Src family kinase potentiates the activity of nicotinic acetylcholine receptor in rat autonomic ganglion innervating urinary bladder

Neuroscience Letters 494 (2011) 190–195

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Src family kinase potentiates the activity of nicotinic acetylcholine receptor in rat autonomic ganglion innervating urinary bladder Na-Hyun Kim a , Kyu-Sang Park b , Seung-Kuy Cha b , Joon-Ho Yoon c , Byung-Il Yeh c , Kyou-Hoon Han d,e , In Deok Kong b,∗ a

Department of Basic Nursing Science, Keimyung University College of Nursing, Daegu, Republic of Korea Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea Department of Biochemistry, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea d Division of Biosystems Research, Korea Research Institute of Bioscience and Biotechnology, Republic of Korea e Department of Bioinformatics, University of Science and Technology, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 19 November 2010 Received in revised form 19 February 2011 Accepted 3 March 2011 Keywords: Major pelvic ganglia Nicotinic acetylcholine receptor Src family kinase

a b s t r a c t Src family kinases (SFKs), one of the tyrosine kinase groups, are primary regulators of signal transductions that control cellular functions such as cell proliferation, differentiation, survival, metabolism, and other important roles of the cell. One of the crucial functions of SFKs is to regulate the activities of various neuronal channels. In this study, we investigated the modulatory action of SFK on nicotinic acetylcholine receptors (nAChRs) expressed in rat major pelvic ganglion (MPG) neurons innervating the urinary bladder. PP1 and PP2 (5 ␮M), selective Src-kinase inhibitors, attenuated ACh-induced ionic currents and [Ca2+ ]i transients in MPG neurons, whereas PP3, an inactive analogue, had no effect. Blocking the tyrosine kinase activity of Src kinase by pp60 c-src inhibitory peptide also reduced the ACh-induced currents. Conversely, sodium orthovanadate (200 ␮M), a tyrosine phosphatase inhibitor, significantly augmented the AChinduced currents. In the kinase assay, the activities of SFKs in MPG neurons were also inhibited by PP2, but not by PP3. These data suggests that SFKs may have a facilitative role on the synaptic transmission in rat pelvic autonomic ganglion. © 2011 Elsevier Ireland Ltd. All rights reserved.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligandgated channels found in the central and peripheral nervous system [27]. Molecular cloning has identified nine ␣ (2–10) and three ␤ (2–4) subunits in neuronal nAChRs, assembled in various combinations to form functional receptors [20]. The homomeric ␣7 and the heteromeric ␣4␤2 nAChRs are abundant in the mammalian brain [27], whereas ␣3␤4 nAChRs are commonly found in autonomic neurons [2,17,25]. It is known that at least three protein kinases can phosphorylate nAChR; protein kinase A phosphorylates the ␥ and ␦ subunits, protein kinase C phosphorylates the ␦ subunit, and a protein tyrosine kinase (PTK) phosphorylates the ␤, ␥, and ␦ subunits of nAChRs [22]. Especially, tyrosine kinases provide the means to regulate most nAChRs, although the functional consequences are specific for each receptor subtype and location [27]. Protein kinases control various biological processes such as cell growth, differentiation, proliferation, survival and apoptosis

Abbreviations: SFKs, Src kinases; PTK, protein tyrosine kinase; ACh, acetylcholine; MPG, major pelvic ganglion; nAChR, nicotinic acetylcholine receptor; [Ca2+ ]i , intracellular calcium ion concentration. ∗ Corresponding author at: Department of Physiology, Institute of Lifelong Health, Yonsei University Wonju College of Medicine, 162 Ilsan-Dong, Wonju, Gangwon-Do 220-701, Republic of Korea. Tel.: +82 33 741 0292; fax: +82 33 745 6461. E-mail address: [email protected] (I.D. Kong). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.009

[5,8,22]. There are approximately 500 protein kinase genes, including 90 tyrosine kinase genes, which implies that about 2% of all human genes encode protein kinases, and about 20% of these protein kinases are tyrosine kinases [10,15]. PTKs can be divided into 58 receptor tyrosine kinases and 32 non-receptor tyrosine kinases [8]; both have been reported as the primary modulators of intracellular signal transduction [24]. In mammals, Src family kinases (SFKs), a member of non-receptor tyrosine kinases, consist of 8 members; Src, Fyn, and Yes are ubiquitously expressed, whereas other members such as Lck, Hck, Fgr, Lyn, and Blk show a more tissue-restricted expression [1,14]. The SFKs play key roles in regulating signal transduction pathways triggered by a diverse set of cell surface receptors, including receptor tyrosine kinases, integrins, G-protein coupled receptors, and antigen receptor [3,19]. Phosphorylation and dephosphorylation of target protein is the key mechanisms employed by SFKs to regulate the activity of integral membrane proteins [5]. Previous studies have shown that SFKs are involved in regulating the activities of neuronal channels, including nAChR [5,22,25], potassium channels [9], NMDA receptors [26,30], and GABA receptors [4]. Heteromeric nAChRs in adrenal chromaffin cells can be activated by SFK-induced phosphorylation, resulting in catecholamine secretion [25]. Conversely, the activity of homomeric ␣7 nAChR is suppressed in response to phosphorylation of the channel by SFKs.

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We previously reported that an ␣3␤4 heteromeric combination act as a major functional nAChR in male rat major pelvic ganglion (MPG) neurons, and its activation induced fast depolarization followed by sustained hyperpolarization [17,18]. In addition, we also found that tyrosine kinases are associated with the functional regulation of nAChR in MPG neurons [13] which play an important physiological role in a variety of autonomic reflexes including micturition [20]. The current study was undertaken to identify the potential role of SFKs in regulating ␣3␤4 nAChR in rat MPG neurons innervating the urinary bladder by using patch clamp, fluorescence measurement, and kinase assay. Our data suggests that tyrosine kinases, in particular Src family kinases, positively regulate the activities of ␣3␤4 nAChRs in MPG neurons thus controlling urination. Sprague–Dawley rats (250 g) were anesthetized with pentobarbital sodium (50 mg/kg i.p.) and then injected with DiI solution (Molecular Probes, Invitrogen, USA) into the urinary bladder after a small incision of abdominal wall. One week later, the rats were anesthetized again and the MPG neurons were isolated, enzymatically dissociated, and cultured as previously described [16,18]. The internal solution used to fill the patch electrode contained (in mM): 20 KCl, 115 K-aspartate, 10 HEPES, 10 EGTA, 2.5 trisphosphocreatine, 5 MgATP, and 0.1 Na2 -GTP (pH 7.2). The external solution contained (in mM): 135 NaCl, 5.0 KCl, 1.8 CaCl2 , 1 MgCl2 , 10 HEPES, and 10 glucose (pH 7.4). The drugs were applied to a single neuron as previously described [18]. The drugs used in the experiments were obtained as follows: fura-2/AM and fluo-3/AM from Invitrogen (Eugene, OR, USA), acetylcholine, sodium orthovanadate, genistein, and genistin from Sigma (St. Louis, MO, USA), PP2 and PP3 from Calbiochem (San Diego, CA, USA), PP1 from Biomol International (Plymouth Meeting, PA, USA) and pp60 c-src peptide from Tocris Bioscience (Ellisville, MO, USA). ␣-Conotoxin AuIB was prepared as previously described [7]. The ionic currents of the MPG neurons were recorded by using the dialyzed whole-cell patch clamp technique. Patch electrodes were fabricated as described previously [17]. The cell membrane capacitance and series resistance were electronically compensated (>80%) by using the patch clamp amplifier (EPC-9, Instrutech, NY, USA). Voltage protocol generation and data acquisition were performed using the Pulse/Pulsefit (v8.50) software (Heka Elektronik, Lambrecht, Germany). Changes in [Ca2+ ]i were assessed by the fluorescence measurement system (Ratiomaster, Photon Technology International, Lawrenceville, NJ, USA). The MPG neurons plated on glass coverslips were loaded with a fluorescent Ca2+ indicator dye, fura-2/AM (5 ␮M) and transferred to a perfusion chamber on a fluorescence microscope (IX-70, Olympus, Tokyo, Japan). The fluorescence signals in the neurons alternately excited at 340 and 380 nm were monitored at an emission wavelength of 510 nm (5 Hz) with dataanalysis software (Felix, Photon Technology International). Tyrosine kinase activity of MPG neurons was measured using Universal tyrosine kinase assay kit (Takara). MPG neurons were harvested and washed with PBS. After centrifugation, pelleted cells were recovered and resuspended in 1 ml extraction buffer by vortexing gently. Cell lysate was centrifuged at 10,000 × g for 10 min at 4 ◦ C, and the resulting supernatant was diluted with the kinase assay reaction buffer. Kinase reaction (or phosphorylation) was performed at 37 ◦ C for 30 min in substrate-coated well plate, containing 40 ␮l of sample with DMSO or PP2 (10 ␮M) or PP3(10 ␮M) with ATP (40 ␮M) as the reaction starter. After kinase reaction (or phosphorylation), the wells were blocked and antiphosphotyrosine-HRP was added to detect the phosphotyrosine. Absorbance was measured at 450 nm with plate reader after substrate (TMBZ) incubation. For quantification of tyrosine kinase activity, the absorbance of PP2 and PP3 reaction was compared with that of control.

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The concentration–response curves, EC50 , and IC50 values were obtained by using Prism (v3.0) software (GraphPad Software, San Diego, CA, USA). Data was presented as means ± SEM. Statistical significance was determined using Student’s t-test or one-way ANOVA followed by a post-hoc test. p < 0.05 was considered significant. The number of neurons innervating the bladder was about 8–10% of the total MPG neurons. MPG neurons were stained with DiI, which was retrogradely transported from the urinary bladder. The DiI stained neurons had a small size with a capacitance of 29.6 ± 17.5 pF of capacitance. T-type Ca2+ currents, a marker of sympathetic neuron, were not evoked in the DiI stained neurons by ramp protocol from −100 to +60 mV in whole cell patch clamp. Data from our previous results demonstrated that 10 ␮M ␣-conotoxin AuIB, a new selective antagonist for ␣3␤4 nAChRs, markedly inhibited the ACh-induced peak current density in rat MPG neurons [7,17]. We further demonstrated that ␣-conotoxin AuIB (0.03–30 ␮M) suppressed ACh-induced currents in a dose-dependent manner in the DiI stained MPG neurons (IC50 = 1.41 ± 0.14 ␮M; n = 8, Fig. 1A and B). Intracellular calcium concentration ([Ca2+ ]i ) was measured in fura-2 loaded MPG neurons after pretreatment with ␣-conotoxin AuIB. In the presence of 10 ␮M ␣-conotoxin AuIB, the [Ca2+ ]i transient after ACh (100 ␮M) application was reduced to less than 20% when compared to that of the control (n = 6, Fig. 1C and D). These inhibitory effects were not significantly different between DiI-stained and unstained MPG neurons (data not shown). Genistein (10 ␮M), a broad-spectrum tyrosine kinase inhibitor, notably decreased ACh-induced inward current in MPG neurons, whereas 10 ␮M genistin, an inactive analogue, had little effect (n = 8, Fig. 2). The inhibitory effect of genistein on ACh-induced inward current was reversed after removal of the drug (Fig. 2B and C). Based on these findings, we examined whether SFKs – members of tyrosine kinase family, are involved in regulating nAChRs in MPG by using specific pharmacological inhibitors. In a voltage-clamp mode, PP1 (5 ␮M) and PP2 (5 ␮M), selective Src kinase inhibitors, markedly attenuated ACh-induced currents in DiI-stained MPG neurons, whereas an inactive analogue, PP3 (5 ␮M) did not show any effects (n = 6, Fig. 3). ACh-induced [Ca2+ ]i transient (0.68 ± 0.12; Fura-2 ratio) was also attenuated by PP2 (0.47 ± 0.04), but not by PP3 (0.64 ± 0.13; n = 5) in a similar manner. ACh-induced currents were blocked by introducing pp60 c-src peptide into the patch pipette, which binds to SH2 domain and inhibits tyrosine kinase activity of SFK (Fig. 3C and D). The action of tyrosine kinase can be opposed by phosphotyrosine phosphatase (PTPase). We added 200 ␮M sodium orthovanadate (Na3 VO4 ), as a PTPase inhibitor, into the patch electrode solution. Peak current amplitudes induced by ACh application was enhanced by ∼60% in the presence of sodium orthovanadate compared to the control which had no PTPase inhibitor (Fig. 4). These results indicate that the activities of ␣3␤4 AChRs are positively regulated by Src family kinase in MPG neurons innervating urinary bladder. To examine the tyrosine kinase activity, we quantified the phosphorylating effect of the enzymes in MPG neurons using PP2, a Src kinase selective inhibitor. As shown in Fig. 5, PP2 reduced the tyrosine phosphorylation (86.0 ± 3.5% of control) whereas PP3 (inactive form of Src kinase inhibitor) had little effect (100.7 ± 7.7%). These results indicate that Src kinase may play a role in the tyrosine kinase activity in MPG neurons. The pelvic ganglia contain functionally different neurons within the same ganglion capsule, for instance neurons innervating the lower bowel, urinary bladder, prostate, and penis [12]. Each of the neuronal groups controls different autonomic reflexes, such as micturition or penile erection. Due to their relative ease of isolation and manipulation, male rat major pelvic ganglia have been used as a good model for studying the neural control of pelvic viscera

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Fig. 1. Effects of a selective blocker for ␣3␤4 nAChR on the amplitude of ACh-induced currents and intracellular Ca2+ concentration ([Ca2+ ]i ) in MPG neurons. (A and B) ␣-Conotoxin AuIB (0.03–30 ␮M), a selective antagonist for ␣3␤4 AChRs, suppressed the peak current amplitude induced by ACh (10 ␮M for 5 s) in a dose-dependent manner (IC50 = 1.41 ± 0.14 ␮M) under voltage clamp mode. (C and D) ␣-Conotoxin AuIB (10 ␮M) elicited complete suppression of ACh-induced [Ca2+ ]i transient. Data are presented as means ± SEM (n = 6–8), and *** p < 0.001.

Fig. 2. The effect of tyrosine kinase inhibitor on the amplitude of ACh-induced peak currents. Pretreatment with genistein (10 ␮M for 3 min), a broad-spectrum tyrosine kinase inhibitor, markedly decreased ACh-induced inward currents (B and C), whereas 10 ␮M genistin, an inactive analogue, had little effect (n = 8; A). Data are presented as means ± SEM (n = 8), and *** p < 0.001.

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Fig. 3. The effect of Src family kinase inhibitors on the amplitude of ACh-induced currents. (A and B) PP1 (5 ␮M) and PP2 (5 ␮M), selective Src kinase inhibitors, notably attenuated ACh-induced currents, whereas PP3 (5 ␮M), an inactive analogue, had little effect (n = 6). (C and D) Dialysis with pp60 c-src inhibitory peptide suppressed ACh-induced currents. Data are presented as means ± SEM, * p < 0.05 and ** p < 0.01.

[12]. The heterogenous population of MPG, however, complicated the interpretation of experimental data from simply dissociated neurons. This is because each population has different electrophysiological properties as well as protein expression profiles [23,28]. Thus, we tried to select the MPG neurons innervating urinary bladder by using retrograde staining with DiI fluorescence dye injected into luminal side of bladder. This helped us to focus on the functional role of kinase modulation in synaptic transmission controlling urinary organ mainly by the detrusor muscle. Among many candidates of tyrosine kinase families, Src family kinases (SFKs) are known to closely regulate the activities of various neuronal channels including nAChRs [11,21,25,29]. The nAChRs

in rat MPG neurons are mainly heteromeric structures composed of ␣3␤4, This is confirmed by the transcript levels [17] as well as blocking effects of a selective antagonist (␣-conotoxin AuIB) on ACh-induced currents and [Ca2+ ]i transients (Fig. 1). We had previously observed that ␣3␤4 nAChRs permit Ca2+ through a Na+ dependent membrane depolarization, as shown by the abolishment of ACh-induced [Ca2+ ]i changes in the absence of extracellular Na+ as well as extracellular Ca2+ [13]. We demonstrated in this study that ACh-induced ionic currents and [Ca2+ ]i transients mediated by ␣3␤4 nAChR in MPG neurons were augmented by tyrosine phosphorylation, implying that SFKs potentiate the synaptic transmission in neurons controlling urinary system. The broad-spectrum

Fig. 4. Sodium orthovanadate (Na3 NO4 ) enhances the amplitude of ACh-induced peak current in MPG neurons. Representative trace (A) and time-dependent changes (B) of ACh-induced currents were measured in the presence or absence of 200 ␮M Na3 NO4 , a broad spectrum PTPase inhibitor. Data are presented as means ± SEM (n = 6), and ** p < 0.01.

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pelvic autonomic ganglia and the functional consequences of these alterations.

Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (R01-2008000-11722-0).

References

Fig. 5. The effect of a specific Src kinase inhibitor PP2 or its inactive analogue PP3 on total tyrosine kinase activity of MPG neurons. The addition of 10 ␮M PP2 to the MPG homogenate reduced the tyrosine phosphorylation, whereas PP3 had little effect. The results expressed as a percentage of the control. The data are mean ± SEM of three independent experiments, ** p < 0.01.

tyrosine kinase inhibitor (genistein) and a selective SFK inhibitor (PP2), successfully decreased the ACh-induced changes. On the other hand, the inactive isoforms genistin and PP3, showed negligible effects on the ACh-induced currents in MPG neurons. It is worth noting that PP2 caused a similar degree of inhibition of ACh-evoked currents as that caused by genistein, implying that other non-SKF tyrosine kinases are less likely to be involved in the functional modulation of nAChRs. The amplitudes of ACh-induced currents were enhanced by a phosphotyrosine phosphatase inhibitor, which augments the phosphorylation level of nAChRs. Overall, these results suggest that the degree of protein tyrosine phosphorylation reflects a dynamic balance between the opposing actions of SFKs and PTPase in regulating the activity of nAChRs and ultimately the excitability of synaptic transmission in autonomic pelvic ganglion. Interestingly, although the same kinase interacts with the various receptors, the functional outcomes could be different depending on the composition of receptor subtypes. Specifically, ␣7 homomeric nAChR in the brain is deactivated by tyrosine phosphorylation, indicating that phosphorylation inhibits channel activity, while dephosphorylation causes a potentiation effect [5,27]. This is contrary to our findings as well as phosphorylation–dephosphorylation effects observed in adrenal chromaffin cells [25]. At present, we do not clearly understand why the regulatory mechanisms are extremely varied among different nAChRs. As described above, tyrosine kinases are known to phosphorylate the ␤ subunit, but not the ␣ subunit of nAChRs, resulting in enhanced channel activity. However, Charpantier et al. reported that the ␣7 subunit can only be phosphorylated by SFK at the surface (not intergral) portion, which inhibits its channel activity [5]. Thus, we guess that different responses depending on the specific isoforms to be phosphorylated could be an explanation for the variable outcomes of SFKs action on the activities of nAChRs. Recently, many studies on SFKs have focused on clinical implications of altered SFK regulation which has been linked to a variety of diseases including several types of human malignancies such as colon, breast, and lung cancer [3]. Src activities are also involved in pathophysiologic progression of neurodegenerative diseases, epilepsy, as well as immune diseases [3,6]. We suggest in this study that SFK can potentiate synaptic transmission in the pelvic ganglia innervating urinary system. Therefore, defective SFK or enhanced phosphatase activity may lead to deterioration of neural control over pelvic organs. Further studies are needed to elucidate the physiological and pathophysiological conditions that alter the activity of SFK or tyrosine phosphatase in

[1] C.L. Abram, S.A. Courtneidge, Src family tyrosine kinases and growth factor signaling, Exp. Cell Res. 254 (2000) 1–13. [2] C.M. Allen, C.M. Ely, M.A. Juaneza, S.J. Parsons, Activation of Fyn tyrosine kinase upon secretagogue stimulation of bovine chromaffin cells, J. Neurosci. Res. 44 (1996) 421–429. [3] D. Benati, C.T. Baldari, SRC family kinases as potential therapeutic targets for malignancies and immunological disorders, Curr. Med. Chem. 15 (2008) 1154–1165. [4] N.J. Brandon, P. Delmas, J. Hill, T.G. Smart, S.J. Moss, Constitutive tyrosine phosphorylation of the GABA(A) receptor gamma 2 subunit in rat brain, Neuropharmacology 41 (2001) 745–752. [5] E. Charpantier, A. Wiesner, K.H. Huh, R. Ogier, J.C. Hoda, G. Allaman, M. Raggenbass, D. Feuerbach, D. Bertrand, C. Fuhrer, Alpha7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases, J. Neurosci. 25 (2005) 9836–9849. [6] J. Chin, J.J. Palop, J. Puolivali, C. Massaro, N. Bien-Ly, H. Gerstein, K. Scearce-Levie, E. Masliah, L. Mucke, Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer’s disease, J. Neurosci. 25 (2005) 9694–9703. [7] J.H. Cho, K.H. Mok, B.M. Olivera, J.M. McIntosh, K.H. Park, K.H. Han, Nuclear magnetic resonance solution conformation of alpha-conotoxin AuIB, an alpha(3)beta(4) subtype-selective neuronal nicotinic acetylcholine receptor antagonist, J. Biol. Chem. 275 (2000) 8680–8685. [8] S.W. Cowan-Jacob, Structural biology of protein tyrosine kinases, Cell. Mol. Life Sci. 63 (2006) 2608–2625. [9] T.C. Holmes, D.A. Fadool, R. Ren, I.B. Levitan, Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain, Science 274 (1996) 2089–2091. [10] T. Hunter, Tyrosine phosphorylation in cell signaling and disease, Keio J. Med. 51 (2002) 61–71. [11] L.V. Kalia, J.R. Gingrich, M.W. Salter, Src in synaptic transmission and plasticity, Oncogene 23 (2004) 8007–8016. [12] J.R. Keast, Unusual autonomic ganglia: connections, chemistry, and plasticity of pelvic ganglia, Int. Rev. Cytol. 193 (1999) 1–69. [13] D.R. Kim, S.W. Ahn, K.S. Park, I.D. Kong, Regulation of nicotinic acetylcholine receptor by tyrosine kinase in autonomic major pelvic ganglion neurons, J. Exp. Biomed. Sci. 41 (2007) 745–752. [14] C.A. Lowell, P. Soriano, Knockouts of Src-family kinases: stiff bones, wimpy T cells, and bad memories, Genes Dev. 10 (1996) 1845–1857. [15] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912–1934. [16] K.S. Park, S.W. Jeong, S.K. Cha, B.S. Lee, I.D. Kong, S.R. Ikeda, J.W. Lee, Modulation of N-type Ca2+ currents by A1-adenosine receptor activation in male rat pelvic ganglion neurons, J. Pharmacol. Exp. Ther. 299 (2001) 501–508. [17] K.S. Park, S.K. Cha, M.J. Kim, D.R. Kim, S.W. Jeong, J.W. Lee, I.D. Kong, An alpha3beta4 subunit combination acts as a major functional nicotinic acetylcholine receptor in male rat pelvic ganglion neurons, Pflugers Arch. 452 (2006) 775–783. [18] K.S. Park, S.K. Cha, M.J. Kim, N.H. Kim, J.W. Lee, S.W. Jeong, I.D. Kong, Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats, Neurosci. Lett. 482 (2010) 167–171. [19] S.J. Parsons, J.T. Parsons, Src family kinases, key regulators of signal transduction, Oncogene 23 (2004) 7906–7909. [20] V.I. Skok, Nicotinic acetylcholine receptors in the neurones of autonomic ganglia, J. Auton. Nerv. Syst. 21 (1987) 91–99. [21] M.M. Sugrue, J.S. Brugge, D.R. Marshak, P. Greengard, E.L. Gustafson, Immunocytochemical localization of the neuron-specific form of the c-src gene product, pp60c-src(+), in rat brain, J. Neurosci. 10 (1990) 2513–2527. [22] S.L. Swope, Z. Qu, R.L. Huganir, Phosphorylation of the nicotinic acetylcholine receptor by protein tyrosine kinases, Ann. N. Y. Acad. Sci. 757 (1995) 197–214. [23] H. Tan, G.M. Mawe, M.A. Vizzard, Electrical properties of neurons in the intact rat major pelvic ganglion, Auton. Neurosci. 134 (2007) 26–37. [24] S.M. Thomas, J.S. Brugge, Cellular functions regulated by Src family kinases, Annu. Rev. Cell Dev. Biol. 13 (1997) 513–609. [25] K. Wang, J.T. Hackett, M.E. Cox, M. Van Hoek, J.M. Lindstrom, S.J. Parsons, Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases, J. Biol. Chem. 279 (2004) 8779–8786. [26] Y.T. Wang, M.W. Salter, Regulation of NMDA receptors by tyrosine kinases and phosphatases, Nature 369 (1994) 233–235.

N.-H. Kim et al. / Neuroscience Letters 494 (2011) 190–195 [27] A. Wiesner, C. Fuhrer, Regulation of nicotinic acetylcholine receptors by tyrosine kinases in the peripheral and central nervous system: same players, different roles, Cell. Mol. Life Sci. 63 (2006) 2818–2828. [28] Y.J. Won, K. Whang, I.D. Kong, K.S. Park, J.W. Lee, S.W. Jeong, Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system, J. Pharmacol. Exp. Ther. 317 (2006) 1064–1071.

195

[29] T. Yagi, Y. Shigetani, N. Okado, T. Tokunaga, Y. Ikawa, S. Aizawa, Regional localization of Fyn in adult brain; studies with mice in which fyn gene was replaced by lacZ, Oncogene 8 (1993) 3343–3351. [30] X.M. Yu, R. Askalan, G.J. Keil II, M.W. Salter, NMDA channel regulation by channel-associated protein tyrosine kinase Src, Science 275 (1997) 674–678.