Plasma membrane redox system protects cells against oxidative stress

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For: Redox signaling


J.C. Rodríguez-Aguilera, G. López-Lluch, C. Santos-Ocaña, J.M. Villalba ¶ and P. Navas*

Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, Ctra. Utrera km 1, E-41013 Sevilla, SPAIN,

Departamento de Biología Celular,

Universidad de Córdoba, Córdoba, SPAIN.

* Corresponding author. Prof. Plácido Navas Laboratorio Andaluz de Biología Universidad Pablo de Olavide Carretera de Utrera, Km 1.0 41013 Sevilla, Spain. Fax: Int + 34 95 434 9238 Phone: Int + 34 95 434 9226 email: [email protected]

INTRODUCTION Aerobic organisms have developed defensive systems to survive in the presence of oxygen and its highly reactive species (ROS). The cellular mechanisms of protection against oxidative injury include specific enzymes such as catalase, glutathione peroxidase and superoxide dismutase; small hydrophilic molecules such as ascorbate, glutathione and uric acid, and hydrophobic agents such as ubiquinone and α-tocopherol in membranes 1 . Among these, coenzyme Q (CoQ) is the only lipid-soluble antioxidant that can be synthesized in all organisms so far studied. Plasma membrane shows a specific redox system (PMRS) that uses NAD(P)H as electron source and recycles oxidized antioxidants back to their reduced physiologically useful forms 2 , 3 . The essential role of coenzyme Q in this system has been previously demonstrated by genetic and biochemical evidence 4 . Here, we report that ubiquinone is a key factor in the maintenance of the plasma membrane redox status and that its levels and PMRS activity are regulated after oxidative damage induced by different agents.

MATERIAL AND METHODS. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and oxidative stress was induced by serum withdrawal as described previously 5 . Yeasts cultures were carried out as described 6 and oxidative stress induced by 2.5 mM H 2 O 2 .

Lipid peroxidation measurements and CoQ levels were determined as described 5 . Enzymatic assays for ascorbate stabilization and NADH-AFR reductase activities were determined spectrophotometrically as described 4 , 6 . Ceramide release was quantified as described by Jayadev et al. 7

RESULTS AND DISCUSSION PMRS is involved in the protection against lipid peroxidation and apoptosis induced by oxidative injury. Enrichment of whole HL-60 cells with CoQ enhanced PMRS activity measured by ascorbate stabilization and decreased lipid peroxidation (Table 1). The same antioxidant effect was found when plasma membrane liposomes were enriched with both CoQ and cytochrome b 5 reductase, an enzyme that reduces CoQ 8 . Quinone analog capsaicin inhibited (30%) PMRS activity, demonstrating the linkage between CoQ and PMRS activity. HL-60 cells undergo apoptosis after serum removal (Table 1). After 48 h of incubation in absence of serum, supplementation with CoQ maintained cell growth and decreased cell death 5 . Cells and plasma membrane liposomes enriched with CoQ impaired the apoptotic signal transduction cascade mediated by ceramide 9 (Table 1).

Also, both, the reduced and the oxidized

forms of CoQ enhanced DNA resistance to H 2 O 2 -induced damage in lymphocytes 1 0 , possibly due to activation of NAD(P)H-CoQ reductases 8 . Under oxidative stress and without CoQ supplementation, human HL-60 cells showed higher levels of PMRS activity measured as ascorbate stabilization (Fig.1). This effect was also found in wild type S. cerevisiae, whereas, in a

CoQ deficient strain, ascorbate stabilization decreases drastically after induction of oxidative stress. The CoQ deficient S. cerevisiae mutant CC301.1 did not translocate CoQ from endomembranes to plasma membrane after oxidative injury and died by a phenomenon that resembles the eukaryotic apoptosis 1 1 . However, both wild type yeast strain W303.1B and human HL-60 cells mobilized CoQ to plasma membrane (Table 1 and Fig.1). We found also a direct relationship between the PMRS activity and the levels of CoQ in plasma membrane before

and after induction. This same

phenomenon has been found in rats feed with antioxidant-deficient diets. In these rats, higher NADH-AFR reductase and NADH-CoQ reductase activity in plasma membrane is accompanied by higher levels of CoQ 9 and CoQ 1 0 and lower levels of lipid peroxidation 1 2 . So, triggering of the antioxidant machinery in plasma membrane by translocation of CoQ and the activation of PMRS seems to be a universal mechanism in eukaryotic cells. CoQ is then a key factor in the cellular mechanism of defense against oxidative damage in plasma membrane. In fact, CoQ has been linked to several oxidative stress related diseases such as Alzheimer 1 3 , Crohn 1 4 , aging 1 5 and isquemia/reperfusion 1 6 . Our results demonstrate that PMRS acts as a defensive mechanism against oxidative damage in cells. Others natural antioxidants such as α-tocopherol and ascorbate also reduce apoptosis in serum deprived cells 1 7 . Then, the maintenance of CoQ in its reduced form and its activity in the recycling of these antioxidants represents the first defensive barrier of the cell against oxidative injury. PMRS prevents lipid peroxidation and apoptosis induced by externally-initiated oxidative stress, after an increase of plasma membrane

ubiquinone content in mammalian and yeast cells. In resume, cells respond to oxidative stress by reinforcing the antioxidant capacity of their first defensive barrier located at the plasma membrane. ACKNOWLEDGEMENTS Supported by the Spanish Dirección General de Educación Superior Grant No. PB98-0329-C02-01.

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Table 1 Plasma membrane CoQ exerts antioxidant function and prevents oxidative injury and apoptosis through the PMRS. Control

CoQ enrichment

P34 enrichment

(50 μM) HL-60 cells Asc. Stabilization (nmol/min/106 cells)

0.8 ± 0.1

1.7 ± 0.1


Lipid peroxidation (units)

4.5 ± 0.5

2.2 ± 0.3


Ceramide release (pmol/nmol Pi)

42.5 ± 4.6

10.0 ± 1.6


Apoptosis (%)

35.5 ± 4.2

15.0 ± 2.1


6.6 ± 0.3

9.1 ± 0.7

10.5 ± 0.3

0.91 ± 0.03

0.42 ± 0.01

0.31 ± 0.01

Plasma membrane liposomes NADH-AFR Rase (nmol/min/mg protein) Lipid peroxidation (units)



* 1.8

Stabilized ascorbate (nmol/min/106 cells)

1.6 1.4

(126) (128)

Control Oxidative damage

* (71)

1.2 1.0 (ND)

0.8 0.6 0.4 0.2

(ND) 0.0 HL-60


CC301.1 S. cerevisiae

Cell lines

Fig. 1. Plasma membrane redox system activity changes after oxidative stress. Serum removal (HL-60) or 2.5 mM H2O2 (S. cerevisiae) were used as oxidative stress models. Data in brackets indicate CoQ levels in plasma membrane in each respective case. A direct relationship can be seen between CoQ levels and PMRS activity before and after oxidative stress induction. Data represent the mean of three independent experiments performed in duplicate. * Significative differences vs. respective control, p
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