SO4 = uptake and catalase role in preconditioning after H2O2-induced oxidative stress in human erythrocytes

Abstract

Preconditioning (PC) is an adaptive response to a mild and transient oxidative stress, shown for the first time in myocardial cells and not described in erythrocytes so far. The possible adaptation of human erythrocytes to hydrogen peroxide (HO)-induced oxidative stress has been here verified by monitoring one of band 3 protein functions, i.e., Cl/HCO exchange, through rate constant for SO uptake measurement. With this aim, erythrocytes were exposed to a mild and transient oxidative stress (30 min to either 10 or 100 μM HO), followed by a stronger oxidant condition (300- or, alternatively, 600-μM HO treatment). SO uptake was measured by a turbidimetric method, and the possible role of catalase (CAT, significantly contributing to the anti-oxidant system in erythrocytes) in PC response has been verified by measuring the rate of HO degradation. The preventive exposure of erythrocytes to 10 μM HO, and then to 300 μM HO, significantly ameliorated the rate constant for SO uptake with respect to 300 μM HO alone, showing thus an adaptive response to oxidative stress. Our results show that (i) SO uptake measurement is a suitable model to monitor the effects of a mild and transient oxidative stress in human erythrocytes, (ii) band 3 protein anion exchange capability is retained after 10 μM HO treatment, (iii) PC response induced by the 10 μM HO pretreatment is clearly detected, and (iv) PC response, elicited by low-concentrated HO, is mediated by CAT enzyme and does not involve band 3 protein tyrosine phosphorylation pathways. Erythrocyte adaptation to a short-term oxidative stress may serve as a basis for future studies about the impact of more prolonged oxidative events, often associated to aging, drug consumption, chronic alcoholism, hyperglycemia, or neurodegenerative diseases.

Band 3 Protein Function and Oxidative Stress in Erythrocytes from Systemic Sclerosis Patients with Interstitial Lung Disease

“…Band 3 protein is the most abundant integral membrane protein of erythrocytes [12,13], responsible for Cl -/HCO 3 exchange, which correlates with ion balance across membrane, osmotic properties, cell shape maintaining, gas exchange and, in turn, oxygenation of the entire organism [14]. These functions are mediated by both membrane and cytoplasmic domains via phosphorylation pathways, as recently shown [15,16]. Other studies have demonstrated a reversible interaction of Band 3 protein with hemoglobin and spectrin [17,18], suggesting that the efficiency of Band 3 protein reflects whole erythrocyte homeostasis.…”

Neuronal‐like differentiated SH‐SY5Y cells adaptation to a mild and transient H2O2‐induced oxidative stress

“…As cell size recovery is involved in PC response in neuronal‐like differentiated SH‐SY5Y cells, the role of ion conductances during adaptation and their use for pharmacological targeting in stroke and brain trauma could be reasonably considered. As a matter of fact, the evidence that 10μM H 2 O 2 –induced PC impacts on membrane transport systems has been recently proven by Morabito and co‐authors, reporting about the role of Band 3 protein in human erythrocytes PC. Nonetheless, this feature does not fall in the scope of the present study and can be the subject of future investigations.…”

“…Unexpectedly, MnSOD levels remained unchanged after both damage and PC, suggesting this antioxidant enzyme may be involved neither in detrimental nor protective H 2 O 2 effect at experimental conditions used here. As a matter of fact, previous investigations tried to verify whether endogenous antioxidant enzymes are key component of PC pathways . An increase in MnSOD has been detected after hypoxic PC both in vivo and in vitro, while Ravati et al showed upregulation for MnSOD but not for Cu/Zn SOD.…”

“…As a matter of fact, previous investigations tried to verify whether endogenous antioxidant enzymes are key component of PC pathways. 17 An increase in MnSOD has been detected after hypoxic PC both in vivo and in vitro, 39 aging and post-ischemic brain trauma. Hence, these outcomes may contribute to define a window for pharmacological therapies, which, hopefully, could be developed to target signal transduction pathways involved in such endogenous neuroprotective strategy.…”

“…As PC involves multiple protective mechanisms 20 that have not been completely understood on neuronal cells, the aim of the present study was to verify whether and how a different cell model, such as neuronal-like differentiated obtained-from-neuroblastoma SH-SY5Y cells, may display more sensitivity to both oxidative stress when compared with other neuronal cells 18 and to PC when preventively exposed to a mild and transient H 2 O 2 -induced oxidative stress in line with what is already demonstrated on other cell types. 4,14,17,19 To this end, H 2 O 2 concentrations lower than those used in previous investigations 18,19 and closer to a physiological range 21 have been assayed by monitoring cell viability, cell size, apoptotic (Bcl-2, Bad) and inflammatory (iNOS) pathways along with antioxidant system (MnSOD). The purpose was also to define a ceiling of H 2 O 2 concentration and a window of protection, as persistence of time of protection correlates with activation of signalling pathways, playing a critical role in PC response after in vivo ischemic episodes.…”