contrast, the M₃ and M₅ receptors are expressed at low levels in the brain compared with M₁ receptor (13, 14).

MAP kinases play a crucial role in the convergence of cell surface signals regulating cell growth and division. Members of the MAP kinase superfamily include the extracellular signal-regulated kinases (ERKs), the Jun NH₂-terminal kinase, and the p38 MAP kinase. ERKs (ERK1 and ERK2) are activated in response to mitogen or growth factor stimulation (15-18). In contrast, the c-Jun NH₂-terminal kinase and p38 MAP kinases are activated by a variety of cellular stresses. Recent studies have shown that mAChRs can induce proliferation by activating the ERK pathway (19). It has been suggested recently that ERK activation may lead to neuronal survival in cortical neurons (20, 21) and may be necessary for and correlated with several forms of synaptic plasticity (22), including long term potentiation in a rat hippocampal slice preparation (23).

Cerebral ischemia, a pathological condition in which brain tissue experiences a shortage of glucose and oxygen, is associated with cerebrovascular disease, brain trauma, epilepsy, drowning, and cardiac arrest. Although many biochemical changes, which may lead to not only neuronal cell death but also dysfunction of central nervous system, are initiated by cerebral ischemia, aspects of intracellular signal transduction via mAChRs after cerebral ischemia are not fully understood. Microsphere embolism is considered to induce small widespread emboli and multiple infarct areas in the brain, which may be comparable with the pathogenesis of multi-infarct dementia (24). It has been shown that a variety of deficits in learning and memory function are induced in animals with ischemic brain damage, such as in rats with four vessel ligation (25), middle cerebral artery occlusion (26), and microsphere embolism (27). It becomes an important objective to determine the nature of intracellular signal transduction pathways via mAChR, particularly the M₁ receptor, and how they are modulated by cerebral ischemia.

In the present study, we investigated changes in signal transduction pathways via mAChR in hippocampal slices after cerebral ischemia. The findings demonstrate that stimulation of mAChR fails to activate ERKs in the ipsilateral, but not in the contralateral, hippocampus of ischemic rats, which may be caused by disturbance in the carbachol-induced tyrosine phosphorylation of G_q/11, association of the G_q/11 with PLC1, and activity of PLC.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Microsphere Embolism-- Male Wistar rats weighing 180-220 g (Charles River Japan Inc., Atsugi, Japan) were used in the present study. The animals were housed in a cage and maintained on a 12-h light/12-h dark cycle at a temperature of 23 ± 1 °C with a humidity of 55 ± 5% throughout the experiment. The animals had free access to food and water according to the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Guideline of Experimental Animal Care issued by the Prime Minister's Office of Japan. All efforts were made to minimize the animals' suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. The study protocol was approved by the Committee of Animal Care and Welfare of our university.

Microsphere-induced cerebral embolism was performed by the method described previously (28) with some modification. In brief, rats were anesthetized intraperitoneally with 40 mg/kg sodium pentobarbital and placed on an operation plate. The right external carotid and pterygo-palatine arteries were temporarily occluded with strings. A needle connected to a polyethylene catheter (3 French, Atom Co., Tokyo) was inserted into the right common carotid artery. 900 microspheres (47.5 ± 0.5 µm in diameter, PerkinElmer Life Sciences), suspended in 20% dextran solution, were injected into the right internal carotid artery through the cannula. The needle was removed, and the puncture wound was repaired with surgical glue. Then the strings occluding the right external carotid and pterygo-palatine arteries were released. It took 2-3 min to restart the blood flow to the brain areas supplied by the right external carotid and pterygo-palatine arteries. The rats that underwent the sham operation received the same volume of vehicle without microspheres.

Examination of Neurological Deficits-- 15 h after the operation, the behavior of operated rats was scored on the basis of paucity of movement, truncal curvature, and forced circling during locomotion, which are considered to be typical symptoms of stroke in rats (29, 30). Each item was rated from 3 to 0 (3 very severe, 2 severe, 1 moderate, 0 little or none). Rats with a total score of 7-9 points were used in the present study.

Rat Hippocampal Slices-- Artificial cerebrospinal fluid (ACSF) contained (in mM): 125 NaCl, 2.4 KCl, 0.83 MgCl₂, 1.1 CaCl₂, 0.5 KH₂PO₄, 0.5 Na₂SO₄, 27 NaHCO₃, 10 glucose, 10 Hepes, pH 7.4. Ca²⁺-free ACSF had the same composition except for MgCl₂, which was increased to 1.93 mM.

The brain was quickly removed and maintained in ice-cold Ca²⁺-free ACSF for 3-5 min before slicing. Rat hippocampal slices (350 µm) were prepared with a McIlewain tissue chopper (Brinkmann, the Mickle Laboratory Engineering Co. Ltd.) and were incubated (three slices/well) for 30 min at 30 °C in 1 ml of Ca²⁺-free ACSF equilibrated at pH 7.4 in O₂/CO₂ (95:5, v/v). They were then incubated for 90 min at 32 °C in 1 ml of ACSF containing 1.1 mM Ca²⁺ and 1 µM tetrodotoxin before pharmacological treatment. The PKC inhibitor chelerythrine (5 µM) and 3 µM atropine were preincubated with the slices for 45 min and 15 min before the carbachol application, respectively. After a 15-min exposure to 100 µM carbachol, 10 µM phorbol 12-myristate 13-acetate (PMA), or 100 µM trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), the slices were immediately frozen in liquid nitrogen and stored at 80 °C until assayed.

Western Immunoblotting-- Rat hippocampal slices were homogenized in ice-cold 20 mM Tris-HCl, pH 7.4; 10 mM NaF; 0.1 mM sodium orthovanadate; 0.1 mM phenylmethylsulfonyl fluoride; 10 mM glycerophosphate; 10 mM p-nitrophenyl phosphate; and 5 µg/ml each antipain, aprotinin, and leupeptin. For immunoblotting, hippocampal homogenates that had been solubilized by boiling for 5 min in SDS sample buffer (10% glycerol, 5% -mercaptoethanol, and 2% SDS, in 62.5 mM Tris-HCl, pH 6.8) were separated on 10% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. Protein blots were incubated with the indicated antibodies, and bound antibody was detected by enhanced chemiluminescence (Amersham Biosciences, Inc.) as described by the manufacturer. Quantification was performed using computerized densitometry and an image analyzer (ATTO). Care was taken to ensure that bands to be semiquantified were in the linear range of response. Immunoblots that had been reacted with anti-phospho-p44/42 MAP kinase (Thr²⁰²/Tyr²⁰⁴) antibodies (New England Biolabs, Beverly, MA) were reprobed with anti-p44/42 MAP kinase antibodies (New England Biolabs) after removing bound antibodies by heating the blots for 30 min at 65 °C in 62.5 mM Tris-HCl buffer, pH 6.8, containing 2% SDS and 0.1 M -mercaptoethanol. The efficacy of the stripping procedure was confirmed by reacting the stripped blot with secondary antibody alone to ensure that no bound antibodies were detected. To quantitate M₁ muscarinic receptor, G_q/11, PLC1, metabotropic glutamate receptor (mGluR) 1, and mGluR5 levels, the contralateral and ipsilateral hippocampi were homogenized in ice-cold 20 mM Tris-HCl, pH 7.4; 10 mM NaF; 0.1 mM sodium orthovanadate; 0.1 mM phenylmethylsulfonyl fluoride; 10 mM glycerophosphate, 10 mM p-nitrophenyl phosphate; and 5 µg/ml each antipain, aprotinin, and leupeptin. The homogenate was centrifuged at 12,000 × g for 20 min at 4 °C. The pellet was suspended in the homogenization buffer and used for immunoblotting analysis. Antibodies used were anti-mAChR M₁ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-G_q/11 antibody (PerkinElmer Life Sciences), anti-PLC1 (Santa Cruz Biotechnology, Inc.), anti-mGluR1 (AB1595, Chemicon), and anti-mGluR5 (AB5232, Chemicon) antibodies.

Immunoprecipitation-- For immunoprecipitation of the G_q/11 subunit, 300-µg hippocampal slice homogenates were solubilized by boiling in 1% SDS containing 1% -mercaptoethanol for 5 min and then diluted 10-fold with 0.05 M Hepes buffer, pH 7.1, containing 1.5% Triton X-100; 0.15 M NaCl; 1.5 mM MgCl₂; 10% glycerol; 1 mM each NaF, sodium orthovanadate, phenylmethylsulfonyl fluoride, ZnCl₂, and EGTA; and 5 µg/ml each antipain, leupeptin, and aprotinin. Samples were preincubated for 60 min with 20 µl of protein G-agarose beads and then centrifuged to remove any proteins that adhered nonspecifically to the protein G-agarose beads. The supernatant was then incubated with anti-G_q/11 antibody overnight at 4 °C. Protein G-agarose beads (20 µl) were added, and the incubation was continued at 4 °C for 1 h. The immune complexes were isolated by centrifugation, washed, and bound proteins were eluted by heating at 100 °C in SDS sample buffer.

For coimmunoprecipitation of the G_q/11 subunit with PLC1, hippocampal slices were exposed to carbachol for 2 min and then treated with 1 mM dithiobis(succinimidyl propionate) for 30 min to induce protein cross-linking. After an exposure to carbachol, the slices were lysed in lysis buffer (1% Triton X-100, 0.5% Nonidet P-40, 50 mM Hepes, pH 7.4, 130 mM NaCl, 50 mM NaF, 1 mM MgCl₂, 1 mM CaCl₂, 15% glycerol, 40 mM KH₂PO₄, and 1 mM orthovanadate). The lysate was centrifuged at 13,000 × g for 15 min. The supernatant was used for coimmunoprecipitation.

Receptor Binding Assay-- The M₁ mAChR binding assay was performed by the method of Ogawa et al. (31) with minor modification. The rats were anesthetized with diethyl ether and decapitated at the appropriate experimental times. The cerebral hemispheres were isolated and separated into the hippocampus. After being weighed, the hippocampus was homogenized in 10 volumes/g, wet tissue, of ice-cold 50 mM sodium potassium phosphate buffer, pH 7.4, with a Teflon-glass homogenizer. The homogenate was centrifuged at 12,000 × g for 20 min at 4 °C. The pellet was washed twice with sodium potassium phosphate buffer and centrifuged again under the same conditions as above. The pellet was finally suspended in the 50 mM sodium potassium phosphate buffer, pH 7.4, and used for radioligand binding assay. To determine the total binding capacity, the sample was incubated at 25 °C for 120 min in 250 µl of 50 mM sodium potassium phosphate buffer, pH 7.4, containing 6 × 10⁷ to 1 × 10⁵ mol/liter [³H]pirenzepine (Japan Isotope Association, Tokyo). The reaction was stopped by filtration of the reaction mixture through a glass fiber filter (GC-50, Advantec Co., Tokyo) and washed twice with 4 ml of the ice-cold buffer as above. After drying, the radioactivity on the filter paper was counted by a liquid scintillation spectrometer (model LSC-1000, Aloka Japan, Tokyo). Nonspecific binding capacity was determined in the presence of 1.0 µM atropine. The specific binding of [³H]pirenzepine was estimated by subtracting nonspecific binding activity from total binding activity. The dissociation constant (K_d) and maximum binding capacity (B_max) of the specific [³H]pirenzepine binding were determined by analysis of Scatchard plots. Protein concentrations were determined by the method of Lowry et al. (32).

Immunofluorescence-- The hippocampal slices that had been treated with carbachol or PMA were fixed for 1 h in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and then placed in 40% sucrose in 0.1 M phosphate buffer overnight. Hippocampal sections of 50 µm were obtained by cryostat sectioning. The hippocampal sections were briefly dried to remove any surface liquid and embedded in OCT (Myers Laboratory). Free floating slices were incubated overnight at 4 °C in the first primary antibodies (anti-rabbit phospho-p44/42 MAP kinase (Thr²⁰²/Tyr²⁰⁴) antibody) in Tris-buffered saline and 0.1% Triton X-100 containing 3% bovine serum albumin. Sections were washed in Tris-buffered saline and 0.1% Triton X-100 and then incubated with the second primary antibodies (anti-mouse neuron-specific antibody (NeuN; Chemicon)) for 2 h at 4 °C. Sections were washed and then incubated for 1 h in a fluorescence secondary antibody mixture containing a tetramethylrhodamine B isothiocyanate-conjugated secondary antibody for the phospho-p44/42 MAP kinase (Thr²⁰²/Tyr²⁰⁴) antibody and a fluorescein isothiocyanate-conjugated secondary antibody for the NeuN antibody. Sections were washed in Tris-buffered saline and then mounted with coverslips on glass slides. Fluorescence was detected using a Bio-Rad MRC 1024 confocal imaging system equipped with a krypton-argon laser and a Nikon ECLIPSE microscope (TE300). Images (512 × 512 pixels) were obtained by averaging six scans and were processed by Adobe PhotoShop (Adobe Systems, Mountain View, CA). The microscopic observations were performed by a person unaware of the study group.

Measurement of IP₃ in Hippocampal Slices-- Carbachol-stimulated hippocampal slices were used for the measurement of IP₃ with the Biotrak radioimmunoassay kit (Amersham Biosciences, Inc.). The hippocampal slices that had been stimulated with carbachol in ACSF containing 10 mM LiCl were immediately frozen in liquid nitrogen. Frozen hippocampal slices were homogenized in ice-cold 4% perchloric acid. Neutralized extracts were used to measure IP₃ levels. Procedures followed the manufacturer's instructions.

Statistics-- The results are expressed as the means ± S.E. Differences between two groups were evaluated statistically with an unpaired Student's t test. Statistical comparison among three groups was evaluated by ANOVA followed by Fisher's protected least significant difference test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We initially determined ERK1/2 activation by the mAChR agonist carbachol in hippocampal slices from naïve rats. In agreement with the results obtained by other investigators, immunoblotting of homogenates from hippocampal slices with anti-phospho-ERK antibodies that recognize the activated form of ERK1/2 showed an increase in the phosphorylation of ERK1/2 after the application of carbachol (ratio of 2.00 ± 0.31 over basal for ERK1 and ratio of 1.54 ± 0.11 over basal for ERK2; n = 4) (Fig. 1, A and B). The muscarinic antagonist atropine blocked the effect of carbachol on ERK1/2 activation (ratio of 1.22 ± 0.06 over basal for ERK1 and ratio of 1.03 ± 0.02 over basal for ERK2; n = 4) (Fig. 1, A and B). The total amounts of ERK1/2 were similar before and after treatment with carbachol (Fig. 1A), indicating that the increase in phosphorylation of ERK1/2 was not because of an overall increase in the concentration of ERK1/2 after the carbachol application.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of carbachol on ERK1/2 phosphorylation in hippocampal slices from naïve rats. Panel A, rat hippocampal slices were incubated with vehicle or 100 µM carbachol for 15 min in ACSF. Slices were homogenized, and the homogenates (20 µg) were analyzed by immunoblotting with anti-phospho-p44/42 MAP kinase (pERK I/II) antibodies in the presence (+) or absence () of carbachol or 3 µM atropine. The level of total ERK1/2 protein was analyzed using anti-p44/42 MAP kinase (ERK I/II) antibodies after a stripping procedure. No differences in the level of ERK1/2 protein were detected. Panel B, individual bands corresponding to phosphorylated ERK1/2 on immunoblots were scanned, and the results are expressed as the mean ratios ± S.E. of the magnitude of ERK1/2 phosphorylation over basal level (n = 4 different animals). * indicates a significant difference from the absence of carbachol, when estimated by ANOVA followed by post hoc Fisher's protected least significant difference test (p < 0.05).

It has been well documented that the hippocampus plays an important role in normal learning and memory and is one of most vulnerable regions to ischemia-induced cell damage. Identification of early molecular events that initiate changes leading to ischemia-induced pathophysiology remains an important objective. Our current study has been targeted primarily to early events after microsphere embolism-induced cerebral ischemia. To determine the nature of intracellular signal transduction pathways in the ischemic hippocampus, we examined ERK1/2 activation by mAChR in hippocampal slices from ischemic rats. The basal phosphorylation of ERK1/2 in the ipsilateral hippocampus was slightly lower than that in the contralateral hippocampus (Fig. 2A). The application of carbachol resulted in increased phosphorylation of ERK1/2 in the contralateral hippocampus (ratio of 2.04 ± 0.40 over basal for ERK1 and ratio of 1.47 ± 0.06 over basal for ERK2; n = 4), but there was no increase in the phosphorylation of ERK1/2 in the ipsilateral hippocampus (ratio of 0.76 ± 0.11 over basal for ERK1 and ratio of 1.00 ± 0.17 over basal for ERK2; n = 4). The magnitude ratio of the phosphorylation of ERK1/2 in the ipsilateral hippocampus was significantly smaller than that in the contralateral hippocampus from ischemic rats or in the hippocampus from naïve rats (Fig. 2, B and C). The total amounts of ERK1/2 in the ipsilateral hippocampus were similar to those in the contralateral hippocampus (Fig. 2A).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effect of carbachol on ERK1/2 phosphorylation in the contralateral (cont) or ipsilateral (ipsi) hippocampal slices from microsphere-embolized rats. Panel A, the homogenates (20 µg) from contralateral or ipsilateral hippocampus were analyzed by immunoblotting with anti-phospho-p44/42 MAP kinase (pERK I/II) antibodies or anti-p44/42 MAP kinase (ERK I/II) antibodies in the presence (+) or absence () of carbachol. Panels B and C, individual bands corresponding to phosphorylated ERK1/2 on immunoblots were scanned, and the results are expressed as the mean ratios ± S.E. of the magnitude of ERK1/2 phosphorylation in the presence and absence of carbachol (n = 4 for each condition) from the contralateral or ipsilateral hippocampus of microsphere-embolized rats (ME) or from the hippocampus of naïve rats (Naïve). * indicates a significant difference from the ratio of the magnitude of ERK1/2 phosphorylation in the ipsilateral hippocampus of microsphere-embolized rats, when estimated by ANOVA followed by post hoc Fisher's protected least significant difference test (p < 0.05).

The altered activation of ERK1/2 by carbachol in the ipsilateral hippocampus suggests that cerebral ischemia might induce changes in the binding capacity and/or affinity of the mAChR. The M₁ subtype is the predominant G_q-coupled muscarinic receptor, which can activate ERK1/2, (10, 11, 14) and has been suggested to play an important role in memory and learning and neuronal plasticity in hippocampus (12). We therefore determined the effects of cerebral ischemia on the M₁ receptor binding of the hippocampus 24 h after the operation. The specific binding capacity of [³H]pirenzepine showed that microsphere embolism-induced cerebral ischemia did not alter the B_max and K_d values in either the contralateral or ipsilateral hippocampus compared with naïve rats (Fig. 3, A and B). We further determined the level of M₁ receptor using anti-M₁ receptor antibodies because pirenzepine has similar affinities for M₁ and M₄ receptors (33). Immunoblotting analysis showed that there were no changes in the level of M₁ receptor between the contralateral and the ipsilateral hippocampus 24 h after the operation (Fig. 3, C and D). Immunoreactivity to the M₁ receptor was not detectable in the cerebellum from naïve animals (Fig. 3C).

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Effects of cerebral ischemia on the muscarinic M1 receptor. Kd (panel A) and Bmax (panel B) values of muscarinic M1 receptor density of the ipsilateral (Right) or contralateral (Left) hippocampus of naïve (Naïve) or microsphere-embolized (ME) rats. There were no significant differences in Kd and Bmax values between naïve and microsphere-embolized rats (p > 0.05). Panel C, immunoblotting analysis using antibody to M1 mAChR of proteins of the contralateral (C) or ipsilateral (I) hippocampus (Hp) from ischemic animals. M1 mAChR of protein migrated on electrophoresis with an apparent molecular mass of 61 kDa. Cb, cerebellum from naïve animal. Panel D, individual bands corresponding to M1 mAChR on immunoblots were scanned, and the results are expressed as the mean optical density units ± S.E. from the contralateral (cont) or ipsilateral (ipsi) hippocampus (n = 4 different animals). No differences in the level of M1 mAChR protein were detected.

Stimulation of G_q-coupled M₁ mAChR activates PLC, which in turn generates two second messengers, IP₃ and diacylglycerol. Diacylglycerol is a physiological activator of PKC. Recently, it has been shown that phorbol esters can activate ERK (34, 35). To determine whether carbachol-stimulated ERK1/2 activation involved PKC, we examined the effect of a specific PKC inhibitor, chelerythrine, on the phosphorylation of ERK1/2 in hippocampal slices. The application of chelerythrine blocked the increase in the phosphorylation of ERK1/2 by carbachol (Fig. 4A). We also confirmed that carbachol-stimulated IP₃ generation was not blocked by chelerythrine (Fig. 4B), indicating that chelerythrine did not inhibit PKC upstream, PLC activity. These results suggest a role of PKC in the response to mAChR stimulation. These results led us to examine the activation of ERK1/2 by PKC activation independent of the mAChR. To determine whether PKC activation induces ERK1/2 phosphorylation in the ipsilateral hippocampus, we examined the effect of PMA on the phosphorylation of ERK1/2 in both ipsilateral and contralateral hippocampi from the ischemic rats. PMA resulted in an increase in the phosphorylation of ERK1/2 in both contralateral and ipsilateral hippocampi (Fig. 4C), whereas carbachol did not elicit any increase in the phosphorylation of ERK1/2 in the ipsilateral hippocampus as indicated above. The average magnitude ratios of ERK1 and 2 phosphorylation by PMA, relative to basal phosphorylation, in the ipsilateral hippocampus were 1.95 ± 0.37 and 1.66 ± 0.27, respectively (Fig. 4D). Similar levels of ERK1/2 were present in the contralateral and ipsilateral hippocampi (Fig. 4C). These results indicate that the PKC remains coupled to ERK1/2 activation in the ipsilateral hippocampus.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Role of PKC on ERK1/2 phosphorylation in contralateral or ipsilateral hippocampal slices from microsphere-embolized rats. Panel A, effects of PKC inhibitor chelerythrine (5 µM) on ERK1/2 phosphorylation induced by carbachol in hippocampal slices from naïve rats. The homogenates (20 µg) from the contralateral or ipsilateral hippocampus were analyzed by immunoblotting with anti-phospho-p44/42 MAP kinase (pERK I/II) antibodies or anti-p44/42 MAP kinase (ERK I/II) antibodies in the presence (+) or absence () of carbachol or chelerythrine. Panel B, IP3 concentration (pmol/slice) was analyzed in the presence (+) or absence () of carbachol or chelerythrine. The values are the means ± S.E. of four separate naïve animals. * indicates a significant difference from the absence of carbachol, when estimated by ANOVA followed by post hoc Fisher's protected least significant difference test (p < 0.05). Panel C, effects of the PKC activator PMA (10 µM) on ERK1/2 phosphorylation in the contralateral or ipsilateral hippocampus of ischemic rats. Homogenates (20 µg) from contralateral or ipsilateral hippocampal slices were analyzed by immunoblotting with anti-phospho-p44/42 MAP kinase (pERK I/II) antibodies or anti-p44/42 MAP kinase (ERK I/II) antibodies in the presence (+) or absence () of carbachol or PMA. Panel D, bands corresponding to phosphorylated ERK1/2 on immunoblots from ipsilateral hippocampal slices of the ischemic rats were scanned, and the results are expressed as the mean ratios ± S.E. of the magnitude of ERK1/2 phosphorylation in the presence and absence of carbachol (Carb) or PMA (n = 4 for each condition). * indicates significant difference from the carbachol application, p < 0.05, Student's t test.

Because cerebral ischemia results in a proliferation of reactive astrocytes, the activation of ERK1/2 by PMA in the ipsilateral hippocampus might occur in astrocytes. To assess this possibility, colocalization of activated ERK1/2 immunoreactivity with the neuronal nuclear marker NeuN immunoreactivity was investigated in hippocampal slices using double immunofluorescence histochemistry. After the carbachol application, ERK1/2 was activated, and the activated ERK1/2 was colocalized with NeuN-positive cells in the contralateral hippocampus (Fig. 5A). NeuN-positive cells were not stained with activated ERK1/2 in the ipsilateral hippocampus after the carbachol application, confirming that the carbachol application did not activate ERK1/2 in the ipsilateral hippocampus (Fig. 5B). In contrast, the application of PMA resulted in an activation of ERK1/2 which was colocalized predominantly with the NeuN-positive cells in both contralateral and ipsilateral hippocampi (Fig. 5B). These results support the results of immunoblotting analysis and suggest that the activation of ERK1/2 by PMA in the ipsilateral hippocampus may occur primarily in neurons.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   The effects of carbachol or PMA on ERK1/2 phosphorylation in contralateral or ipsilateral hippocampal slices were detected by immunofluorescence colocalization study. Phosphorylation of ERK1/2 (pERK I/II, red) by carbachol or PMA application was detected in contralateral hippocampal slices (panel A), whereas phosphorylated ERK1/2 was only detected by PMA application in the ipsilateral hippocampal slices (panel B). The neuron-specific marker (NeuN, green) was detected in both contralateral and ipsilateral hippocampal slices. Phosphorylated ERK1/2 was colocalized (Merge, yellow) with NeuN-positive neuron in both contralateral and ipsilateral hippocampal slices.

Recently, it has been shown that stimulation of receptors coupled to G_q/11 induces phosphorylation on a tyrosine residue of the G_q/11 subunit, and this tyrosine phosphorylation event was essential for G_q/11 activation (36). We next examined if stimulation of the mAChR with carbachol induced tyrosine phosphorylation of the G_q/11 subunit in the ipsilateral hippocampus. To assess this, the G_q/11 subunit was immunoprecipitated with G_q/11 subunit antibodies followed by immunoblotting with anti-tyrosine phosphate antibodies. Application of carbachol to the contralateral hippocampal slices resulted in an increase in the tyrosine phosphorylation of G_q/11, whereas no increase in the tyrosine phosphorylation of the G_q/11 subunit occurred in the ipsilateral hippocampus (Fig. 6A). Additional immunoblotting experiments showed that the levels of the precipitated G_q/11 protein in the ipsilateral hippocampus were similar to those in the contralateral hippocampus (Fig. 6A). The altered tyrosine phosphorylation of G_q/11 after the application of carbachol was not the result of a change in the level of G_q/11 protein after ischemia. The average increases in the tyrosine phosphorylation of the G_q/11 subunit in the contralateral and ipsilateral hippocampi, relative to basal phosphorylation, were 1.70 ± 0.08-fold and 0.81 ± 0.12-fold (p < 0.05), respectively (Fig. 6B).

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Effects of carbachol on tyrosine phosphorylation of Gq/11 subunit in ipsilateral and contralateral hippocampal slices. Panel A, homogenates (300 µg) from contralateral or ipsilateral hippocampal slices underwent immunoprecipitation (IP) with antibodies specific for Gq/11 subunit, and the immunoprecipitates were analyzed by immunoblotting (IB) with anti-tyrosine phosphate antibodies (PY) in the presence (carb) or absence (basal) of carbachol. Blots were stripped and reprobed with antibodies specific for the Gq/11 subunit (IB, Gq/11). Panel B, bands corresponding to the tyrosine-phosphorylated Gq/11 subunit (42 kDa) on immunoblots were scanned, and the results are expressed as the mean ratios ± S.E. of the magnitude of tyrosine phosphorylation of the Gq/11 subunit in the presence and absence of carbachol (n = 3 for each condition). * indicates a significant difference from the contralateral hippocampus, p < 0.05, Student's t test.

The failure of the tyrosine phosphorylation of G_q/11 after the carbachol application in the ipsilateral hippocampus may contribute to receptor-G_q- effector uncoupling. IP₃ is a downstream molecule generated by PLC activity. To determine the effects of cerebral ischemia on mAChR-stimulated activation of PLC, we next examined IP₃ generation in response to carbachol in both contralateral and ipsilateral hippocampal slices. The carbachol application significantly increased IP₃ generation compared with the basal level in the contralateral hippocampus, whereas there was no increase in IP₃ generation by carbachol in the ipsilateral hippocampus (Fig. 7). This result suggests that cerebral ischemia diminishes the ability of G_q/11 to interact with PLC1 in response to carbachol stimulation. To assess this possibility, we further examined changes in the interaction of the G_q/11 with PLC1 after carbachol stimulation in both contralateral and ipsilateral hippocampus. We initially determined levels of G_q/11 and PLC1 in the contralateral and ipsilateral hippocampi from ischemic rats. There were no changes in the total levels of G_q/11 and PLC1 between the contralateral and the ipsilateral hippocampus (Fig. 8A). Carbachol stimulation increased an association of G_q/11 with PLC1 in the contralateral hippocampus. In contrast, there was no alteration of the carbachol-induced association of G_q/11 with PLC1 in the ipsilateral hippocampus (Fig. 8B). Similar amounts of G_q/11 were immunoprecipitated from the contralateral and the ipsilateral hippocampus, indicating that alteration of the agonist-induced association of G_q/11 with PLC1 did not reflect to changes in the total concentration of G_q protein and PLC1, and in the precipitated G_q in the coimmunoprecipitation analysis.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effects of carbachol on IP3 generation in ipsilateral and contralateral hippocampal slices. The IP3 concentration (pmol/slice) of the contralateral (cont) or ipsilateral (ipsi) hippocampus was analyzed in the presence (+) or absence () of carbachol. The values are the means ± S.E. of four separate ischemic animals. * indicates a significant difference from the absence of carbachol, p < 0.05, Student's t test.

View larger version (58K): [in this window] [in a new window]   Fig. 8.   Effects of carbachol on interaction of Gq/11 with PLC1 in ipsilateral and contralateral hippocampal slices. Panel A, immunoblotting analysis using antibody to Gq/11 (42 kDa) or PLC1 (140 and 150 kDa) of proteins of the contralateral (cont) or ipsilateral (ipsi) hippocampus from ischemic animals. No differences in the level of Gq/11 and PLC1 protein were detected. Panel B, lysates from contralateral or ipsilateral hippocampal slices after protein cross-linking underwent immunoprecipitation (IP) with antibodies specific for the Gq/11 subunit, and the immunoprecipitates were analyzed by immunoblotting (IB) with anti-PLC1 antibodies in the presence (carb) or absence (basal) of carbachol. Blots were stripped and reprobed with antibodies specific for the Gq/11 subunit (IB, Gq/11). Similar results were obtained in three separate ischemic animals.

Finally, we determined whether activation of ERK by other G_q/11-coupled receptors was altered by cerebral ischemia. The group I mGluRs (mGluR1 and mGluR5) couple to G_q/11 and to the activation of PLC (37). The application of ACPD, an agonist for the mGluR, resulted in an increased phosphorylation of ERK1/2 in the contralateral hippocampus (ratio of 1.30 ± 0.06 over basal for ERK1 and ratio of 1.22 ± 0.06 over basal for ERK2; n = 4). The ratio of magnitude of the phosphorylation of ERK1/2 in the ipsilateral hippocampus was significantly smaller than that in the contralateral hippocampus from ischemic rat (Fig. 9, A and B). The total amounts of ERK1/2 in the ipsilateral hippocampus were similar to those in the contralateral hippocampus (Fig. 9A). Additional immunoblotting analysis showed that there were no changes in the levels of mGluR1 and mGluR5 between the contralateral and ipsilateral hippocampus 24 h after the operation (Fig. 9, C and D).

View larger version (45K): [in this window] [in a new window]   Fig. 9.   Effect of ACPD on ERK1/2 phosphorylation in the contralateral (cont) or ipsilateral (ipsi) hippocampal slices from microsphere-embolized rats. Panel A, the homogenates (20 µg) from contralateral or ipsilateral hippocampus were analyzed by immunoblotting with anti-phospho-p44/42 MAP kinase (pERK I/II) antibodies or anti-p44/42 MAP kinase (ERK I/II) antibodies in the presence (+) or absence () of ACPD. Panel B, individual bands corresponding to phosphorylated ERK1/2 on immunoblots were scanned, and the results are expressed as the mean ratios ± S.E. of the magnitude of ERK1/2 phosphorylation in the presence and absence of ACPD (n = 4 for each condition) from the contralateral or ipsilateral hippocampus of microsphere-embolized rats. * indicates a significant difference from the ratio of the magnitude of ERK1/2 phosphorylation in the contralateral hippocampus of microsphere-embolized rats, p < 0.05, Student's t test. Panel C, immunoblotting analysis using antibody to mGluR1 or mGluR5 of proteins of contralateral or ipsilateral hippocampus from four separate ischemic animals. mGluR1 and mGluR5 of protein migrated on electrophoresis with an apparent molecular mass of 140 kDa. Panel D, individual bands corresponding to mGluR1 or mGluR5 on immunoblots were scanned, and the results are expressed as the mean optical density units ± S.E. from contralateral or ipsilateral hippocampus. No differences in the level of mGluR1 or mGluR5 protein were detected.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have shown previously that microsphere-induced cerebral embolism in the right hemisphere of rats resulted in a marked decrease in cerebral blood flow and a disturbance of energy metabolism in the brain and was capable of inducing progressive and sustained cerebral ischemia (28, 38) which was followed by the development of multi-infarct in the ipsilateral hemisphere. Although studies on the development of infarct in several cerebral ischemic models have been well documented, identification of early events that initiate the changes in intracellular signal transduction leading to ischemia-induced pathophysiology remains an important objective. Therefore, our current study has primarily been targeted to early events after embolism-induced cerebral ischemia at 24 h, when no obvious infarct area was detected by the 2,3,5-triphenyltetrazolim chloride staining study (38).

In the present study, the application of carbachol failed to phosphorylate ERK1/2 in the ipsilateral hippocampus of ischemic rats. The M₁ subtype is a G_q-coupled muscarinic receptor most abundantly localized in brain, which can activate ERK1/2 (10, 11, 14), although M₂ and M₄ receptors that preferentially couple to the inhibition of adenylyl cyclase via G_i protein, may activate ERK1/2 in some cell types (2-4). Furthermore, mice lacking the M₁ receptor have shown that the M₁ receptor is the main subtype of mAChR responsible for activation of PLC and ERK1/2 in the forebrain (39). Therefore, we focused on signal transduction via M₁ mAChR in the hippocampus after ischemia. Analysis of M₁ mAChR binding suggests that failure in ERK1/2 phosphorylation is not the result of an alteration in the pharmacological property of M₁ mAChR after cerebral ischemia. Because pirenzepine has similar affinities for M₁ and M₄ receptors (33), we further examined the immunoblotting analysis to determine the level of M₁ receptor using anti-M₁ receptor antibodies. The results showed that the level of M₁ mAChR was unchanged by ischemia. Therefore, it is conceivable that ischemia-induced alteration of ERK1/2 phosphorylation is attributed to changes in intracellular signal transduction through the M₁ mAChR coupled with the G_q/11 subunit. Although it has been shown that M₁ mAChR represents the majority of muscarinic receptors in the rat hippocampus (10, 11), we cannot completely rule out a contribution of the M₃ and M₄ receptors, which show nearly identical affinities for carbachol (40) and/or nicotinic receptors. We demonstrated that the selective PKC inhibitor chelerythrine blocked the carbachol-induced increase in ERK1/2 phosphorylation without changes in PLC activity in hippocampal slices. This is consistent with the finding of Roberson et al. (41) that endogenous PKC activation by carbachol is involved in ERK1/2 phosphorylation. However, activation of ERK1/2 by G protein-coupled receptors has shown to be PKC-dependent (9), PKC-independent (42), or partially PKC-dependent (43). A recent study showed that carbachol-induced ERK1/2 phosphorylation is independent on PKC activity in primary cortical neurons and COS-7 cells expressing M₁ mAChR (44). Although the contribution of PKC to ERK1/2 activation through the mAChR is still controversial, the distinction may be attributed to differences in cell type and in stimulus.

Because we confirmed in the current study that PKC was one of the upstream components of ERK1/2 phosphorylation through mAChR in hippocampal slices, we next examined the effects of exogenous activation of PKC by PMA on ERK1/2 phosphorylation. The application of PMA to the contralateral hippocampus increased the phosphorylation of ERK1/2. This is consistent with the finding that the PKC activator phorbol diacetate results in an increase in the phosphorylation of ERK in the CA1 region of the hippocampal slice (41). We have shown in previous studies the marked reduction in high energy phosphate content (35-40% decrease) and mitochondrial oxidative phosphorylation activity (35-65% decrease) on the 1st day after the embolism (28, 45). These findings raise the possibility that in the ipsilateral hippocampus, there would be little ATP available for the increase in phosphorylation of ERK1/2 after the carbachol application. With respect to this, it is noteworthy that the application of PMA to the ipsilateral hippocampus resulted in an increase in the phosphorylation of ERK1/2. The magnitude ratio of increase in the ERK1/2 phosphorylation between the contralateral and ipsilateral hippocampi was similar. These results suggest that ischemia-induced ATP depletion might be independent of ERK1/2 phosphorylation in the pathway downstream of PKC. In contrast, the low basal level of phosphorylated ERK1/2 might contribute to an ischemia-induced ATP depletion but not reflect an overall decrease in ERK1/2 concentration in the ipsilateral hippocampus because similar levels of the ERK1/2 were present in both sides of ischemic rats. A recent study has shown that phosphorylation of ERK1/2 increased as early as 30 min after permanent middle cerebral artery occlusion, peaked at 2 h, and decreased to control levels by 6 h (46). Therefore, sustained cerebral ischemia would cause a reduction in the basal phosphorylation of ERK1/2 because of a disturbance of energy metabolism.

Analysis of the localization of phosphorylated ERK demonstrated that the increase in phosphorylation of the ERK1/2 after the application of PMA was associated with NeuN-positive neurons in both ipsilateral and contralateral hippocampi. Thus, the results of Western blotting analysis would reflect changes in the phosphorylation of ERK1/2 in neuron. However, we cannot fully exclude the possibility of an increase in the ERK1/2 phosphorylation after the application of PMA in other glial cells (e.g. microglia and oligodendroglia). The results of Western blotting and immunohistochemical analysis suggest that in the early stage of cerebral embolism, the PKC downstream pathway still retains the ability to phosphorylate ERK1/2, even in the ipsilateral hippocampus.

The G_q/11 subunit is phosphorylated on tyrosine by M₁ mAChR stimulation in MEF cells expressing M₁ mAChR (36). It is interesting that tyrosine phosphorylation of G_q/11 may be essential for the activation of the G_q/11 subunit by agonist stimulation (36). The present study indicates that the failure of ERK1/2 phosphorylation in response to carbachol stimulation in the ipsilateral hippocampus may be involved in dysfunction of elements upstream of PKC. Therefore, we investigated the tyrosine phosphorylation of the G_q/11 subunit after the application of carbachol in both contralateral and ipsilateral hippocampi. The results showed that there were no changes in the tyrosine phosphorylation in the ipsilateral hippocampus, despite enhanced tyrosine phosphorylation of the G_q/11 subunit after the carbachol application in the contralateral hippocampus. Because purified M₁ receptors, G_q protein, and PLC can be reconstituted with functional activity (47), tyrosine phosphorylation of the G_q protein may not be an absolute requirement for activation of PLC. However, receptor-G protein-effector coupling would be more multiply regulated in vivo and/or ex vivo situations. Indeed, tyrosine phosphorylation of G_q/11 increases its ability to activate PLC (48). In this sense, we found the failure of carbachol-stimulated IP₃ generation in the ipsilateral hippocampus. The tyrosine phosphorylation of G_q/11 may make it more susceptible to activation of PLC coupled with mAChR in vivo and/or ex vivo. Furthermore, we also found the failure of the carbachol-induced interaction of G_q/11 with PLC1 in the ipsilateral hippocampus. It is conceivable that failure of appropriate tyrosine phosphorylation of G_q/11 in response to agonist stimulation may contribute to a defect in receptor-G protein-effector coupling, which may be an initial step in the dysfunction of ERK signaling, although direct evidences for a contribution of tyrosine phosphorylation of G_q/11 to an interaction with PLC1 is lacking. The specific tyrosine residue phosphorylated by mAChR stimulation and the tyrosine kinases involved in the tyrosine phosphorylation of the G_q/11 subunit in response to mAChR stimulation also remain to be determined in hippocampal slices.

The phosphorylation of ERK1/2 by mAChR in the ischemic brain in situ might differ from that in the hippocampal slices because the microsphere embolism-induced cerebral ischemia decreased the ACh content of the hippocampus 24 h after the operation (49). A decrease in the ACh content is observed frequently in ischemic models such as bilateral carotid artery-occluded gerbils (50), four vessel-ligated rats (51) and middle cerebral artery-occluded rats (52). Therefore, the ERK1/2 phosphorylation through mAChR in the ischemic brain in situ may also depend on synaptic cholinergic transmission. Changes in in situ protein phosphorylation under pathological conditions, such as cerebral ischemia, may also be dependent upon localization of protein kinases, phosphatases, and other proteins that are involved in the regulation of protein phosphorylation in intracellular spaces.

Further experiments will be required to determine the pathophysiologic consequences of failure of ERK1/2 activation by carbachol after cerebral ischemia. ERK1/2 is generally thought to contribute to proliferation and differentiation. Increased evidence also suggests that ERK1/2 activation may be necessary for associated memories in the mammalian nervous system. In addition, recent studies have shown that M₁ agonists ameliorate age-related learning and memory impairments, suggesting that M₁ agonists may be an effective therapy for reducing the cognitive deficits that accompany normal aging and/or Alzheimer's disease (53). Therefore, it is interesting to consider that failure of tyrosine phosphorylation of G_q/11, uncoupling of the receptor-G protein-effector, and a defect in ERK1/2 activation by mAChR observed in the current study may be involved in the early stage of impairment of learning and memory function after cerebral ischemia. It has been shown that G_q-coupled mGluRs also play an important role in regulating hippocampal function (54). Although the ratios of the magnitude of ERK1/2 phosphorylation by ACPD in the contralateral hippocampus were smaller than that by carbachol in the present study, stimulation of mGluRs by ACPD also failed to phosphorylate ERK1/2 in the ipsilateral hippocampus of ischemic rats. Therefore, cerebral ischemia may induce dysfunction of G_q-coupled signal transduction because of the failure of appropriate tyrosine phosphorylation of G_q/11 and a defect in receptor-G protein-effector coupling. Our results suggest that the tyrosine phosphorylation of the G_q/11 subunit in response to receptor stimulation may be an appropriate target to regulate the short term and/or long term effects of ischemia on neuronal function which is associated with learning and memory function.

	ACKNOWLEDGEMENT

We thank Dr. James W. Gurd for a critical review of this manuscript.

	FOOTNOTES

^* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-426-76-4584; Fax: 81-426-76-4584; E-mail: takagino@ps.toyaku.ac.jp.

Published, JBC Papers in Press, November 19, 2001, DOI 10.1074/jbc.M108081200

	ABBREVIATIONS

The abbreviations used are: mAChR, muscarinic acetylcholine receptor(s); ACPD, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid; ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; ERK, extracellular signal-regulated kinase; G protein, guanine nucleotide-binding protein; IP₃, inositol 1,4,5-trisphosphate; MAP, mitogen-activated protein; mGluR, metabotropic glutamate receptor; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES