Uysal, Murat Giris, Feti Tuiubas,
Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul
University, Capa 34093, Istanbul, Turkey
Supported by the Istanbul University Research Foundation, No.
Correspondence to: Necla Kocak-Toker, Department of
Biochemistry, Istanbul Faculty of Medicine, Istanbul University,
Capa 34093, Istanbul, Turkey. firstname.lastname@example.org
(ONOO) is a powerful oxidant shown to damage membranes. In the
present study, the effect of taurine on changes of liver plasma
induced by ONOO was investigated.
plasma membrane was exposed to ONOO
with or without taurine. Na+,
activity and lipid peroxidation as thiobarbituric acid reactive
substances (TBARS) levels were measured.
Different concentrations of ONOO (100, 200, 500, and 1 000 mmol/L)
were found to decrease liver plasma membrane Na+,
activity significantly. The depletion of enzyme activity was not
concentration dependent. Effects of different concentrations of
taurine on liver plasma membrane Na+,
activity were also measured. Taurine did not cause any increase in
enzyme activity. When plasma membranes were treated with 200 mmol/L
ONOO with different concentrations of taurine, a restoring effect of
taurine on enzyme activity was observed. TBARS levels were also
measured and taurine was found to decrease the elevated values.
Taurine is observed to act as an antioxidant of ONOO
to decrease lipid peroxidation and thus
affect liver plasma membrane Na+,
by restoring its activity.
The WJG Press and Elsevier Inc. All rights reserved.
Key words: Na+,
Kocak-Toker N, Giris M, Tuiubas
F, Uysal M, Aykac-Toker G. Peroxynitrite induced decrease in Na+,
activity is restored by taurine. World J Gastroenterol
2005; 11(23): 3554-3557
Taurine (2-aminoethanesulfonic acid) is present as a free amino acid
in mammalian tissues like liver, heart, brain, and leukocytes.
Although it does not take part in protein structure, it has a
pivotal role in maintenance of various cellular functions like
osmoregulator, neuromodulator, and membrane stabilizer.
Besides its hypolipidemic and antiatherosclerotic effects, it is
proved to be an effective antioxidant[3-6].
ONOO is a powerful oxidant that can be formed by
the reaction of superoxide anion and nitric oxide in significant
amount in vivo. It has been shown to be associated with both
beneficial and harmful effects. Its contribution to the host-defense
response to bacterial invasion is known to be due to its production
by neutrophils and macrophages. On the other hand, the role of ONOO
in ischemia-reperfusion has been
implicated to cause myocardial dysfunction and infarction. ONOO can react with DNA leading to mutations and strand
breaks. It can cause oxidation of thiol groups, proteins and low
molecular antioxidants and induce lipid peroxidation.
It is also known to nitrate macromolecules to contribute to various
is a plasma membrane-bound enzyme that provides the necessary
electrochemical gradients of Na+
to maintain the cell volume and thus it plays a crucial role in
It functions by exporting intracellular Na+
and importing extracellular K+
across the plasma membrane to provide energy for
membrane transport of various metabolites taking part in
special cell functions. Therefore, dysfunction of the enzyme may
lead to severe consequences.
has been known to be a good target of free radical induced membrane
Lipid peroxidation induced by free radicals has been shown to
inactivate the enzyme by particularly modifying the active site for
binding of the substrates.
ONOO has also been known to disrupt membrane fluidity and decrease
activity by oxidation of its thiol groups and probably partially due
to nitration of its aromatic amino acids[12,13].
In this study we investigated the effects of
taurine on changes of liver plasma membrane Na+,
induced by ONOO.
MATERIALS AND METHODS
Bovine liver obtained from slaughterhouse from freshly slaughtered
animal was rapidly rinsed with ice-cold physiological saline and
frozen in liquid nitrogen. It was stored at -80 ℃.
All reagents used were analytically pure.
Liver plasma membrane preparation
Frozen liver portions were homogenized in ice-cold buffer containing
8% saccharose, 0.1 mmol/L phenylmethane sulfonyl fluoride, 1 mmol/L
EDTA and 30 mmol/L imidazole-HCl, pH 7.4.
The homogenate was centrifuged at 120 r/min for 5 min, 6 800 r/min
for 15 min and 48 000 g for 30 min using Sorvall centrifuge
with AH-650 rotor. The obtained pellet was resuspended in 8%
saccharose and 30 mmol/L imidazole-HCl, pH 7.4 and stored at -80 ℃
Five milliliters of 0.6 mol/L NaNO2
and 5 mL 0.6 mol/L H2O2
in 0.7 mol/L HCl were filled in two syringes separately.
They were immersed in ice for about 30 min. A beaker containing 5 mL
1.2 mol/L NaOH solution with a magnetic stirrer was also cooled on
ice. The syringes, after being cooled, were held with a T-piece
above the NaOH solution that was in ice. Both plungers were rapidly
pressed down at the same time. Excess H2O2
was removed by using granular MnO2
(2 g) at 4 ℃.
Concentration of ONOO was determined by measuring the absorbance at
302 nm using the extinction coefficient of 1 670/M.cm.
ONOO solution was kept at -80 ℃.
Preparation of decomposed ONOO–
Samples of the ONOO solution were allowed to decompose overnight in
imidazole-HCl buffer to control the effect of decomposition
products, nitrite and nitrate, and H2O2.
Treatment of liver plasma membrane with ONOO and taurine
One hundred microliters of plasma membrane samples (30 mg protein)
were incubated with 5 mL of 100, 200, 500, and 1 000 mmol/L
ONOO solutions at room temperature. The incubations were done with
decomposed ONOO as well. Following incubations, membrane Na+,K+-ATPase
activity and thiobarbituric acid reactive substances (TBARS) levels
One hundred microliters of plasma membrane
samples (30 mg protein) were incubated with taurine (1, 2, and 5
mmol/L) and 200 mmol/L ONOO (5 mL) plus 10 mL of taurine (1, 2, and
5 mmol/L). Following incubations, membrane Na+,
activity and TBARS levels were measured.
Assay of Na+,
Enzymatic activity was measured in triplicate by the inorganic
released from ATP in the presence or absence of 1 mmol/L ouabain.
Membrane preparations (20 mg)
were added to the medium containing 150 mmol/L NaCl, 5 mmol/L KCl,
2.5 mmol/L MgCl2
and 20 mmol/L imidazole-HCl buffer, pH 7.4. After 8 min of
preincubation at 37 ℃,
2.5 mmol/L ATPNa2
was added to make the final volume of 0.5 mL and to start the
reaction. The samples were incubated at 37 ℃
for 30 min. The reaction was stopped by the addition of 100 mL of
35% ice-cold trichloroacetic acid. The amount of liberated Pi
was measured in the supernatant by using FeSO4-ammonium
molybdate solution. The mixtures were kept for 5 min in the dark and
the absorbances were measured at 700 nm.
Determination of lipid peroxidation
The level of lipid peroxidation was assessed by the determination of
Following incubation with ONOO, membrane samples were reacted with
TBA to yield a pink colored product. Absorbances were measured at
532 nm and the amount of TBARS was calculated by using the
extinction coefficient of 1.56105/M.cm.
Protein determinations were done by the method of
Lowry et al., using bovine serum albumin as a standard.
Ten experiments were performed separately. All results were
expressed as mean±SD.
Statistically significant differences between groups were analyzed
by one-way analysis of variance (ANOVA) followed by Tukey's
honestly significant difference
post hoc test (THS test).
Effect of ONOO
on liver plasma membrane Na+,
When plasma membrane was treated with 100, 200, 500, and 1 000 mmol/L
ONOO solutions, significant depletion of enzyme activity was
observed with all ONOO
concentrations (all P<0.05).
with a concentration above 200 mmol/L
produced no additional decreasing effect (Figure 1). Plasma membrane
samples were also incubated with decomposed ONOO. This incubation
was made to eliminate the effect of decayed products and
contaminants. Decomposed ONOO solutions were also found to cause
significant decreases in enzyme activity (P<0.05).
(PDF) Effects of decomposed ONOO
and ONOO on liver plasma membrane Na+,
activity. Values are mean±SD,
n = 10. Values not sharing a common superscript letter are
significantly different by ANOVA (THS test), aP<0.05
vs controls. Control (black), decomposed ONOO-
(gray lines), ONOO (white lines).
Effect of taurine on liver plasma membrane Na+,
The effect of taurine on Na+,
is shown in Table 1. After incubation of liver plasma membrane with
1, 2, and 5 mmol/L taurine, Na+,
activities were unchanged.
Table 1 Effect
of taurine on liver plasma membrane Na+,
activity (values are mean±SD, n = 10)
activity (nmol Pi/mg
taurine on ONOO induced inhibition of liver plasma membrane Na+,
Since ONOO at concentration >200 mmol/L
has not caused additional depletion in enzyme activity, 200 mmol/L
was chosen to observe the effect of different concentrations of
taurine on the change in enzyme activity (Table 2). With all chosen
taurine concentrations, significant activity increases from the
depleted values by decomposed ONOO and ONOO were observed (all P<0.05).
Table 2 Effects of
200 mmol/L decomposed ONOO and ONOO-
with and without taurine on liver plasma membrane Na+,
activity (values are mean±SD,
n = 10)
activity (nmol Pi/mg
ONOO (200 mmol/L)
ONOO (200 mmol/L)
+taurine (1 mmol/L)
ONOO (200 mmol/L)
+taurine (2 mmol/L)
ONOO (200 mmol/L)
+taurine (5 mmol/L)
sharing a common superscript letter are significantly different by
ANOVA (THS test), aP<0.05
Effect of taurine on liver plasma membrane lipid peroxidation
TBARS levels were measured when liver plasma membrane was incubated
with 200 mmol/L
decomposed ONOO and 200 mmol/L
ONOO with or without taurine (1, 2, and 5 mmol/L) (Table 3). The
elevation of lipid peroxide levels was decreased with taurine in a
concentration-dependent manner (P<0.05).
Table 3 Effects of taurine, 200 mmol/L
with and without taurine on liver plasma membrane. TBARS levels
(values are mean±SD, n = 10)
(nmol MDA/mg Pr)
sharing a common superscript letter are significantly different by
ANOVA (THS test), aP<0.05
In this study, ONOO at 200 mmol/L
was found to significantly decrease Na+,
activity, when liver plasma membrane was treated with different
concentrations of ONOO. Increasing ONOO concentration did not appear
to cause an additional decreasing effect. In our previous works,
human sperm Na+,
located in plasma membrane was also found to be significantly
decreased when treated with 100 mmol/L
with increasing lipid peroxide levels and decreasing total
Indeed, ONOO releasing agents like 3-morpholinosy-dnonimine (SIN-1)
have been found to inhibit Na+,K+-ATPase
activity by interacting with a sulfhydryl group at the active site.
Incubation of liver plasma membrane with SIN-1 reduced Na+,
activity and it was suggested that ONOO caused inhibition both by
oxidizing thiol groups and in part by decreasing membrane fluidity.
Rat erythrocyte membrane Na+,
was also found to be inhibited by ONOO, which was considered to be
compatible with oxidation of thiol groups either directly by being
involved in ATP binding or of those located outside the active site
being important for enzyme activity.
Taurine has been found to restore depletion of
activity due to ozone exposure or cholesterol enrichment, thus
considered both as an antioxidant to prevent lipid peroxidation and
as a membrane stabilizer to maintain the environment for Na+,
to function properly.
Glucose-induced lipid peroxidation and protein glycosylation have
been lowered and erythrocyte Na+,
activities preserved by taurine treatment, implicating the
inhibition of development of diabetic complications.
Chronic taurine supplementation has also been proved to decrease
lipid peroxidation and preserve retinal Na+,
activity in diabetic rats.
The scavenging activity of taurine against
superoxide anion and peroxides is under debate[24-27].
There are studies suggesting the absence of a direct reaction of
taurine and oxygen-derived radicals, as well as taurine protection
of hepatocytes against H2O2-stimulated
While protection provided by taurine against reactive oxygen species
has been assumed as an indirect action, its reaction with
hypochlorous acid is clearly shown.
In the study of Redmond et al., however, treatment of
cultured hepatocytes with 4 mmol/L taurine has been found to reduce
apoptosis and necrosis by inhibiting nitric oxide and oxy radicals
due to suppression of nitric oxide synthase mRNA.
Likewise in another study, it has been demonstrated that 1 mmol/L
taurine could not reduce ONOO formation by SIN-1 treatment of
cerebellar granular cells.
Indeed in the study of Mehta and Dawson, taurine at concentrations
above 30 mmol/L has been shown to be only a modest scavenger of ONOO
produced from SIN-1.
Our study suggests that in vitro taurine
treatment can protect liver plasma membrane against oxidative damage
caused by ONOO, by acting as an antioxidant and thus contribute to
restoring of Na+,
GB, Park E. Taurine:new implications for an old amino acid. FEMS
Microbiol Lett 2003; 226: 195-202
HH. The protective effect of taurine against cyclosporine
A-induced oxidative stress and hepatotoxicity
in rats. Toxicol Lett 2004; 151:
J, Kanbagll , Hatipoglu A, Kucuk
U, Aykac-Toker G, Uysal M. Improving effect of dietary
supplementation on the oxidative stress and lipid levels in the
plasma, liver and aorta of rabbits fed on
high-cholesterol diet. Biosci Biotechnol Biochem 2002; 66:
S, Aykac-Toker G, Uysal M. Effects of added dietary taurine on
erythrocyte lipids and oxidative
in rabbits fed a high
cholesterol diet. Biosci Biotechnol Biochem 2002; 66:
J, Dogru-Abbasoglu S, Kanbagll , O
U, Ayka-Toker G, Uysal M. Taurine has a protective effect
thioacetamide-induced liver cirrhosis by decreasing oxidative
stress. Hum Exp Toxicol 2001; 20: 251-254
J, Kanbagli , Aykac-Toker G, Uysal M. Taurine treatment reduces
hepatic lipids and oxidative stress in
ethanol-treated rats. Biol Pharm Bull 2002; 25:
J. Physiological effects of peroxynitrite. Circ Res 2000;
H, Al-Mehdi AB. Peroxynitrite-mediated oxidative protein
modifications. FEBS Lett 1995; 364: 279-282
G, Hasler U, Beggah AT, Yu C, Modyanov NN, Horisberger JD, Lelievres
L, Geering K. Transport
pharmacological properties of nine different human Na,K-ATPase
isozymes. J Biol Chem 2000; 275: 1976-1986
M, Stark G, Apell HJ. Effects of free radicals on partial
reactions of the Na,K-ATPase. J Membrane Biol
1997; 156: 63-71
OP, Delivoria-Papadopoulos M, Cahillane G, Wagerle LC. Lipid
peroxidation as the mechanism of modification
affinity of the Na+,K+-ATPase
active sites for ATP, K+,Na+,
and strophanthidin in vitro. Neurochem Res
P, Sandoval G. Nitric oxide and peroxynitrite anion modulate
liver plasma membrane fluidity
activity. Nitric Oxide 2000; 4: 333-342
G, Ayka-Toker G. The role of Na,K-ATPase in human sperm motility. Int
J Androl 2002;
S, Gerbi A, Duran MJ, Benkoel L, Pierre S, Lambert R, Dodero F,
Chamlian A, Vague P, Maixent JM.
quantitative immunocytochemical study of Na+,K+-ATPase
in rat hepatocytes after STZ-induced diabetes and
fish oil supplementation. J Histochem Cytochem 1999; 47:
JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of
peroxynitrite. Method Enzymol 1994;
H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues
by thiobarbituric acid reaction. Anal Chem
OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with
the Folin phenol reagent. J Biol Chem
Kervancloglu E, Kocak
N, Ayka-Toker G. In vitro effects of peroxynitrite
human spermatozoa. Andrologia 1999; 31: 195-198
T, Kamata Y, Irifune M, Nishikawa T. Inhibition of purified (Na+,K+)-ATPase
activity from porcine cerebral cortex
generating drugs. Brain Res 1995; 704: 117-120
P, Castaneda G, Ortega M, Noel F. Insights into the mechanism of
oxide and peroxynitrite anion. J Appl Toxicol 2003; 23:
21 Qi B,
Yamagami T, Naruse Y, Sokejima S, Kagamimori S. Effects of
taurine on depletion of erythrocyte membrane
ATPase activity due to ozone exposure or cholesterol enrichment. J
Nutr Sci Vitaminol 1995; 41: 627-634
TA, Anuradha CV. Inhibition of lipid peroxidation, protein
glycation and elevation of membrane ion
activity by taurine in RBC exposed to high glucose. Clin Chim
Acta 2003; 336: 129-135
di MAS, Santini SA, Cercone S, Lepore D, Silveri NG, Caputo S,
GrecoAV, Giardina B, Franconi F, Ghirlanda
Chronic taurine supplementation ameliorates oxidative stress and Na+K+ATPase
impairment in the retina of
rats. Amino Acids 2002; 23: 401-406
S, Azuma J, Takahashi K, Mozaffari M. Why is taurine
cytoprotective? Adv Exp Med Biol 2003; 526: 307-321
PK, Lau-Cam CA. Protection by taurine and structurally related
sulfur-containing compounds against
membrane damage by hydrogen peroxide. Adv Exp Med Biol 2000; 483:
I, Nakamura T, Ogasawara M, Nemoto M, Yoshida T. The protective
effect of taurine on biomembrane
damage produced by the oxygen radical. Adv Exp Med Biol 1992;
T, Ogasawara M, Koyama I, Nemoto M, Yoshida T. The protective
effect of taurine on the
against damage produced by oxygen radicals. Biol Pharm Bull
1993; 16: 970-972
K, Koyama I, Nakamura T, Yoshida T, Umeda M, Inoue K.
Effectiveness of taurine in protecting
against oxidant. Chem Pharm Bull 1990; 38:
HP, Wang JH, Bouchier-Hayes D. Taurine attenuates nitric oxide-
and reactive oxygen
dependent hepatocyte injury. Arch Surg 1996; 131:
AA, Jonhson P, Wei Y, Tan Y, Carpenter DO. Carnosine and taurine
protect rat cerebellar granular cells
free radical damage. Neurosci Lett 1999; 263:
TR, Dawson R Jr. Taurine is a weak scavenger of peroxynitrite
and does not attenuate sodium
toxicity to cells in culture. Amino Acids 2001; 20:
Editor Zhu LH and Guo SY Language
Editor Elsevier HK