Nitric oxide (NO) is a biological signaling molecule that plays an important role in vascular regulation, immune responses, and neuronal signal transduction. The importance of NO as a biological signaling molecule was highlighted by a Nobel Prize in 1998, which was shared by a UCLA researcher, Dr. Louis Ignarro. This molecule is produced from the amino acid arginine in many cell types. The regulation of NO in physiological systems is complex and involves many aspects of its production and degradation. We are currently investigating the reactions of NO with hemoglobin in red blood cells, and have discovered novel mechanisms that regulate these reactions. This problem is crucial to the design of artificial blood substitutes and the treatment of cardiovascular diseases. This work contributes significantly to the fundamental understanding of the biological regulation of NO and the pathophysiology of NO related diseases.
Supplemental Information for Huang et al. PNAS 2001, 11771-11776
Competition Assay Protocol and Competition Assay Program
Erythrocyte Consumption of Nitric Oxide
Han TH, Qamirani E, Nelson AG, Hyduke DR, Chaudhuri G, Kuo L, and Liao JC (2003) “Regulation of Nitric Oxide Consumption by Hypoxic Red Blood Cells” Proc. Natl. Acad. Sci. USA, 100. 12504–12509
Huang, K.T.; Han, T. H.; Hyduke, D. R.; Vaughn, M. W.; Van Herle, H.; Hein, T. W.; Zhang, C.; Kuo, L.; and Liao, J.C. (2001) “Modulation of Nitric Oxide Bioavailability by Erythrocytes” Proc. Natl. Acad. Sci. USA. 98, 11771-11776
Liao, J.C., T.W. Hein, M. W. Vaughn, K.T. Huang, and L. Kuo (1999) “ Intravascular Flow Decreases Erythrocyte Consumption of Nitric Oxide” Proc. Natl. Acad. Sci. USA, 96:8757-8761.
Vaughn, M.W., K.T Huang, L. Kuo, and J. C. Liao (1999) “Erythrocytes Possess an Intrinsic Barrier to Nitric Oxide Consumption” J. Biol. Chem. 275, 2342-2348.
It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (KHb ~107 M-1 s-1) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, KRBC, would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the KRBC approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.
Intravascular Flow Decreases Erythrocyte Consumption of Nitric Oxide
J. C. Liao, T.W. Hein, M. W. Vaughn, K.T. Huang, and L. Kuo (1999) “ Intravascular Flow Decreases Erythrocyte Consumption of Nitric Oxide” Proc. Natl. Acad. Sci. USA, 96:8757-8761.
Nitric oxide (NO) produced by the endothelium diffuses both into the lumen and to the smooth muscle cells according to the concentration gradient in each direction. The extremely high reaction rate between NO and hemoglobin (Hb), kHb= 3-5 × 107 M-1·s-1, suggests that most of the NO produced would be consumed by Hb in the red blood cells (RBCs), which then would block the biological effect of NO. Therefore, specific mechanisms must exist under physiological conditions to reduce the NO consumption by RBCs, in which the Hb concentration is very high (24 mM heme). By using isolated microvessels as a bioassay, here we show that physiological concentrations of RBCs in the presence of intravascular flow does not inhibit NO-mediated vessel dilation, suggesting that RBCs under this condition are not an NO scavenger. On the other hand, RBCs (50% hematocrit) without intravascular flow reduce NO-mediated dilation to serotonin by 30%. In contrast, free Hb (10 µM) completely inhibits NO-mediated dilation with or without intravascular flow. The effect of flow on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which is caused by hydrodynamic forces on particles. Intravascular flow does not affect the reaction rate between NO and free Hb in the lumen, because the latter forms a homogeneous solution and is not subject to the hydrodynamic separation. However, intravascular flow only partially contributes to the reduced consumption of NO by RBCs, because without the flow, the NO consumption by RBCs is already about 3 orders of magnitude slower than free Hb.
Mathematical Modeling of NO Diffusion and Reaction
Diffusion Distance
El-Farra , N.H. ;P. D. Christofides and J. C. Liao (2003) "Analysis of Nitric Oxide Transport Barriers in Blood Vessels Using a Distributed Multi-cellular Model” Annals of Biomed Engineering, Annals of Biomedical Engineering, Vol. 31, pp. 294–309
Vaughn, M.W., L. Kuo, and J.C. Liao (1998) "Effective diffusion distance of nitric oxide in microcirculation" Am.J. Physiol. 274:H1705-1714.
Despite its well-documented importance, the mechanism for nitric oxide (NO) transport in vivo is still unclear. In particular, the effect of hemoglobin-NO interaction and the range of NO action have not been characterized in the microcirculation, where blood flow is optimally regulated. Using a mathematical model and experimental data on NO production and degradation rates, we investigated factors that determine the effective diffusion distance of NO in the microcirculation. This distance is defined as the distance within which NO concentration is greater than the equilibrium dissociation constant (0.25 µM) of solubleguanylyl cyclase, the target enzyme for NO action. We found that the size of the vessel is an important factor in determining the effective diffusion distance of NO. In ~30- to 100-µm-ID microvessels the luminal NO concentrations and the abluminal effective diffusion distance are maximal. Furthermore, the model suggests that if the NO-erythrocyte reaction rate is as fast as the rate reported for the in vitro NO-hemoglobin reaction, the NO concentration in the vascular smooth muscle will be insufficient to stimulate smooth muscle guanylyl cyclase effectively. In addition, the existence of an erythrocyte-free layer near the vascular wall is important in determining the effective NO diffusion distance. These results suggest that 1) the range of NO action may exhibit significant spatial heterogeneity in vivo, depending on the size of the vessel and the local chemistry of NO degradation, 2) the NO binding/reaction constant with hemoglobin in the red blood cell may be much smaller than that with free hemoglobin, and 3) the microcirculation is the optimal site for NO to exert its regulatory function. Because NO exhibits vasodilatory function and antiatherogenic activity, the high NO concentration and its long effective range in the microcirculation may serve as intrinsic factors to prevent the development of systemic hypertension and atherosclerotic pathology in microvessels.
Production and Reaction Rates
Vaughn, M.W., L. Kuo, and J.C. Liao (1998) “Estimation of nitric oxide production and reaction rates in tissue using a mathematical model" Am.J. Physiol. 274:H2163-H2176.
Nitric oxide (NO) produced by the vascular endothelium is an important biologic messenger that regulates vessel tone and permeability and inhibits platelet adhesion and aggregation. NO exerts its control of vessel tone by interacting with guanylyl cyclase in the vascular smooth muscle to initiate a series of reactions that lead to vessel dilation. Previous efforts to investigate this interaction by mathematical modeling of NO diffusion and reaction have been hampered by the lack of information on the production and degradation rate of NO. We use a mathematical model and previously published experimental data to estimate the rate of NO production, 6.8 × 1014
µmol · µm2 · s-1; the NO diffusion coefficient, 3,300 µm2 s1; and the NO consumption rate coefficient in the vascular smooth muscle, 0.01 s-1 (1st-order rate expression) or 0.05 µM-1 · s-1 (2nd-order rate expression). The modeling approach is discussed in detail. It provides a general framework for modeling the NO produced from the endothelium and for estimating relevant physical parameters.
NOS Regulation
Protein tyrosine kinase-dependent protein activation
Huang, K.T., Kuo, L. and J.C. Liao (1998) Lipopolysaccharide activates endothelial nitric oxide synthase through protein tyrosine kinase, Biochem. Biophys. Res. Comm. 245, 33-37.
Vascular endothelial cell injury or activation by lipopolysaccharide (LPS) plays an important role in the pathogenesis of endotoxin shock. However, the effect of LPS on NO production from vascular endothelial cells (ECs) is incompletely understood. In this study, bovine coronary venular ECs were treated with LPS and the release of NO and expression of the endothelial NO synthase (ecNOS) were examined. We found that the ecNOS activity is transiently enhanced by LPS within the time scale of about 10 h due to the interplay between two LPS-induced mechanisms. Within the first 10 h of LPS treatment, the specific activity of ecNOS is increased by a post-translational modification mediated through a protein tyrosine kinase cascade. After about 10 h of treatment, however, LPS destabilizes the transcript of ecNOS and thus decreased the expression level and total activity.
Transcript Degradation
Lu, J-T. Schmiege, L.M. III. Kuo, L. and J.C Liao (1996) Downregulation of Endothelial Constitutive Nitric Oxide Synthase Expression by Lipopolysaccharide , Biochem. Biophys. Res. Comm. 225, 1-5.
Lipopolysaccharide (LPS), a causal agent of sepsis, has been shown to induce systemic nitric oxide (NO) synthesis through complex mechanisms. However, the effect of LPS on endothelial cells is incompletely understood. To investigate the mechanism by which LPS influences the release of NO from endothelial cells, the effect of this compound on endothelial constitutive nitric oxide synthase (ecNOS) was studied in cultured bovine coronary venular endothelial cells. Western and Northern analyses showed that LPS decreased ecNOS expression at the protein and mRNA levels in a time-dependent and dose-responsive manner. Concurrent treatment of the endothelial cells with LPS and a transcription inhibitor, actinomycin D, resulted in decreased ecNOS mRNA within 8 hours. In contrast, treatment with actinomycin D had only a relatively insignificant effect on the ecNOS transcript level. This result suggests that the reduction of ecNOS by LPS resulted from an increased degradation rate of its transcript.
Related Publications
Han TH, Qamirani E, Nelson AG, Hyduke DR, Chaudhuri G, Kuo L, and Liao JC (2003) “Regulation of Nitric Oxide Consumption by Hypoxic Red Blood Cells” Proc. Natl. Acad. Sci. USA, 100. 12504–12509
El-Farra, N.H.;P. D. Christofides and J. C. Liao (2003) “Analysis of Nitric Oxide Transport Barriers in Blood Vessels Using a Distributed Multi-cellular Model” Annals of Biomed Engineering, Annals of Biomedical Engineering, Vol. 31, pp. 294–309
Liao, JC (2002) “Blood feud: Keeping hemoglobin from nixing NO”, Nature Medicine, 8: 1350-1351.
Han, T.H., Hyduke, D.R., Vaughn, M.W.,. Fukuto, J.M., and Liao, J.C. (2002) Nitric oxide reaction with red blood cells and hemoglobin under heterogeneous conditions. Proc. Natl. Acad. Sci. USA 99: 7763–7768.
Joshi, M.S., Ferguson, T.B., Han,T.H., Hyduke, D.R., Liao,J.C., Rassaf, T., Bryan, N., Feelisch, M., and Lancaster, J.R. (2002) Nitric Oxide is Consumed, Rather than Conserved, by Reaction with Oxyhemoglobin under Physiological Conditions. Proc. Natl. Acad. Sci. USA, 99:10341-6.
Huang, K.T.; Han, T. H.; Hyduke, D. R.; Vaughn, M. W.; Van Herle, H.; Hein, T. W.; Zhang, C.; Kuo, L.; and Liao, J.C. (2001) “Modulation of Nitric Oxide Bioavailability by Erythrocytes” Proc. Natl. Acad. Sci. USA. 98, 11771-11776
Chang, C.-I., J.C. Liao, and L. Kuo (2001) “Macrophage Arginase Promotes Tumor Cell Growth and Suppresses Nitric Oxide-Mediated Tumor Cytotoxicity, Cancer Research, 61, 1100-1106.
Vaughn, M.W., Kuang-Tse Huang, Lih Kuo, and J. C. Liao (2001), “Erythrocyte Consumption of Nitric Oxide: Competition Experiment and Model Analysis” Nitric Oxide Biology and Chemistry, 5, 18-31.
Hein, T.W., J. C. Liao, and L.Kuo (2000) “Oxidized LDL specifically impairs endothelium-dependent, nitric oxide-mediated dilation of coronary microvessels” Am. J. Physiol. Heart Circ. Physiol. 278:H175-H183.
Chang, C.I.; B. Zoghi; J. C. Liao; and L. Kuo (2000) “Interleukin-13 inhibits nitric oxide production through arginase induction in activated macrophages: Involvement of cAMP/PKA, tyrosine kinase, and p38 mitogen-activated protein kinase” J. Immunology. 165:2134-2141
J. C. Liao, T.W. Hein, M. W. Vaughn, K.T. Huang, and L. Kuo (1999) “ Intravascular Flow Decreases Erythrocyte Consumption of Nitric Oxide” Proc. Natl. Acad. Sci. USA, 96:8757-8761.
Vaughn, M.W., K.T Huang, L. Kuo, and J. C. Liao (1999) “Erythrocytes Possess an Intrinsic Barrier to Nitric Oxide Consumption” J. Biol. Chem. 275, 2342-2348.
Huang, K.T., Kuo, L. and J.C. Liao (1998) Lipopolysaccharide activates endothelial nitric oxide synthase through protein tyrosine kinase, Biochem. Biophys. Res. Comm. 245, 33-37.
Chang, C.I., J.C. Liao, and L. Kuo, (1998) “Arginase modulates nitric oxide production in activated macrophages” Am. J. Physiol. 274 (Heart Circ. Physiol.43): H342-348.
Vaughn, M.W., L. Kuo, and J.C. Liao (1998) "Effective diffusion distance of nitric oxide in microcirculation" Am.J. Physiol. 274:H1705-1714.
Vaughn, M.W., L. Kuo, and J.C. Liao (1998) “Estimation of nitric oxide production and reaction rates in tissue using a mathematical model" Am.J. Physiol. 274:H2163-H2176.
Liao, J.C., and L. Kuo. (1997) " Interaction between adenosine and flow-induced dilation in coronary microvascular network" Am. J. Physiol. (Heart Circ.Physiol. 41):H1571-H1581.