J Physiol Sci (2017) 67:313–323 DOI 10.1007/s12576-016-0464-1 ORIGINAL PAPER Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells 1,2 1 2 1 Yuki Arai • Hirotaka Ishii • Makito Kobayashi • Hitoshi Ozawa Received: 10 May 2016 / Accepted: 9 June 2016 / Published online: 24 June 2016 Ó The Physiological Society of Japan and Springer Japan 2016 Abstract GnRH neurons form a final common pathway DMEM Dulbecco’s modified Eagle’s medium for the central regulation of reproduction. Although the EIA Enzyme immunoassay involvement of acetylcholine in GnRH secretion has been GnRH Gonadotropin-releasing hormone reported, direct effects of acetylcholine and expression IBMX Isobutylmethylxanthine profiles of acetylcholine receptors (AChRs) still remain to mAChR Muscarinic acetylcholine receptor be studied. Using immortalized GnRH neurons (GT1-7 MLA Methyllycaconitine cells), we analyzed molecular expression and functionality nAChR Nicotinic acetylcholine receptor of AChRs. Expression of the mRNAs were identified in the order a7 [ b2 = b1 ] a4 ] a5 = b4 = d [ a3 for nicotinic acetylcholine receptor (nAChR) subunits and Introduction m4 [ m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that a7 nAChRs Acetylcholine is the first neurotransmitter identified in the contributed to Ca2? influx and GnRH release and that m2 nervous systems and plays pivotal roles in a wide variety of and m4 mAChRs inhibited forskolin-induced cAMP pro- physiological processes such as muscle contraction, trans- duction and isobutylmethylxanthine-induced GnRH secre- mission of autonomic signaling, and learning and memory. tion. These findings demonstrate the molecular profiles of There are two categories of receptors that bind acetyl- AChRs, which directly contribute to GnRH secretion in choline and transmit its signaling: nicotinic acetylcholine GT1-7 cells, and provide one possible regulatory action of receptors (nAChRs) and muscarinic acetylcholine receptors acetylcholine in GnRH neurons. (mAChRs) named after the effective agonists, nicotine and muscarine, respectively [1, 2]. Keywords Acetylcholine Á Acetylcholine receptors Á NAChRs belong to the cis-loop family of ligand-gated Gonadotropin-releasing hormone Á GnRH neurons Á GT1-7 ion channels that include ionotropic GABA, glycine, and 5-HT3 receptors. Sixteen subunits of nAChR (a1–7, 9, 10, Abbreviations b1–4, c, e, and d) have been identified in the mammals and BSA Bovine serum albumin assemble into a variety of receptor subtypes each con- 2? [Ca ]i Intracellular calcium concentration taining five homologous subunits [3, 4]. nAChRs in the nervous system are formed from ab combination of a2–a6 and b2–b4 subunits. In addition, a7 and a9 subunits are & Hirotaka Ishii capable of forming homomeric nAChRs, and a10 forms a [email protected] heteromer with a9. These ion channels are permeable to Na? and K?, and in some neuronal types to Ca2?.Some 1 Department of Anatomy and Neurobiology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan subunit compositions such as a7 homomeric receptors are highly permeable to Ca2? [5]. mAChRs are G-protein 2 Department of Life Science, International Christian University, 3-10-2, Osawa, Mitaka-shi, Tokyo 181-8585, coupled receptors, and are classified as metabotropic Japan receptors. There are five muscarinic acetylcholine receptor 123 314 J Physiol Sci (2017) 67:313–323 subtypes, designated m1-5. They are further divided into mAChR subtypes and their functional characteristics in two broader families based on coupling to different GT1-7 cells. We comprehensively analyzed the expression G-proteins. m1, m3, and m5 receptors are coupled to Gq/11- of nAChR subunit and mAChR subtype mRNAs in GT1-7 proteins and activate phospholipase C, while m2 and m4 cells using RT-PCR. Expression levels were quantified receptors are coupled to Gi/o-proteins and inhibit adenylyl using real-time PCR. The functional characteristics of cyclases [6]. nAChRs were evaluated from Ca2? influx using Ca2? Gonadotropin-releasing hormone (GnRH) neurons rep- imaging, while mAChRs were assessed by measurement of resent the final common pathway for the central regulation cAMP formation using enzyme immunoassays (EIAs). In of reproduction. A prior study using the bovine median addition, the effects of both nAChR and mAChR agonists eminence implicated the involvement of acetylcholine in on GnRH secretion were assessed using GnRH assay. GnRH release [7]. However, relatively few studies have reported a physiological role of acetylcholine in function of GnRH neurons [8–11]. Richardson et al. [12] documented Materials stimulation of GnRH release by acetylcholine from hypothalamic organ cultures, which was blocked by a Animals nAChR antagonist, hexamethonium and not by a mAChR antagonist, atropine, suggesting the involvement of Adult male and pregnant female C57BL/6 J mice were nAChRs in GnRH release. A recent immunohistochemical purchased from CLEA Japan (Tokyo, Japan). The mice study of Turi et al. [13] demonstrated the existence of were maintained for 1–2 weeks under a 12-h light/dark cholinergic afferents to GnRH neurons of the preoptic area illumination with free access to food and water. Ten- in the rat. DNA microarray analysis of Todman et al. [14] week-old male and neonatal mice were used. The animals documented the presence of nicotinic b1, b2, and c sub- were deeply anesthetized and killed by decapitation. units and muscarinic m1 subtype in mouse GnRH neurons. Adult and neonatal mouse skeletal muscles and adult However, direct effects of acetylcholine on GnRH neurons mouse brains were removed. The brain regions containing and detailed expression profiles of acetylcholine receptors the preoptic area and hypothalamus were dissected. The in the neurons still remain to be analyzed. skeletal muscles and brain sections were frozen in liquid GnRH neurons are relatively a few in numbers, and are nitrogen until use. Experiments using the animals were scattered in regions including the diagonal band of Broca, conducted in adherence to the Guidelines for the Care and medial septum, medial preoptic area and suprachiasmatic Use of Laboratory Animals of Nippon Medical School. nucleus, anterior and lateral hypothalami making complex The experimental procedures were approved by the neural network with other neurons [15]. These in vivo Committee for Animal Experimentation of Nippon Med- characteristics greatly hinder the endocrinological and ical School. morphological investigation that directly target GnRH neurons. An immortalized cell line, GT1-7, derived from Cell culture mouse hypothalamus synthesizes and releases GnRH [16]. Furthermore, owing to plentiful cell numbers and the GT1-7 cells [16] (kindly provided by Dr. Richard Weiner) ability to manipulate the cells in vitro, this cell line pro- were grown in Dulbecco’s modified Eagle’s medium vides a suitable model for GnRH neurons. GT1-7 cells also (DMEM), containing 10 % fetal bovine serum (Equitech- express functional neurotransmitter receptors, which per- Bio, Kerrville, TX, USA), 4.5 g/l D-glucose, 586 mg/l L- mits their molecular and pharmacological properties to be glutamine, 2.0 g/l NaHCO3 and 110 mg/l sodium pyruvate. studied. A pioneering study by Krsmanovic et al. [17] Cells were cultured at 37 °C under 5 % CO2,95% pharmacologically demonstrated the presence of nAChRs atmosphere. The culture medium was changed every and mAChRs coupled with both Gq/11- and Gi/o-proteins in 3–4 days, and the cells were passaged every 1 week, and GT1-7 cells, and characterized modulatory effects of used in experiments within ten passages. acetylcholine on GnRH release from GT1-7 cells. Deter- mination of subunit expression and the detailed composi- RT-PCR tion of acetylcholine receptors in GnRH neurons are required to further the understanding of the modulatory Total RNA was extracted from GT1-7 cells, the skeletal effects of acetylcholine on GnRH secretion; however, such muscles, and the brain sections using RNAiso plus (Takara studies have been limited. Bio, Tokyo, Japan) following the manufacturer’s instruc- Therefore, as a preliminary study to investigate acetyl- tions. Total RNA was treated with Turbo DNase (Thermo choline receptors in GnRH neurons, this paper aimed to Fisher Scientific, Waltham, MA, USA) and re-purified. determine the expression profiles of nAChR subunits and RNA concentration was quantified by absorption at 123 J Physiol Sci (2017) 67:313–323 315 260 nm. Total RNA was reverse-transcribed with oligo-dT Ca21 imaging primers to produce first-strand cDNA. The reaction mix- tures (total volume, 20 ll) contained 5 lg of total RNA, GT1-7 cells were seeded on poly-D-lysine-coated glass- 5 1 9 RT buffer, 1 mM dNTP mixture, 500 ng oligo-(dT)15, bottom dishes at a concentration of 2 9 10 cells/dish and 20 U RNA inhibitor (Promega, Madison, WI, USA) and cultured for 48 h. The cells were washed with DMEM and 100 U ReverTra Ace (Toyobo, Osaka, Japan). The reaction loaded for 45 min with 3 lM Fluo-4-AM (Dojindo, was carried out at 42 °C for 60 min, and stopped by Kumamoto, Japan) in DMEM containing 0.04 % Pluronic heating to 75 °C for 15 min. cDNA was treated with 4 U of F-127 (Thermo Fisher Scientific). After loading, the med- RNase H (Takara Bio). ium was replaced with recording solution [126 mM NaCl, PCR was performed in 25 ll of reaction mixtures 5 mM KCl, 10 mM CaCl2, 0.8 mM MgCl2, 10 mM glu- comprising cDNA corresponding to 200 ng of total RNA, cose, 20 mM HEPES, 0.6 mM NaHCO3, and 0.1 % bovine 1 9 PCR buffer, 0.2 mM dNTP mixture, 0.2 lM forward serum albumin (BSA) pH 7.4]. The cells were incubated at and reverse primers, and Blend Taq polymerase (Toyobo). 37 °C for 15–30 min, and then analyzed for fluorescence PCR conditions consisted of an appropriate number of using a confocal laser scanning microscope (LSM 710 Carl cycles (25–33 cycles for acetylcholine receptor subunits Zeiss, Jena, Germany) and LSM Software ZEN 2009 (Carl and subtypes, 16 cycles for Gnrh1, and 17 cycles for Actb Zeiss).