Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression
Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription–polymerase chain reaction study
Introduction
In recent years, extracellular nucleotides have been identified as an important class of signal molecule that mediate diverse biological effects in a variety of cells and tissues. Thus the nucleotides ATP, UTP, ADP and UDP are known to play key roles in such diverse tissue functions as fast excitatory neurotransmission, platelet aggregation/haemostasis, pulmonary function, astroglial cell function, bone and cartilage disease, the apoptotic cascade, nociception, metastasis formation and many more [1]. The biological actions of these extracellular nucleotides result from the activation of cell surface P2 receptors [2]. P2 receptors have been divided into two distinct groups, P2X and P2Y. P2X receptors are ATP-gated ionotropic channels which mediate selective permeability to some cations. In contrast, P2Y receptors are G protein-coupled receptors that are preferentially activated by adenosine and uridine nucleotides. Agonist stimulation of most P2Y receptor subtypes generally results in mobilisation of intracellular Ca2+ ions from internal stores via Gq/11-mediated phospholipase C activation. However, the P2Y12 receptor is coupled solely to Gi/o-mediated adenylyl cyclase inhibition, while the P2Y11 receptor couples to both phospholipase C and adenylyl cyclase stimulation.
To date, at least six distinct human P2Y receptor subtypes have been identified (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and P2Y12) that can be distinguished by their specific pharmacological selectivities for different nucleotides. The P2Y1 receptor is highly selective for both ADP and ATP [3], [4], [5], [6]. The triphosphate-preferring P2Y2 receptor is activated equipotently by both ATP and UTP [7], [8]. The P2Y4 receptor is activated preferentially by UTP, while both UDP and ADP have little or no effect [8], [9], [10]. UDP potently activates the pyrimidine-preferring P2Y6 receptor, while UTP and ADP are low potency agonists [8], [11]. The P2Y11 receptor is potently activated by ATP and certain ATP analogues [12], [13]. Finally, ADP is a potent agonist at the P2Y12 receptor [14], [15]. Previous studies have shown that P2Y receptors are widely expressed throughout many diverse human tissues. In terms of receptor expression throughout the brain, only the P2Y1 receptor subtype has been investigated in some detail in the rat, chick and human CNS [16], [17], [18], [19]. Some data also suggests the expression of certain P2Y receptor subtypes in a variety of human cell lines.
In order to characterise the precise physiological roles played by the various P2Y receptor subtypes in the human body, a thorough expression profile is required throughout peripheral tissues and brain regions. In addition, many cell lines are commonly used as vital tools for elucidating both receptor pharmacology and function, and thus determining the endogenous expression of P2Y receptor subtypes in such cell lines is clearly of important value. To this end we have investigated the expression of five P2Y receptor subtypes (P2Y1,2,4,6,11) in a number of human peripheral tissues and brain regions from multiple individuals, both male and female, and also in human cell lines and primary cells using TaqMan-quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis.
Section snippets
Generation of samples for TaqMan mRNA analysis [20]
Human tissue or RNA was purchased (Biochain, San Leandro, USA; Invitrogen, Leek, The Netherlands; Clontech, Palo Alto, CA, USA) or donated (Netherlands Brain Bank, Amsterdam, The Netherlands) and poly(A)+ RNA was prepared by the PolyATract method according to manufacturers instructions (Promega, Madison, WI, USA). The poly(A)+ RNA samples from 20 body tissues (Masterplate II) and 18 brain regions (TaqBrain plate) from four individuals per tissue (two males/two females, except prostate) were
Results
Quantitative RT–PCR analysis (TaqMan) was used to measure the mRNA expression levels of five distinct human P2Y receptor subtypes in RNA samples derived from a collection of human peripheral tissues, brain regions, cell lines and primary cells. In addition, the mRNA expression levels of three housekeeping genes were also measured in these human RNA samples in order to ensure the quality, quantity and integrity of each preparation. For each gene measured, a combination of transcript-specific
Discussion
In this study, we have determined and compared the mRNA expression levels of five cloned P2Y receptor subtypes in a vast array of human peripheral tissues and brain regions from multiple individuals, both male and female, and also human cell lines and primary cells. We employed TaqMan-quantitative RT–PCR analysis to measure receptor mRNA expression levels since specific radioligands or antibodies are currently unavailable for most P2Y receptor subtypes to measure cell surface receptor numbers.
Acknowledgements
The authors would like to thank Dr. R. Ravid (Netherlands Brain Bank, The Netherlands) for arrangement/donation of human brain tissue. D.J.M. is grateful for funding from the BBSRC (UK) and GlaxoSmithKline (Harlow, UK). The authors would also like to thank to Dr. D.J. Cousens (GlaxoSmithKline, Stevenage, UK) for critical reading of the manuscript.
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