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Department of Surgery, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Experiments were
designed to determine whether normal fluctuations in endogenous sex
steroid hormones and/or gender affect endothelium-dependent
relaxations in coronary arteries, and, if so, to identify
endothelium-derived factors contributing to these differences. Coronary
arteries from sexually mature, gonadally intact male and female pigs or
ovariectomized pigs were prepared either for study of isometric force
in organ chambers or for measurement of prostanoids and activity of
nitric oxide (NO) synthase. In organ chamber studies,
neither the magnitude nor the sensitivity of endothelium-dependent
relaxations correlated with endogenous estrogen or progesterone in
female pigs. However, relaxations to bradykinin and UK-14304 were
significantly greater and/or shifted leftward in arterial rings
from female compared with male pigs. Indomethacin
(10
5 mol/l) increased
endothelium-dependent relaxations only in arteries from male pigs.
NG-monomethyl-L-arginine reduced
endothelium-dependent relaxations to a similar extent in coronary
arteries from either sex. Neither production nor response to
thromboxane A2 or prostacyclin
differed in coronary arteries from male compared with female pigs.
Activity for calcium-dependent or -independent NO synthase was similar in both sexes. These results suggest that normal fluctuations in
endogenous sex steroid hormones do not affect endothelium-dependent relaxations in coronary arteries from female pigs. There are, however,
gender differences in endothelium-dependent relaxations that are
indomethacin sensitive and may be due to cyclooxygenase products other
than thromboxane A2 or
prostacyclin.
arachidonic acid; eicosanoids; nitric oxide synthase; prostacyclin; estrogen; thromboxane
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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BEFORE MENOPAUSE women have a lower incidence of
coronary artery disease compared with men of the same age. After
menopause this difference persists only if estrogen replacement therapy is initiated. Such data suggest that female sex steroid hormones prevent the development of coronary artery disease (see Refs. 7 and 23
for review). Mechanisms accounting for this cardioprotective action of
estrogen may include differences in regulation and production of
endothelium-derived factors. For example, estrogen treatment of whole
animals after ovariectomy enhances endothelium-dependent relaxations in
isolated arteries to some, but not all, endothelium-dependent agonists
(10, 18). Similarly, overnight incubation of isolated porcine coronary
arteries with physiological concentrations of 17
-estradiol enhances
relaxations to the Ca2+ ionophore
A-23187 but not adenosine 5'-diphosphate or nitric oxide (4).
Other studies suggest that progesterone and testosterone also alter
endothelium-dependent relaxation of coronary arteries (1, 9, 19). All
of these studies involve depletion of nearly all endogenous sex steroid
hormones followed by replacement of one, or at best two, sex steroid
hormones. Such an approach does not reflect potential interactions
between the sex steroid hormones during the normal ovulatory cycle,
when a milieu of sex steroid hormones are present and fluctuating in a
coordinated manner. An understanding of the regulation of
endothelium-derived factors and coronary vasomotion during the normal
ovulatory cycle is critical because this is when developmental
differences in coronary artery disease occur in humans. Experiments
were therefore designed to determine whether normal endogenous
fluctuations in sex steroid hormones and/or gender affect
endothelium-dependent relaxations and, if so, to identify
endothelium-derived factors accounting for the differences.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Sexually mature pigs were chosen for this study because the estrous
cycle in pigs occurs monthly and the hormonal profile is analogous to
women. At no time were either pigs or isolated preparations treated
with exogenous sex steroid hormones. Animals were grouped according to
gender and endogenous plasma concentrations of sex steroid hormone when
they were killed. Sexually mature, gonadally intact Yorkshire male (130 ± 10 kg, n = 19) and
female (87 ± 1 kg, n = 32) or laparoscopically ovariectomized (6) pigs (79 ± 2 kg,
n = 5) were anesthetized by an
intramuscular injection of a ketamine-xylazine-butorphanol mixture (30, 6, and 0.3 mg/kg, respectively). Weight gain in maturing male
and female pigs is not the same. Therefore, animals were age matched to
assure groups of comparable sexual maturity. Ovariectomy was performed on sexually mature female pigs (5-6 mo of age), and experiments were performed after 4 wk. All other animals were studied between 5 and
7 mo of age. Blood samples were collected from the femoral artery and
analyzed for plasma 17-estradiol, progesterone, and testosterone at
the Clinical Steroid Laboratory of Mayo Medical Laboratories. The
detection limit for plasma sex steroid hormones was 0.04 pg/ml for
progesterone and 10 pg/ml for 17-estradiol and testosterone. Serum
cholesterol and lipoprotein profiles were determined by Mayo Medical
Laboratories. Hearts were removed and immediately placed in ice-cold
modified Krebs-Ringer bicarbonate solution (control solution in mmol/l,
118.3 NaCl, 4.7 KCl, 2.5 CaCl2,
1.2 MgSO4, 1.2 KH2PO4,
25.0 NaHCO3, 0.026 calcium
disodium edetate, 11.1 glucose, pH 7.4, and aerated with 95%
oxygen-5% carbon dioxide). The right circumflex coronary artery was
excised and prepared either for study in organ chambers or
radioimmunoassay of eicosanoid release or frozen for subsequent
homogenization for assessment of nitric oxide synthase (NOS) assay.
Animal care was conducted in accordance with both the
Principles of Laboratory Animal Care
formulated by the National Society for Medical Research and the
Guide for the Care and Use of Laboratory
Animals [DHHS Publication No. (NIH) 86-23,
Revised 1985].
Organ chamber experiments.
The right circumflex coronary artery was excised, cleaned of connective
tissue, and cut into 4-mm rings for study in organ chambers. One-half
of the arterial rings were denuded mechanically of endothelium by
gently scraping the lumen with fine-tipped forceps. Removal of the
endothelium was confirmed by the absence of relaxation to
endothelium-dependent vasodilators described below. Pairs of rings with
and without endothelium were suspended between a fixed stirrup and
force transducer for measurement of isometric force in 25-ml organ
chambers filled with control solution. Each ring was stretched to the
optimal point on its length-tension curve as determined by tension
developed to 20 mmol/l KCl at each level of stretch. Once the optimal
tension was set, 60 mmol/l KCl was added to each bath to determine
maximal response to KCl. To some of the baths either indomethacin
(105 mol/l, dissolved in
sodium carbonate, final bath concentration sodium carbonate 2 × 105 mol/l) or
NG-monomethyl-L-arginine
(L-NMMA,
104 mmol/l), or both, was
added 45 min before administration of the various agonists. Rings were
contracted with prostaglandin
F2
(2 × 106 mol/l) or endothelin-1
(107 mol/l) and cumulative
concentration responses to bradykinin
(1010 to
107 mol/l), UK-14304
(5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline, 108 to
106 mol/l), the calcium
ionophore A-23187
[109 to
106 mol/l, dissolved in
dimethyl sulfoxide (DMSO); final bath concentration DMSO 8.2 × 103 mol/l] or nitric oxide
(3 × 108 to
105 mol/l) determined. Once
indomethacin and/or
L-NMMA was added to an organ
bath they remained in the baths for the duration of the experiment.
9 to
106 mol/l) or prostacyclin
(109 to
105 mol/l, dissolved in
sodium bicarbonate; final bath concentration sodium bicarbonate 8.8 × 104 mol/l, pH 8.5)
were obtained. In preliminary studies prostacyclin did not
significantly relax arteries contracted with prostaglandin F2 (2 × 106 mol/l), and therefore
responses to prostacyclin were performed in rings set at their optimal
basal tension.
Activity of NOS. Activity of NOS was determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in cellular homogenates according to previously described methods with minor modifications (20). Right and left circumflex and left anterior descending coronary arteries with endothelium were cleaned of fat and connective tissue, combined and pulverized in liquid nitrogen, and suspended in six volumes of ice-cold homogenization buffer of the following composition: 320 mmol/l sucrose, 50 mmol/l tris(hydroxymethyl)aminomethane · HCl, 0.1 mmol/l EDTA, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain, pH 7.8. The suspension was homogenized three times for 10 s each using a Tekmar homogenizer (Tekmar, Cincinnati, OH). Homogenates were centrifuged at 2,000 g for 15 min at 4°C to remove cellular debris. The supernatant was passed through a 213-µm nylon sieve onto an equilibrated 10-DG desalting column (Bio-Rad, Hercules, CA) and eluted according to the manufacturer's directions. A small aliquot was used to determine protein concentrations using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) with bovine serum albumin as standard.
To quantitate NOS activity, duplicate reactions were carried out in the presence of calcium (total activity), in the absence of calcium plus ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) (calcium-independent activity), and in the absence of
calcium plus EGTA in the presence of
L-NMMA (nonspecific activity). Reactions were started by adding 150 µl protein homogenate to 150 µl cofactor mix such that the final concentrations were as follows:
14.7 nmol/l
L-[3H]arginine
(0.3 µCi specific activity at 68 Ci/mmol), 54 mmol/l L-valine, 1.2 mmol/l
MgCl2, 1.0 mmol/l NADPH, 2 µmol/l FAD, 5 µmol/l
L-arginine, 10 µmol/l
tetrahydrobiopterin, 50 U/ml calmodulin, and, as described above, with
or without 0.83 mmol/l CaCl2, 1 mmol/l EGTA, and 2 mmol/l
L-NMMA. The reaction was carried
out in a shaker bath at 27°C for 1 h and terminated by the addition of 1.5 ml ice-cold stop buffer (20 mmol/l
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 8 mmol/l EDTA, pH 5.5). Separation of
L-[3H]arginine
from
L-[3H]citrulline
was accomplished using affinity columns containing a resin that retains
the charged species of
L-[3H]arginine
while allowing
L-[3H]citrulline
to pass through. The assay mixture was then passed over Poly-Prep
chromatography columns (Bio-Rad) loaded with 1 ml of equilibrated AG50
WX-8 Na+ form 200-400 mesh
molecular biology grade Dowex resin (Bio-Rad), and the eluate was
collected into 18-ml OptiFluor (Packard, Meriden, CT). The column was
washed with 2 ml of water while continuing to collect into the
scintillation fluid.
L-[3H]citrulline
activity was determined using a Beckman 6800 liquid scintillation
counter. Blank incubations contained 150 µl protein-free homogenization buffer previously passed over a desalting column as
described. Activity calculations account for scintillation counting
efficiency and the ratio of
L-[3H]arginine
to nonradioactive L-arginine in
the incubation mixture. Nitric oxide produced by NOS is presumably in a
1/1 molar ratio with
L-citrulline, and thus NOS
activity is expressed as picomoles of
L-[3H]citrulline
produced per milligram of protein per hour. Calcium-dependent activity
equaled total activity minus calcium-independent activity after
correcting for nonspecific activity.
Eicosanoid production.
Rings of right coronary artery (4 mm) with endothelium from male
(n = 8) or female
(n = 7) pigs were placed in 1.5-ml
tubes containing 1 ml control solution at 37°C continuously aerated with 95% oxygen-5% carbon dioxide. After a 30-min equilibration period, the control solution was replaced with fresh solution. Two
minutes later prostaglandin F2
(2 × 106 mol/l) was
added to two of the rings, and solvent (water, 10 µl) was added to
two other rings. Ten minutes later UK-14304
(106 mol/l) was added to
one of the tubes containing prostaglandin F2 and to one tube not
containing prostaglandin F2. Exactly 3 min later the arterial rings were removed, and the test tube
containing incubation solution was immediately frozen in liquid
nitrogen for subsequent radioimmunoassay of 6-ketoprostaglandin F1 (stable metabolite of
prostacyclin) and thromboxane B2 (stable metabolite of thromboxane
A2). The arterial rings were dabbed dry with tissue paper and weighed. Radioimmunoassays were performed according to the manufacturer's instructions for the individual assay kit.
Histology. Right and left circumflex and left anterior descending coronary arteries were placed in 10% formaldehyde for a minimum of 24 h before paraffin embedding and sectioning (6 µm). Sections mounted on silanized slides were stained with hematoxylin and eosin and examined by light microscopy.
Data analysis.
All data are expressed as means ± SE;
n equals the number of pigs from which
arteries were taken. For organ chamber studies, data are expressed as
percent change in tension from contraction to prostaglandin
F2 or endothelin-1. Maximal
relaxations and effective concentration producing half-maximal
relaxation (EC50) were
calculated for individual concentration-response curves.
Drugs and chemicals.
Nitric oxide was prepared by the method of Palmer et al. (21).
Indomethacin was dissolved in an aqueous solution of sodium carbonate
(bath concentration 20 µmol/l). A-23187 was dissolved in DMSO, final
bath concentration 8.2 mmol/l, and diluted with distilled water. This
concentration of DMSO has been shown to not affect smooth muscle (17).
All other drugs used in organ chamber studies were prepared in
distilled water. All drugs were from Sigma Chemical (St.
Louis, MO) except as follows:
L-[3H]arginine
(Amersham, Arlington Heights, IL), NADPH and calmodulin (Boehringer
Mannheim, Indianapolis, IN), tetrahydrobiopterin (Research Biochemicals
International, Natick, MA), antipain, leupeptin, and pepstatin A
(Peptides International, Louisville, KY),
L-NMMA (Calbiochem, La Jolla,
CA), UK-14304 (Pfizer Research Central, Sandwich, UK). Radioimmunoassay
kits for 6-ketoprostaglandin F1 and thromboxane B2 were from
DuPont-NEN (Boston, MA). All other reagents were from Sigma Chemical.
All concentrations are expressed as the final molar (mol/l)
concentration in the organ bath or incubation solution.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Blood chemistry and histology.
Gonadally intact female pigs exhibited cyclic variation in hormonal
status as observed by external examination of the genitalia and direct
measure of plasma estrogen. Female pigs were grouped according to
plasma 17-estradiol as either low (<10 pg/ml) or high (>10
pg/ml) at the time of the experiments. Plasma levels of 17-estradiol
were significantly higher in high-estrogen females compared with
ovariectomized and low-estrogen females (Table
1). Males had significantly higher plasma
levels of 17-estradiol than either the ovariectomized or
low-estrogen females. Progesterone levels were not significantly
different among all groups (Table 1). Plasma levels of testosterone
were significantly higher in males compared with all female groups
(Table 1). Total serum cholesterol was lower in males compared with
either high- or low-estrogen females (Table 1). There was no
significant difference in either high- or low-density lipoprotein among
groups (Table 1). There was no consistent evidence of
atherosclerosis in histological sections of arteries from either sex
(data not shown).
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Organ chamber experiments.
In arteries from female pigs, there was no significant difference in
relaxations to any of the drugs tested when the data were grouped
according to endogenous plasma concentrations of 17-estradiol,
progesterone, or testosterone (Fig. 1).
Neither were there significant correlations between maximal relaxations and plasma estrogen concentrations in either male or female pigs. Therefore, all data from female pigs were combined for analysis by
gender. Contractions to potassium chloride (60 mmol/l) in arteries with
or without endothelium were not significantly different among arteries
from all pigs (males, n = 8, with
endothelium 24.9 ± 2.4 g and without endothelium 21.8 ± 3.3 g;
females, n = 22, with endothelium 23.8 ± 1.2 g and without endothelium 21.1 ± 1.3 g). The addition of
indomethacin to organ chambers did not cause a change in tension in
rings with or without endothelium from pigs of either sex. However, the
addition of indomethacin plus
L-NMMA caused similar increases
from baseline tension in arteries with endothelium in 5 of 8 (62.5%)
arteries from male pigs (1.54 ± 0.73 g,
n = 5) and in 14 of 22 (63.3%)
arteries from female pigs (1.43 ± 0.31 g,
n = 14). There were no statistically
significant differences in contraction to prostaglandin
F2 among groups (range
13-24 g in all groups). Contractions to endothelin-1
(107 M) were significantly
greater in arteries from females (high estrogen 27.8 ± 2.2 g,
n = 6; low estrogen 24.1 ± 1.6 g
and ovariectomized 23 ± 1.9 g, n = 5) compared with male pigs (16.6 ± 2.6 g,
n = 8).
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UK-14304.
UK-14304 (108 to
106 mol/l) caused
concentration-dependent relaxations in rings with endothelium in
arteries contracted with prostaglandin
F2 (2 × 106 mol/l) from male
(n = 8) and female
(n = 22) pigs.
Endothelium-dependent relaxations to UK-14304 in arteries
from female pigs were significantly shifted leftward
(EC50 6.93 ± 0.08 
log
mol/l female vs. 6.33 ± 0.33 log mol/l male) compared with
rings from male pigs (Fig. 2A).
Maximal relaxations to UK-14304 were not significantly different between genders. In arteries from male but not female pigs indomethacin potentiated relaxations to UK-14304 (males, maximum 41.7 ± 11.2% control vs. 68.1 ± 9.2% + indomethacin, Fig.
3). In the presence of indomethacin
(105 mol/l) relaxations
were reduced by L-NMMA
(104 mol/l) to the same
extent in both sexes (Fig. 3). In the
presence of indomethacin
(105 mol/l) or indomethacin
plus L-NMMA
(104 mol/l),
endothelium-dependent relaxations to UK-14304 were not different
between arteries from male and female pigs (Fig. 3).
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Bradykinin.
In coronary arterial rings contracted with prostaglandin
F2 (2 × 106 mol/l), bradykinin
(1010 to
107 mol/l) caused
concentration-dependent relaxations only in rings with endothelium
(Fig. 2B).
Endothelium-dependent relaxations to bradykinin in arteries from female
(n = 22) pigs were shifted significantly leftward (EC50 8.27 ± 0.07 log mol/l female vs. 7.74 ± 0.13 log mol/l
male) with significantly greater maximal relaxation (maximum
98.9 ± 0.8% female vs. 83.9 ± 8.9% male) compared with rings from male (n = 8)
pigs. In the presence of indomethacin
(105 mol/l) or indomethacin
plus L-NMMA
(104 mol/l),
endothelium-dependent relaxations to bradykinin were not statistically
different between male and female pigs (Fig. 4). In arteries from male
pigs, indomethacin did not significantly change relaxations to
bradykinin (Fig. 4).
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A-23187.
A-23187 (109 to
106 mol/l) caused
concentration-dependent relaxations in coronary arterial rings with
endothelium contracted with endothelin-1
(107 mol/l) from male
(n = 8) and female
(n = 22) pigs (Fig.
2C). Relaxations to A-23187
were not statistically different between arteries from male and female
pigs (EC50 6.77 ± 0.07 log mol/l female vs. 6.52 ± 0.13 log mol/l male;
maximum 54.2 ± 5.4% female vs. 44.3 ± 11.2%
male, Fig. 2C). In the presence of indomethacin (105 mol/l) or indomethacin
plus L-NMMA
(104 mol/l),
endothelium-dependent relaxations to A-23187 were the same for arteries
from male and female pigs. In arteries from male but not female pigs,
indomethacin significantly potentiated maximal relaxations to
A-23187 (males maximum 44.3 ± 11.2% control vs.
66.3 ± 12.5% + indomethacin, Fig. 5).
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Nitric oxide.
In coronary arterial rings without endothelium contracted with
endothelin-1 (107 mol/l),
nitric oxide (3 × 108
to 105 mol/l) produced
concentration-dependent relaxations in rings from male
(n = 8) and female
(n = 22) pigs. Relaxations to nitric oxide were not different in arteries without endothelium between male
and female pigs (maximum 65.7 ± 6.8% males, 64.2 ± 5.1% females). For either sex endothelium-independent
relaxations to nitric oxide were not significantly altered by
indomethacin (105 mol/l) or
indomethacin plus L-NMMA
(104 mol/l) (indomethacin,
maximal relaxation 50.8 ± 8.5% males, 69.2 ± 5.8% females or indomethacin plus
L-NMMA, maximal relaxation 60.3 ± 6.6% males, 66.1 ± 5.6% females).
U-46619 and prostacyclin. The thromboxane A2 mimetic U-46619 caused concentration-dependent increases in tension in coronary arteries with and without endothelium from both female (n = 7) and male (n = 6) pigs (Fig. 6A). Rings without endothelium were significantly more sensitive to U-46619 compared with rings with endothelium from both male and female pigs. There were no significant gender differences in the responses to U-46619 in rings with or without endothelium. Maximal responses to U-46619 were the same in rings with and without endothelium from either sex.
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9 to
107 mol/l) elicited a small
relaxation (maximum relaxation, 2.50 ± 0.69 g female,
2.74 ± 1.14 g male, P = not
significant) before increasing tensions at higher concentrations.
NOS activity.
There were no significant correlations between endogenous
concentrations of sex steroid hormones and activity of NOS derived from
membrane homogenates of coronary arteries with endothelium from female
pigs. There were no gender differences in either calcium-dependent (male 47 ± 6 and female 44 ± 7 pmol
L-[3H]citrulline · mg
protein1 · h1)
or -independent (male 7 ± 2 and female 11 ± 2 pmol
L-[3H]citrulline · mg
protein1 · h1)
NOS activity of coronary artery homogenates.
Eicosanoid production.
Prostaglandin F2 but not
UK-14304 significantly increased both thromboxane
B2 and 6-ketoprostaglandin
F1 in the incubation medium of
coronary arteries from male (n = 8)
and female (n = 7) pigs (Fig.
7). The amounts of both thromboxane
B2 and 6-ketoprostaglandin
F1 detected in the incubation
medium of unstimulated (solvent control), UK-14304-stimulated, or
prostaglandin F2-stimulated
coronary arterial rings were not statistically greater in arteries from
male compared with female pigs. The amounts of thromboxane
B2 and 6-ketoprostaglandin
F1 in rings stimulated with
UK-14304 plus prostaglandin F2
were the same for arteries from male and female pigs. Endogenous
concentrations of sex steroid hormones of female pigs did not correlate
with either thromboxane B2
or 6-ketoprostaglandin
F1 production.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Results of the present study suggest that physiological fluctuations in estrogen in the presence of other endogenous sex steroid hormones do not significantly affect agonist-stimulated endothelium-dependent relaxations of isolated coronary arteries from female pigs. This seems at variance with other studies when acute or chronic estrogen treatment potentiates endothelium-dependent relaxations (4, 10, 15, 22). However, studies of estrogen replacement do not address changes in estrogen that occur in conjunction with fluctuations in other endogenous sex steroid hormones during the estrous cycle. Interactions among the sex hormones are unclear (9, 19). A limitation of the present study is that estrogen grouping was based on measurable estrogen as related to the sensitivity of the assay. It could not be determined whether a given estrogen value represented a rising or falling phase of the estrus cycle. The lack of difference in responses between intact females and ovariectomized females is surprising and may relate to the age at which ovariectomy was performed and the time from ovariectomy to study of the arteries.
In healthy women flow-mediated endothelium-dependent vasodilatation of the brachial artery is enhanced in the follicular and luteal phases of menstruation when endogenous estrogen is elevated (12). In the present study no estrogen was present in the incubation medium of the organ baths as would be in the blood, and there is an absence of mechanical stimulation by flow. Flow-mediated coupling for release of endothelium-derived relaxing factors may differ from that of direct agonist stimulation and may be more sensitive to regulation by endogenous hormones.
An important finding of the present study is that gender differences in agonist-stimulated endothelium-dependent relaxations may not be related to estrogen levels alone. This conclusion is supported by the observation that plasma concentrations of estrogen in male pigs were greater than those of female pigs, probably as a result of metabolism of testosterone by aromatase in the adipose tissue. Therefore, it is not possible from the present study design to determine whether testosterone alone or estrogen-to-testosterone ratio is important in affecting production of endothelium-derived substances. Systemic evaluation of affects of testosterone on vascular responses in male animals warrants further study. However, such studies should take into consideration the timing of castration, because castration before puberty (as would be the case in the studies of male arteries obtained from the abattoir) may not represent the same developmental conditions as removal of gonads from animals that experienced puberty.
Metabolism of arachidonic acid by cyclooxygenase may be responsible for
gender difference in agonist-stimulated endothelium-dependent relaxations because indomethacin potentiates relaxations in arteries from male but not female pigs. One metabolite of arachidonic acid is
thromboxane A2, which elicits
contractions that are sexually dimorphic in some animals (5, 8).
Prostacyclin is another potential mediator of contractions, because
male pigs used in this study had relatively high 17-estradiol plasma
levels and chronic estrogen treatment may increase sensitivity of
smooth muscle to prostacyclin contractions (18). In the present study there was a tendency for isolated arteries of male pigs to have a
greater sensitivity to both thromboxane
A2 (U-46619) and prostacyclin (Fig. 5). There was also a trend toward greater basal and
agonist-stimulated release of both thromboxane
B2 and 6-ketoprostaglandin
F1 from arteries of male
compared with female pigs (Fig. 6). Neither of these observations alone
was statistically significant; however, it is possible that the
combination of these two effects in the arterial ring may account for
the indomethacin-sensitive gender differences in endothelium-dependent
relaxation.
Any of a number of endothelium-derived factors other than eicosanoids may contribute to gender differences in relaxations, including nitric oxide, hyperpolarizing factor(s), locally produced peptides (2), cytochrome P-450 products (11, 16), and free radicals (13). Several studies suggest that endothelium-derived nitric oxide is a mediator accounting for gender differences in endothelium-dependent relaxations. For example, relaxations to acetylcholine are greater in the aorta of female compared with male rats (13, 14). However, unlike the current study, indomethacin does not eliminate gender differences in acetylcholine-mediated relaxations in rat aorta (14). This suggests that mechanisms of gender differences in endothelium-dependent relaxations may differ by agonist, species, and/or anatomic origin of the artery. Results of the present study do not eliminate the possibility of gender differences in production of nitric oxide. The lack of gender differences in the biochemical assessment of NOS activity is limited because this assay reflects the enzyme under optimized conditions with all cofactors present. Therefore, potential gender-hormonal regulation of NOS cofactors and/or their availability or even endogenous pathways of NOS activation are not accounted for in this assay. However, the results confirm that the response of the smooth muscle to nitric oxide is the same in coronary arteries of male and female because both relaxations to exogenously administered nitric oxide and/or relaxations to endothelium-dependent agonists in the presence of indomethacin plus L-NMMA were similar between sexes.
Basal myogenic tone was not determined in these experiments. Therefore,
it is not possible to evaluate differences in contractions or
relaxations relative to contribution of myogenic tone. Differences in
relaxations cannot be attributed to the level of contraction of
arteries with prostaglandin F2
because these were similar among groups. Contractions to endothelin-1
are greater in females than males (3). However, a greater level of
contraction would be expected to decrease relaxations in females
relative to males, a result opposite to what was observed.
In summary, the present study demonstrates that normal fluctuations in endogenous sex steroid hormones do not change endothelium-dependent relaxations of isolated porcine coronary arteries from female pigs. There are, however, gender differences in endothelium-dependent relaxations that are indomethacin sensitive and not due to differences in NOS activity. This indomethacin-sensitive component of the relaxations is not due solely to either differences in sensitivity of coronary arteries to thromboxane A2 and/or prostacyclin or to differences in production and/or release of thromboxane A2 and/or prostacyclin.
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ACKNOWLEDGEMENTS |
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We thank Marylou Stewart, Kevin Rud, Sandra Severson, and Rod Bolterman for dedicated technical support.
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FOOTNOTES |
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This work was supported by the National Heart, Lung, and Blood Institute Grants HL-51736 and HL-07111 and by the Mayo Clinic and Mayo Foundation.
Address for reprint requests: V. M. Miller, Medical Sciences, 4-57, Mayo Clinic & Foundation, 200 First St., S. W., Rochester, MN 55905.
Received 6 December 1996; accepted in final form 15 July 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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1.
Adams, M. R.,
J. K. Williams,
and
J. R. Kaplan.
Effects of androgens on coronary artery atherosclerosis and atherosclerosis-related impairment of vascular responsiveness.
Arterioscler. Thromb. Vasc. Biol.
15:
562-570,
1995
2.
Barber, D. A.,
J. C. Burnett, Jr.,
and
V. M. Miller.
Gender differences in relaxations evoked by C-type natriuretic peptide in porcine coronary arteries (Abstract).
Endothelium
2:
S2,
1995.
3.
Barber, D. A.,
G. C. Sieck,
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2-adrenergic agonist UK-14304
(A), bradykinin
(B), and calcium ionophore A-23187
(C) in rings of right coronary
arteries with endothelium from high-estrogen females, low-estrogen
females, and ovariectomized pigs. For UK-14304 and bradykinin, data are
expressed as percent change in tension from contraction to
prostaglandin F2
