Role of Heme metabolism in the oxidative state of NO‐receptor soluble guanylyl cyclase (sGC) in aging mouse brain

Abstract

IntroductionAging is an important risk factor for cardiovascular diseases and neurodegeneration. Heme synthesis and metabolism decline with age. Heme moiety is essential for proper function of the Nitric Oxide receptor soluble guanylyl cyclase (sGC). Evidence suggests the involvement of NO/cGMP signaling in human neurological disorders. sGC also an important role in maintenance of cerebral blood flow, vascular homeostasis and platelet function. NO binds to the ferrous heme moiety of sGC and activates cGMP‐forming activity hundred fold. Therefore, the integrity of reduced sGC heme moiety is essential for proper transduction of NO signal.Methods and ResultsWe investigated if the oxidative state and the relative content of sGC heme moiety changes with age in mouse brain tissue. Three groups of animals have been used in our studies: young, 3.5 months old, and mature, 15 months old, and old, 24 months, C57Bl6 mice. sGC‐dependent synthesis of cGMP in protein homogenates from mouse brains in response to sGC regulators was evaluated by quantifying the amount P32‐labeled cGMP using thin layer chromatography. To estimate the changes in the amount of heme‐deficient sGC, protoporphyrin IX (PPIX) was used as a heme‐independent sGC activator. NO‐independent activator BAY 58‐2667, which has a higher affinity for sGC with ferric heme, was used to evaluate the changes in the amount of sGC with oxidized heme. NO donor DEA‐NO was used to evaluate the level of sGC with ferrous heme. For each individual sample the ratios of sGC‐[HemeFe3+] vs sGC‐[HemeFe2+] activities (BAY 58‐2667/DEA‐NO) and heme‐deficient vs sGC‐[HemeFe2+] activities (PPIX/DEA‐NO) were determined. Our analysis demonstrated that the relative amount of heme‐deficient and heme‐oxidized sGC increased in both males and females of older age. Decline in sGC protein expression in oldest animal groups was detected by Western blot analysis. We also investigated if the accumulation of heme‐deficient sGC or sGC with ferric heme in ageing brain correlates with changes in the level of proteins involved in iron and heme metabolism. We found no difference in the level of HSP90 chaperone needed for sGC heme insertion and unchanged level in the iron chaperone PCBP2, which is essential for iron trafficking. The expression of divalent metal ion transporter (DMT1) was also not affected by age. However, we observe a decrease in the expression of Heme Oxygenase 1 (HO1) and Aconitase 2 proteins involved in maintenance of cellular oxidative balance in old animals of both genders.ConclusionOur data indicate that changes in heme homeostasis might contribute to the decline of NO/sGC in brain with aging.