Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia

Abstract

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)(-/-) mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS(-/-) mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS(-/-) mice, despite comparable reductions in tissue PO(2). Paradoxically, nNOS(-/-) mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Fig. 1.

Differential role of nNOS in the survival of acutely anemic and hypoxic mice. (A) WT (n = 21, black), nNOS^−/− (n = 10, red), eNOS^−/− (n = 10, blue), and iNOS^−/− (n = 10, green) mice were hemodiluted (acute anemia) until mortality. (B) WT (n = 18, black), nNOS^−/− (n = 14, red), eNOS^−/− (n = 14, blue), and iNOS^−/− (n = 12, green) were exposed to hypoxia (5% O₂). In both cases, mortality was assessed by the cessation of breathing. P < 0.05 WT vs. nNOS^−/−.

Fig. 2.

nNOS regulates HIF-α–dependent genes in acute anemia, but not in hypoxia. HIF-α–dependent mRNA levels were measured in brain samples of WT (open bars) and nNOS^−/− in anemic (A–D; filled bars) and hypoxic mice after 6 h (E–H; gray bars) (n = 6 per group). Relative mRNA levels were normalized to the respective baseline. *P < 0.05 vs. baseline. #P < 0.05 vs. WT.

Fig. 3.

nNOS regulates HIF-1α protein level in acute anemia, but not in hypoxia. Immunoblots of nNOS, HIF-1α, and HIF-2α protein were assessed in anemic (A–C) and hypoxic (D–F) WT (open bars) and nNOS^−/− brain samples (black or gray bars), respectively. Representative blots of n = 6 per group were shown. Protein levels were normalized to control (Ctrl) group and α-tubulin. *P < 0.05 vs. baseline.

Fig. 4.

Anemia leads to increased HIF-1α expression in HIF-α(ODD)-luciferase mice in WT (open bars) but not in nNOS^−/− (filled bars) mice. Representative dorsal (A) and ventral (B) images of WT and nNOS^−/− mice (n = 6) were obtained at baseline and up to 24 h anemia. Total body radiance was normalized to each animal's baseline. Color bar indicated photons (cm² × sec × steradian) with minimum and maximum threshold values. (C) Extracted brain tissue was assessed for luciferase activity in vitro at baseline and after 6 h of anemia (n = 6). *P < 0.05 vs. baseline; #P < 0.05 vs. WT.

Fig. 5.

Anemia leads to increased S-nitrosylated pVHL (SNO-pVHL) that depends on nNOS. (A) SNO-pVHL levels in WT mouse brain at baseline or 1, 6, and 24 h anemia (n = 4). (B) SNO-pVHL levels in control (C), 6 h anemic (A), or 6 h hypoxia (Hyp) vs. normoxia (norm) brain in WT and nNOS ^−/− (KO) mice (n = 5). *P < 0.05 vs. baseline.