嘌呤和嘧啶核苷酸对人嗜酸性粒细胞胞内钙离子的影响：嘌呤能P2Y受体的激活

Effects of purine and pyrimidine nucleotides on intracellular Ca2+ in human eosinophils: activation of purinergic P2Y receptors.

作者信息

Mohanty J G, Raible D G, McDermott L J, Pelleg A, Schulman E S

机构信息

Division of Pulmonary/Critical Care, Department of Medicine, MCP Hahnemann University, Philadelphia 19102, USA.

出版信息

J Allergy Clin Immunol. 2001 May;107(5):849-55. doi: 10.1067/mai.2001.114658.

DOI:10.1067/mai.2001.114658

PMID:11344352

Abstract

BACKGROUND

Extracellular adenosine 5'-triphosphate (ATP) increases human eosinophil intracellular Ca(2+) concentration; the mechanism of action is not fully known. ATP, a physiologic regulator, acts through 2 purinergic receptor types: cation channels (P2X) and G protein-coupled receptors (P2Y).

OBJECTIVE

This study is aimed at identifying the functional purinergic receptors in human eosinophils.

METHODS

The relative potency of ATP, uridine (UTP), cytidine (CTP), and inosine (ITP) 5'-triphosphates (P2Y agonists); 2-methylthio-ATP (P2Y(1) agonist); and 2 P2X agonists, alpha,beta-methylene-ATP and beta,gamma-methylene-ATP on intracellular Ca(2+) concentration was examined in Ca(2+)-sensitive Fura-2-labeled human eosinophils. For comparison, ATP effects were similarly studied in human neutrophils. P2X/P2Y mRNA expression in cells was examined by reverse transcription and PCR.

RESULTS

The nucleotide potency order was UTP > or = ATP > ITP >>> 2-methylthio-ATP > alpha,beta-methylene-ATP = beta,gamma-methylene-ATP = CTP = 0 in eosinophils. Pertussis toxin (500 ng/mL) pretreatment abolished the effect of lower (10(-6) mol/L) but not higher (10(-5) mol/L) concentrations of ATP in eosinophils, whereas it attenuated the effects of 10(-4) mol/L ATP in neutrophils. The phospholipase C inhibitor U73122 (2 micromol/L) partially inhibited the effect of ATP in eosinophils but totally blocked it in neutrophils. Both cells constitutively express mRNA for P2X(1), P2X(4), P2X(5), P2Y(1), and P2Y(2), but not P2X(7), with much weaker expressions of P2X(4) and P2X(5) in neutrophils. Eosinophils cultured with the T(H)1 cytokine, IFN-gamma, expressed mRNA for P2X(7), a receptor linked to apoptosis.

CONCLUSIONS

These results suggest that the P2 purinergic receptor signal transduction pathways in eosinophils and neutrophils are different and are mediated by more than 1 subtype of functional P2Y receptors.

摘要

背景

细胞外5'-三磷酸腺苷（ATP）可增加人类嗜酸性粒细胞内钙离子浓度；其作用机制尚不完全清楚。ATP作为一种生理调节因子，通过两种嘌呤能受体发挥作用：阳离子通道（P2X）和G蛋白偶联受体（P2Y）。

目的

本研究旨在鉴定人类嗜酸性粒细胞中功能性嘌呤能受体。

方法

在钙离子敏感的Fura-2标记的人类嗜酸性粒细胞中，检测ATP、尿苷三磷酸（UTP）、胞苷三磷酸（CTP）和肌苷三磷酸（ITP）（P2Y激动剂）、2-甲硫基ATP（P2Y(1)激动剂）以及两种P2X激动剂α,β-亚甲基ATP和β,γ-亚甲基ATP对细胞内钙离子浓度的影响。为作比较，在人类中性粒细胞中对ATP的作用进行了类似研究。通过逆转录和PCR检测细胞中P2X/P2Y mRNA的表达。

结果

在嗜酸性粒细胞中，核苷酸的效力顺序为UTP≥ATP＞ITP＞＞2-甲硫基ATP＞α,β-亚甲基ATP = β,γ-亚甲基ATP = CTP = 0。百日咳毒素（500 ng/mL）预处理消除了低浓度（10⁻⁶ mol/L）但未消除高浓度（10⁻⁵ mol/L）ATP对嗜酸性粒细胞的作用，而它减弱了10⁻⁴ mol/L ATP对中性粒细胞的作用。磷脂酶C抑制剂U73122（2 μmol/L）部分抑制了ATP对嗜酸性粒细胞的作用，但完全阻断了其对中性粒细胞的作用。两种细胞均组成性表达P2X(1)、P2X(4)、P2X(5)、P2Y(1)和P2Y(2)的mRNA，但不表达P2X(7)，中性粒细胞中P2X(4)和P2X(5)的表达较弱。用Th1细胞因子γ干扰素培养的嗜酸性粒细胞表达与凋亡相关的受体P2X(7)的mRNA。