Protein kinase C (PKC)-mediated regulation of the mitogen-activated protein kinases (MAPK) may play a role in the protection afforded by ischemic preconditioning (PC). Nitric oxide (NO) can influence MAPK activation via interaction with PKC or farnesylation of low-molecular-weight (LMWT) G proteins. However, we have recently reported the mechanism of NO-induced cardioprotection to be a PKC-independent process. Therefore, we investigated the role of LMWT G proteins and MAPK signaling in NO-induced cardioprotection against simulated ischemia-reoxygenation (SI-R) injury. Neonatal rat cardiomyocytes treated for 90 min with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) 1 mM were protected against 6 h of SI (hypoxic conditions at 37 degreesC with 20 mM lactate, 16 mM KCl at pH 6:2) and 24 h reoxygenation under normal culture conditions. NO-induced protection was blocked by the G protein inhibitor alpha -hydroxyfarnesylphosphonic acid (alpha HFP) 10 muM. We studied the time course of p42/44 and p38 MAPK dual-phosphorylation hourly during SI using phosphospecific antibodies. p38 was phospholylated during SI and the peak phosphorylation was significantly delayed by SNAP pretreatment. The p38 inhibitor SB203580 1 muM, given during SI, protected against injury. Thus the delay in peak p38 activation may contribute to, rather than be the effect of, NO-induced cardioprotection. We have shown that p38 beta does not contribute to the total p38 signal in our extracts. Thus there is no detectable beta isoform. We conclude that the main isoform present in these cells and thought to be responsible for the observed phenomenon, is the a isoform.