© 2000 American Heart Association, Inc.
Vascular Biology |
Simultaneously Increased TxA2 Activity in Isolated Arterioles and Platelets of Rats With Hyperhomocysteinemia
From the Institute of Pathophysiology (Z.U., E.S.-N., Z.B., L.S., A.K.), Semmelweis University of Medicine, Budapest, Hungary, and the Department of Physiology (A.K.), New York Medical College, Valhalla, NY.
Correspondence to Akos Koller MD, PhD, Department of Physiology, New York Medical College, Valhalla, New York 10595. E-mail kolako{at}net.sote.hu
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Abstract—We aimed to elucidate the effect of hyperhomocysteinemia (HHcy) on the synthesis of prostaglandins in rat skeletal muscle arterioles and platelets. Male Wistar rats were divided into 2 groups: (1) control rats, with plasma Hcy levels of 6.5±0.5 µmol/L (n=50) and (2) rats with HHcy, induced by daily intake of 1 g/kg body weight methionine in the drinking water for 4 weeks (plasma Hcy levels were 20.6±3.0 µmol/L, P<0.01 versus controls; n=50). Arterioles (diameter
130 µm) were isolated from the gracilis
muscle, cannulated, and pressurized (at 80 mm Hg), and changes in
their diameters were followed by video microscopy. Constrictions to
bradykinin (BK; 10-10 to
10-7 mol/L) were significantly greater in HHcy
than in control rat arterioles (at 10-9 mol/L
BK, changes were 11±3% in control and 41±9% in HHcy rats). The
cyclooxygenase inhibitor
indomethacin (10-5 mol/L), the
prostaglandin
H2/thromboxane
A2
(PGH2/TxA2) receptor
antagonist SQ 29,548 (10-6 mol/L),
or the TxA2 synthase inhibitor
furegrelate (5x10-6 mol/L) significantly
decreased constrictions to BK in both groups but more so in HHcy
arterioles, thus eliminating the difference between responses of HHcy
and control arterioles. Constrictions to U46619 (a
TxA2 analogue) were significantly greater in HHcy
than in control arterioles (at 10-8 mol/L
U46619, values for controls were 33±2% and 54±3% for HHcy).
Endothelium removal or indomethacin
treatment attenuated constrictions to U46619 in HHcy arterioles and
eliminated the difference in responses. Also, aggregation of
platelets from HHcy rats to collagen and ADP was significantly
enhanced compared with controls (with 5 µg/mL collagen: controls,
23±5%; HHcy, 49±5%; with 10-7 mol/L ADP:
controls, 25±3%; HHcy, 35±3%). Indomethacin or SQ
29,548 caused greater inhibition of aggregation of HHcy platelets
compared with controls, thereby eliminating the differences between the
2 groups. Thus, HHcy enhances TxA2 synthesis both
in the arteriolar endothelium and platelets. By
promoting vascular constriction and platelet aggregation
simultaneously, these alterations are likely to contribute
to the atherothrombotic vascular diseases described in HHcy.
Key Words: homocysteine • methionine • endothelium • arterioles • platelet aggregation
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Introduction |
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Several epidemiological studies have shown an association between elevated plasma homocysteine (Hcy) concentrations and atherothrombotic vascular diseases.1 2 3 4 In the general human population, mild hyperhomocysteinemia (HHcy) is rather common (1:70) and is found in
30% of individuals with coronary,
cerebrovascular, or peripheral atherothrombotic
disease.3 4 Hcy is a thiol-containing amino acid
that is formed from the essential amino acid methionine. Plasma Hcy
concentration may increase in different
pathophysiological conditions, including deficiency
of vitamins such as folic acid, cyanocobalamin, and pyridoxal
phosphate; and in the presence of various enzyme abnormalities
(cystathionine ß-synthetase, temperature-sensitive
methylenetetrahydrofolate reductase;
for further information, see Reference 2 ), all of
which participate in the metabolism of methionine and Hcy. The mechanism(s) by which elevated Hcy promotes atherothrombotic vascular diseases is still not clearly elucidated and is likely to be multifactorial.5 6 It has been shown that HHcy stimulates smooth muscle proliferation7 8 and promotes LDL oxidation.9 There are reports demonstrating that HHcy impairs the vasoactive function of the endothelium as well. In experimental animals, severe HHcy resulted in endothelial cell loss.10 The adverse effect of Hcy was also confirmed by in vitro studies on cultured endothelial cells.11 12 In conduit arteries of patients with HHcy, vasodilation associated with reactive hyperemia is impaired,13 14 15 16 whereas reduced acetylcholine (ACh)-induced dilation was demonstrated in carotid arteries isolated from HHcy monkeys.17 18
Less is known, however, of how the function of microvessels is altered in HHcy. We have recently demonstrated that elevated levels of Hcy in rats interfere with skeletal muscle arteriolar responses, which are mediated or modulated by endothelium-derived NO.19 Alterations of NO synthesis/release are frequently accompanied by enhanced constrictor responses due to changes in other endothelial pathways, such as an increased synthesis of prostaglandin H2/thromboxane A2 (PGH2/TxA2).20 Interestingly, previous studies found an enhanced platelet activation to infusion of collagen, which caused a marked reduction of hindlimb blood flow in HHcy compared with control monkeys.17 The underlying mechanism for this alteration could be due to increased TxA2 synthesis of platelets in HHcy.21 Thus, it is likely that in HHcy, there are changes in vascular arachidonic acid metabolism as well, yet the effect of HHcy on endothelial prostaglandin synthesis in arterioles has not been elucidated.
On the basis of these findings, we hypothesized that in HHcy, the synthesis of PGH2 and/or TxA2 is simultaneously upregulated in vascular tissue and platelets. To test this hypothesis, we measured the arteriolar responses of control and HHcy rats to bradykinin (BK), an agent that is known to initiate the synthesis of arachidonic acid metabolites, such as PGI2 and TxA2, in the endothelium.22 23 24 BK has pathological implications as well, since it is an important mediator of various inflammatory processes that are also associated with atherosclerotic vascular diseases.25 Also, we investigated the arteriolar responses to intraluminal pressure and U46619, a stable TxA2 analogue, and the aggregation of platelets from the same animals in response to ADP and collagen, which are known to depend, at least in part, on TxA2 synthesis.
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Methods |
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In male Wistar rats weighing
150 g (purchased from Charles
River Co), moderate HHcy was induced by administration of
L-methionine (1 g · kg body
weight-1 · d-1)
and succinylsulfathiazole (0.1 g · kg body
weight-1 · d-1) in
the drinking water for a period of 4 weeks (n=50), as described
previously.17 18 19 Succinylsulfathiazole was used to avoid
bacterial proliferation and subsequent folate neoproduction.
The doses administered were based on average daily fluid intake.
Control animals (n=50) had free access to water. Animals were housed
separately, fed standard rat chow, and were weighed at the start and
end of the 4-week period.
Determination of Serum Hcy
Blood was collected from the aortas of fasted rats. It was
immediately cooled on ice and centrifuged at 3000g
for 20 minutes at 4°C to limit the release of Hcy from blood cells.
Serum was then stored at -20°C until assayed. Total Hcy
concentrations were measured by a high-performance liquid
chromatography technique with fluorometric
detection.19 26 In brief, 240 µL of serum and 60
µL of internal standard
(N-acetyl-L-cysteine, 50 µmol/L
final concentration) were reduced for 30 minutes at 4°C with 30 µL
of tri-n-butylphosphine (10%). Deproteinization was
performed with 300 µL of 10% trichloroacetic acid. After
centrifugation, 100 µL of clear supernatant was mixed
with 20 µL of 1.55 mol/L NaOH, 250 µL of 0.125 mol/L borate buffer
(pH 9.5), and 50 µL of 1 mg/mL
7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. After derivatization at
60°C (1 hour), the sample was analyzed on a
high-performance liquid chromatograph (JASCO
International Co Ltd) equipped with a fluorescence detector (LC
1255, GBC Scientific Equipment Pty Ltd). Separation was carried out on
a 200x4.6-mmx5-µm Nucleosil C18 column. The eluent was 0.1 mol/L
acetate buffer (pH 4.0) containing 2% methanol. The
fluorescence intensities were measured with excitation at 386
nm and emission at 516 nm.
Analysis of Plasma Fatty Acid Content, Lipid Peroxidation
Products, and Lipoprotein Susceptibility to Oxidation
Plasma lipids were extracted27 and the extracts
were transmethylated with BF3/methanol. The fatty
acid methyl esters were separated by capillary gas
chromatography (Chrompack CP 9000 column:
wall-coated open tubular fused-silica 50-mx0.25-mm CP-SIL88).
Plasma lipid peroxidation was measured as thiobarbituric acid–reactive
substances and expressed in malondialdehyde equivalents.27
In brief, each plasma sample (200 µmol/L) was mixed with
thiobarbituric acid reagent (1 mL), heated (100°C, 20 minutes), and
centrifuged, and the absorbance of the supernatant fraction was
measured at 532 nm. LDL and VLDL were isolated by
ultracentrifugation and were tested for their
susceptibility to in vitro copper-induced oxidation.27 In
brief, after isolation, lipoproteins were dialyzed for 20 hours at
4°C in the dark. against PBS (pH 7.4, purged with
N2). The kinetics of LDL oxidation to
CuCl2 (15 µmol/L) was determined by
monitoring the change in diene absorption at 234 nm on a UV
spectrophotometer.
Isolation of Arterioles
Experiments were conducted on isolated arterioles (130
µm active and 180 µm passive diameters at 80 mm Hg)
of rat gracilis muscle as described previously.19 20 28 In
brief, on the fourth week, the rats were fasted overnight and then
anesthetized with an intraperitoneal
injection of sodium pentobarbital (50 mg/kg). The gracilis muscle was
exposed and isolated from surrounding tissues. The muscle then was
dissected out; placed in a silicone-lined Petri dish containing cold
(0°C to 4°C) physiological saline composed of
(in mmol/L) 110 NaCl, 5.0 KCl, 2.5 CaCl2,
1.0 MgSO4, 1.0
KH2PO4, 10.0 dextrose, and
24.0 NaHCO3; and equilibrated with a gas mixture
of 10% O2 and 5% CO2,
balanced with N2, at pH 7.4. With the use of
microsurgery instruments and an operating microscope, a 1.5- mm-long
segment of the first-order arteriole running intramuscularly was
isolated and transferred into an organ chamber containing 2 glass
micropipettes filled with physiological saline
solution. From a reservoir, the vessel chamber (15 mL) was continuously
supplied with physiological saline solution at a
rate of 20 mL/min. After the vessel had been mounted on the proximal
micropipette and was secured with sutures, the perfusion pressure was
raised to 20 mm Hg to clear the red blood cells from the lumen.
Then the other end of the vessel was mounted on the distal pipette.
Both micropipettes were connected with silicone tubing to an adjustable
physiological saline solution reservoir. Pressures
on both sides were measured by electromanometers. The perfusion
pressure was slowly (over 1 minute) increased to 80 mm Hg. The
temperature was set at 37°C by a temperature controller (Grant
Instruments), and the vessel was allowed to equilibrate for 1
hour.
Experimental Protocols
After the equilibration period, changes in the diameter of
arterioles in response to increases in perfusion pressure (from 10 to
20 and from 20 to 140 mm Hg in 20-mm Hg steps) were measured
under zero-flow conditions.29 The pressure was
maintained for 5 minutes at each pressure step to allow the vessel to
reach a steady-state diameter. At the conclusion of each experiment,
the suffusion solution was changed to a Ca2+-free
physiological saline solution, which contained
sodium nitroprusside (SNP, 10-4 mol/L) and EGTA
(1.0 mmol/L); the vessel was incubated for 10 minutes and the
pressure steps were repeated to obtain the maximum passive diameter at
each pressure value (pressure–passive diameter relationship). The
diameter was measured with a microangiometer and recorded on a
chart recorder (Radelkis).
In preliminary studies, we selected doses of BK that elicited constrictions of gracilis arterioles. Responses of arterioles of control and HHcy rats to BK (10-9 to 10-7 mol/L) were compared in the absence and presence of indomethacin (10-5 mol/L), an inhibitor of prostaglandin synthesis; the specific TxA2 receptor antagonist SQ 29,548 (10-6 mol/L); or the specific TxA2 synthase inhibitor furegrelate30 (U63557A, 5x10-6 mol/L).
In separate experiments, the constrictor responses of arterioles of
control and HHcy rats to the stable TxA2 analogue
U46619 (10-11 to 5x10-6
mol/L) were compared before and after endothelium
removal. The endothelium of the arteriole was removed
by perfusion of the vessel with air for 1 minute at a perfusion
pressure of 20 mm Hg. The arteriole was then perfused with
physiological saline solution to clear the debris.
The perfusion pressure was then raised to 80 mm Hg for 30 minutes
to establish a stable tone. The efficacy of endothelial
denudation was ascertained by arteriolar responses to ACh
(10-7 mol/L, an
endothelium-dependent agent) and SNP
(10-7 mol/L, an
endothelium-independent agent) before and after
administration of the air bolus.29 The infusion of air
resulted in loss of function of the endothelium, as
indicated by the absence of dilation to ACh, whereas dilation to SNP
remained intact.
In further experiments, the constrictor responses of arterioles of control and HHcy rats to U46619 were compared in the presence and absence of SQ 29,548 (10-8, 2x10-8, 10-7, and 10-6 mol/L). The U46619 (10-10 to 10-6 mol/L) and SQ 29,548 (10-8 and 2x10-8 mol/L) interaction being apparently competitive, the negative logarithm (pA2) of the equilibrium dissociation constant (Ke) was calculated for the antagonist.31 32 The Ke was calculated from the equation Ke=a/(DR -1), where a is the molar concentration of antagonist, and DR, the dose ratio, is the measure of the rightward shift of the agonist dose-response curve. In separate experiments, the constrictor responses of arterioles of control and HHcy rats to U46619 (10-11 to 5x10-6 mol/L) were compared in the absence and presence of indomethacin (10-5 mol/L) or furegrelate (5x10-6 mol/L).
All drugs were added to the vessel chamber, and final concentrations are reported. After responses to each drug subsided, the system was flushed with physiological saline solution. Changes in diameter were expressed as a percentage of baseline.
Platelet Aggregation Studies
Aggregation of platelets was studied as described
previously.21 33 34 In brief, heparinized blood was
removed from the aorta of the same rats that were used to study
isolated arterioles. Platelet-rich plasma (PRP) was obtained by
centrifugation of blood at 250g for 10
minutes. Platelet-poor plasma (PPP) was prepared from the remaining
volume of blood by centrifugation at 1600g
for 10 minutes.33 The concentration of platelets
in PRP was determined by light microscopy in a counting chamber and was
adjusted to 0.3x109/mL by diluting it with PPP.
Both PRP and PPP were kept at 22°C and used within 1.5 hours of blood
collection. Cuvettes containing 0.5 mL of PRP were placed in a
turbidimetric aggregometer (Mikron M304) under stirring (1000 rpm at
37°C). Aggregation was started by addition of collagen (2.5 to 20
µg/mL) or ADP (10-8 to
3x10-6 mol/L). Where indicated,
indomethacin (5x10-5 mol/L) or
SQ 29,548 (10-5 mol/L) was given 6 minutes
before the start of platelet activation. The extent of aggregation
was expressed as a percentage of the maximal change in optical density
represented by autologous PPP.
U46619 was obtained from Cayman Chemical Co, and SQ 29,548 was obtained from Bristol-Myers Squibb; all other salts and chemicals were obtained from Sigma-Aldrich Co. Solutions were prepared on the day of the experiment. Data are expressed as mean±SEM. Statistical analyses were performed by 2-way ANOVA for repeated measures followed by Tukey’s post hoc test or Student’s t test, as appropriate. P<0.05 was considered statistically significant.
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Results |
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The methionine-rich diet elicited a significantly greater concentration of Hcy in the plasma of HHcy compared with control rats (20.6±3.0 and 6.5±0.5 µmol/L, respectively; P<0.01). There was no difference in the body weight between control rats and methionine-fed rats (382±10 and 365±15 g, respectively; NS).
Plasma Fatty Acid Composition and Lipid Peroxidation
Products
The 20:4(n-6) arachidonic acid content was
significantly increased in plasma lipids of HHcy (n=6) compared with
control (n=6) rats (24.6±2.5% and 16.3±1.7%, respectively;
P<0.05). The lipoprotein oxidation rate was 17.0±4.0 and
17.5±8.0 nmol · min-1 · mg LDL
protein-1; NS), and the thiobarbituric
acid–reactive substances content was 12.5±1.5 and 11.8±2.0 nmol/mL
(NS) in control and HHcy rats, respectively.
Pressure-Diameter Relationships of Arterioles
Isolated arterioles of gracilis muscle from both control and HHcy
rats developed active tone in response to step increases in
intraluminal pressure (10 to 140 mm Hg) without the use of any
vasoactive agent (Figure 1
). Initially,
the diameter of these vessels increased from 100 to 150 µm
in response to an increase in intravascular pressure from 10 to 40
mm Hg. Beyond this point, further increases in pressure resulted in
constrictions of arterioles. There was no significant difference in
pressure-induced responses of arterioles from the 2 groups of rats. In
the absence of Ca2+ and in the presence of
10-4 mol/L SNP, the pressure–passive diameter
relationship in each arteriole was also obtained (Figure 1). In
this condition, step increases in pressure elicited increases in
diameter of arterioles, reaching a plateau at 100 mm Hg
pressure.
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Arteriolar Responses to BK and U46619
In a dose-dependent manner, BK (10-9 to
10-7 mol/L) elicited significantly greater
constrictions in arterioles from HHcy than those from control rats
(Figure 2A). To elucidate the nature of
mediators released in response to BK, we tested arteriolar responses in
the presence of the prostaglandin synthesis
inhibitor indomethacin and the specific
PGH2/TxA2 receptor
inhibitor SQ 29,548. Incubation with
indomethacin or SQ 29,548 elicited significantly
greater inhibition of BK-induced constrictions in arterioles of HHcy
than of control rats and eliminated the difference between the
responses in the 2 groups (Figures 2B and 2C). To further
identify the constrictor prostanoid released in response to BK, we
utilized the specific TxA2 synthase
inhibitor furegrelate. Furegrelate also elicited
significantly greater inhibition of BK-induced constrictions in
arterioles of HHcy than of control rats and eliminated the difference
between the responses in the 2 groups (Figure 2D).
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In a dose-dependent manner, the stable TxA2
analogue U46619 (10-9 to
10-6 mol/L) elicited significantly greater
constrictions of arterioles from HHcy than those from control rats
(Figure 3A). Increasing doses of SQ
29,548 (10-8 to 10-7
mol/L) elicited gradual decreases in arteriolar constrictions to
U46619, whereas 10-6 mol/L abolished the
responses. A selected dose of SQ 29,548 (10-8
mol/L) caused a parallel rightward shift of U46619 dose-response curves
in both control and HHcy arterioles (Figure 3B). From the
parallel shift and assuming a competitive interaction, a
pA2 of 8.69±0.18 and 8.56±0.17 (in control and
HHcy arterioles, respectively; NS) was calculated.
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Indomethacin significantly decreased constrictions to
U46619 in arterioles of HHcy rats. In the presence of
indomethacin, constrictions to U46619 were not
significantly different between control and HHcy arterioles (Figure 3C
). To elucidate the cellular source of enhanced release of
TxA2, the endothelium of
arterioles was removed. Endothelium removal
significantly attenuated constrictions to U46619 in arterioles of HHcy
rats. In the absence of the endothelium, there was no
significant difference between responses to U46619 in arterioles from
control and HHcy rats (Figure 3D).
Platelet Aggregation Studies
Next we investigated the effect of collagen on aggregation of
platelets isolated from the blood of HHcy and control rats. We
found that collagen-induced aggregation of platelets from HHcy rats
was significantly enhanced compared with that of controls (at 5
µg/mL, controls=23±5% and HHcy=49±5%; Figure 4A). Similarly, we found that ADP
elicited significantly greater aggregation in HHcy than in control
platelets (at 10-7 mol/L, controls=25±3%
and HHcy=35±3%; Figure 4B). Indomethacin or SQ
29,548 caused greater inhibition of collagen- and ADP-induced
aggregation of HHcy platelets compared with controls (Figure 5).
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Discussion |
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The salient findings of this study are that elevated plasma Hcy concentration in rats resulted in enhanced arteriolar constrictions to BK and the stable TxA2 analogue U46619 and an augmented collagen- and ADP-induced aggregation of platelets. The underlying mechanism for these alterations is likely an increased TxA2 synthesis in the arteriolar endothelium and platelets in HHcy.
Epidemiological studies have revealed that an elevated level of Hcy is an independent risk factor of human atherosclerosis.1 2 3 4 Previous studies suggested an impaired endothelium-associated dilation in conduit arteries of patients with HHcy.13 14 15 16 In addition, the endothelium-dependent relaxation of carotid artery and an increase in hindlimb circulation to ACh were shown to be reduced in monkeys with diet-induced HHcy.18 The endothelium is important in the local control of blood flow,20 29 35 and in several cardiovascular diseases, such as hypertension20 and atherosclerosis,29 an impaired endothelial regulation of microvascular tone has been documented. Recently, we have demonstrated that NO-mediated endothelial responses are impaired in skeletal muscle arterioles of HHcy rats,19 suggesting an important alteration in endothelial regulation of arteriolar tone.
Interestingly, impairment of endothelial NO
synthesis is often associated with alterations of vascular synthesis of
prostaglandins.36 37 For example, in
hypertension, decreased NO synthesis and increased
PGH2/TxA2
production are present simultaneously in
skeletal muscle arterioles.20 38 Thus, we hypothesized
that the impaired endothelial NO bioavailability in
skeletal muscle arterioles would also lead to enhanced
PGH2/TxA2 synthesis in
HHcy. The methionine diet utilized in the present
study19 21 34 39 increased plasma Hcy levels by 3-fold,
reaching a concentration similar to what was shown to be associated
with an increased risk of vascular disease in
humans.1 2 3 4
In the present experiments, we found no significant
differences between the pressure-diameter curves of the 2 groups of
arterioles (Figure 1), suggesting that the myogenic tone and the
contractile activity of arteriolar smooth muscle are not affected in
general by this model of HHcy, and that the observed alterations in
arteriolar responses are not due to changes in the function of smooth
muscle.
To test the role of
PGH2/TxA2 in the impaired
responses of arterioles, we investigated responses to BK, which are
known to be mediated by multiple pathways, including eicosanoids.
Previous studies in isolated porcine iliac arteries23 and
renal afferent arterioles24 demonstrated that
10-9 to 10-7 mol/L BK
elicited constriction, primarily due to the release of
cyclooxygenase products from the
endothelium,23 24 whereas higher
concentrations (>10-7 mol/L) of BK exerted a
direct effect on the vascular smooth muscle.40 We
confirmed these findings by demonstrating that in rat gracilis muscle
arterioles, BK-induced constrictions were mediated primarily by
constrictor prostanoids, since both inhibition of
prostaglandin synthesis with indomethacin
and blocking the PGH2/TxA2
receptors with SQ29,548 (Figures 2B and 2C) inhibited these
responses. We found that HHcy significantly enhanced BK-induced
constrictions of arterioles (Figure 2A), which were likely due
to an increased synthesis of
PGH2/TxA2, as both
indomethacin and SQ 29,548 substantially inhibited the
responses, thereby eliminating the difference between HHcy and control
arterioles (Figures 2B and 2C). Furthermore, the findings that
the specific TxA2 synthase inhibitor
furegrelate30 inhibited BK-induced constrictions and also
eliminated the difference between responses in control and HHcy
arterioles (Figure 2D) indicate that TxA2
is the primary constrictor prostaglandin synthesized to BK
in arterioles of HHcy rats.
We also found that constrictions to the stable
TxA2 analogue U46619 were enhanced in HHcy
(Figure 3A). Because SQ 29,548 caused a similar rightward shift
in the dose-response curves of control and HHcy arterioles to U46619
(as reflected by the calculated pA2 values;
Figure 3B), we assumed that an altered
TxA2 receptor sensitivity was unlikely to have
contributed to the increased constriction to BK and U46619 in HHcy.
Previously, it had been shown that vascular responses to U46619 were
modulated by a further release of endothelial factors,
such as TxA2.41 42 Thus, the increased
constriction to U46619, as well as to BK in HHcy, is likely due to an
additional release of TxA2 from the arteriolar
endothelium. Indeed, the findings that
indomethacin or removal of the
endothelium decreased constrictions to U46619 in HHcy
but not in control arterioles support this hypothesis (Figures 3C and 3D). Thus, arteriolar release of
TxA2 may contribute to the increased urinary
excretion of TxB2, the metabolite of
TxA2, that has been demonstrated in patients with
genetic HHcy.43 44 45 In the present study, we aimed to
elucidate whether alterations in platelet function were also
related to increased TxA2 activity in HHcy,
independent of vascular mechanisms.
To that purpose, platelets were isolated from the blood of
control and HHcy rats used for vascular studies, and agonist-induced
platelet aggregation in the 2 groups was compared. Collagen and ADP
are known to elicit platelet aggregation by a mechanism that
involves TxA2
synthesis/release.33 46 We found that collagen- and
ADP-induced platelet aggregation was significantly enhanced in HHcy
rats (Figure 5) and that either inhibition of
cyclooxygenase or blocking of the
TxA2 receptors (Figure 5) eliminated the
difference between aggregation of platelets from HHcy and control
rats. These results indicate that the increased platelet
aggregation in HHcy is most likely due to an enhanced formation of
TxA2 in platelets. Earlier
studies21 also demonstrated an increased ADP-induced
aggregation of platelets associated with an enhanced
TxA2 biosynthesis in rats with HHcy.
Collectively, these studies suggest that simultaneous
elevation of TxA2 synthesis in
endothelial cells and platelets is responsible for
the increased urinary excretion of TxB2
demonstrated in human HHcy.43 44 45
One of the mechanisms that might favor the formation of TxA2 is an increased formation of arachidonic acid. Indeed, we confirmed earlier findings21 by showing an increased level of arachidonic acid in the plasma of HHcy rats. This could be due to an elevated level of reactive oxygen species shown to be present in HHcy.21 Homocysteine, by the autooxidation of the sulfhydryl group, may promote the generation of oxygen free radicals, resulting in oxidative stress.12 In addition, homocysteine may decrease the intracellular level of glutathione and glutathione peroxidase, which are responsible for the elimination of oxygen free radicals.47 48 An enhanced level of reactive oxygen species is known to interfere with NO, a mechanism that may be responsible for the impaired arteriolar responses to ACh and histamine in HHcy.19 Also, NO and superoxide can form peroxynitrite, which may interfere with PGI2 synthase promoting the elevation of TxA2,49 especially when the level of its precursor arachidonic acid is elevated.
In conclusion, in HHcy, the reduced endothelial release of NO19 together with the simultaneously increased TxA2 synthesis in arterioles and platelets could interfere with endothelial regulation of blood flow and enhance platelet aggregation, thereby predisposing the circulatory system to atherothrombotic alterations.
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Acknowledgments |
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This study was supported by grants from the Hungarian National Scientific Research Foundation (OTKA T-023863 and T-033117), the Health Science Council (ETT/524), and the National Heart, Lung, and Blood Institute, Bethesda, Md (HL-46813). The authors are indebted to Katalin Rischák for homocysteine measurements.
Received June 25, 1999; accepted February 7, 2000.
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