Post-conditioning (Post-C) induced cardioprotection involves activation of guanylyl-cyclase. In the ischemic preconditioning scenario, the downstream targets of cGMP include mitochondrial ATP-sensitive K(+) (mK(ATP)) channels and protein kinase C (PKC), which involve reactive oxygen species (ROS) production. This study tests the hypothesis that mK(ATP), PKC and ROS are also involved in the Post-C protection. Isolated rat hearts underwent 30 min global ischemia (I) and 120 min reperfusion (R) with or without Post-C (i.e., 5 cycles of 10 s R/I immediately after the 30 min ischemia). In 6 groups (3 with and 3 without Post-C) either mK(ATP) channel blocker, 5- hydroxydecanoate (5-HD), or PKC inhibitor, chelerythrine (CHE) or ROS scavenger, N-acetyl-cysteine (NAC), were given during the entire reperfusion (120 min). In other 6 groups (3 with and 3 without Post-C), 5-HD, CHE or NAC were infused for 117 min only starting after 3 min of reperfusion not to interfere with the early effects of Post-C and/or reperfusion. In an additional group NAC was given during Post-C maneuvers (i.e., 3 min only). Myocardial damage was evaluated using nitro-blue tetrazolium staining and lactate dehydrogenase (LDH) release. Post-C attenuated myocardial infarct size (21 +/- 3% vs. 64 +/- 5% in control; p < 0.01). Such an effect was abolished by 5-HD or CHE given during either the 120 or 117 min of reperfusion as well as by NAC given during the 120 min or the initial 3 min of reperfusion. However, delayed NAC (i.e., 117 min infusion) did not alter the protective effect of Post- C (infarct size 32 +/- 5%; p < 0.01 vs. control, NS vs. Post-C). CHE, 5-HD or NAC given in the absence of Post-C did not alter the effects of I/R. Similar results were obtained in terms of LDH release. Our data show that Post-C induced protection involves an early redox-sensitive mechanism as well as a persistent activation of mK(ATP) and PKC, suggesting that the mK(ATP)/ROS/PKC pathway is involved in post-conditioning.

NO produced in tumors can either positively or negatively regulate growth. To examine this dichotomy, effects of NO concentration and duration on the posttranslational regulation of several key proteins were examined in human breast MCF7 cells under aerobic conditions. We found that different concentration thresholds of NO appear to elicit a discrete set of signal transduction pathways. At low steady-state concentrations of NO (<50 nM), extracellular signal-regulated kinase (ERK) phosphorylation was induced via a guanylate cyclase-dependent mechanism. Hypoxic inducible factor 1␣ (HIF-1␣) accumulation was associated with an intermediate amount of NO (>100 nM), whereas p53 serine 15 phosphorylation occurred at considerably higher levels (>300 nM). ERK phosphorylation was transient during NO exposure. HIF-1␣ stabilization paralleled the presence of NO, whereas p53 serine 15 phosphorylation was detected during, and persisted after, NO exposure. The dose-dependent effects of synthetic NO donors were mimicked by activated macrophages cocultured with MCF7 cells at varying ratios. ERK and HIF-1␣ activation was similar in breast cancer cell lines either mutant (MB231) or null (MB157) in p53. The stabilization of HIF-1␣ by NO was not observed with increased MCF7 cell density, demonstrating the interrelationship between NO and O 2 consumption. The findings show that concentration and duration of NO exposure are critical determinants in the regulation of tumor-related proteins.cancer ͉ hypoxia ͉ macrophage ͉ inducible NO synthase N itric oxide (NO) has been shown to participate in numerous physiological functions important to tumor survival and propagation. Over the last decade, many reports have presented both positive and negative aspects of NO in tumor biology. Whereas NO was found to be either cytostatic or cytotoxic (1-5), other reports provide evidence that NO promotes cancer propagation by increasing tumor growth angiogenesis and metastasis (6-16). NO appears to be two-sided: beneficial or deleterious. In vivo transfection models have demonstrated inducible NO synthase (iNOS) can slow the growth in murine melanoma cells (17), whereas human colon DLD-1 cells showed a more aggressive phenotype (18). It was found that the relative iNOS activities from these melanoma cells were considerably higher than those reported for the DLD-1 cell line. Because of the complex chemistry of NO, its concentration and the cellular redox environment can dictate the biological outcome (19,20). These factors suggest that perhaps the rate of NO production is a critical determinant. Most of the current data has focused on whether NO can regulate specific signal transduction pathways. Little is known regarding NO dose and concentration effects on these mechanisms. Because in vivo NO concentrations can vary considerably depending on the location and conditions of its production, we felt that it was of great importance to investigate this aspect on the regulation of key proteins. In this study, we have examined the concentration and temporal ef...

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