Elsevier

Journal of Hepatology

Volume 45, Issue 2, August 2006, Pages 236-245
Journal of Hepatology

Purinergic P2Y2 receptors promote hepatocyte resistance to hypoxia

https://doi.org/10.1016/j.jhep.2006.02.017Get rights and content

Background/Aims

ATP stimulation of purinergic P2 receptors (P2YR and P2XR) regulates several hepatic functions. Here we report the involvement of ATP-mediated signals in enhancing hepatocyte tolerance to lethal stress.

Methods

The protection given by purinergic agonists was investigated in rat hepatocytes exposed to hypoxia.

Results

ATP released after hypotonic stress (200 mOsm/L) as well as P2YR agonists prevented hepatocyte killing by hypoxia with efficiency ranking UTP > ATPγS > ADPβS, whereas the P2XR agonist, methylene-adenosine-5′-triphosphate, was ineffective. Adenosine-5′-O-3-thiotriphosphate (ATPγS; 100 μmol/L) also prevented Na+-overload in hypoxic cells by inhibiting the Na+/H+ exchanger, without interfering with hypoxic acidosis. ATPγS activated Src and promoted a Src-dependent stimulation of both ERK1/2 and p38MAPK. Blocking p38MAPK with SB203580 reverted the protection given by ATPγS on both cell viability and Na+ accumulation, whereas ERK1/2 inhibition with PD98058 was ineffective. An increased phosphorylation of ERK1/2 was also evident in untreated hypoxic hepatocytes. PD98058 ameliorated Na+ accumulation and cell death caused by hypoxia. Hepatocyte pre-treatment with ATPγS reverted ERK1/2 activation in hypoxic cells. SB203580 blocked the effects of ATPγS on both ERK1/2 and Na+/H+ exchanger.

Conclusions

The activation of p38MAPK by P2Y2R increases hepatocyte resistance to hypoxia by down-modulating ERK1/2-mediated signals that promote Na+ influx through the Na+/H+ exchanger.

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.

References (41)

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