Mol. Cells 2015; 38(7): 616-623 http://dx.doi.org/10.14348/molcells.2015.2357 Molecules and Cells http://molcells.org Established in 1990 The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells Tae Hoon Kwon1, Hyunwoo Jung1, Eun Jeong Cho1, Ji Hoon Jeong2, and Uy Dong Sohn1,* P2 receptors are membrane-bound receptors for extracel- per esophageal sphincter (UES), are under voluntary control. lular nucleotides such as ATP and UTP. P2 receptors have The lower esophageal sphincter (LES) is a bundle of muscles been classified as ligand-gated ion channels or P2X recep- at the low end of the esophagus, where it connects the stom- tors and G protein-coupled P2Y receptors. Recently, ach. When the LES is closed, it prevents acid and stomach purinergic signaling has begun to attract attention as a content reflux. The LES consists of smooth muscle like the rest potential therapeutic target for a variety of diseases espe- of the digestive tract. Smooth muscle in the gastrointestinal cially associated with gastroenterology. This study deter- tract is controlled by the autonomic nervous system. Contrac- mined the ATP and UTP-induced receptor signaling mech- tion or relaxation of smooth muscle has a key role in gastroin- anism in feline esophageal contraction. Contraction of testinal motility. Digestive motility disorders can lead to impaired dispersed feline esophageal smooth muscle cells was peristalsis resulting in slow contractions, rapid contractions, or measured by scanning micrometry. Phosphorylation of combination of both slow and fast contractions (Vantrappen et MLC20 was determined by western blot analysis. ATP and al., 1986). UTP elicited maximum esophageal contraction at 30 s and Recently, physiological and pharmacological investigation 10 μM concentration. Contraction of dispersed cells treat- showed that neurotransmitters other than acetylcholine or nor- ed with 10 μM ATP was inhibited by nifedipine. However, adrenaline are involved in peripheral autonomic transmission. contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM These neurotransmitters are commonly referred to as non- UTP was decreased by U73122, chelerythrine, ML-9, PTX adrenergic, non-cholinergic (NANC) neurotransmitters (Burnstock and GDPβS. Contraction induced by 0.1 μM ATP and UTP et al., 1997). Representative NANC neurotransmitters are com- was inhibited by Gαi3 or Gαq antibodies and by PLCβ1 or posed of the peptide mediators including calcitonin gene- PLCβ3 antibodies. Phosphorylated MLC20 was increased by related peptide (CGRP) and substance P, and the non-peptide ATP and UTP treatment. In conclusion, esophageal con- mediators including nitric oxide (NO) and nucleotides such as traction induced by ATP and UTP was preferentially medi- ATP and UTP (Lundberg, 1996). The nucleotidesin the form of ated by P2Y receptors coupled to Gαi3 and Gα q proteins, the nucleoside triphosphates ATP and UTP play important roles which activate PLCβ1 and PLCβ3. Subsequently, increased as essential energy sources and enzyme cofactors in cellular intracellular Ca2+ and activated PKC triggered stimulation metabolism. Furthermore, ATP and UTP also function as extra- of MLC kinase and inhibition of MLC phosphatase. Finally, cellular messengers inducing intracellular signaling through increased pMLC20 generated esophageal contraction. activation of distinct cell surface receptors of the P2 receptor family (Ralevic and Burnstock, 1998). The P2 receptors for extracellular nucleotides are ubiquitously INTRODUCTION distributed in various organs of the body and involved in regula- 1 tion of almost all physiological processes such as the immune, The esophagus is a muscular tube that connects the back of inflammatory, cardiovascular, muscular, and central and pe- the pharynx to the top of the stomach and ranges from approx- ripheral nervous systems (Abbracchio et al., 2006; Surprenant imately 18-25 cm in length, and 1-2 cm in diameter. The mus- and North, 2009). The term “P2” implies that purine and pyrimi- cles in the upper portion of the esophagus, also called the up- dine nucleotides can act as selective ligands of various receptor subtypes (Fredholmet al., 1997). The P2 receptors exist as 2 1 2 distinct families. P2X receptors are ligand-gated ionotropic Department of Pharmacology, College of Pharmacy, Department of + + - 2+ Pharmacology, College of Medicine, Chung-Ang University, Seoul 156- channel family, gating primarily Na , K , Cl and Ca . They are 756, Korea composed of 7 subtypes (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, *Correspondence:[email protected] and P2X7) (Jiang et al., 2003). P2Y receptors are metabotropic, heptahelical G-protein coupled receptor (GPCR) family mem- Received 30 December, 2014; revised 7 April, 2015; accepted 16 April, bers with 8 identified subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, 2015; published online 27 May, 2015 P2Y12, P2Y13, and P2Y14 (Abbracchio et al., 1997; Fredholm et al 1997; Lee et al., 2000). Recently, purinergic signaling has Keywords: ATP, contraction, Esophagus, P2Y receptor, UTP begun to attract attention as a potential therapeutic target for a eISSN: 0219-1032 The Korean Society for Molecular and Cellular Biology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. Mechanisms of Esophageal Contraction Induced by ATP and UTP Tae Hoon Kwon et al. variety of diseases (Burnstock, 2006). Exploratory studies fo- and then incubated in this solution at 31°C, which was gassed cused on purinergic receptors as the future therapeutic targets with 95% O2-5% CO2. The cells were allowed to dissociate of GI diseases (Burnstock, 2008; Yiangou et al., 2001). freely for 10 to 20 min. Before beginning the experiment, the It was previously demonstrated that both P2X and P2Y re- cells were kept at 31°C for at least 10 min to relax the cells. ceptors exist in esophageal smooth muscle and mediate Throughout the procedure, care was taken not to agitate the esophageal contraction (Cho et al., 2010). However, the de- fluid in order to avoid cell contraction in response to mechanical tailed signaling mechanism of ATP- and UTP- induced contrac- stress. tion via P2X and P2Y receptors in feline esophageal smooth The experiments were performed in accordance with the muscle has not been studied. Understanding the signaling guidelines of the Institutional Animal Care and Use Committee mechanism of purinergic receptors on esophagus can contrib- of Chung-Ang University (No.14-0045). ute to the treatment of esophageal diseases. In addition, coex- istence of P2X and P2Y receptors raises the question of which Preparation of permeabilized smooth muscle cells receptor subtype preferentially mediates the action of the en- Cells were permeabilized, when required, to diffuse agents dogenous ligand. The purpose of this study was to investigate such as G protein antibodies and PLC isozyme antibodies, which receptor preferentially induces contraction activated by which do not diffuse across intact cell membrane. The process ATP and UTP and the signaling mechanism involved in feline of preparation of permeabilized cells did not affect cell contrac- esophageal smooth muscle cells. The nucleotides ATP and tion (Cao et al., 2001; Horowitz et al., 1996; Murthy et al., 2003; UTP were utilized to activate P2 receptors and to identify pref- Shim et al., 2002;Sohn et al., 1997). After completion of the erentially activated receptor and the signaling pathways. Selec- enzymatic phase of the digestion process, the partly digested tive G-protein antibody was utilized to identify the coupling of muscle tissue was washed with an enzyme-free cytosolic buffer specific G-proteins to effector enzymes, and selective inhibitors of the following composition: 20 mM NaCl, 100 mM KCl, 5.0 were used to characterize the pathways involved in MLC20 (20 mM MgSO4; 0.96 mM NaH2PO4; 1.0 mM EGTA and 0.48 mM kDa, regulatory light chain of Myosin II) phosphorylation and CaCl2 and 2% bovine serum albumin. The cytosolic buffer was esophageal smooth muscle cell contraction. equilibrated with 95% O2-5% CO2 to maintain pH 7.2 at 31°C. The muscle cells were dispersed spontaneously in this medium. MATERIALS AND METHODS The cytosolic buffer contained 0.48 mM CaCl2 and 1 mM EGTA, yielding 0.18 mM free Ca2+ (Fabiato and Fabiato, 1979). Materials and reagents After dispersion, the cells were permeabilized by incubation G protein antibodies (Gαi1, Gαi2, Gαi3, Gαq, Gαs, Gαo, and Gβ) and for 5 min in cytosolic buffer containing saponin (75 μg/ml). After PLC antibodies (β1, β3, γ1) from Santa Cruz Biotechnology exposure to saponin, the cell suspension was spun at 350 g, (USA); Chelerythrine chloride from Research Biochemicals and the resulting pellet was washed with saponin-free modified (USA); goat anti-rabbit IgG-HRP from Bethyl Laboratories Inc. cytosolic buffer that contained antimycin A (10 μM), ATP (1.5 (USA); rainbow molecular weight marker from Amersham mM) and an ATP-regenerating system that consisted of crea- (USA); enhanced chemiluminescence (ECL) agents from tine phosphate (5 mM) and creatine phosphokinase (10 PerkinElmer Life Sciences (USA); sodium dodecyl sulfate units/ml) (Bitar et al., 1986). The procedure was repeated twice (SDS) sample buffer from Owl scientific Inc. (USA); nitrocellu- to ensure complete removal of saponin. After the cells were lose membrane, Tris/Glycine/SDS