Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, 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 and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32835 |
| Date | 31 August 2012 |
| Creators | Tsui, Albert King-Yeung |
| Contributors | Hare, Gregory, Marsden, Philip A. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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