Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

Abstract

In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 μM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 μM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that 78% of NO metabolism in SMCs is Cygb-dependent. Of this, 87% was B5R-and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (V NO), while depletion of ascorbate had no effect. Kinetic analysis of V NO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that V NO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.

Vascular nitric oxide resistance in type 2 diabetes

“…NO 2 likely contributes to the NO• stores via conversion into NO•, probably via the action of metalloproteins, including Hb, myoglobin (Mb), cytoglobin ([Cygb), xanthine oxidase, cytochrome c oxidase, and eNOS, that is favored by hypoxia and low pH [ 127 ]. Cygb [i.e., a globin expressed at μM levels (~3.5–5.0 μM [ 144 ]) and co-localized with myosin heavy chain [ 145 ]] regulates NO• bioavailability within the VSM [ 146 ]; under a normal O 2 level, Cygb metabolizes excessive amount of NO• by dioxygenation (converting NO• to NO 3 , a rate of 11.6 ± 0.6 nM/s in mouse aorta); in contrast, under a hypoxic condition, it generates NO• from NO 2 (referred to as O 2 -dependent NO•-dioxygenase and NO 2 -reductase, respectively) [ 146 ], and that NO• binds to sGC in the VSM, making the vessel to be relaxed [ 147 ]. About 78% of NO• metabolism in VSMCs is Cygb-dependent [ 144 ].…”

“…Cygb [i.e., a globin expressed at μM levels (~3.5–5.0 μM [ 144 ]) and co-localized with myosin heavy chain [ 145 ]] regulates NO• bioavailability within the VSM [ 146 ]; under a normal O 2 level, Cygb metabolizes excessive amount of NO• by dioxygenation (converting NO• to NO 3 , a rate of 11.6 ± 0.6 nM/s in mouse aorta); in contrast, under a hypoxic condition, it generates NO• from NO 2 (referred to as O 2 -dependent NO•-dioxygenase and NO 2 -reductase, respectively) [ 146 ], and that NO• binds to sGC in the VSM, making the vessel to be relaxed [ 147 ]. About 78% of NO• metabolism in VSMCs is Cygb-dependent [ 144 ]. In human VSMCs, Cygb-mediated NO• production from NO 2 (at the physiological intracellular level of NO 2 ~ 10 μM) is estimated to be ~7 and 35 pM/s in VSM, at pH 7.0 and 5.5, respectively); an amount that can rise to 10-fold ( ~ 350 pM/s) under acidic condition (pH=5.5) and chronic hypoxia (i.e., intracellular Cygb concentration of ~350 μM) [ 147 ].…”

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations

“…Therefore, for these activities to be physiologically relevant, Cygb requires a rapid cellular reduction system to recycle the ferrous form. Cytochrome b5, cytochrome b5 reductase, NADH, and ascorbate all rapidly reduce ferric Cygb in vitro, potentially fitting the role of a cellular reduction system [ 22 , 24 , 25 ].…”