Atrial natriuretic peptide (
ANP) reduces
ischemia and/or
reperfusion damage in several organs, but the mechanisms involved are largely unknown. We used freshly isolated rat hepatocytes to investigate the mechanisms by which
ANP enhances hepatocyte resistance to
hypoxia. The addition of
ANP (1 micromol/L) reduced the killing of hypoxic hepatocytes by interfering with intracellular Na(+) accumulation without ameliorating
adenosine triphosphate (
ATP) depletion and pH decrease caused by
hypoxia. The effects of
ANP were mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (cGMP) and were associated with the activation of cGMP-dependent
kinase (cGK), suggesting the involvement of
guanylate cyclase-coupled
natriuretic peptide receptor (NPR)-A/B
ANP receptors. However, stimulating NPR-C receptor with des-(Gln(18), Ser(19),Gly(20),Leu(21),Gly(22))-ANP fragment 4-23
amide (C-
ANP) also increased hepatocyte tolerance to
hypoxia. C-
ANP protection did not involve cGK activation but was instead linked to the stimulation of
protein kinase C (PKC)-delta through G(i)
protein- and
phospholipase C-mediated signals. PKC-delta activation was also observed in hepatocytes receiving
ANP. The inhibition of
phospholipase C or PKC by
U73122 and
chelerythrine, respectively, significantly reduced
ANP cytoprotection, indicating that
ANP interaction with NPR-C receptors also contributed to cytoprotection. In
ANP-treated hepatocytes, the stimulation of both cGK and PKC-delta was coupled with dual phosphorylation of
p38 mitogen-activated protein kinase (MAPK). The
p38 MAPK inhibitor
SB203580 abolished
ANP protection by reverting
p38 MAPK-mediated regulation of Na(+) influx by the
Na(+)/H(+) exchanger. In conclusion,
ANP recruits 2 independent signal pathways, one mediated by cGMP and cGK and the other associated with G(i)
proteins,
phospholipase C, and PKC-delta. Both cGK and PKC-delta further transduce
ANP signals to
p38 MAPK that, by maintaining Na(+) homeostasis, are responsible for
ANP protection against hypoxic injury.