Mechanism Of No Decay In Vascular Smooth Muscle Cells: Role Of Cytoglobin And Identification Of Its Cellular Reducing System

Background In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. However, major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. Therefore, we performed studies to assess the intracellular concentrations of Cygb and its reducing systems in vascular smooth muscle cells (SMCs), and their role in the process of NO decay. Methods Cygb and its reducing systems, cytochrome B5 reductase (B5R), cytochrome B5 (B5) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Murine aortic smooth muscle cells (SMCs) were purchased from American Type Culture Collection (ATCC; CRL-2797™) and passaged. For depleting Cygb, B5R or B5, siRNAs were used. SMCs were transfected with Cygb siRNA, B5R siRNA, B5 siRNA or CPR siRNA using Lipofectamine RNAiMAX (Invitrogen). The rate of NO metabolism by normal SMCs of different cell numbers was assessed using a Clark-type NO electrode (NOCHM-4, WPI) utilizing two ports on the side wall of a four-port water-jacketed electrochemical chamber (NOCHM-4, WPI) and additionally another oxygen electrode was placed through second port on the side wall of the chamber. For protein assays, Cygb, B5R and B5 were expressed and purified using reported procedures. Quantitative immunoblotting was used to estimate the cellular concentrations of Cygb and each reducing system protein from comparison to standards of known concentration for each purified protein. Results 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 1 µM NO to air-equilibrated cells. The NO dioxygenation rate (VNO) with 3.5 x 106 cells•ml-1 was 12.7 ± 0.3 nM•s-1. siRNA-mediated knockdown experiments indicated that ~78% of NO metabolism in SMCs was Cygb-dependent. Of this, ~87 % was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in VNO, while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5R/B5/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Conclusions B5R/B5 was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs and studies were performed to experimentally model and predict the alterations of VNO with changes in cellular B5R/B5/Cygb levels.