Lung epithelial cells release ATP during ozone exposure: Signaling for cell survival

Abstract

The common air pollutant ozone causes acute toxicity to human airways. In primary and transformed epithelial cells from all levels of human or rat airways, ozone levels relevant to air pollution (50–200 ppb) increased extracellular [ATP] within 7–30 min. A human bronchial epithelial cell line (16HBE14o^-) that forms electrically resistant polarized monolayers had up to 10-fold greater apical than basolateral surface extracellular [ATP] within 7 min of ozone exposure. Increased extracellular [ATP] appeared due to ATP secretion or release because (1) inhibition of ectonucleotidase (cell surface enzyme(s) which degrade ATP) by ozone did not occur until >120 min of ozone exposure and (2) brefeldin A, a secretory inhibitor, eliminated elevation of extracellular [ATP] without affecting intracellular ATP. Extracellular ATP protected against ozone toxicity in a P2Y receptor-dependent manner as (1) removal of ATP and adenosine by apyrase and adenosine deaminase, respectively, potentiated ozone toxicity, (2) extracellular supplementation with ATP, a poorly hydrolyzable ATP analog ATPγS, or UTP inhibited apoptotic and necrotic ozone-mediated cell death, and (3) ATP-mediated protection was eliminated by P2 and P2Y receptor inhibitors suramin and Cibacron blue (reactive blue 2), respectively. The decline in glucose uptake caused by prolonged ozone exposure was prevented by supplemental extracellular ATP, an effect blocked by suramin. Further, Akt and ERK phosphorylation resulted from exposure to supplemental extracellular ATP. Thus, extracellularly released ATP signals to prevent ozone-induced death and supplementation with ATP or its analogs can augment protection, at least in part via Akt and /or ERK signaling pathways and their metabolic effects.

Introduction

Extracellular signaling through purinoreceptors mediates important physiological events in a variety of cell types [1], [2], [3]. Cells release a fraction of their endogenous ATP either basally or upon stimulation. Once released, extracellular ATP binds to P2 receptors on adjacent cells (paracrine) or of the same cell (autocrine) to activate important signaling pathways. Previously we reported that hypoxia causes lung endothelial cells and adventitial fibroblasts to release ATP [4]. Hypoxia along with extracellular ATP acted synergistically to modulate growth and proliferation of these cells via activation of P2 receptors. The role of extracellular nucleotides, if any, during oxidant stresses remains unclear. There are reports that indicate decreased intracellular ATP contents in oxidative stress [5], [6]. A delayed catabolism of extracellular nucleotides due to diminished ectonucleotidase activity in the presence of reactive oxygen species also has been suggested [7]. More recently we reported that acute hyperoxic exposure of human lung microvascular endothelial cells (HLMVECs)¹ caused enhanced ATP concentration in the extracellular media [8]. This extracellular ATP activated key cell survival-related protein kinases and was critical for survival during hyperoxia. In this paper we investigate the release and role of extracellular ATP during exposure of lung epithelial cells to ozone, another important oxidant gas.

Ozone is formed by photochemical action on exhausts of internal combustion engines and other industrial sources. It can cause exacerbation of asthma and other chronic lung diseases [9], [10], [11]. Unlike high oxygen concentrations, ozone exerts its effects in the lung through endogenous chemical reactions with superficial target molecules in the airway lining. Pulmonary epithelial lining fluid (ELF) contains surfactant rich in glycerophosphocholine lipids. Ozone treatment of pulmonary surfactant results in the production of oxidized lipids including 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn glycero-3-phosphocholine (9-al), an oxidized glycerophosphocholine product [12]. This ozone-oxidized lipid molecule causes a host of biologic activities including interleukin-8 release, loss of mitochondrial dehydrogenase activity, and enhanced cytotoxicity in lung macrophages as well as epithelial cells [12]. Exposure to ozone itself also causes enhanced toxicity to A549 cells. Herein, using human and rat lung epithelial cells, we demonstrate that ATP rapidly accumulated in the extracellular medium during acute ozone exposure of epithelial cells from lungs of humans or rats. Extracellular ATP decreased ozone-induced apoptotic and necrotic cell death, at least in part, by preserving glucose uptake and activating ERK and Akt survival pathways. Our findings demonstrate that extracellular ATP, through purinergic receptors, modulates important signaling events and plays a critical role in epithelial cell survival during toxic ozone exposure.