Abstract
Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.
Highlights
Heme oxygenase (HO), residing at the endoplasmic reticulum membrane, is the rate-limiting enzyme in the degradation of heme, yielding equivalent amounts of carbon monoxide (CO), ferrous iron (Fe2+), and biliverdin (BV)
In the first part of the study we focused on the improvement of the HO assay, since determination of HO activity using BR quantification was compromised by higher protein concentrations [19]
Are components exclusively formed by the HO/BV reductase (BVR) system and generally tissue or cell homogenates possess sufficient BVR activity assuring the complete conversion of BV to BR and allowing the determination of BR by means of HPLC [20] or by photo spectroscopy [15,17]
Summary
Heme oxygenase (HO), residing at the endoplasmic reticulum membrane, is the rate-limiting enzyme in the degradation of heme, yielding equivalent amounts of carbon monoxide (CO), ferrous iron (Fe2+), and biliverdin (BV). Determination of HO activity by means of BR quantification following the classical photometric extraction assay [15,16,17] is sensitive to higher protein concentrations making direct comparison between different sample types difficult Improvements of these assays should take the high affinity of lipophilic BR to proteins into account. Increased levels of BR are formed during enhanced HO activity [5] and may target intracellular structures It is not clear whether such an interaction would contribute to protective effects of the HO reaction or whether it may compromise cellular function and thereby limit the cytoprotective properties of the HO reaction. (6) Which subcellular structure in the hepatocyte is sensitive to increased levels of BR?
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
