IRUCAA@TDC : Nerve growth factor and brain-derived neurotrophic factor attenuate angiotensin-II-induced facilitation of calcium channels in acutely dissociated nucleus tractus solitarii neurons of the rat

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(1)Title. Nerve growth factor and brain-derived neurotrophic factor attenuate angiotensin-II-induced facilitation of calcium channels in acutely dissociated nucleus tractus solitarii neurons of the rat. Author(s) Alternative. Endoh, T; Sato, D; Wada, Y; Ishihara, K; Hashimoto, S; Yoshinari, M; Matsuzaka, K; Tazaki, M; Inoue, T. Journal URL. Archives of Oral Biology, 53(12): 1192-1201 http://hdl.handle.net/10130/635. Right. Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/.

(2) Archives of Oral Biology (AOB-D-07-00364R1) Re-Revised version. Nerve growth factor and brain-derived neurotrophic factor attenuate angiotensin Ⅱ -induced facilitation of calcium channels in acutely dissociated nucleus tractus solitarii neurons of the rat. Takayuki Endoh*, Daisuke Sato, Yoshiyuki Wada, Kazuyuki Ishihara, Sadamitsu Hashimoto, Masao Yoshinari, Kenichi Matsuzaka, Masakazu Tazaki and Takashi Inoue. Oral Health Science Center HRC7, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan *Corresponding author. E-mail address: [email protected] (T. Endoh). 30 pages 6 figures and figure legends, 1 Table. Keywords: Nerve growth factor, Brain -derived neurotrophic factor, Angiotensin Ⅱ, Voltage-dependent Ca2+ channels current.

(3) ABSTRACT. Objective: Neurotrophins, such as Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), promote neuronal development and neuronal survival. The mechanisms, however, remain controversial. The aim of the present study is to investigate the hypothesis that NGF and BDNF interferes with angiotensin Ⅱ- and glutamate-induced facilitation of voltage-dependent Ca2+ channels (VDCCs) in the nucleus tractus solitarius (NTS). Design: The profile of NGF and BDNF actions in acutely dissociated rat NTS has been studied using the whole-cell configuration of the patch clamp technique. Results: Pretreatment with NGF and BDNF attenuated the Angiotensin Ⅱ-induced facilitation of VDCCs but not glutamate-induced facilitation of L-type VDCCs current in NTS. NGF-induced attenuations were antagonized by pretreatment with a tyrosine kinase A (TrkA) receptor antagonist K-252a. Conclusions: NGF attenuated the angiotensin Ⅱ-induced facilitation of L-type VDCCs mediated by a TrkA receptor in NTS.. 2.

(4) 1. Introduction. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) play critical roles in the development and neuronal survival of the peripheral and central nervous system. 1,2.. NGF is also able to contribute to the neuronal differentiation, neuronal. survival, and neuronal long-term maintenance 3. Angiotensin Ⅱ (Ang Ⅱ), an important mediator of the renin -angiotensin system, does not only exert actions on blood pressure control and body fluid homeostasis but also contributes to cell growth demonstrated. that. Ang. Ⅱ. 4,5. and neuronal differentiation. induces neurite formation and. 6.. It has been. potentiates. the. NGF-mediated effect on differentiation 7. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity, and gene expression 8,9. VDCCs also play a variety roles in many cell processes including neuronal proliferation, neuronal differentiation, and programmed cell death 10-16.. The nucleus tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic, and swallowing functions. 3. 17,18.. In.

(5) addition, caudal NTS is believed to belong to the swallowing pattern generators, whereas the rostal NTS is part of the taste pathways. 19.. The NTS appears not to be a. simple ‘relay’ nucleus, rather it performs complex integration of information from multiple synaptic inputs of both peripheral and central origins. Previously, we and other groups have demonstrated that Ang Ⅱ facilitates VDCCs in NTS. 20-22.. Therefore, this study was designed to test the hypothesis that NGF and. BDNF interferes with Ang Ⅱ-induced facilitation of VDCCs in NTS. 2. Material and Methods. 2.1. Cell preparation. The study was carried out according to “The guideline for the treatment of experimental animals in Tokyo Dental College”. The animals were individually housed with a 12-h dark/light cycle and free access to food and water. 125 animals were used in all experiment. Young Wistar rats (7-18 days old) were decapitated and their brains were quickly removed and submerged in ice cold artificial cerebrospinal fluid (aCSF) saturated with 95% O2 and 5% CO2 of the following composition (in mM): 126 NaCl, 26.2. 4.

(6) NaHCO3, 1 NaH2PO4, 3 KCl, 1.5 MgSO 4, 1.5 CaCl2 and 30 glucose; pH 7.4. The medulla was removed and prepared for in vitro recording as we described previously. 22.. The. dorsal portion of a block extending from approximately 5 mm caudal to 10 mm rostral to the calamus scriptorius was cut into 400 μm slices with a vibratome (DTK-1000; Dosaka EM Co., Ltd, Kyoto) under oxygenated aCSF. After being sectioned, 3-5 slices obtained from a single brain were transferred to a holding chamber and stored in oxygenated aCSF at room temperature for at least 40 min before use. Slices were then transferred to a conical tube containing gently bubbled aCSF at 36 ℃ to which 1.8 U/ml dispase (gradeⅠ; 0.75 ml/slice) 23 was added. After 60 min incubation, slices were rinsed with enzyme-free aCSF. Under a dissecting microscope, the NTS region was micropunched and placed on a poly-l-lysine-coated coverslip. The cells were then dissociated by trituration using progressively smaller diameter pipettes and allowed to settle on a coverslip for 20 min. After the dissociated neurons had adhered to the bottom of the coverslip (usually within 20 min) the electrophysiological measurements were started. Whole-cell patch clamp recordings were performed 30-50 min after dissociation.. 2.2 Whole-cell patch-clamp recordings. 5.

(7) Voltage-clamp recordings were conducted using the whole-cell configuration of the patch-clamp technique. 24.. Fabricated recording pipettes (2-3 MΩ) were filled with the. internal solution of the following composition (in mM): 100 CsCl, 1 MgCl2, 10 HEPES, 10 BAPTA, 3.6 MgATP, 14 Tris2phosphocreatine (CP), 0.1 GTP, and 50 U/ml creatine phosphokinase (CPK). The pH was adjusted to 7.2 with CsOH. After the formation of a giga seal, in order to record VDCCs currents (ICa) carried by Ba 2+ (I Ba), the external solution was replaced from aCSF solution to a solution containing the following (in mM): 151 tetraethylammonium (TEA) chloride, 5 BaCl2, 1 MgCl 2, 10 HEPES and 10 glucose. The pH was adjusted to 7.4 with TEA-OH. The IBa were isolated by blocking potassium channels with a CsCl (100 μM) in the internal solution and TEA (151 μM) in the external solution. Using Ba2+ instead of Ca2+ in the external solution in these experiments acted to block Ca2+-dependent potassium channels. Command voltage protocols were generated with a computer software pCLAMP version 8 (Axon Instruments, Union City, CA, U.S.A.) and transformed to an analogue signal using a DigiData 1200 interface (Axon Instruments, Union City, CA, U.S.A.). The command pulses were applied to cells through an L/M-EPC7 amplifier (HEKA Elektronik, Lambrecht, Germany). The currents were recorded with the amplifier and a computer software pCLAMP 8 acquisition system. Access resistance (＜ 15 MΩ) was determined. 6.

(8) by transient responses to voltage commands. Access resistance compensation was not used. To ascertain that no major changes in the access resistance had occurred during the recordings a 5 mV, 10 msec pulses were used before IBa was evoked. The temperature of the superfusing extracellular solution was maintained at 20-26℃. The neurons were constantly superfused with external solution by gravity flow at a rate of approximately 1 ml/min. All drugs were applied by superfusion; time to reach equilibrium concentrations in the bath was 90-120 s, with 1-2 min exposure times. To avoid desensitization, all drugs were randomly applied to 85 different neurons and non-overlaped drugs were applied to a single neuron.. 2.3. Materials. Ang Ⅱ , ω-conotoxin GⅥA (ω-CgTx GⅥA) and ω-agatoxin ⅣA (ω-Aga ⅣA) were purchased from Peptide Institute (Osaka, Japan). NGF was purchased from ICN. (RS)-3,5-dihydroxyphenylglycine (DHPG) was purchased from Tocris (Avonmouth, U.K.). BDNF and nifedipine (Nif) were purchased from Sigma (Tokyo, Japan). K-252a was purchased from Calbiochem (La Jolla, CA, U.S.A.).. 7.

(9) 2.4. Data analysis and statistics. All data analysis was performed using the pCLAMP 8.0 acquisition system. Values in text and figures are expressed as mean ± SEM. Statistical analysis was made by student t-tests for comparisons between pairs of groups and by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Probability (p) values of less than 0.05 were considered significant.. 3. Results. 3.1. AngⅡ-induced facilitation of IBa was attenuated by NGF. Whole-cell patch recordings were obtained from 85 neurons in 420 preparation of 400 μM thick slices of rat brainstem. The average peak IBa value for neurons recorded was 451.9 ± 156.6 pA (n ＝85). The average capacitance for neurons recorded was 8.2 ± 0.4 pF (n＝85). Any drug did not change the capacitance value. An example of Ang Ⅱ-induced facilitation of IBa is shown in Fig. 1. IBa was evoked every 20 sec with a 100 msec depolarizing voltage step to 0 mV from a holding potential. 8.

(10) of −80 mV. As shown in Fig. 1A and B, application of 1 μM Ang Ⅱ rapidly and reversibly facilitates IBa. To evaluate the possible contribution of NGF to the AngⅡ -induced facilitation of IBa, the effects of Ang Ⅱ on I Ba in neurons treated with NGF (38 μM) were investigated. In 7 neurons tested, treatment with NGF attenuated the Ang Ⅱ-induced facilitation of IBa (55.2 ± 4.0 pA/pF for control, 73.1 ± 5.8 pA/pF for Ang Ⅱ only, and 58.4 ± 4.4 pA/pF for Ang Ⅱ in neuron treated with NGF, n＝18, 12 and 7, respectively, P < 0.05). These results indicated that NGF attenuated the Ang Ⅱ-induced facilitation of IBa in NTS. An example of glutamate-induced facilitation of IBa is shown in Fig. 2. As shown in Fig. 2A and B, application of 100 μM DHPG rapidly and reversibly facilitates IBa. To evaluate the possible contribution of NGF to the glutamate-induced facilitation of IBa, the effects of glutamate on IBa in neurons treated with NGF (38 μM) were investigated. In 7 neurons tested, treatment with NGF did not attenuate the glutamate-induced facilitation of IBa (55.2 ± 4.0 pA/pF for control, 78.9 ± 5.3 pA/pF for DHPG only, and 78.4 ± 4.8 pA/pF for DHPG in neuron treated with NGF, n＝18, 7 and 7, respectively). These results indicated that NGF did not attenuate the glutamate-induced facilitation of IBa in NTS.. 9.

(11) 3.2. AngⅡ-induced facilitation of IBa was attenuated by BDNF. To evaluate the possible contribution of BDNF to the AngⅡ-induced facilitation of IBa, the effects of Ang Ⅱ on I Ba in neurons treated with BDNF (35 μM) were investigated. In 8 neurons tested, treatment with BDNF attenuated the Ang Ⅱ-induced facilitation of IBa (55.2 ± 4.0 pA/pF for control, 73.1 ± 5.8 pA/pF for Ang Ⅱ only, and 56.4 ± 4.3 pA/pF for Ang Ⅱ in neuron treated with BDNF, n＝18, 12 and 8, respectively, Figs. 3 and 4C, P < 0.05). To evaluate the possible contribution of BDNF to the glutamate-induced facilitation of IBa, the effects of glutamate o n IBa in neurons treated with BDNF (35 μM) were investigated. In 7 neurons tested, treatment with BDNF did not attenuate the glutamate-induced facilitation of IBa (55.2 ± 4.0 pA/pF for control, 78.9 ± 5.3 pA/pF for DHPG only, and 76.9 ± 4.0 pA/pF for DHPG in neuron treated with BDNF, n＝18, 7 and 7, respectively). These results indicated that BDNF also attenuated the Ang Ⅱ-induced facilitation of IBa but not glutamate-induced facilitation of IBa in NTS. As shown in Figs. 1 and 3, differences of kinetics of IBa were observed. One possible. 10.

(12) explanation is that there is a variation in that VDCCs subtypes distribution in each neuron. L-type VDCCs has long lasting large conductance, whereas N-, P- and Q-types VDCCs show rapid inactivation during voltage step. 2 5.. It can be considered that. different distribution has s influence on the differences in the kinetics of the IBa.. 3.3. NGF- and BDNF-induced attenuation of AngⅡ-induced facilitation of IBa was mediated by a TrKA receptor. NGF binds to two types of receptors: the low-affinity receptor p75 high-affinity receptor tyrosine kinase A. 27.. 26. and the. The kinase inhibitor K-252a blocks TrkA. receptor autophosphorylation and thus prevents NGF signaling through the TrkA receptor 28. To evaluate the possible contribution of a TrkA receptor to the NGF-induced attenuation of AngⅡ-induced facilitation of IBa, the effects K-252a on NGF-induced attenuation of Ang Ⅱ-induced facilitation of IBa were investigated. In 7 neurons tested, treatment with K-252a antagonized NGF-induced attenuation of the Ang Ⅱ-induced facilitation of IBa (58.4 ± 4.4 pA/pF for Ang Ⅱ in neuron treated with NGF, 74.4 ± 6.0 pA/pF for Ang Ⅱ in neuron treated with K-252a＋NGF, n＝7 and 7, respectively, Fig. 4, P < 0.05).. 11.

(13) To evaluate the possible contribution of a TrkA receptor to the BDNF-induced attenuation of AngⅡ-induced facilitation of IBa, the effects K-252a on BDNF-induced attenuation of Ang Ⅱ-induced facilitation of IBa were investigated. In 9 neurons tested, treatment with K-252a did not antagonize BDNF-induced attenuation of the Ang Ⅱ -induced facilitation of IBa (56.4 ± 4.3 pA/pF for Ang Ⅱ in neuron treated with BDNF, 60.2 ± 5.8 pA/pF for Ang Ⅱ in neuron treated with K-252a＋BDNF, n＝8 and 9, respectively, Fig. 4). These results indicated that NGF attenuated the Ang Ⅱ-induced facilitation of IBa mediated by a TrkA receptor in NTS.. 3.4. NGF- and BDNF-induced facilitation of IBa. Note that both NGF and BDNF facilitate IBa (Fig.1D, 2D, 3B and 3D). The IBa current density-voltage relations measured before and during application of NGF (38 μM) and BDNF (38 μM) are shown in Fig. 5A and 5B, respectively. From a holding potential of −80 mV, the IBa was activated after −30 mV with a peak current amplitude at 0 mV. As shown in Fig. 5, application of NGF and BDNF resulted in a slight, but non-significant, facilitated peak I Ba current density. Both NGF (38 μM) and BDNF (35. 12.

(14) μM) did not shift the IBa current density-voltage relations (n＝5). VDCCs were split into two groups on the basis of activation: low voltage activated (LVA)-T-type and high voltage activated (HVA)-L-, N-, P/Q- and R-types. We previously defined pharmacologically the distinct HVA VDCCs in NTS. Mean percentages of L-type IBa components (IBa-L), N-type IBa components (IBa-N), P/Q-type IBa components (IBa-P/Q) and R-type IBa components (IBa- R) of total IBa is 42.2 ± 3.8%, 28.4 ± 3.4%, 19.3 ± 3.2% and 10.1 ± 1.4%, respectively in NTS. 29.. It was then investigated about which. types of the VDCCs were facilitated by NGF. When ω-CgTx GⅥA (N-type VDCCs blocker, 1 μM) ＋ ω-Aga ⅣA (P/Q-type VDCCs blocker, 1 μM) were applied first, the resistant IBa , i.e. L ＋ R components, were facilitated by a subsequent application of NGF. On the other hand, Nif (L-type VDCCs blocker, 10 μM) ＋ ω-Aga ⅣA (1 μ M) and Nif ＋ω-CgTx GⅥA (1 μM) were applied first, the resistant IBa , i.e. N ＋ R components and P/Q ＋ R components, respectively, were not affected by a subsequent application of NGF (38 μ M) (Fig.6, Table 1). These results indicated that NGF facilitated L-type IBa.. 4. Discussion. 13.

(15) This study has shown that NGF and BDNF attenuate the Ang Ⅱ-induced facilitation of VDCCs mediated in NTS. Especially, NGF attenuated the Ang Ⅱ-induced facilitation of VDCCs mediated by a TrkA receptor in NTS. As mentioned above, VDCCs serve as crucial mediators of membrane excitability of neurons and Ca2+-dependent functions such as neurotransmitter release, enzyme activity, and gene expression. Additionally, there is some evidence that Ca2+ mediates growth factor actions in neurons. In sympathetic neurons, for example, Ca2+ is involved in the effect of NGF on neurite outgrowth. 30.. In addition, the ability of NGF to protect. hippocampal neurons against hypoglycemic damage involves modulation of VDCCs Ca2+ is also involved in the control of many types of synaptic plasticity. 25,. 31.. and changes. in Ca2+ conductance can regulate neuronal excitability. In the present study, we demonstrated that signal transduction of NGF and BDNF attenuate Ang Ⅱ effects in NTS involving Ca2+ current modulation. Interaction between receptor tyrosine kinases (RTK) and G-protein coupled receptors (GPCRs) has recently been recognized as important in the regulation of signaling and the regulation of cellular responses, such as neuronal proliferation and neuronal migration. 32.. Additionally, it has been demonstrated that activation of GPCRs increases the activation/phosphorylation of a TrkA receptors in the presence of NGF, resulting in the. 14.

(16) blockade of apoptosis and enhanced neuronal differentiation and growth 33. It is known that survival signals produced by receptor activation travel retrogradely along to cell bodies. Several studies focused on the retrograde transport of NGF and a TrkA receptor which is also retrogradely transported TrkA are found in endosomes. 36.. 34,35.. In addition, both NGF and phosphorylated. It has also been reported that K252a blocks retrograde. NGF signaling and neuronal survival in sympathetic neurons. 37.. BDNF is also an. important signaling molecule that regulates neuroplasticity in multiple brain regions. 38.. The mechanism of Ang Ⅱ and NGF interaction is not known, but it might involve mitogen activated protein kinases (MAPK). It has been shown that Ang Ⅱ stimulation inhibits NGF-stimulated p42/p44 MAPK phosphorylation 39,40. Previously, we and Wang et al. have demonstrated that Ang Ⅱ facilitates L-type VDCCs via Src tyrosine kinase, p38 MAPK kinase and Nox2-dependent reactive oxygen species (ROS) production mediated by AT 1 receptors 20-22. p38 MAPK has multiple actions in neuronal and other tissues. Its initial characterization was as a signaling pathway activated by pathological conditions such as osmotic stress or inflammation mediated by interleukin-1 and tumor necrosis factor. 41.. For this reason, p38 MAPK is an active focus of study in chronic. disease as rheumatoid arthritis and neurogenic pain syndromes 41-43. Physiological roles of p38 MAPK in neurons have begun to follow that of its better characterized MAPK. 15.

(17) family, ERK1/2, which in addition to its initial discovery as a mediator of mitogenic stimulation has also been found to modulate a number of ion channels and neuronal plasticity processes. 44,45.. Additionally, in this study we also observed that NGF did not. attenuate the glutamate-induced facilitation of VDCCs in NTS. We have previously reported that glutamate facilitates L-type VDCCs involving protein kinase C (PKC) including inositol-1,4,5-trisphosphate (IP3) formation. 46.. This data supports the notion. that NGF and BDNF modulate Ang Ⅱ-induced facilitation of VDCCs via MAPK and/or tyrosine kinase pathways, but not protein kinase pathway. In this study, treatment with K-252a, TrkA receptor inhibitor, antagonized NGF-induced attenuation, but not BDNF-, of the Ang Ⅱ-induced facilitation of VDCCs. In fact, NGF preferentially activates a TrkA receptors and BDNF is selective for a TrkB receptors. 47,48.. In the basal forebrain, neurotransmitter synthesis as well as neuronal. survival after injury are regulated by NGF survival. 51.. 49,50,. while BDNF influences neuronal. BDNF activate intracellular signaling pathways, which include PLC-γ,. ERK1/2 and phosphatidylinositol-3 (PI3)-kinase signaling pathway. 52.. Interestingly, it. has been reported that TrkA receptor induces neuroprotection involving PI3-kinase/Akt pathways, whereas MAPK and Erk1/2 are not involved 53. Several studies have demonstrated that neurotrophins can alter the expression of. 16.

(18) specific ion channel proteins associated with action potential characteristics but relatively few. studies have. reported immediate effects of. neurotrophins on. electrophysiological properties. For example, NGF prolongs the action potential duration of sensory neurons from mature mice neonatal CNS neurons. 55.. 54. and BDNF rapidly depolarizes. Levine et al. demonstrated that NGF facilitates ICa in. cultured basal forebrain neurons. In contrast, BDNF did not modulate ICa in this neuron 56.. As shown in the present study, both NGF and BDNF facilitate ICa in NTS.. During differentiation, L-type VDCCs-mediated Ca2+ influx is essential for neurite outgrowth, synapse formation, neuronal survival, and the shift to the mature action potential profile. 57.. In addition, L-type VDCCs also have been implicated in synaptic. plasticity 58 and regulation of gene expression 59. In conclusion, the findings present new evidence suggesting that NGF and BDNF can not only promote VDCCs modulation, but also can further long-lasting neuronal changes including cell growth, neuronal differentiation, neuronal survival and long-term maintenance. It can be considered that NGF- and BDNF-induced attenuation of Ang Ⅱ-induced facilitation of VDCCs is important role in these events in NTS. NTS are divided into subtypes: rostal, medial and caudal NTS. Comparison between rostal and caudal NTS neurons indicate that they have comparable intrinsic properties,. 17.

(19) which suggests some similarities in the processing of sensory information. However, caudal NTS are believed to belong to the respiratory or swallowing central pattern generators, whereas the rostal NTS are part of the taste and somatosensory pathways. However, it is not possible to identify distinct NTS subtypes in this study. Therefore the NTS subtypes mechanisms discrepancy must be investigated in a further study.. Acknowledgements. This research was supported by Oral Health Science Center Grant HRC7 from the Tokyo Dental College by a “High-Tech Research Center” Project for Private Universities and matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) of Japan and 2007-2010 (No.19592414). The authors would like to thank Mr. Bryan Loudon for his assistance with the writing of the English manuscript.. 18.

(20) REFERENCES. 1. Levi-Montalcini R, Angeletti PU. Nerve growth factor. Physiol Rev 1968;48:534-569.. 2. Thoenen H, Bandtlow C, Heumann R. The physiological function of nerve growth factor in the central nervous system: comparison with the periphery. Rev Physiol Biochem Pharmacol 1987;109:145-178.. 3. Davies AM. The neurotrophic hypothesis: Where does it stand? Philos Trans R Soc Lond B Biol Sci 1996;351:389-394.. 4. Stoll M, Stechelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest 1995;95:651-657.. 5. Tsuzuki S, Eguchi S, Inagami T. Inhibition of cell proliferation and activation of protein tyrosine tyrosine phosphatase mediated by angiotensin Ⅱ type 2 (AT2) receptor in R3T3 cells. Biochem Biophys Res Commun 1996;228:825-830.. 19.

(21) 6. Laflamme L, Gasparo M, Gallo JM, Payet MD, Gallo-Payet N. Angiotensin Ⅱ induction of neurite outgrowth by AT2 receptors in NG108-15 cells. Effect counteracted by the AT1 receptors. J Biol Chem 1996;271:22729-22735.. 7. Meffert S, Stoll M, Steckelings UM, Bottari SP, Unger T. The angiotensin Ⅱ AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells. Mol Cell Endocrinol 1996;122:59-67.. 8. Tsien RW, Lipscombe D, Madison DV, Bley KR, Fox AP. Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 1988;11:431-438.. 9. Sheng M, Greenberg ME. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 1990;4:477-485.. 10. Spitzer NC, Gu X, Olson E. Action potentials, calcium transients and the control of differentiation of excitable cells. Curr Opin Neurobiol 1994;4:70-77.. 20.

(22) 11. Bito H, Deisseroth K, Tsien RW. Ca2+-dependent regulation in neuronal gene expression. Curr Opin Neurobiol 1997;7:419-429.. 12. Nicotera P, Orrenius S. The role of calcium in apoptosis. Cell Calcium 1998;23:173-180.. 13. Marshall J, Dolan BM, Garcia EP, Sathe S, Tang X, Mao Z, Blair LA. Calcium channel and NMDA receptor activities differentially regulate nuclear C/EBPβ levels to control neuronal survival. Neuron 2003;39:625-639.. 14. Weick JP, Groth RD, Isaksen AL, Mermelstein PG. Interaction with PDZ proteins are required for L-type calcium channels to activate cAMP response element-binding protein-dependent gene expression. J Neurosci 2003;23:3446-3456.. 15. Grassi C, D’Ascenzo M, Torsello A, Martinotti G, Wolf F, Cittadini A, Azzena GB. Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation. of. neuroendocrine. cell. proliferation. 2004;35:307-315.. 21. and. death.. Cell. Calcium.

(23) 16. Toescu EC, Verkhratsky A, Landfield PW. Ca2+ regulation and gene expression in normal brain aging. Trends Neurosci 2004;27:614-620.. 17. Jean A. Brain stem control of swallowing: neuronal network and cellular mechanisms. Physiol Rev 2001;81:929-969.. 18. Lawrence A, Jarrott B. Neurochemical modulation of cardiovascular control in the nucleus tractus solitarius. Prog Neurobiol 1996;48:21-53.. 19. Paton JFR. The Sharpey-Schafer prize lecture. Nucleus tractus solitarii: integrating structures. Exp Physiol 1999;84:815-833.. 20. Wang G, Anrather J, Huang J, Speth RC, Pickel VM, Iadecola C. NADPH oxidase contributes to angiotensinⅡ signaling in the nucleus tractus solitarius. J. Neurosci. 2004;24:5516-5524.. 21. Wang G, Anrather J, Glass MJ, Tarsitano J, Zhou P, Frys KA, Pickel VM, Iadecola C.. 22.

(24) Nox2, Ca 2+, and protein kinase C play a role in angiotensin Ⅱ-induced free radical production in nucleus tractus solitarius. Hypertension 2006;48:482-489.. 22. Endoh T. Involvement of src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin Ⅱ. J Physiol 2005;568:851-865.. 23. Rhim H, Miller R. Opioid receptors modulate diverse types of calcium channels in the nucleus tractus solitarius of the rat. J Neurosci 1994;14:7608-7615.. 24. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfugers Arch 1981;391:85-100.. 25. Endoh T. Modulation of voltage-dependent calcium channels by neurotransmitters and neuropeptides in parasympathetic submandibular ganglion neurons. Arch Oral Biol 2004;49:539-557.. 23.

(25) 26. Casaccia-Bonnefil P, Gu C, Khursigara G, Chao MV. p75 neurotrophin receptor as a modulator of survival and death decisions. Microsc Res Tech 1999;45:217-224.. 27.. Barbacid. M.. The. Trk. family. of. neurotrophin. receptors.. J. Neurobiol. 1994;25:1386-1403.. 28. Berg MM, Sternberg DW, Parada LF, Chao MV. K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J Biol Chem 1992;267:13-16.. 29. Endoh T. Pharmacological characterization of inhibitory effects of postsynaptic opioid- and cannabinoid-receptors on calcium currents in neonatal rat nucleus tractus solitarius. Br J Pharmacol 2006;147:391-401.. 30. Rogers M, Hendry I. Involvement of dihydropyridine-sensitive calcium channels in nerve growth factor-dependent neurite outgrowth by sympathetic neurons. J Neurosci Res 1990;26:447-454.. 24.

(26) 31. Cheng B, McMahon DG, Mattson MP. Modulation of calcium current, intracellular calcium levels and cell survival by glucose deprivation and growth factors in hippocampal neurons. Brain Res 1993;607:275-285.. 32. Piiper A, Zeuzem S. Receptor tyrosine kinases are signaling intermediates of G protein-coupled receptors. Curr Pharm Des 2004;10:3539-3545.. 33. Arthur DB, Akassoglou K, Insel PA. P2Y2 and TrkA receptors interact with Src family. kinase. for. neuronal. differentiation.. Biochem. Biophys. Res. Commun. 2006;347:678-682.. 34. Ehlers MD, Kaplan DR, Price DL, Koliatsos VE. NGF-stimulated retrograde transport of trkA in the mammalian nervous system. J Cell Biol 1995;130:149-156.. 35.. Tsui-Pierchala. BA,. Ginty. DD.. Characterization. of. an. NGF-P-TrkA. retrograde-signaling complex and age-dependent regulation of TrkA phosphorylation in sympathetic neurons. J Neurosci 1999;19:8207-8218.. 25.

(27) 36. Delcroix JD, Valletta JS, Wu C, Hunt SJ, Kowal AS, Mobley WC. NGF signaling in sensory neurons: evidence that endosomes carry NGF retrograde signals. Neuron 2003;39:69-84.. 37. Ye H, Kuruvilla R, Zweifel LS, Ginty DD. Evidence in support of signaling endosome-based retrograde survival of sympathetic neurons. Neuron 2003;39:57-68.. 38. Gomez-Pinilla F, Ying Z, Roy RR, Molteni R, Edgerton VR. Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity. J Neurophysiol 2002;88:2187-2195.. 39. Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, Horiuchi M, Pratt RE, Dzau VJ. The angiotensin Ⅱ type 2 (AT 2) receptor antagonizes the growth effects of the AT 1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci USA 1995;92:10663-10667.. 40. Yamada T, Horiuchi M, Dzau V.. Angiotensin Ⅱ type 2 receptor mediates. programmed cell death. Proc Natl Acad Sci USA 1996;93:156-160.. 26.

(28) 41. Kumar S, Boehm J, Lee JC. p38 MAP kinases: key signaling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2003;2:717-726.. 42. Ji RR. Peripheral and central mechanisms of inflammatory pain, with emphasis on MAP kinases. Curr Drug Targets Inflamm Allergy 2004;3:299-303.. 43. Saklatvala J. The p38 MAP kinases pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol 2004;4:372-377.. 44. Yuan LL, Adams JP, Swank M, Sweatt JD, Johnston D. Protein kinase modulation of dendritic K+ channels in hippocampus involves a mitogen-activated protein kinase pathway. J Neurosci 2006;22:4860-4868.. 45. Sweatt JD. Mitogen-activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 2004;14:311-317.. 46. Endoh T. Characterization of modulatory effects of postsynaptic metabotropic. 27.

(29) glutamate receptors on calcium currents in rat nucleus tractus solitarius. Brain Res 2004;1024:212-224.. 47. Chao MV. Neurotrophins and their receptors: a convergence point for many signaling pathways. Nat Rev Neurosci 2003;4:299-309.. 48. Kalb R. The protean of neurotrophins and their receptors on the life and death of neurons. Trends Neurosci 2005;28:5-11.. 49. Hefti F, Hartikka J, Eckenstein F, Gnahn H, Heumann R, Schwab M. Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons. Neuroscience 1985;14:55-68.. 50. Hefti F. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transactions. J Neurosci 1986;6:2155-2162.. 51. Friedman WJ, Ibanez CF, Hallbook F, Persson H, Cain LD, Dreyfus CF, Black IB. Differential actions of neurotrophins in the locus coeruleus and basal forebrain. Exp. 28.

(30) Neurol 1993;119:72-78.. 52. Impey S, Obrietan K, Storm DR. Making new connections: role of ERK/MAP kinase signaling in neuronal plasticity. Neuron 1999;23:11-14.. 53. Culmsee C, Gerling N, Lehmann M, Nikolova-karakashian M, Prehn JHM, Mattson MP, Krieglstein J. Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TrkA and requires the common neurotrophin receptor p75. Neuroscience 2002;115:1089-1108.. 54. Shen KF, Crain SM. Nerve growth factor rapidly prolongs the action potential of mature sensory ganglion neurons in culture, and this effect requires activation of Gs-coupled. excitatory. kappa-opioid. receptors. on. these. cells.. J. Neurosci. 1994;14:5570-5579.. 55. Kafitz KW, Rose CR, Thoenen H, Konnerth A. Neurotrophin-evoked rapid excitation through Trk-B receptors. Nature 1999;401:918-921.. 29.

(31) 56. Levine ES, Dreyfus CF, Black IB, Plummer MR. Diffrential effects of NGF and BDNF on voltage-gated calcium currents in embryonic basal forebrain neurons. J Neurosci 1995;15:3084-3091.. 57. Spitzer NC. Spontaneous Ca2+ spikes and waves in embryonic neurons: signaling systems for differentiation. Trend Neurosci 1994;17:115-118.. 58. Kullman DM, Perkel DJ, Manabe T, Nicoll RA. Ca2+ entry via postsynaptic voltage-sensitive Ca2+ channels can transiently potentiate excitatory synaptic transmission in the hippocampus. Neuron 1992;9:1175-1183.. 59. Bading H, Ginty DD, Greenberg ME. Regulation of gene expression in hipocampal neurons by distinct calcium signaling pathways. Science 1993;260:181-186.. 30.

(32) Fig. 1. NGF attenuated Ang Ⅱ-induced facilitation of IBa. (A) Typical superimposed IBa traces at the times indicated in the time course graph B. IBa were evoked from a holding potential of −80 mV by a 100 msec voltage step to 0 mV at 20 sec intervals. (B) Typical time course of Ang Ⅱ-induced facilitation of IBa current density (pA/pF). The peak IBa was divided by cell capacitance to obtain the current density. Ang Ⅱ (1 μM) was bath-applied during the time indicated by the filled bar. (C) Typical superimposed IBa traces at the times indicated in the time course graph D. (D) Typical time course of NGF-induced attenuation of Ang Ⅱ-induced facilitation of IBa current density. NGF (38 μM) and Ang Ⅱ (1 μM) were bath-applied during the time indicated by the open and filled bar, respectively.. 31.

(33) Fig. 2. NGF did not attenuate glutamate-induced facilitation of IBa. (A) Typical superimposed IBa traces at the times indicated in the time course graph B. (B) Typical time course of DHPG-induced facilitation of IBa current density. DHPG (100 μM) was bath-applied during the time indicated by the filled bar. (C) Typical superimposed IBa traces at the times indicated in the time course graph D. (D) Typical time course of the effect of NGF on DHPG-induced facilitation of IBa current density. NGF (38 μM) and DHPG (100 μM) were bath-applied during the time indicated by the open and filled bar, respectively.. 32.

(34) Fig. 3. BDNF attenuated Ang Ⅱ-induced facilitation of IBa. (A) Typical superimposed IBa traces at the times indicated in the time course graph B. (B) Typical time course of the effect of BDNF on Ang Ⅱ-induced facilitation of IBa current density. BDNF (35 μ M) and Ang Ⅱ (1 μM) were bath-applied during the time indicated by the open and filled bar, respectively. (C) Typical superimposed IBa traces at the times indicated in the time course graph D. (D) Typical time course of the effect of BDNF on DHPG-induced facilitation of IBa current density. BDNF (35 μ M) and DHPG (100 μ M) were bath-applied during the time indicated by the open and filled bar, respectively.. 33.

(35) Fig. 4. K-252a blocked NGF-induced attenuation of the Ang Ⅱ-induced facilitation of IBa. (A) Typical superimposed IBa traces at the times indicated in the time course graph B. (B) Typical time course of the effect of K-252a and NGF on Ang Ⅱ-induced facilitation of IBa current density. K-252a (10 μM), NGF (38 μM) and Ang Ⅱ (1 μM) were bath-applied during the time indicated by the stripe, open and filled bar, respectively. (C) Histogram demonstrating the degree of IBa current density, control (untreated neurons), after Ang Ⅱ (1 μM), after NGF (38 μM) ＋ Ang Ⅱ, after BDNF (35 μM) ＋ Ang Ⅱ, after K-252a (10 μM) ＋ NGF ＋ Ang Ⅱ, and after K-252a ＋ BDNF ＋ Ang Ⅱ.Numbers in parentheses indicate the number of neurons tested. *＜0.05 compared with control, ANOVA.. 34.

(36) Fig. 5. NGF and BDNF facilitated IBa density. (A) IBa current density-voltage relations evoked by a series of voltage steps from a holding potential of −80 mV to test potentials between −80 and ＋40 mV in ＋10 mV increments in the absence (opened points) and presence (filled points) of NGF (38 μM). (B) IBa current density-voltage relations evoked by a series of voltage steps from a holding potential of −80 mV to test potentials between −80 and ＋40 mV in ＋10 mV increments in the absence (opened points) and presence (filled points) of BDNF (35 μM). Values of IBa are the averages of five neurons.. 35.

(37) Fig. 6. NGF and BDNF facilitated L-type IBa density. Histogram demonstrating the degree of IBa current density by NGF (38 μM) and BDNF (35 μM) on L ＋ R-types (after treatments with ω-CgTx GⅥA ＋ ω-Aga ⅣA), N ＋ R-types (after treatments with Nif ＋ ω-Aga ⅣA) and P/Q ＋ R-types (after treatments with Nif ＋ω-CgTx G ⅥA) VDCCs. Numbers in parentheses indicate the number of neurons tested. *＜0.05 compared with L ＋ R-types, ANOVA.. 36.

(38) Table 1. Comparison of IBa density in NTS. Values are mean ± SEM.. 37.

(39) Fig.1. A. B. 0 mV -80 mV. 1 2. 500 pA 100 ms. C. D. 1. 3. 2. 4. 500 pA 100 ms. 1.

(40) Fig.2. A. B. 1 2. 500 pA. 100 ms. C. D. 1 2 3 4. 200 pA 100 ms. 2.

(41) Fig.3. A. B. 1 2. 3 4. 500 pA 100 ms. D. C. 1. 3. 2 4. 500 pA 100 ms. 3.

(42) Fig.4. A. B. 1 2. 500 pA 100 ms. C * (12) (18). * (7) (7) (8). (9). 4.

(43) Fig.5. A. B. 5.

(44) Fig.6. L+R (6) (5). * *. N+R. (11). P/Q + R (8) (4) (4). (4) (4) (8). 6.

(45) Table.1 IBa d e n s i t y ( p A / p F ). n. Control. 55.2 ± 4.0. 18. AngⅡ. 73.1 ± 5.8. 12. DHPG. 78.9 ± 5.3. 7. No Response. 3. NGF ＋ AngⅡ. 58.4 ± 4.4. 7. NGF ＋ D H P G. 78.4 ± 4.8. 7. BDNF ＋ AngⅡ. 5 6 . 4 ± 4.3. 8. BDNF ＋ D H P G. 7 6 . 9 ± 4.0. 7. K-252a ＋ NGF ＋ AngⅡ K-252a ＋ BDNF ＋ AngⅡ. 74.4 ± 6 . 0. 7. 60.2 ± 5 . 8. 9. To t a l. 85.

(46)