Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver

Abstract

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.

Figures

Figure 1

Nitrite therapy in hepatic I/R injury. (A) Experimental protocol for murine model of hepatic I/R injury. (B) Serum AST levels in mice after hepatic I/R. *P < 0.05 vs. vehicle (0 μM); **P < 0.01 vs. vehicle (0 μM). (C) Serum ALT levels in mice after hepatic I/R. (D–G) Representative photomicrographs of hepatic histopathology after 45 minutes of ischemia and 24 hours of reperfusion. Sham-treated (D) and I/R-injured mice treated with saline (E), nitrite (F), or nitrate (G) are shown. (H) Hepatic tissue sample pathology scores after 45 minutes of ischemia and 24 hours of reperfusion. (I) Hepatocellular apoptosis as measured by TUNEL staining after 45 minutes of ischemia and 24 hours of reperfusion. ***P < 0.001 vs. I/R alone. Numbers inside the bars indicate the number of mice investigated in each group. In D–I, 48 nmol nitrate and nitrite were administered intraperitoneally.

Figure 2

Nitrite therapy in myocardial I/R injury. (A) Experimental protocol for myocardial I/R studies in mice. Asterisk indicates in vivo AAR as shown by Evans blue injection, with infarct size 1.0% of 2,3,5-triphenyltetrazolium chloride (TTC). (B) Representative photomicrographs of murine hearts after 30 minutes of myocardial ischemia (MI) and 24 hours of reperfusion. Areas of the myocardium that appear blue (i.e., Evans blue dye) represent the areas of myocardium not at risk for infarction. In contrast, the areas of myocardium that stain red (i.e., TTC-positive) represent viable myocardium that was at risk for infarction. Myocardium that appears pale (i.e., TTC-negative) indicates areas of myocardium at risk that are necrotic (i.e., infarcted). Nitrite treatment significantly reduced myocardial infarction after 30 minutes of myocardial ischemia and 24 hours of reperfusion. (C) Myocardial AAR per LV for mouse hearts receiving nitrate (48 nmol) or doses of nitrite ranging from 2.4 to 1,920 nmol. The myocardial AAR per LV was similar for all of the study groups (P = NS between groups). (D) Myocardial infarct size per AAR for mouse hearts receiving nitrate (48 nmol) or doses of nitrite ranging from 2.4 to 1,920 nmol. Nitrite therapy (2.4–960 nmol) significantly reduced myocardial infarct size compared with nitrate therapy (**P < 0.001). (E) Blood levels of iron-nitrosylated hemoglobin after intraventricular injection of 48 nmol nitrite. (F) Myocardial AAR per LV and infarct size (INF) per AAR and per LV in mice treated with the NO scavenger PTIO prior to nitrite therapy (48 nmol). Nitrite-mediated cardioprotection was not observed in the presence of PTIO (P = NS, PTIO versus PTIO + nitrite). (G) Myocardial AAR per LV and INF per AAR and per LV in mice treated with the HO-1 inhibitor ZnDPGG prior to nitrite therapy (48 nmol). Treatment with ZnDPGG did not attenuate the protective effects of nitrite therapy. Numbers inside bars indicate the number of animals that were investigated in each group.

Figure 3

Blood and liver tissue levels of nitrite, RSNO, and RxNO. (A) Liver nitrite, RSNO, and RxNO levels (μmol/l) in animals (n = 3–5 per group) subjected to sham hepatic I/R (Sham) or to hepatic ischemia plus either 1 minute (1 Rep) or 30 minutes (30 Rep) of reperfusion. Experiments are saline-treated controls without nitrite. **P < 0.001 vs. baseline. (B) Liver tissue nitrite levels in mice (n = 3–5 per group) subjected to hepatic I/R injury. (C) Liver tissue RSNO levels (μmol/l) in mice (n = 3–5 per group) subjected to hepatic ischemia and varying periods of reperfusion. (D) Hepatic tissue RxNO levels (μmol/l) after hepatic ischemia and reperfusion in mice (n = 3–5 per group). Experiments in B–D compare saline-treated controls to those with nitrite treatment (48 nmol). Numbers inside bars indicate the number of mice investigated in each group.

Figure 4

Nitrite-mediated hepatoprotection and the NO and HO-1 signaling pathways. (A) Serum AST levels after hepatic I/R injury in mice receiving saline vehicle, nitrite (48 nmol), the NO scavenger PTIO, or nitrite (48 nmol) plus PTIO. **P < 0.01 vs. vehicle. (B) Serum levels of AST in eNOS-deficient (eNOS–/–) mice receiving saline vehicle or sodium nitrite (48 nmol). (C) Serum levels of AST in mice receiving ODQ followed by nitrite (nitrite; 48 nmol) or the sGC inhibitor ODQ and in mice receiving ODQ pretreatment after nitrite (48 nmol; Nitrite + ODQ). (D) Hepatic tissue protein levels at 6 hours after ischemia determined using Western blot methods in animals subjected to sham and I/R with saline, nitrite, or nitrate. Hepatic tissue mRNA levels of HO-1 were determined using quantitative real-time RT-PCR methods in animals subjected to hepatic I/R. (E) Serum AST levels in mice treated with nitrite (48 nmol) or the HO-1 inhibitor ZnDPBG in the setting of hepatic I/R injury. (F) Serum AST and ALT levels in HO-1–/– mice treated with and without 48 nmol of nitrite.