Purinergic P2Y2 receptors promote hepatocyte resistance to hypoxia
Introduction
In the recent years there has been a growing interest about the role played by purinergic receptors in modulating epithelial cell functions [1], [2]. Purine receptors are divided in two main classes: P1 or adenosine receptors and P2 receptors which recognise as ligands ATP, ADP, UTP and UDP [3]. The P2 receptors are further divided in P2X and P2Y sub-groups. P2X receptors (P2XR) are ATP-gated non-selective cation channels, while P2Y (P2YR) are heptahelical receptors that couple with heterotrimeric Gi proteins and phospholipase C [3]. In the liver, the stimulation of purinergic P2 receptors regulates volume recovery, glycogenolysis and bile secretion [4], [5], [6], [7]. Moreover, Thevananther and co-workers have reported that extracellular ATP activates c-Jun N-terminal kinase and induces hepatocyte proliferation [8].
The stimulation of P1 receptors by adenosine released during transient episodes of tissue ischemia is now recognized to play a main role in the development of acquired resistance to hypoxia/re-oxygenation injury that characterizes the phenomenon known as “ischemic preconditioning” [9], [10], [11]. We have previously shown that the stimulation of adenosine A2 receptors activates a network of intracellular kinases including phosphatidylinositol 3-kinase (PI3K), protein kinase A (PKA), protein kinase C (PKC) and p38 mitogen-activated protein kinase (p38MAPK) that enhances the tolerance against hypoxic hepatocyte damage [12], [13]. Studies in the heart have shown that, beside adenosine, myocardiocytes exposed to brief hypoxia release appreciable amounts of ATP [14], [15] that contribute to the onset of ischemic preconditioning [16], [17]. Moreover, recent evidence indicates that P2-mediated signals are involved in protecting against oxidative stress of astrocytes and lung microvascular endothelial cells [18], [19]. These observations along with the importance of autocrine/paracrine ATP secretion in regulating hepatocyte functions [5], [6], [8] prompted us to investigate whether and by which mechanism the stimulation of purinergic P2 receptors by extracellular ATP might improve hepatocyte tolerance to lethal stress.
Section snippets
Materials
Collagenase (Type I), N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) (HEPES), chelerythrine, phenylmethylsulfonyl fluoride, propidium iodide, leupeptin, aprotinin, adenosine-5′-O-(3-thiotriphosphate), (ATPγS), UTP, adenosine-5′-(β-thio)-diphosphate (ADP-βS), methylene-adenosine-5′triphosphate (AMP-CPP), wortmannin, suramin, apyrase (Grade VII), PP2, AG-1478, Reactive Blue 2, pertussis toxin, nigericine, 5,5′dimethylamiloride, U73127, SB203580 were purchased from Sigma Chemical Co.
Stimulation of purinergic P2 receptors increases hepatocyte tolerance to hypoxia
Previous studies have shown that adenosine increased hepatocyte tolerance to hypoxic injury [12], [20]. In order to avoid the possible interference of ATP degradation products, the role of purinergic P2 receptors in modulating hepatocyte killing by hypoxia was investigated using the non-hydrolyzable ATP analogue adenosine-5′-O-(3-thiotriphosphate) (ATPγS). Fifteen minutes pre-treatment of freshly isolated rat hepatocytes with ATPγS (10–100 μmol/L) protected in a dose-dependent manner against
Discussion
Increasing evidence indicates hepatic preconditioning as an efficient means to improve ischemia/reperfusion injury during liver surgery [12], [26], [27]. In this contest there is a growing interest in investigating pharmacological agents capable of mimicking the protective effects of preconditioning. Here we report that ATP promotes preconditioning effects, as detected by an increased hepatocyte tolerance to hypoxic injury. Such effect is mediated by the activation of purinergic P2 receptors
Acknowledgements
This work has been supported by grants from: University “Amedeo Avogadro” of East Piedmont, the Regional Government of Piedmont and the Italian Ministry for Instruction, University and Scientific Research (Research Program: Liver preconditioning: molecular mechanisms responsible for the modification of tolerance to ischemia/reperfusion and clinical application in liver resection and transplantation). G.B. is financially supported by the Centro Ricerche “E. Menni” Brescia, Italy.
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