Effect
of functional pinealectomy on hippocampal lipid peroxidation,
antioxidant enzymes and N-methyl-D-aspartate receptor subunits
2A and 2B in young and old rats
Namik
Delibas, Nalan Tuzmen, Zafer Yonden &
Irfan Altuntas
Suleyman
DemirelUniversity,
School of Medicine, Department of Biochemistry and Clinical
Biochemistry, Isparta, Turkey.
Key
words:
melatonin; lipid peroxidation; antioxidant enzymes; NMDA
receptors; rat
Submitted:
July 29, 2002
Accepted: July 30, 2002
ABSTRACT
OBJECTIVES:
To investigate the effects of pinealectomy on lipid peroxidation,
antioxidant status and NMDA receptor subunits 2A and 2B concentrations
in hippocampus.
DESIGN:
Forty-eight male Wistar-albino rats were used.
SETTINGS:
Animals were divided into three groups: 24 h dark throughout
the study (highest melatonin release), 24 h light exposure
(light-induced functional pinealectomy) and 12 h light/12
h dark exposure (control group). Thereafter, each group was
divided into two groups as young and old animals.
RESULTS:
There was an increase in NR2A and NR2B concentration in DY
group compared to all other treatments. CuZn and Mn SOD activities
were found to be increased in CO compared to CY group. Continual
light exposure for 4 weeks did not change neural CuZn and
Mn SOD activities. In old rats, light exposure reduced the
activities of both CuZn and Mn SOD relative to those in the
young animals. In addition, CuZn and Mn SOD activities were
higher in dark exposed rats than in those in the continual
light exposed or LD 12:12 rats. GSH-Px activity was found
elevated in the DY rats compared to the CY groups. MDA levels
were significantly higher in the CO than in the CY group.
CONCLUSIONS:
NR2A and NR2B receptor concentrations in hippocampus of the
rats maintained in dark showed significant increases compared
to the control and functional pinealectomy groups but there
was no significant increase in lipid peroxidation.
ABBREVIATIONS
ROS – Reactive oxygen species
NMDAR – N-methyl D-aspartat receptors
CNS – Central nervous system
NR2A – NMDA receptor subunit 2A
NR2B – NMDA receptor subunit 2B
Mn SOD – Mn superoxide dismutase
CuZn SOD – CuZn superoxide dismutase
MDA – Malondialdehyde
CAT – Catalase
GSH – Glutathione
GSH-Px – Glutathione peroxidase
NR1 – NMDA receptor subunit 1
NR2 – NMDA receptor subunit 2
BCIP/NBT – 5-bromo-4-chloro-3-indolyl phosphate/nitroblue
tetrazolium
EGTA – Ethylene glycol-bis (b-aminoethyl ether)-
N,N,N',N'-tetraacetic acid
PVDF – Polyvinylidene difluoride
CY – Control young
CO – Control old
LY – Light young
LO – Light old
DY – Dark young
DO – Dark old
PBS – Phosphate buffered saline
EDTA – Ethylenediaminetetraacetic acid
SDS/PAGE – Sodium dodecyl sulfate/polyacrylamide gel electrophoresis
TBST – Tris-buffered saline with Tween 20
BSA – Bovine serum albumin
LTP – Long-term potentiation
O.– – Superoxide
Introduction
Of
all the organs in the body, the central nervous system (CNS)
takes more than its share of oxidative abuse. The major reasons
for this are its high utilization of O2, its relatively poorly
developed antioxidant network, and the fact that it contains
large amounts of easily oxidizable fatty acids [1]. Antioxidant
defense system becomes insufficient in advanced ages and results
in various diseases and symptoms of aging [2]. Melatonin is
a secretory product mainly synthesized by the pineal gland and
secreted primarily at night, when blood levels reach levels
10 times higher than those present in the daytime [3]. Melatonin
is a well-known antioxidant that protects DNA, lipids, and proteins
from free radical damage. Melatonin not only scavenges free
radicals such as superoxide radicals and hydroxyl radicals,
but also activates some antioxidant enzymes such as catalase
(CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase
(SOD) [1,3,4]. Furthermore, melatonin has been reported to increase
mRNA levels for SOD [5]. Since endogenous melatonin levels fall
markedly in advanced ages, the loss of this antioxidant may
contribute to the incidence or severity of some age-associated
neurodegenerative diseases [6].
The N-methyl-D-aspartate (NMDA) receptor is a member of the
group ionotropic glutamate receptors. The NMDA receptor is a
heteromeric protein composed of two classes of subunits, NR1
and NR2. Four separate genes encode NR2 subunits, NR2A to NR2D.
While NR2 subunits cannot form functional channels when expressed
alone, they can alter NMDAR channel properties when complexed
with NR1 subunits [7]. This receptor is involved in a wide variety
of processes in the central nervous system (CNS) including synaptogenesis
and synaptic plasticity. Additionally, the NMDA receptor has
been implicated in excitotoxicity, neurodegenerative disorders
and aging [8–11]. Thus, a greater understanding of the
modulation of this receptor is likely to be important to the
understanding of the physiology and pathophysiology of these
processes.
Though there are many reports regarding the effect of melatonin
on lipid peroxidation and antioxidant enzymes [1,3–5],
the effects of continuous light (low melatonin) and dark (high
melatonin) on NMDA receptor concentration in the hippocampus
is lacking. Also, it is not known how NMDA receptor concentrations
in hippocampi of young and old animals change in response to
light and dark. Hence, we have undertaken this study to investigate
lipid peroxidation, antioxidant status and NMDA receptor subunits
2A and 2B concentrations in hippocampus of young and old rats
exposed to 24h light (functional pinealectomy), 24 h dark and
12h light/12h dark for 4 weeks.
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