Full text of DOI:10.1093/gerona/56.8.B350

Journal of Gerontology: BIOLOGICAL SCIENCES
Copyright 2001 by The Gerontological Society of America
2001, Vol. 56A, No. 8, B350–B355
Gene Expression of Cyclooxygenase in the
Aging Heart
Jung Won Kim,1 Bong Sook Baek,1 Yun Kyung Kim,1 Jeremiah T. Herlihy,2 Yuji Ikeno,2
Byung Pal Yu,2 and Hae Young Chung1
1Department of Pharmacy, Pusan National University, Korea.
2Department of Physiology, the University of Texas Health Science Center, San Antonio.
Cyclooxygenase (COX) is the key rate-limiting enzyme in the prostaglandin synthetic pathway.
Two isoforms of COX have been identified: a constitutive COX-1 and an inducible COX-2,
which is activated in response to various stimuli. We investigated the changes of COX-1 and
COX-2 in rat heart during aging. We measured the age-related changes in the mRNA and pro-
tein levels of COX by using reverse-transcription polymerase chain reaction and Western blot-
ting, respectively. COX-2 mRNA and protein levels increased with age, whereas those of COX-1
showed no change. The COX activity determined by prostaglandin E2 production increased
with age. Because the COX-catalyzed arachidonate cascade is an important source of reactive
oxygen species (ROS) generation, changes in ROS generation and lipid peroxidation were also
assessed. The amount of ROS generated by the COX pathway increased with age, as did the to-
tal ROS generation and lipid peroxidation. These results show that COX-2 activity increases
with age, partially because of elevated transcriptional expression and protein content, and they
suggest that increased COX-2 can play a role in oxidative alterations in the aged heart.
HE oxidative stress theory of aging proposes that the
progressive deterioration and time-associated changes
Previous work from this laboratory (15) and others (16)
showed that aging leads to elevated oxidative damage in the
heart. The source and oxidative processes of the ROS have
not been defined. One possibility that has not been investi-
gated is that an age-related activation of COX may contrib-
ute to the appearance of oxidation stress in the aging heart.
The aim of the present study was to determine whether
changes occur in the COX activity of the two isoforms that
could contribute to the age-related oxidative damage to the
heart.
T
observed during aging are the cumulative result of contin-
ual reactive oxygen species (ROS) production occurring
in the course of normal cellular metabolism (1,2). Under
normal conditions, the mitochondria are considered the
major loci for free radical production, because most of the
oxygen-derived species arise during mitochondrial respi-
ration (3). Although less explored, the arachidonate cas-
cade, in which cyclooxygenase (COX) is the key rate-lim-
iting enzyme, could be a potentially important intracellular
source of ROS, particularly under various pathological
conditions (4).
Prostaglandins (PGs), produced by diverse cell types,
modulate a variety of physiological and pathophysiological
processes (5). COX is the key rate-limiting enzyme in the
conversion of arachidonic acid to PGs (6). COX exists in
two isoforms: cyclooxygenase-1, or COX-1 (7–9), a consti-
tutive isoform located in the endoplasmic reticulum that is
responsible for normal prostaglandin production, and a mi-
togen-inducible cyclooxygenase-2, or COX-2, located both
in the endoplasmic reticulum and in the nuclear membrane
(10). COX-2 is expressed highly upon appropriate stimula-
tion with proinflammatory agents, such as lipopolysaccha-
rides (11) and cytokines (12).
MATERIALS AND METHODS
Tissue Preparation
Hearts from male, specific pathogen free, Fischer 344
rats that were raised in the barrier facilities at the Depart-
ment of Physiology in San Antonio, TX, were used in the
study. Complete descriptions of the housing, care, and feed-
ing of the animals have been reported elsewhere (17). At 6,
12, 18, and 24 months of age, the rats were decapitated,
their chests were opened, and their hearts were quickly ex-
cised and immersed in ice-cold isotonic saline. Six rats per
age group were used in the study. The atria were removed.
The ventricles were rapidly frozen between aluminum tongs
cooled to the temperature of liquid nitrogen and stored at
that temperature until assayed.
The arachidonate cascade catalyzed by COX contributes
to ROS generation in two ways: first, oxygen radical gener-
ation during the catalytic conversion of prostaglandin G2 to
H2 (13) and second, PG-facilitated oxidative alterations of
the inflammatory process (14).
The ventricles were individually homogenized with a
polytron homogenizer in 7 vol (vol/wt) of ice-cold homoge-
nization solution of the following composition: 50 mM
phosphate buffer (pH 7.4), 0.5 mM phenylmethylsulfonyl
B350

CHANGES IN CYCLOOXYGENASE IN THE AGING HEART
B351
fluoride (PMSF), 1 mM ethylenediamine tetra-acetic acid
(EDTA), 80 mg/l of trypsin inhibitor, and 1 ꢀM leupeptin.
In order to obtain a mitochondrial fraction, the homogenate
was centrifuged at 900ꢁ g for 15 minutes at 4°C, and the
supernatant was centrifuged at 12,000ꢁ g for 15 minutes at
4°C. The supernatant was regarded as a postmitochondrial
fraction, and the pellet was resuspended in homogenization
buffer as a mitochondrial fraction.
was discarded. Ethanol (75%) was added to the pellet and
the mixture was centrifuged for an additional 5 minutes.
The supernatant was discarded; the pellet was dried for 20
minutes and then dissolved in DEPC-treated water.
Polymerase chain reaction.—The primer pairs for COX-2
(19) were as follows: sense, 5ꢄ-CAAGCAGTGGCAAAG-
GCCTCCATT-3ꢄ; antisense, 5ꢄ-TAGTCTGGAGTGGGA-
GGCACTTGC-3ꢄ. For COX-1, the primer pairs were as
follows: sense, 5ꢄ-CTGCATGTGGCTGATGTCATC-3ꢄ; an-
tisense, 5ꢄ-AGGACCCGTCATCTCCAGGGTAATC-3ꢄ. The
expected product sizes for COX-1 and COX-2 are 441 and
474 base pairs (bp), respectively.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
primers were used in separate polymerase chain reactions to
control for the efficiency of cDNA synthesis in each sam-
ple. The primer pairs were as follows: sense, 5ꢄ-GGGT-
GATGCTGGTGCTGAGTATGT-3ꢄ; antisense, 5ꢄ-AAGA-
ATGGGAGTTGCTGTTGAAGTC-3ꢄ. The GAPDH primer
set yields a polymerase chain reaction (PCR) product of 700
bp in length.
Cyclooxygenase Activity
The COX activity was measured in the postmitochondrial
fraction. Forty microliters (ꢀ0.3 mg of protein) of the su-
pernatant fraction were incubated in the presence of 0.6 ꢀM
arachidonate at 37°C for 30 minutes. At the end of the incu-
bation period, the mixture was boiled for 3 minutes and cen-
trifuged for 15 minutes at 10,000ꢁ g. The prostaglandin E2
content of the supernatant was assayed by using a commer-
cially available enzyme immunoassay kit (Amersham,
Bucks, UK) (18).
Reverse-Transcription Polymerase Chain Reaction
As a way to carry out the PCR, 25 ꢀl of PCR master mix
was added to each tube directly. Fifty nanograms of sense
primer and 35 ng of antisense primer were used per reac-
tion. Deoxynucleotides were added to a final concentration
of 0.2 mM; 10ꢁ PCR buffer (Perkin Elmer, Gaithersburg,
MD) was diluted to a ratio of 1:10. Taq DNA polymerase
(Promega, Madison, WI; 1.25 U) and 32P-labeled primer
(0.25 ꢀl) was added to each tube. Reaction conditions con-
sisted of 38 cycles for rat COX-2, 33 cycles for rat COX-1,
and 20 cycles for GAPDH of 94°C for 30-second denatur-
ation, 54°C for 30-second annealing, and 72°C for 1-minute
extension. Electrophoresis was performed in 5% polyacryl-
amide gel. After suitable separation was achieved, the gel
was vacuum dried for autoradiography and exposed to Fuji
x-ray film.
Preparation of RNA.—RNA samples were isolated from
three rat tissues per each age. Tissue samples were homoge-
nized in the presence of RNAzol B (2 ml/100 mg of tissue;
Tel-Test, Inc., Friendwood, TX) with a Polytron homoge-
nizer. Chloroform (0.2 ml) was added to 2 ml of homoge-
nate. The samples were covered tightly, shaken vigorously
for 15 seconds, and placed on ice for 5 minutes. The suspen-
sion was centrifuged at 12,000ꢁ g at 4°C for 15 minutes.
The aqueous phase was transferred to a fresh tube, an equal
volume of isopropanol was added, and the sample was
cooled to 4°C for 15 minutes. Samples were centrifuged a
second time as above. The supernatant was removed and the
RNA pellet was washed once with 75% ethanol by vortex-
ing. After a final centrifugation at 7500ꢁ g at 4°C for 8
minutes, the pellet was dried for 10–15 minutes and dis-
solved in diethylpyrocarbonate (DEPC)-treated water.
Western Blotting
Reverse transcription.—The first strand cDNA was syn-
thesized from 2 ꢀg total RNA. DEPC-treated water and 250
ng of random primer were added, and the mixture was incu-
bated at 75°C for 5 minutes. The mixture was placed on ice
for 5 minutes. Aliquots containing 2 ꢀl of 0.1 M dithiothre-
itol (DTT), 4 ꢀl of 5ꢁ buffer, 4 ꢀl of 2.5 mM deoxynucleo-
side triphosphate (dNTP), 100 U of reverse transcriptase
(Gibco-Bethesda Research Laboratory, Gaithersburg, MD)
and 16.5 U of ribonuclease (RNase) inhibitor were added
and incubated at 37°C for 2 hours. The reaction was stopped
by boiling at 100°C for 2 minutes, and the cDNA was stored
at ꢂ20°C until use.
Samples were boiled for 5 minutes with gel loading
buffer, consisting of 0.125 M Tris-Cl, 4% sodium dodecyl
sulfate (SDS), 10% 2-mercaptoethanol, pH 6.8, and 0.2%
Bromphenol Blue, at a ratio of 1:1. Total protein equiva-
lents for each sample were separated on a 10% SDS–
polyacrylamide minigel by using a Laemmli buffer sys-
tem and were transferred to a polyvinylidene difluoride
membrane (Millipore, Hertfordshire) at 15 V for 1 hour in a
semidry transfer system. The membrane was immediately
placed in blocking buffer (1% nonfat milk) in 10 mM Tris,
pH 7.5, 100 mM NaCl, and 0.1% Tween 20. The blot was
allowed to block at room temperature for 1 hour. The mem-
brane was incubated with specific rabbit polyclonal anti-
COX-2 antibody (Oxford Biomedical, Oxford, MI; 1:2,000)
or goat polyclonal anti-COX-1 antibody (Santa Cruz Bio-
technology, Santa Cruz, CA; 1:200) for 1 hour at 25°C fol-
lowed by a horseradish peroxidase-conjugated donkey anti-
rabbit antibody (Amersham; 1:2,000) or antigoat antibody
(Serotec, Kidlington, Oxford, England; 1:3,000) for 1 hour
at 25°C. Antibody labeling was detected by using enhanced
chemiluminescence (Amersham) per the manufacturer’s in-
Preparation of radiolabeled primer.—One microgram
of sense primer, 1 ꢀl of 10ꢁ T4 kinase buffer, 10 U of T4
kinase, 2.5 ꢀl of ꢃ-32P-adenosine triphosphate (10 ꢀCi/ꢀl,
3000 Ci/mmol), and distilled water were mixed, incubated
at 37°C for 1 hour, and then left at 75°C for 10 minutes.
DEPC-treated water, 8 M ammonium acetate, and ethanol
were added and left at ꢂ80°C for 1 hour. The mixture was
centrifuged at 12,000ꢁ g for 25 minutes and the supernatant

B352
KIM ET AL.
structions. Prestained blue protein markers were used for
molecular weight determination (20).
groups were assessed by the Fischer’s Protected LSD post
hoc Test.
RESULTS
Measurement of ROS
COX activity, measured as the production of prostaglan-
din E2, increased with age (Figure 1). The activity at 24
months of age was significantly higher than that observed at
6 months of age. As a way to determine whether the in-
crease in COX activity was due to enhanced gene expres-
sion of COX-2 or of COX-1, their mRNA and protein levels
were examined, using RT-PCR and Western blotting, re-
spectively. Aging was associated with an increase in the
mRNA for COX-2 (Figure 2A) and an elevation in the
COX-2 protein content (Figure 2B). At 6 and 12 months of
age, very low levels of COX-2 mRNA were present, but
significant levels were detected at 18 and 24 months of age.
The mRNA and protein levels of COX-1 remained unaf-
fected by aging (Figures 3A and 3B), indicating that the
age-related increase in COX activity is due to higher COX-2
expression.
The effect of age on total ROS generation measured by
the DCF method is shown in Figure 4. ROS production sig-
nificantly increased from 85.4 ꢅ 12.0 nmol DCF (formed/
min)/mg protein at 6 months of age to 160.3 ꢅ 12.3 nmol
DCF (formed/min)/mg protein at 24 months of age. COX-
derived ROS production contributes to the age-related in-
crease in the overall ROS production. COX-derived ROS
production (Figure 5) increased from 23.92 ꢅ 2.55 nmol
DCF (formed/min)/mg protein at 6 months of age to 32.84 ꢅ
1.71 nmol DCF (formed/min)/mg protein at 24 months of age.
Comparable results were obtained for lipid peroxidation.
The lipid peroxidation of the cardiac postmitochondrial
fraction increased with age (Figure 6). The concentration of
AAPH-induced TBARS rose from 2851 ꢅ 246 (pmol/30
min)/mg protein at 6 months of age to 3740 ꢅ 180 (pmol/30
min)/mg protein at 24 months of age.
For total ROS generation in the postmitochondrial frac-
tions, the various oxygen species, including superoxide, hy-
drogen peroxide, hydroxyl radicals, and lipid hydroperox-
ides, were quantified by using previously published methods
(21–23), using the probe, 2ꢄ,7ꢄ-dichlorofluorescein-diac-
etate (DCFDA). One hundred micrograms of protein were
added to ice-cold potassium phosphate buffer (50 mM,
pH 7.4) containing 25 ꢀM DCFDA. The final volume of
the mixture was 250 ꢀl. The changes in fluorescence in-
tensity were measured at 10, 15, 20, 25, and 30 minutes on a
Fluorescence Microplate Reader (FL500, Bio-Tek Instru-
ments), with excitation and emission wavelengths set at 485
nm and 530 nm, respectively.
For COX-derived ROS generation, quantitation of reac-
tive species generation was carried out as described above,
except that 1 mM indomethacin was included in the reaction
mixture. The difference in ROS generation in the presence
and absence of indomethacin was considered the COX-
derived ROS generation.
Lipid Peroxidation
Aliquots of the supernatant fraction were incubated in the
presence of 0.5 mM 2,2ꢄ-azobis(2-amidinopropane) dihy-
drochloride (AAPH) for 30 minutes at 37°C. To the incu-
bate was added 0.5 ml of the assay mixture containing 1.2%
thiobarbituric acid (TBA) solution, 8.1% SDS solution, and
20% acetic acid at a ratio of 20:4:30, respectively. The reac-
tion mixture was boiled at 94°C for 30 minutes and cooled.
The TBA reactive substance (TBARS) was extracted by bu-
tanol and the entire mixture was centrifuged for 10 minutes
at 900ꢁ g. The fluorescence intensity of the butanol frac-
tion was measured at an excitation wavelength of 530 nm
and an emission wavelength of 590 nm (24).
DISCUSSION
COX plays a crucial role in the arachidonic acid cascade
in a number of tissues and cells. However, little is known
Protein Assay
The concentration of protein was measured by Lowry’s
method; bovine serum albumin was used as a standard (25).
Materials
The DCFDA was from Molecular Probes, Inc. (Eugene,
OR). The ꢃ-32P-ATP (250 ꢀCi) and enzyme immunoas-
say kit for the thromboxane B2, prostaglandin E2, and leu-
kotriene B4 were purchased from Amersham. RNAzol B
was obtained from Tel-Test. Primers for reverse-transcrip-
tion PCR (RT-PCR) were synthesized by Bioneer (Daejeon,
Korea). Antibodies of COX-1 and COX-2 for Western blot-
ting were obtained from Santa Cruz Biotechnology and Ox-
ford Biomedical, respectively. Polyvinylidene difluoride
(PVDF) membranes were obtained from Millipore Corpora-
tion (Bedford, MA). Other chemicals were purchased from
Sigma (St. Louis, MO).
Statistical Analysis
Results were analyzed statistically by a one-factor analy-
sis of variance. Differences between the means of individual
Figure 1. Effects of age on cyclooxygenase activity in rat heart.
Each value represents the mean ꢅ standard error for six rats. *p ꢆ
.05 vs 6 months of age.

CHANGES IN CYCLOOXYGENASE IN THE AGING HEART
B353
Figure 2. A, Effects of age on cardiac mRNA levels of COX-2. B,
Changes of cardiac protein levels of COX-2. Rats of 6, 12, 18, and 24
months of age were used. COX-2 ꢇ cyclooxygenase-2; GAPDH ꢇ glyc-
eraldehyde-3-phosphate dehydrogenase. **p ꢆ .01 vs 6 months of age.
Figure 3. A, Cardiac mRNA levels of COX-1 with age. B, Cardiac
protein levels of COX-1. Rats of 6, 12, 18, and 24 months of age were
used. COX-1 ꢇ cyclooxygenase-1; GAPDH ꢇ glyceraldehyde-3-phos-
phate dehydrogenase.
about the expression and regulation of COX in the heart
during aging. We observed a significant increase in COX-2
mRNA (Figure 2A), COX-2 protein (Figure 2B), and COX
enzyme activity (Figure 1) in the heart with age. This indi-
cates that the basis for the age-related changes in COX is at
the level of gene expression. The possibility that the ele-
vated concentration of COX-2 could also arise from a de-
cline in the removal of the molecule cannot be ruled out.
Because the mRNA for COX-1 did not change with age, in
our study, it is reasonable to assume that changes in the ex-
pression of the mRNA for COX-1 appear not to play any
significant role in normal aging process.
One salient finding of our study is that a substantial
amount (ꢁ25%) of the total ROS generated in the superna-
tant fraction arises from COX activity (Figures 4 and 5).
ROS generation in the heart increases with age (Figure 4)
and a significant fraction of that increase arises from en-
hanced COX activity. As the aging heart suffers a substan-
tial amount of oxidative damage (15,16) and according to
the oxidative theory of aging (1,2), this damage that was ex-
acerbated by the increased ROS generated by means of a
COX reaction could contribute significantly to the func-
tional decline observed in aging organisms (26). The in-
crease in the levels of lipid peroxidation shown in this study
(Figure 6) further confirms this notion of enhanced oxida-
tive stress in the aging heart (15,16). Much of the previous
work in the aging heart utilized a mitochondria fraction. The
salient finding in this study is that a nonmitochondrial frac-
tion can contribute substantially to the increase in oxidative
damage seen in the aged heart.
It is noteworthy that COX activity is regulated by lipid
peroxides (27), the products of oxidative stress. Conse-
quently, an increase in the concentration of lipid peroxides
should enhance COX activity. From the point of view of a
positive feedback phenomenon, the stimulatory role of the
lipid peroxidation in inflammatory process and other tissue
damage can be substantial in the aged organisms. Results
from this study (Figure 6) and others (2,15) have shown that
the degree of lipid peroxidation increases with age. It is pos-
sible that this increase leads to elevated COX activity, en-

B354
KIM ET AL.
Figure 4. Effects of age on ROS generation in heart postmitochon-
drial fraction. Each value represents the mean ꢅ standard error for six
rats. **p ꢆ .01 vs 6 months of age. DCF ꢇ 2ꢄ7ꢄ-dichlorofluorescein.
Figure 6. Effects of age on AAPH-induced lipid peroxidation in
heart postmitochondrial fraction. TBARS, thiobarbituric acid reac-
tive substance; AAPH, 2,2ꢄ-azobis(2-amidinopropane) dihydrochlo-
ride). Each value represents the mean ꢅ standard error for six rats.
*p ꢆ .05 vs 6 months of age.
hanced ROS production, and finally greater lipid peroxida-
tion—thus completing the positive feedback loop.
In summary, the present experiments demonstrate that the
heart exhibits an increase in the COX-2 with age and this in-
crease is associated with elevations in ROS. An age-related
elevation in COX-2 activity may be due to an increased
mRNA expression and higher levels of the enzyme itself.
Acknowledgments
This study was supported by a grant from the Ministry of Health and
Welfare (HMP-98-M-3-0044). We are grateful to the Aging Tissue Bank
for the supply of the aging tissue. We thank Corinne Price for checking the
manuscript.
Address correspondence to Hae Young Chung, Department of Pharmacy,
Pusan National University, Kumjung-Ku, Pusan 609-735, Korea. E-mail:
hyjung@hyowon.pusan.ac.kr
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Received August 9, 2000
Accepted March 15, 2001
Decision Editor: John A. Faulkner, PhD
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