P2Y11 receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors
Introduction
Nucleotides, such as ATP, ADP and the nucleoside, adenosine exert a wide range of physiological effects by activating two families of cell surface purinoceptors. Based on pharmacological properties, these receptors have been divided into the P1 and P2 receptors Burnstock, 1996, Dubyak and El-moatassim, 1993. P1 receptors have been further subdivided into three main types: A1 adenosine receptors, which inhibit adenylyl cyclase, A2A and A2B receptors, which stimulate adenylyl cyclase and A3 receptors (Jacobson et al., 1996). The P2 family includes P2X receptors which form ionic channels and the P2Y receptors which, in general, couple via G-proteins to phospholipase-C. Cloning studies have revealed seven subtypes of P2X receptors, the most recent member, P2X7, being a unique pore-forming receptor which is strongly expressed on human macrophages and lymphocytes Falzoni et al., 1995, Rassendren et al., 1997, Wiley and Dubyak, 1989. P2Y receptors are also expressed on cells of the immune system since P2Y2 (formerly termed P2U) is found on neutrophils and monocyte-macrophages (Cowen et al., 1989). In a recent RT-PCR based survey of haematological cells, several phospholipase C-linked P2Y receptors were detected including P2Y1, P2Y2, P2Y4 and P2Y6 receptors (Jin et al., 1998). Lymphocytes, however, are thought to lack P2Y receptors or their associated PLC signalling pathway since extracellular nucleotides fail to elicit release of Ca2+ from intracellular stores (Wiley and Dubyak, 1989). However, the P2Y11 receptor which couples either to phospholipase-C or adenylyl cyclase is expressed at high levels in the spleen (Communi et al., 1997) raising the possibility that this receptor is normally expressed by B- and/or T-lymphocytes.
Intracellular cAMP levels exert a powerful influence on the fate of B-lymphocytes. For example, elevated cAMP enhances proliferation of B-lymphocytes in the presence of the key growth factor interleukin-4 (Gantner et al., 1998). However, in the absence of growth factors, elevated cAMP promotes apoptosis of B-lymphocytes Mentz et al., 1996, Mentz et al., 1999, Kim and Lerner, 1998. Lymphocyte cAMP may be influenced by many physiological factors including plasma adenosine. Thus, incubation with adenosine elevates cAMP levels in peripheral blood lymphocytes Smith et al., 1971, Wolberg et al., 1975 presumably by A2A receptors which have been identified recently on human T-lymphocytes (Koshiba et al., 1999). In the current work, we set out to determine whether purinoceptors might also stimulate cAMP accumulation in circulating B-lymphocytes from subjects with CLL. The work provides evidence for the expression of cAMP-coupled P1 and P2 receptors.
Section snippets
Materials
Ficoll–Paque (d.1.077) was obtained from Pharmacia (Uppsala, Sweden). ATP, ADP, adenosine, UTP, α,β-methylene ATP, EGTA, forskolin, IBMX (3-isobutyl-1-methyl xanthine) and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Xanthine amine congener, 8-(p-sulfophenyl) theophylline, 1-[N, O-bis(5-isoquinoline-sulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62), the A2A/A2B agonist 5′-N-ethylcarboxamidoadenosine (NECA), the A2A-selective agonist CGS-21680 and the A2A
Effects of extracellular ATP, ATP analogs and ATP breakdown products
Basal levels of cAMP in B-lymphocytes from 17 patients with chronic lymphocytic leukemia ranged from 13.1 to 29.2 pmol/106 cells with a mean value of 19.9±5.8 (SD) pmol/106 cells. The addition of extracellular ATP (1 mM) induced a rapid, approximately fivefold, increase in intracellular cAMP levels that reached a maximum value after 10 min (Fig. 1); half-maximal responses occurred at approximately 2 min. Although there was variability between patient samples, the ATP-induced elevation in cAMP
Discussion
cAMP is an important second messenger for the control of lymphocyte proliferation, differentiation and apoptosis Gantner et al., 1998, Mentz et al., 1995. Hence, receptors that stimulate cAMP accumulation have potentially important roles in the control of lymphocyte maturation, disposition and cell density. The novel cAMP-linked P2Y11 receptor was shown to be expressed at high levels in the spleen (Communi et al., 1997) suggesting the hypotheses that lymphocytes express P2Y11 receptors at high
Acknowledgements
This work was supported by the Leo and Jenny Leukemia and Cancer Research Foundation, the NSW Cancer Council, the University of Sydney Cancer Research Fund and the Cecilia Kilkeary Foundation.
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