Signal Pathway Involved in the Development of Hypoxic Preconditioning in Rat Hepatocytes

Abstract

Ischemic preconditioning improves liver resistance to hypoxia and reduces reperfusion injury following transplantation. However, the intracellular signals that mediate the development of liver hypoxic preconditioning are largely unknown. We have investigated the signal pathway leading to preconditioning in freshly isolated rat hepatocytes. Hepatocytes were preconditioned by 10-minute incubation under hypoxic conditions followed by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Preconditioning reduced hepatocyte killing by hypoxia by about 35%. A similar protection was also obtained by preincubation with chloro-adenosine or with A 2A -adenosine receptor agonist CGS21680, whereas A 1 -adenosine receptor agonist N-phenyl-isopropyladenosine (R-PIA) was inactive. Conversely, the development of preconditioning was blocked by A 2 -receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), but not by A 1 -receptor antagonist 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX). In either preconditioned or CGS21680-treated hepatocytes a selective activation of ␦ and protein kinase C (PKC) isoforms was also evident. Inhibition of heterotrimeric G i protein or of phospholypase C by, respectively, pertussis toxin or U73122, prevented PKC activation as well as the development of preconditioning. MEK inhibitor PD98509 did not interfere with preconditioning that was instead blocked by p38 MAP kinase inhibitor SB203580. The direct activation of p38 MAPK by anisomycin A mimicked the protection against hypoxic injury given by preconditioning. Consistently, an increased phosphorylation of p38 MAPK was observed in preconditioned or CGS21680-treated hepatocytes, and this effect was abolished by PKC-blocker, chelerythrine. We propose that a signal pathway involving A 2A -adenosine receptors, G i -proteins, phospholypase C, ␦-and -PKCs, and p38 MAPK, is responsible for the deve- The term ischemic preconditioning refers to the resistance to ischemic injury acquired by tissue following one or more brief periods of ischemia followed by reperfusion. 1,2 Ischemic preconditioning was first described in the myocardium, 1 but has been shown in several other organs, including the brain, the skeletal muscles, and the small intestine. 3 In the heart, ischemic preconditioning occurs in 2 phases: an early phase (early preconditioning) that immediately follows the transient hypoxia and lasts 2 to 3 hours and a late phase (late preconditioning), which begins 12 to 24 hours from the transient ischemia and lasts for about 3 to 4 days. 3 Recent studies have shown that the same phenomenon could also be observed in the liver. [4][5][6][7][8][9][10] In particular, 10-minute interruption of liver blood supply in anesthetized rats followed by 10 minutes of reperfusion reduces transaminases released during a subsequent 90-minute period of ischemia and 90-minute reoxygenation. 5 A similar effect has also been observed in steatosic livers following heat shock preconditioning. 7 Furthermore, ischemic preconditioning before cold preserva...

Mechanisms of hepatocyte protection against hypoxic injury by atrial natriuretic peptide

“…Collagenase (type I), HEPES, chelerythrine, phenylmethylsulfonyl fluoride, propidium iodide, leupeptin, aprotinin, curcumin, 8-bromo-cGMP, ANP, NP receptor (NPR)-C agonist des-(Gln 18 , Ser 19 ,Gly 20 ,Leu 21 ,Gly 22 )-ANP fragment 4-23 amide (C-ANP), and SB203580 were purchased from Sigma Chemical Co. (St. Louis, MO). KT5823 was from ICN Biomedicals (Costa Mesa, CA), and PD98059 was from Calbiochem-Novabiochem (San Diego, CA).…”

Preconditioning-induced cytoprotection in hepatocytes requires Ca2+-dependent exocytosis of lysosomes

“…Studies performed on isolated and perfused liver have shown that resistance to hypoxia induced by preconditioning is associated with the release of nitric oxide and adenosine (Peralta et al, 1997;Peralta et al, 1999), downregulation of caspase 3 activity (Yadav et al, 1999) and decreased production of TNFα by Kupffer cells (Peralta et al, 2001). We have shown that the preconditioning-induced cytoprotection can be reproduced in isolated rat hepatocytes by a short cycle of hypoxiareoxygenation or by direct stimulation of the adenosine A2A-receptor with the agonist CGS21680 (Carini et al, 2001a). The signaling pathway was shown to involve a trimeric G-inhibitor protein, the phospholipase C, PKC isoenzymes and the p38-MAPK (Carini et al, 2000;Carini et al, 2001a).…”

Redox therapeutics in hepatic ischemia reperfusion injury

“…Mechanistic studies have revealed that adenosine, bradykinin, or perhaps other circulating factors signal through G-protein coupled receptors [26] . This initiates a signaling cascade involving activation of protein kinase C [27] , heat shock factor 1 [28] , and mitogen-activated protein kinase [29] . Activation of these receptors during preconditioning result in up regulation of multiple systems capable of attenuating IRI including superoxide dismutase, heat shock proteins, and eNOS [30][31][32] .…”