P2Y11 receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors

doi:10.1016/S0014-2999(01)01222-5

European Journal of Pharmacology

Volume 426, Issue 3, 31 August 2001, Pages 157-163

https://doi.org/10.1016/S0014-2999(01)01222-5 Get rights and content

Abstract

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC₅₀=120±20 μM)>ADP≫AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, α,β-methylene ATP (EC₅₀=110±15 μM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y₂ receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A_2A/A_2B receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5′-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and α,β-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y₁₁ receptor as well as A_2A and A_2B receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A_2B receptors would appear to be relatively ineffective because the A_2A selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A_2A-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration–response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y₁₁ receptors whereas adenosine induces cAMP accumulation via the activation of A_2A receptors. Coordinate activation of P2Y₁₁ and A_2A receptors may influence the developmental fate of normal B-lymphocytes.

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, A_2A and A_2B 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 A_2A 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 A_2A/A_2B agonist 5′-N-ethylcarboxamidoadenosine (NECA), the A_2A-selective agonist CGS-21680 and the A_2A

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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P2Y11 receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors

Abstract

Introduction

Section snippets

Materials

Effects of extracellular ATP, ATP analogs and ATP breakdown products

Discussion

Acknowledgements

Biochem. Pharmacol.

J. Biol. Chem.

Cell. Immunol.

Trends Pharmacol. Sci.

Biochem. Biophys. Res. Commun.

Blood

J. Biol. Chem.

Blood

J. Biol. Chem.

J. Biol. Chem.

Clin. Biochem.

Blood

Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

Br. J. Pharmacol.

P2 purinoceptors: localization, function and transduction mechanism

Pharmacological characterization of the human P2Y11 receptor

Br. J. Pharmacol.

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated H L-60 leukemia cells

Br. J. Pharmacol.

P2Y₁₁ receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors

Pharmacological characterization of the human P2Y₁₁ receptor