kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2011.15.1.61 kjpp-15-61 Original Article Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells KimNa-Hyun 1 ParkKyu-Sang 2 SohnJoon Hyung 3 YehByung-Il 3 KoChang Mann 4 KongIn Deok 2 Department of Basic Nursing Science and Institute for Nursing Science, Keimyung University, Daegu 704-701, Korea Department of Physiology, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea Department of Biochemistry, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea Department of Pharmacology, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea Corresponding to: In Deok Kong, Department of Physiology, Yonsei University Wonju College of Medicine, 162, Ilsan-dong, Wonju 220-701, Korea. (Tel) 82-33-741-0292, (Fax) 82-33-745-6461, (E-mail) kong@yonsei.ac.kr 022011 18022011 15 1 61 66 11022011 16022011 18022011 Copyright © 2011 Korean J Physiol Pharmacol 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used Ca2+ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP (10 μM) elicited strong but transient [Ca2+]i increase in a concentration-dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking [Ca2+]i transients were 2MeS-ATP> ATP>> UTP=αβ-MeATP, which was compatible with the subclass of P2Y1 receptor. The [Ca2+]i transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of P2Y1 selective blocker (MRS 2179; 30 μM). P2Y1 receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, P2Y1 receptor is mainly expressed in a retinoblastoma cell, which elicits Ca2+ release from internal Ca2+ storage sites via the phospholipase C-mediated pathway. P2Y1 receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic [Ca2+]i signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

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ATP-evoked Ca2+ signaling in WERI-Rb-1 cells. (A) Original trace of Ca2+ responses evoked by four different concentrations of ATP in the same group of cells. (B) Dose-response curve fitting the peak responses obtained from five different groups of cells. F0: basal fluorescence after Fluo 3-AM loading, F: change in fluorescence after introducing ATP, n=total cell number, N=number of experiment.

Concentration-response curve of P2Y receptor agonists in WERI-Rb-1 cells. Each curve shows the peak calcium responses of increasing concentrations of 2MeS-ATP, a selective P2Y1 agonist, αβ-MeATP, a P2X agonist, and UTP, a P2Y2/P2Y4/P2Y6 agonist. Both αβ-MeATP and UTP did not induce calcium rise. Values are means±S.E.M. n=total cell number, N=number of experiments.

Differential effect of P2Y agonists on [Ca2+]i in WERI-Rb-1 cells. Original traces (A) and graph (B) showing calcium transient evoked by ATP (10 μM), a putative P2Y agonist, and its suppression after application of 30 μM MRS2179, a selective P2Y1 antagonist. F0: basal fluorescence after Fluo 3-AM loading, F: changes in fluorescence after introducing agonist or antagonist, n=total cell number, N=number of experiment. ∗∗∗Denotes p<0.001.

Effect of P2Y1 antagonist on 2-MeS-ATP-induced [Ca2+]i changes in WERI-Rb-1 cells. Original traces (A) and graph (B) showing calcium transient evoked by 2-MeS-ATP (1 μM), a P2Y1 agonist, and its suppression after application of the 30 μM MRS2179, a selective P2Y1 antagonist. F0: basal fluorescence after Fluo 3-AM loading, F: changes in fluorescence after introducing agonist or antagonist, n=total cell number, N=number of experiment, ∗∗∗denotes p<0.001.

Immunoblots of P2Y receptor subtypes in WERI-Rb-1 cells. P2Y1 purinoceptor was identified by the band at around 70 kDa, which is compatible with its deduced protein size.