Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes

Ischemic preconditioning improves liver resistance to hypoxia and reduces r eperfusion injury following transplantation. However, the intracellular sig nals that mediate the development of liver hypoxic preconditioning are larg ely unknown. We have investigated the signal pathway leading to preconditio ning in freshly isolated rat hepatocytes. Hepatocytes were preconditioned b y 10-minute incubation under hypoxic conditions followed by 10 minutes of r eoxygenation and subsequently exposed to 90 minutes of hypoxia. Preconditio ning reduced hepatocyte killing by hypoxia by about 35%. A similar protecti on was also obtained by preincubation with chloro-adenosine or with A(2A)-a denosine receptor agonist CGS21680, whereas Al-adenosine receptor agonist N -phenyl-isopropyladenosine (R-PIA) was inactive. Conversely, the developmen t of preconditioning was blocked by A(2)-receptor antagonist 3,7-dimethyl-1 -propargylxanthine (DMPX), but not by A(1)-receptor antagonist 8-cyclopenth yl-1,3-dipropylxanthine (DPCPX), In either preconditioned or CGS21680-treat ed hepatocytes a selective activation of delta and epsilon protein kinase C (PKC) isoforms was also evident. Inhibition of heterotrimeric Gi protein o r of phospholypase C by, respectively, pertussis toxin or U73122, prevented PKC activation as well as the development of preconditioning. MEK inhibito r PD98509 did not interfere with preconditioning that was instead blocked b y p38 MAP kinase inhibitor SB203580, The direct activation of p38 MAPK by a nisomycin A mimicked the protection against hypoxic injury given by precond itioning, Consistently, an increased phosphorylation of p38 MAPK was observ ed in preconditioned or CGS21680-treated hepatocytes, and this effect was a bolished by PKC-blocker, chelerythrine. We propose that a signal pathway in volving A(2A)-adenosine receptors, G(i)-proteins, phospholypase C, delta- a nd epsilon -PKCs, and p38 MAPK, is responsible for the development of liver ischemic preconditioning.