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SAPK and p38 signal transduction pathways
Regulatory Peptides 109 (2002) 83 – 88 www.elsevier.com/locate/regpep
Pituitary adenylate cyclase-activating polypeptide (PACAP) prevents hippocampal neurons from apoptosis by inhibiting JNK/SAPK and p38 signal transduction pathways Kenji Dohi a,b,d,*, Hidekatsu Mizushima b, Shigeo Nakajo c, Hirokazu Ohtaki b,d, Seiji Matsunaga b, Tohru Aruga a, Seiji Shioda b,d a
Department of Critical Care Medicine, School of Medicine, Showa University, Tokyo, Japan b Department of Anatomy, Showa University School of Medicine, Tokyo, Japan c Laboratory of Biological Chemistry, Showa University School of Pharmaceutical Science, Tokyo, Japan d The Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology (JST), Japan
Abstract We have demonstrated that ischemic neuronal death (apoptosis) of rat CA1 region of the hippocampus was prevented by infusing pituitary adenylate cyclase-activating polypeptide (PACAP) either intracerebroventricularly or intravenously. We have also demonstrated that the activity of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, was increased in the hippocampus within 1 – 6 h after brain ischemia. The molecular mechanisms underlying the PACAP anti-apoptotic effect were demonstrated in this study. Ischemic stress had a strong influence on MAP kinase family, especially on JNK/SAPK and p38. PACAP inhibited the activation of JNK/SAPK and p38 after ischemic stress, while ERK is not suppressed. These findings suggest that PACAP inhibits the JNK/SAPK and p38 signaling pathways, thereby protecting neurons against apoptosis. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Ischemia; ERK; PACAP
1. Introduction Brain ischemia induces various degrees of neuronal damage depending on its intensity and duration [1]. It is believed that an increase in intracellular Ca2 + concentration, stimulated by several bioactive substances and others, induces neuronal cell death (apoptosis) in the hippocampal CA1 [2,3]. Rat CA1 pyramidal cells are selectively vulnerable to transient ischemic insult, and ischemia causes severe neuronal damage to the CA1 neurons which start dying 2 or 3 days after the reperfusion [3,4]. A number of neurotrophic factors and nerve growth factors prevent the ischemia-induced degeneration of hippocampal CA1 neurons. Among them, PACAP is the potent anti-apoptotic neurotrophic factor. PACAP has been
* Corresponding author. Department of Critical Care Medicine, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel.: +81-3-3784-8744; fax: +81-3-3784-6880. E-mail address: [email protected] (K. Dohi).
found to stimulate the outgrowth of neurites from PC12 cells and to enhance the survival of sympathetic ganglion cells [7,8]. PACAP prevents the neuronal cell death in the dorsal root ganglia in chick embryo and also increases the survival of lumbar spinal motoneurons [9]. PACAP also prevents apoptosis in cultured cerebellar granule cells [10,11]. In addition, hippocampal neuronal cell death by gp120 in vitro was completely prevented by PACAP at extremely low concentration as vasoactive intestinal peptide (VIP) [9]. Contrary to VIP, the dose – response curve is bimodal, with the greatest cytoprotective activity at 0.1 pM and 0.1 nM, suggesting that PACAP may act at two different receptors: PACAP and VIP/PACAP. In in vitro studies, brain-derived neurotrophic factor (BDNF) mediates the neuroprotective effect of PACAP38 on rat cortical neurons [12]. PACAP38 increased survival of cerebellar neurons in a dose-dependent manner by decreasing the extent of apoptosis estimated by DNA fragmentation in primary culture of rat cerebellar granule cells [11]. The signaling pathways that lead to apoptosis are beginning to be defined, and a number of proteins that
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induce or prevent apoptosis have been identified. There are molecular mechanisms that regulate apoptosis, the contribution to cell death of mitogen-activated protein (MAP) kinase family members, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK; also termed SAPK or stress-activated protein kinase) and p38 [5]. It is known that the activation of JNK/SAPK and p38 and the concurrent inhibition of ERK are critical for the induction of apoptosis in PC12 cell [6]. We have demonstrated that JNK/SAPK, p38 and ERK activities increased during the first 6 h and decreased at 12 h after ischemia –reperfusion in hippocampus of rat cardiac arrest model [24]. PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A (PKA)/protein kinase C (PKC)-dependent inhibition of caspase-3 activity [13]. They also reported that PACAP38 induced activation of the ERK-type of MAP kinase through a cAMP-dependent pathway [11]. In in vivo study, it has been reported that PACAP prevents ischemiainduced neuronal death in global and focal ischemic models [14,15]. The precise molecular mechanism of neuroprotection and contribution of PACAP in regulating MAP kinase family members have not been clarified, though PACAP has the effect of neuroprotection in vivo. In the present study, using rat global ischemia model (cardiac arrest model), we investigated the effect of intracerebroventricular (i.c.v.) administration of PACAP to the expression of activated MAPK family members (p-JNK, pp38 and p-ERK) in the hippocampus after ischemia – reperfusion [3,16].
2. Materials and methods 2.1. Animal preparations Adult male Sprague – Dawley rats (250 – 300 g; Saitama Experimental Animal Center, Saitama, Japan) were housed in a temperature- and light-controlled room (lights on at 0600 and off at 1800) and supplied with standard laboratory chow and water ad libitum. The Institutional Animal Care and Use Committee of Showa University approved all experimental procedures involving animals. The rats were anesthetized with 3.5% halothane and maintained with 2% sevoflurance in 70% N2O and 30% O2. Body temperature was monitored with a rectal thermometer and maintained at 37 jC. A polyethylene catheter was inserted into the left femoral artery to monitor blood pressure during all the of the procedures. The circulation was interrupted completely by compressing the major cardiac vessels. After 5 min of transient global ischemia, cardiac massage was started for resuscitation under artificial ventilation with a tidal volume of 2.5 ml at 200 times per min (100% O2) [16]. After a recovery period of 30 min, the animals could be disconnected from the respirator. For intracerebroventricular
(i.c.v.) administration of PACAP, PACAP was infused through the cannula at 1 pmol/h using an osmotic pump (ALZET, Palo Alto, CA). The animals were infused with PACAP for 2 days before the ischemia – reperfusion experiment. 2.2. Tissue preparation Apoptotic and morphological changes in the CA1 region of the hippocampus in the rat cardiac arrest model were studied with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method (n = 3 in each group). Following recirculation periods of 1 and 7 days, the animals were perfusion-fixed with 4% paraformaldehyde and 0.4% glutaraldehyde in 0.1 M phosphate buffer (PB) for hematoxylin – eosin staining and the TUNEL method. After the brains were removed, they were postfixed in the same fixative overnight at 4 jC. The brains were cryoprotected in PB containing 20% sucrose for 2 days at 4 jC. Briefly, frontal cryostat sections (10 Am thick) were treated with RNase (20 mg/ml) and incubated with proteinase K (20 mg/ml) for 15 min at room temperature. Endogenous peroxidase activity was inactivated by covering the sections with 2% H2O2 for 10 min at room temperature. TdT (0.3 e.u./ ml) and biotinylated dUTP in TdT buffer were then added to cover the sections and were incubated in humid atmosphere at 37 jC for 60 min. The reaction was terminated by transferring the slides to citrate buffer (30 mM sodium chloride and 30 mM sodium citrate) for 15 min at room temperature. After a 30-min immersion in 2% bovine serum albumin, the sections were incubated with the streptavidin – biotin – peroxidase complex for 2 h at room temperature. Labeling was developed with 3,3V-diaminobenzidine-4 HCl. Labeled DNA was not observed in sections when either TdT or its biotinylated substrate was omitted. 2.3. Measurement of MAPK activities Hippocampus were lysed in 10 volumes of lysis buffer consisting of 10 mM Tris – HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 5 Ag/ml aprotinin, 5 Ag/ml leupeptin, 5 Ag/ml pepstatin, 5 Ag/ml antipain, 0.5 mM phenylmethylsufonylfluoride, 50 mM NaF, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.15 M NaCl and 1% Triton-100. The lysate was centrifuged at 15,000 g for 20 min and the supernatant (50 Ag of protein) was subjected to immunoblot analysis using antibodies (Cell Signaling Technology) that recognize active forms.
3. Results 3.1. Physiological examinations Body temperature rose in each animal 15– 20 min after the ischemia, but it could be maintained within the normal
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Table 1 Physiological parameters after ischemia – reperfusion with intracerebroventricular (i.c.v) administration of PACAP at 1 pmol/h
Before 5 min 15 min 30 min 60 min 120 min 180 min
pH
pCO2 (mm Hg)
7.40 F 0.02 7.46 F 0.02 * 7.43 F 0.02 7.44 F 0.04 7.45 F 0.01 7.45 F 0.01 7.44 F 0.02
44.1 F 5.5 27.9 F 5.0 * 31.8 F 3.8 32.9 F 4.5 29.6 F 11.3 34.7 F 2.4 * 28.9 F 11.0 *
pO2 (mm Hg) 79.9 F 8.7 105.8 F 5.7 * 97.9 F 6.3 * 98.1 F 18.3 94.3 F 18.2 * 93.6 F 18.2 92.3 F 2.3 *
BS (mg/dl) 176.0 F 58.0 317.0 F 69.4 * 358.7 F 28.5 * 350.0 F 78.7 219.0 F 10.6 * 293.7 F 25.3 * 189.7 F 83.0 *
BE (nmol/l) 1.2 F 2.0 3.2 F 2.3 * 2.7 F 0.9 1.2 F 2.3 * 2.9 F 5.7 0.2 F 0.5 * 0.9 F 2.3
O2 SAT (%)
HCO3 (nmol/l)
95.5 F 1.4 98.2 F 0.3 * 97.6 F 0.4 97.5 F 1.0 96.8 F 1.3 96.7 F 1.2 97.4 F 0.3
26.4 F 2.7 19.2 F 3.1 * 20.5 F 1.6 22.0 F 2.7 19.8 F 7.4 23.5 F 0.9 * 19.0 F 7.0 *
The changes with hyperventilation under artificial ventilation were observed transiently. Transient hyperglycemia also observed from 5 to 120 min after ischemia. Values are means F S.E.M. * p < 0.05.
range by cooling the animal. The biological changes caused by hyperventilation under artificial ventilation were observed transiently. Transient hyperglycemia also observed from 5 to 120 min after ischemia. These data were improved 180 min after ischemia (Table 1).
3.2. TUNEL-positive pyramidal neurons in the CA1 region At 1 day after ischemia –reperfusion, a few TUNELpositive cells were apparent in the CA1 region in both groups (Fig. 1A and B). Many TUNEL-positive pyramidal
Fig. 1. Staining with the TUNEL (in situ nick-end labeling) method in the CA1 region of the rat hippocampus at 1 day (A, B) and 7 days (C, D) after 5 min of ischemia – reperfusion. In control animal (A, C), particularly numerous TUNEL-positive neurons are visible 7 days after ischemia (B). In the PACAP treatment group (B, D), the number of the TUNEL-positive neuron was less than the control group (C) 7 days after ischemia – reperfusion (D). Bars 200 Am.
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Fig. 2. Effect of PACAP on MAP kinase signaling pathway in the hippocampus after ischemia – reperfusion. In control group, p-JNK 1, p-p38 and ERK activations were observed after ischemia – reperfusion (A, B, C). For the contrariety, p-ERK1/2, no p-JNK 1 and p-p38 activations were observed after ischemia – reperfusion (A, B). For the contrariety, p-ERK1/2 was activated obviously from 1 to 3 h after ischemia – reperfusion (C).
cells were found in the CA1 region 7 days after reperfusion in the control group (Fig. 1C). In PACAP-administrated group, TUNEL-positive pyramidal cells were clearly suppressed at 7 days after ischemia (Fig. 1D). 3.3. Measurement of MAPK families activity Contribution of activated MAPK family members, including p-JNK/SAPK, p-p38 and p-ERK, were examined in the ischemic hippocampus with PACAP (1 pmol/h) infusion (i.c.v.). In the control group, p-JNK/SAPK, p-p38 and ERK activities were detected clearly within the first 6 h (Fig. 2). No activations of p-JNK/SAPK and p-p38 were observed within the first 6 h in PACAP-treated animals. Instead, p-ERK 1 and 2 activities were increased in 1 –3 h after ischemia –reperfusion (Fig. 2). These data indicate that PACAP not only inhibits the activation of p-JNK and p-p38; it also does not suppress p-ERK 1 and 2 after ischemia –reperfusion stress. These findings suggest that PACAP protects against apoptosis in CA1 pyramidal neurons through the MAP kinase-signaling pathway.
4. Discussion The pathophysiological mechanisms of delayed neuronal cell death in the CA1 region of the hippocampus have been studied precisely and delayed neuronal cell death is regarded as programmed cell death (apoptosis) but not necrosis [3,17,18]. Apoptotic changes in the CA1 region after transient ischemia were reported using the two-vessel occlusion model [4,17], the four-vessel occlusion model [18] and other models [19]. The morphological features of
the ultrastructural apoptotic changes are cytoplasmic condensation, cytoplasmic vacuoles, fragmented DNA and apoptotic bodies [3,20]. The TUNEL method is by far the most sensitive and reliable method for detecting apoptosis [21]. We found that TUNEL-positive cells were detected as stained nuclei of CA1 pyramidal neurons after ischemia – reperfusion [3]. The results suggest that the delayed death of the CA1 pyramidal cells in the cardiac arrest model is not necrotic but apoptotic. The cytoprotective effect of PACAP appears to be mediated by direct and indirect actions, the latter possibility via astrocytes. Neurons in the CA1 region of the hippocampus are vulnerable to global forebrain ischemia, and this model has been widely used for evaluating neuroprotective agents [3,22]. Intracerebroventricular infusion of PACAP38 into the ischemic animals prevented the otherwise total loss of pyramidal cells and their dendritic processes throughout CA1 [14,15]. Ischemic death of rat CA1 neurons was prevented by infusing PACAP38 either intracerebroventricularly (1 pmol/h) or intravenously (16 – 160 pmol/h) [14]. Immunohistochemistry showed only a small amount of PAC1-R-L1 in the CA1 region in the normal rat brain [23]. After transient cerebral ischemia, astrocytes identified by staining for glial fibrillary acidic protein near the CA1 pyramidal cell layer expressed PAC1-R-LI. Both pyramidal cells and astrocytes increased their expression of PAC1-R mRNA [14]. In the hippocampus of the rat cardiac arrest model including the CA1 region, JNK and p38 activities increased during 1 – 12 h after ischemia – reperfusion [24]. Taken together, these observations suggest that the activation of JNK or p38 MAP kinase, or both, may contribute to the induction of apoptosis in CA1 neurons [6,24 – 27]. In con-
K. Dohi et al. / Regulatory Peptides 109 (2002) 83–88
trast, in PACAP-treated animals, only small increases of pJNK and p-p38 activities were detected within the first 6 h of this study. This data indicate that PACAP suppresses the activation of p-JNK and p-p38 after ischemia in comparison with the untreated animals [24]. It was reported that p38 inhibition of cytokines such as IL-1 and TNF in the brain is a response to injury [28]. PACAP also regulates the release of cytokines from microglia [29]. PACAP may regulate inflammatory cytokines through inhibition of p38 activation. On other hand, it is considered that ERK is the MAPK member leading to the protection and reconstruction of neurons [6,28]. We demonstrate that PACAP did not suppress p-ERK1/2 activation 1 h after ischemia –reperfusion. These data indicate that PACAP acts synergistically to inhibit the p-JNK and p-p38 signaling pathways, not the p-ERK signaling pathway, and protects neurons against neuronal cell death. In conclusion, PACAP directly or indirectly protects against hippocampal neuronal death in rat cardiac arrest model. During the process of the cytoprotective action of PACAP, PACAP may regulate the dynamic balance between the growth factor-activated ERK and stress-activated JNK – p38 pathways.
Acknowledgements This study was supported in part by grants to S.S. from the Ministry of Education, Science, Sports, and Culture of Japan, and the High-Technology Research Center Project of the Ministry of Education, Science, Sports, and Culture of Japan.
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[9] Arimura A, Somogyvari-Vigh A, Weill C, Fiore RC, Tatsuno I, Bay V, et al. PACAP functions as a neurotrophic factor. Ann N Y Acad Sci 1994;739:228 – 43. [10] Cavallaro S, Copani A, D’Agata V, Musco S, Petralia S, Ventra C, et al. Pituitary adenylate cyclase activating polypeptide prevents apoptosis in cultured cerebellar granule neurons. Mol Pharmacol 1996;50: 60 – 6. [11] Villalba M, Bockaert J, Journot L. Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. J Neurosci 1997;17:83 – 90. [12] Frechilla D, Garcia-Osta A, Palacios S, Cenarruzabeitia E, Del Rio J. BDNF mediates the neuroprotective effect of PACAP-38 on rat cortical neurons. NeuroReport 2001;12:919 – 23. [13] Vaudry D, Gonzalez BJ, Basille M, Pamantung TF, Fontaine M, Fournier A, et al. The neuroprotective effect of pituitary adenylate cyclaseactivating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase 3/CPP32. Proc Natl Acad Sci U S A 2000;97:13390 – 5. [14] Uchida D, Arimura A, Somogyvari-Vigh A, Shioda S, Banks WA. Prevention of ischemia-induced death of hippocampal neurons by pituitary adenylate cyclase activating polypeptide. Brain Res 1996; 736:280 – 6. [15] Reglodi D, Somogyvari-Vigh A, Vigh S, Kozicz T, Arimura A. Delayed systemic administration of PACAP38 is neuroprotective in transient middle cerebral artery occlusion in the rat. Stroke 2000;31: 1411 – 7. [16] Kawai K, Nitecka L, Ruetzler CA, Nagashima G, Joo F, Mies G, et al. Global cerebral ischemia associated with cardiac arrest in the rat: I. Dynamics of early neuronal changes. J Cereb Blood Flow Metab 1992;12:238 – 49. [17] MacManus JP, Buchan AM, Hill IE, Rasquinha I, Preston E. Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain. Neurosci Lett 1993;164:89 – 92. [18] Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, et al. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci 1995;15:1001 – 11. [19] Beilharz EJ, Williams CE, Dragunow M, Sirimanne ES, Gluckman PD. Mechanisms of delayed cell death following hypoxic – ischemic injury in the immature rat: evidence for apoptosis during selective neuronal loss. Brain Res Mol Brain Res 1995;29:1 – 14. [20] Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251 – 306. [21] Li Y, Chopp M, Jiang N, Zhang ZG, Zaloga C. Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. Stroke 1995;26:1252 – 7. [22] Pulsinelli WA, Brierley JB. A new model of bilateral hemisphere ischemia in the unanesthetized rat. Stroke 1979;10:267 – 72. [23] Shioda S, Shuto Y, Somogyvari-Vigh A, Legradi G, Onda H, Coy DH, et al. Localization and gene expression of the receptor for pituitary adenylate cyclase-activating polypeptide in the rat brain. Neurosci Res 1997;28:345 – 54. [24] Ozawa H, Shioda S, Dohi K, Matsumoto H, Mizushima H, Zhou CJ, et al. Delayed neuronal cell death in the rat hippocampus is mediated by the mitogen-activated protein kinase signal transduction pathway. Neurosci Lett 1999;262:57 – 60. [25] Herdegen T, Claret FX, Kallunki T, Martin-Villalba A, Winter C, Hunter T, et al. Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J Neurosci 1998;18:5124 – 35. [26] Barone FC, Irving EA, Ray AM, Lee JC, Kassis S, Kumar S, et al. Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia. Med Res Rev 2001;21: 129 – 45. [27] Takagi Y, Nozaki K, Sugino T, Hattori I, Hashimoto N. Phosphorylation of c-Jun NH(2)-terminal kinase and p38 mitogen-activated pro-
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tein kinase after transient forebrain ischemia in mice. Neurosci Lett 2000;294:117 – 20. [28] Irving EA, Barone FC, Reith AD, Hadingham SJ, Parsons AA. Differential activation of MAPK/ERK and p38/SAPK in neurones and glia following focal cerebral ischaemia in the rat. Brain Res Mol Brain Res 2000;77:65 – 75.
[29] Kim WK, Kan Y, Ganea D, Hart RP, Gozes I, Jonakait GM. Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-alpha production in injured spinal cord and in activated microglia via a cAMP-dependent pathway. J Neurosci 2000;20:3622 – 30.
Pituitary adenylate cyclase-activating polypeptide (PACAP) prevents hippocampal neurons from apoptosis by inhibiting JNK/SAPK and p38 signal transduction pathways Kenji Dohi a,b,d,*, Hidekatsu Mizushima b, Shigeo Nakajo c, Hirokazu Ohtaki b,d, Seiji Matsunaga b, Tohru Aruga a, Seiji Shioda b,d a
Department of Critical Care Medicine, School of Medicine, Showa University, Tokyo, Japan b Department of Anatomy, Showa University School of Medicine, Tokyo, Japan c Laboratory of Biological Chemistry, Showa University School of Pharmaceutical Science, Tokyo, Japan d The Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology (JST), Japan
Abstract We have demonstrated that ischemic neuronal death (apoptosis) of rat CA1 region of the hippocampus was prevented by infusing pituitary adenylate cyclase-activating polypeptide (PACAP) either intracerebroventricularly or intravenously. We have also demonstrated that the activity of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, was increased in the hippocampus within 1 – 6 h after brain ischemia. The molecular mechanisms underlying the PACAP anti-apoptotic effect were demonstrated in this study. Ischemic stress had a strong influence on MAP kinase family, especially on JNK/SAPK and p38. PACAP inhibited the activation of JNK/SAPK and p38 after ischemic stress, while ERK is not suppressed. These findings suggest that PACAP inhibits the JNK/SAPK and p38 signaling pathways, thereby protecting neurons against apoptosis. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Ischemia; ERK; PACAP
1. Introduction Brain ischemia induces various degrees of neuronal damage depending on its intensity and duration [1]. It is believed that an increase in intracellular Ca2 + concentration, stimulated by several bioactive substances and others, induces neuronal cell death (apoptosis) in the hippocampal CA1 [2,3]. Rat CA1 pyramidal cells are selectively vulnerable to transient ischemic insult, and ischemia causes severe neuronal damage to the CA1 neurons which start dying 2 or 3 days after the reperfusion [3,4]. A number of neurotrophic factors and nerve growth factors prevent the ischemia-induced degeneration of hippocampal CA1 neurons. Among them, PACAP is the potent anti-apoptotic neurotrophic factor. PACAP has been
* Corresponding author. Department of Critical Care Medicine, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel.: +81-3-3784-8744; fax: +81-3-3784-6880. E-mail address: [email protected] (K. Dohi).
found to stimulate the outgrowth of neurites from PC12 cells and to enhance the survival of sympathetic ganglion cells [7,8]. PACAP prevents the neuronal cell death in the dorsal root ganglia in chick embryo and also increases the survival of lumbar spinal motoneurons [9]. PACAP also prevents apoptosis in cultured cerebellar granule cells [10,11]. In addition, hippocampal neuronal cell death by gp120 in vitro was completely prevented by PACAP at extremely low concentration as vasoactive intestinal peptide (VIP) [9]. Contrary to VIP, the dose – response curve is bimodal, with the greatest cytoprotective activity at 0.1 pM and 0.1 nM, suggesting that PACAP may act at two different receptors: PACAP and VIP/PACAP. In in vitro studies, brain-derived neurotrophic factor (BDNF) mediates the neuroprotective effect of PACAP38 on rat cortical neurons [12]. PACAP38 increased survival of cerebellar neurons in a dose-dependent manner by decreasing the extent of apoptosis estimated by DNA fragmentation in primary culture of rat cerebellar granule cells [11]. The signaling pathways that lead to apoptosis are beginning to be defined, and a number of proteins that
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induce or prevent apoptosis have been identified. There are molecular mechanisms that regulate apoptosis, the contribution to cell death of mitogen-activated protein (MAP) kinase family members, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK; also termed SAPK or stress-activated protein kinase) and p38 [5]. It is known that the activation of JNK/SAPK and p38 and the concurrent inhibition of ERK are critical for the induction of apoptosis in PC12 cell [6]. We have demonstrated that JNK/SAPK, p38 and ERK activities increased during the first 6 h and decreased at 12 h after ischemia –reperfusion in hippocampus of rat cardiac arrest model [24]. PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A (PKA)/protein kinase C (PKC)-dependent inhibition of caspase-3 activity [13]. They also reported that PACAP38 induced activation of the ERK-type of MAP kinase through a cAMP-dependent pathway [11]. In in vivo study, it has been reported that PACAP prevents ischemiainduced neuronal death in global and focal ischemic models [14,15]. The precise molecular mechanism of neuroprotection and contribution of PACAP in regulating MAP kinase family members have not been clarified, though PACAP has the effect of neuroprotection in vivo. In the present study, using rat global ischemia model (cardiac arrest model), we investigated the effect of intracerebroventricular (i.c.v.) administration of PACAP to the expression of activated MAPK family members (p-JNK, pp38 and p-ERK) in the hippocampus after ischemia – reperfusion [3,16].
2. Materials and methods 2.1. Animal preparations Adult male Sprague – Dawley rats (250 – 300 g; Saitama Experimental Animal Center, Saitama, Japan) were housed in a temperature- and light-controlled room (lights on at 0600 and off at 1800) and supplied with standard laboratory chow and water ad libitum. The Institutional Animal Care and Use Committee of Showa University approved all experimental procedures involving animals. The rats were anesthetized with 3.5% halothane and maintained with 2% sevoflurance in 70% N2O and 30% O2. Body temperature was monitored with a rectal thermometer and maintained at 37 jC. A polyethylene catheter was inserted into the left femoral artery to monitor blood pressure during all the of the procedures. The circulation was interrupted completely by compressing the major cardiac vessels. After 5 min of transient global ischemia, cardiac massage was started for resuscitation under artificial ventilation with a tidal volume of 2.5 ml at 200 times per min (100% O2) [16]. After a recovery period of 30 min, the animals could be disconnected from the respirator. For intracerebroventricular
(i.c.v.) administration of PACAP, PACAP was infused through the cannula at 1 pmol/h using an osmotic pump (ALZET, Palo Alto, CA). The animals were infused with PACAP for 2 days before the ischemia – reperfusion experiment. 2.2. Tissue preparation Apoptotic and morphological changes in the CA1 region of the hippocampus in the rat cardiac arrest model were studied with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method (n = 3 in each group). Following recirculation periods of 1 and 7 days, the animals were perfusion-fixed with 4% paraformaldehyde and 0.4% glutaraldehyde in 0.1 M phosphate buffer (PB) for hematoxylin – eosin staining and the TUNEL method. After the brains were removed, they were postfixed in the same fixative overnight at 4 jC. The brains were cryoprotected in PB containing 20% sucrose for 2 days at 4 jC. Briefly, frontal cryostat sections (10 Am thick) were treated with RNase (20 mg/ml) and incubated with proteinase K (20 mg/ml) for 15 min at room temperature. Endogenous peroxidase activity was inactivated by covering the sections with 2% H2O2 for 10 min at room temperature. TdT (0.3 e.u./ ml) and biotinylated dUTP in TdT buffer were then added to cover the sections and were incubated in humid atmosphere at 37 jC for 60 min. The reaction was terminated by transferring the slides to citrate buffer (30 mM sodium chloride and 30 mM sodium citrate) for 15 min at room temperature. After a 30-min immersion in 2% bovine serum albumin, the sections were incubated with the streptavidin – biotin – peroxidase complex for 2 h at room temperature. Labeling was developed with 3,3V-diaminobenzidine-4 HCl. Labeled DNA was not observed in sections when either TdT or its biotinylated substrate was omitted. 2.3. Measurement of MAPK activities Hippocampus were lysed in 10 volumes of lysis buffer consisting of 10 mM Tris – HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 5 Ag/ml aprotinin, 5 Ag/ml leupeptin, 5 Ag/ml pepstatin, 5 Ag/ml antipain, 0.5 mM phenylmethylsufonylfluoride, 50 mM NaF, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.15 M NaCl and 1% Triton-100. The lysate was centrifuged at 15,000 g for 20 min and the supernatant (50 Ag of protein) was subjected to immunoblot analysis using antibodies (Cell Signaling Technology) that recognize active forms.
3. Results 3.1. Physiological examinations Body temperature rose in each animal 15– 20 min after the ischemia, but it could be maintained within the normal
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Table 1 Physiological parameters after ischemia – reperfusion with intracerebroventricular (i.c.v) administration of PACAP at 1 pmol/h
Before 5 min 15 min 30 min 60 min 120 min 180 min
pH
pCO2 (mm Hg)
7.40 F 0.02 7.46 F 0.02 * 7.43 F 0.02 7.44 F 0.04 7.45 F 0.01 7.45 F 0.01 7.44 F 0.02
44.1 F 5.5 27.9 F 5.0 * 31.8 F 3.8 32.9 F 4.5 29.6 F 11.3 34.7 F 2.4 * 28.9 F 11.0 *
pO2 (mm Hg) 79.9 F 8.7 105.8 F 5.7 * 97.9 F 6.3 * 98.1 F 18.3 94.3 F 18.2 * 93.6 F 18.2 92.3 F 2.3 *
BS (mg/dl) 176.0 F 58.0 317.0 F 69.4 * 358.7 F 28.5 * 350.0 F 78.7 219.0 F 10.6 * 293.7 F 25.3 * 189.7 F 83.0 *
BE (nmol/l) 1.2 F 2.0 3.2 F 2.3 * 2.7 F 0.9 1.2 F 2.3 * 2.9 F 5.7 0.2 F 0.5 * 0.9 F 2.3
O2 SAT (%)
HCO3 (nmol/l)
95.5 F 1.4 98.2 F 0.3 * 97.6 F 0.4 97.5 F 1.0 96.8 F 1.3 96.7 F 1.2 97.4 F 0.3
26.4 F 2.7 19.2 F 3.1 * 20.5 F 1.6 22.0 F 2.7 19.8 F 7.4 23.5 F 0.9 * 19.0 F 7.0 *
The changes with hyperventilation under artificial ventilation were observed transiently. Transient hyperglycemia also observed from 5 to 120 min after ischemia. Values are means F S.E.M. * p < 0.05.
range by cooling the animal. The biological changes caused by hyperventilation under artificial ventilation were observed transiently. Transient hyperglycemia also observed from 5 to 120 min after ischemia. These data were improved 180 min after ischemia (Table 1).
3.2. TUNEL-positive pyramidal neurons in the CA1 region At 1 day after ischemia –reperfusion, a few TUNELpositive cells were apparent in the CA1 region in both groups (Fig. 1A and B). Many TUNEL-positive pyramidal
Fig. 1. Staining with the TUNEL (in situ nick-end labeling) method in the CA1 region of the rat hippocampus at 1 day (A, B) and 7 days (C, D) after 5 min of ischemia – reperfusion. In control animal (A, C), particularly numerous TUNEL-positive neurons are visible 7 days after ischemia (B). In the PACAP treatment group (B, D), the number of the TUNEL-positive neuron was less than the control group (C) 7 days after ischemia – reperfusion (D). Bars 200 Am.
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Fig. 2. Effect of PACAP on MAP kinase signaling pathway in the hippocampus after ischemia – reperfusion. In control group, p-JNK 1, p-p38 and ERK activations were observed after ischemia – reperfusion (A, B, C). For the contrariety, p-ERK1/2, no p-JNK 1 and p-p38 activations were observed after ischemia – reperfusion (A, B). For the contrariety, p-ERK1/2 was activated obviously from 1 to 3 h after ischemia – reperfusion (C).
cells were found in the CA1 region 7 days after reperfusion in the control group (Fig. 1C). In PACAP-administrated group, TUNEL-positive pyramidal cells were clearly suppressed at 7 days after ischemia (Fig. 1D). 3.3. Measurement of MAPK families activity Contribution of activated MAPK family members, including p-JNK/SAPK, p-p38 and p-ERK, were examined in the ischemic hippocampus with PACAP (1 pmol/h) infusion (i.c.v.). In the control group, p-JNK/SAPK, p-p38 and ERK activities were detected clearly within the first 6 h (Fig. 2). No activations of p-JNK/SAPK and p-p38 were observed within the first 6 h in PACAP-treated animals. Instead, p-ERK 1 and 2 activities were increased in 1 –3 h after ischemia –reperfusion (Fig. 2). These data indicate that PACAP not only inhibits the activation of p-JNK and p-p38; it also does not suppress p-ERK 1 and 2 after ischemia –reperfusion stress. These findings suggest that PACAP protects against apoptosis in CA1 pyramidal neurons through the MAP kinase-signaling pathway.
4. Discussion The pathophysiological mechanisms of delayed neuronal cell death in the CA1 region of the hippocampus have been studied precisely and delayed neuronal cell death is regarded as programmed cell death (apoptosis) but not necrosis [3,17,18]. Apoptotic changes in the CA1 region after transient ischemia were reported using the two-vessel occlusion model [4,17], the four-vessel occlusion model [18] and other models [19]. The morphological features of
the ultrastructural apoptotic changes are cytoplasmic condensation, cytoplasmic vacuoles, fragmented DNA and apoptotic bodies [3,20]. The TUNEL method is by far the most sensitive and reliable method for detecting apoptosis [21]. We found that TUNEL-positive cells were detected as stained nuclei of CA1 pyramidal neurons after ischemia – reperfusion [3]. The results suggest that the delayed death of the CA1 pyramidal cells in the cardiac arrest model is not necrotic but apoptotic. The cytoprotective effect of PACAP appears to be mediated by direct and indirect actions, the latter possibility via astrocytes. Neurons in the CA1 region of the hippocampus are vulnerable to global forebrain ischemia, and this model has been widely used for evaluating neuroprotective agents [3,22]. Intracerebroventricular infusion of PACAP38 into the ischemic animals prevented the otherwise total loss of pyramidal cells and their dendritic processes throughout CA1 [14,15]. Ischemic death of rat CA1 neurons was prevented by infusing PACAP38 either intracerebroventricularly (1 pmol/h) or intravenously (16 – 160 pmol/h) [14]. Immunohistochemistry showed only a small amount of PAC1-R-L1 in the CA1 region in the normal rat brain [23]. After transient cerebral ischemia, astrocytes identified by staining for glial fibrillary acidic protein near the CA1 pyramidal cell layer expressed PAC1-R-LI. Both pyramidal cells and astrocytes increased their expression of PAC1-R mRNA [14]. In the hippocampus of the rat cardiac arrest model including the CA1 region, JNK and p38 activities increased during 1 – 12 h after ischemia – reperfusion [24]. Taken together, these observations suggest that the activation of JNK or p38 MAP kinase, or both, may contribute to the induction of apoptosis in CA1 neurons [6,24 – 27]. In con-
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trast, in PACAP-treated animals, only small increases of pJNK and p-p38 activities were detected within the first 6 h of this study. This data indicate that PACAP suppresses the activation of p-JNK and p-p38 after ischemia in comparison with the untreated animals [24]. It was reported that p38 inhibition of cytokines such as IL-1 and TNF in the brain is a response to injury [28]. PACAP also regulates the release of cytokines from microglia [29]. PACAP may regulate inflammatory cytokines through inhibition of p38 activation. On other hand, it is considered that ERK is the MAPK member leading to the protection and reconstruction of neurons [6,28]. We demonstrate that PACAP did not suppress p-ERK1/2 activation 1 h after ischemia –reperfusion. These data indicate that PACAP acts synergistically to inhibit the p-JNK and p-p38 signaling pathways, not the p-ERK signaling pathway, and protects neurons against neuronal cell death. In conclusion, PACAP directly or indirectly protects against hippocampal neuronal death in rat cardiac arrest model. During the process of the cytoprotective action of PACAP, PACAP may regulate the dynamic balance between the growth factor-activated ERK and stress-activated JNK – p38 pathways.
Acknowledgements This study was supported in part by grants to S.S. from the Ministry of Education, Science, Sports, and Culture of Japan, and the High-Technology Research Center Project of the Ministry of Education, Science, Sports, and Culture of Japan.
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