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Vol. 283, Issue 1, 75-81, 1997
Department of Pharmacology, New York Medical College, Valhalla, New York
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Rings of thoracic aortae taken from rats made hypertensive by aortic
coarctation express a calcium-dependent basal tone. We investigated
whether this basal tone is mediated by prostanoids. To this end, we
contrasted the effects of indomethacin, an inhibitor of cyclooxygenase,
and of ifetroban, an antagonist of thromboxane A2/prostaglandin endoperoxide H2 receptors, on
basal tone in aortic rings taken from normotensive and hypertensive
rats. Rings with endothelium from normotensive rats were unaffected by
indomethacin and ifetroban. However, in endothelium-intact rings from
hypertensive rats, the basal tone was reduced 65 to 75% by
indomethacin and ifetroban, but not by CGS13080, an inhibitor of
thromboxane synthase. The reductions in tone elicited by indomethacin
and ifetroban in rings from hypertensive rats were eliminated upon
removal of the endothelium and were attenuated when the rings were
pretreated with an inhibitor of nitric oxide synthase
(N
-nitro-L-arginine methyl ester or
N-nitro-L-arginine) or an inhibitor of soluble guanylate
cyclase. Neither indomethacin nor ifetroban affected tissue cGMP levels
or nitrite release in aortic rings taken from hypertensive rats.
However, sodium nitroprusside offset the inhibitory effects of
N-nitro-L-arginine methyl ester, on the relaxant
responses to indomethacin and ifetroban. These data suggest that a
constrictor prostanoid other than thromboxane A2,
presumably prostaglandin endoperoxide H2 contributes to the
implementation of the basal tone in rings from hypertensive rats and
that part of the relaxant response to indomethacin and ifetroban is
linked to nitric oxide.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In
rats with aortic coarctation-induced hypertension as well as in other
models of AngII-dependent hypertension, antagonists of
TxA2/PGH2 receptors reduce
blood pressure (Boussairi et al., 1994
; Lin et
al., 1991; Mistry and Nasjletti, 1988, 1990). An antagonist of
TxA2/PGH2 receptors, but
not an inhibitor of thromboxane synthase, was shown to inhibit
contractile responses to AngII, calcium ionophore and arachidonic acid
in rings of thoracic aortae taken from rats with aortic
coarctation-induced hypertension (Lin et al., 1994; Lin and
Nasjletti, 1992, 1991). From these observations it was inferred that a
prostanoid-mediated pressor mechanism contributes to the development of
aortic coarctation-induced hypertension. This pressor mechanism appears
to be related to activation of the
TxA2/PGH2 receptor in
arterial smooth muscle by a constrictor prostanoid other than
TxA2, presumably PGH2.
Rings of thoracic aortae taken from rats with aortic
coarctation-induced hypertension, but not from normotensive rats,
display a high level of active basal tone in the absence of exogenous vasoconstrictors (Pucci et al., 1995). This tone is
implemented by a constrictor mechanism that relies on calcium and
protein kinase C activity and is subject to inhibitory regulation by
endogenous and exogenous nitric oxide (Pucci et al., 1995).
The present study was undertaken to test the hypothesis that a
constrictor prostanoid contributes to the mechanism underlying the
active basal tone displayed by aortic rings of rats with aortic
coarctation-induced hypertension.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Drugs and Solutions
Indomethacin, sodium nitroprusside, sodium nitrite, L-NA, L-NAME and nifedipine were obtained from Sigma Chemical Co., St. Louis, MO; CGS13080 was from CIBA-GEIGY, Summit, NJ; ifetroban was from Bristol Myers Squibb, Princeton, NJ; DAN reagent was from LC Laboratories, Woburn, MA; PGH2 was from BIOMOL Research Laboratories Inc, Plymouth Meeting, PA; ODQ was from Calbiochem, LaJolla, CA.
Pyrogen-free deionized water was used in the preparation of all
solutions and buffers. A stock solution of CGS13080 was made in 50 mM
Na2CO3 and stored at
20°C. Stock solutions of ifetroban and sodium nitroprusside were
made in distilled water and stored at 20°C. Stock solutions of ODQ
and nifedipine were made in dimethyl sulfoxide and stored at 20°C.
Stock solutions of indomethacin and L-NA were freshly
prepared in 50 mM Na2CO3
and L-NAME in Krebs' bicarbonate buffer. The DAN reagent
was first diluted in 0.3 N HCl (5 mg/ml) and immediately before usage
rediluted in 0.62 N HCl (0.05 mg/ml). The DAN reagent was protected
from light. PGH2 was dried under nitrogen
immediately before use and was dissolved in 0.1 M Tris buffer.
The composition of Krebs' bicarbonate buffer was (mol/l): NaCl, 118.5; KCl, 4.7; CaCl2, 2.8; KH2PO4, 1.2; MgSO4, 1.1; NaHCO3, 25.0; and dextrose, 11.1. The composition of calcium-free Krebs' bicarbonate buffer was as above except that CaCl2 was omitted.
Animals and Surgical Procedures
Experiments were conducted on male Sprague-Dawley rats (Charles
River, Wilmington, MA) weighing 300 to 325 g according to protocols approved by the Institutional Animal Care and Use Committee. To induce the development of hypertension, rats were anesthetized with
sodium pentobarbital (50 mg/kg i.p.) and subjected to aortic coarctation according to a published procedure (Fernandes et
al., 1976). The abdominal cavity was exposed through a midline
incision, a silk ligature no. 000 was passed around the aorta at a
point below the right renal artery and above the left renal artery, just below the origin of the superior mesenteric artery, and the aorta
was completely ligated.
On the day of the experiment, 7 to 14 days after aortic coarctation, the right carotid artery of rats anesthetized with metofane (Pitman Moore, Mundelein, IL) was cannulated with polyethylene tubing (PE-50) and connected to a pressure transducer (model P231D, Statham Division, Gould Inc., Oxnard, CA) for recording of mean blood pressure on a polygraph (model 7D, Grass Instrument Co., Quincy, MA). Animals were allowed to completely recover from anesthetic before the measurement of blood pressure. A rat was considered hypertensive if mean blood pressure was greater than 150 mm Hg. The mean blood pressure of rats with aortic coarctation-induced hypertension (171 ± 3 mm Hg; n = 93) surpassed (P < .01) that of unoperated normotensive rats (103 ± 3 mm Hg; n = 8). After blood pressure measurement, the rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.), the thoracic cavity was exposed and the descending thoracic aorta was excised, transferred to a dish filled with ice-cold Krebs' bicarbonate buffer, cleared of periadventitial tissue and cut transversely into ring segments (2.0-3.0 mm in length). The aortic rings were used immediately in the following protocols.
Protocols to Investigate the Contribution of Prostanoids to the Resting Tension of Aortic Rings
Each aortic ring was placed inside a water-jacketed 5-ml tissue
bath filled with Krebs' bicarbonate buffer (37°C), bubbled with 95%
O2-5% CO2 and attached to
a force-displacement transducer (model FT03C, Grass) coupled to a
polygraph (model 7D, Grass) for measurement of isometric tension as
described previously (Lin and Nasjletti, 1991). The rings of thoracic
aortae were allowed to equilibrate at room temperature at a resting
tension of 2 g for approximately 30 min (previous experiments from
our laboratory have shown that 2 g of resting tension is optimal
for the expression of potassium chloride-induced contraction of aortic
rings obtained from normotensive and hypertensive rats). After this
initial equilibration period, the Krebs' bicarbonate buffer was
gradually heated to 37°C. Sixty minutes later, unless otherwise
indicated, the rings were exposed to calcium-free Krebs' bicarbonate
buffer for 15 min followed by reexposure to regular Krebs' bicarbonate
buffer. The amount of isometric tension developed in the presence of
regular buffer after exposure to calcium-free buffer was defined as
calcium-dependent tone. In some experiments, aortic rings never before
exposed to calcium-free buffer were challenged with the calcium channel
blocker nifedipine (10
6 mol/l) and ensuing
changes in isometric tension were taken as a reflection of the
calcium-dependent tone.
After a stable base line had been reached, the effect on isometric
tension of indomethacin (105 mol/l) to
inhibit cyclooxygenase (Flower, 1974), ifetroban
(106 mol/l) to block
TxA2/PGH2 receptors
(Tesfamariam, 1994) or CGS13080 (105
mol/l) to inhibit thromboxane synthase (MacNab et al., 1984) was evaluated. In hypertensive rats, some of these experiments were
conducted in vascular preparations in which the endothelium was removed
by gently rubbing the lumen with forceps. The functionality or lack of
functionality of the endothelium was established by examining whether
or not acetylcholine (106 mol/l) was
effective in relaxing aortic rings precontracted with phenylephrine.
The effect of indomethacin and ifetroban on resting tone in aortic
rings of hypertensive rats also was evaluated in preparations
pretreated and not pretreated with an inhibitor of nitric oxide
synthase (L-NAME or L-NA, both at 3 × 104 mol/l) or an inhibitor of soluble
guanylate cyclase (ODQ, 105 mol/l). [This
concentration of ODQ was found to completely inhibit the relaxant
responses to sodium nitroprusside (106
mol/l) in aortic rings from hypertensive rats.] In these experiments, the inhibitors of nitric oxide synthase or of guanylate cyclase were
present in the organ bath from the onset and were readministered any
time the buffer was changed during the study. In complementary experiments, the effects of indomethacin
(105 mol/l) and ifetroban
(106 mol/l) on the basal tone of aortic
rings from hypertensive rats was examined in preparations pretreated
with L-NAME (3 × 104
mol/l) and bathed in media with or without enough sodium nitroprusside (5.84 ± 0.92 nmol/l) to replace for the loss of endogenous nitric oxide. In these experiments, the rings were exposed to calcium-free Krebs' bicarbonate buffer followed by reexposure to regular Krebs' bicarbonate buffer. Once the rings had recontracted, sodium
nitroprusside was administered until a 15 to 20% reduction in
calcium-dependent tone was obtained. After a new base line had been
reached, the effects of indomethacin (105
mol/l) and ifetroban (106 mol/l) were
examined. Results are expressed as the change in prevailing
calcium-dependent tone after exposure to sodium nitroprusside.
Protocols to Investigate the Effect of Indomethacin and Ifetroban on Aortic Content of cGMP and Release of Nitrite
To evaluate the effect of indomethacin and ifetroban on aortic
content of cGMP, rings of thoracic aortae were taken from rats with
aortic coarctation-induced hypertension and calcium-dependent tone was
assessed as described above. Thirty minutes after reexposure to regular
Krebs' bicarbonate buffer, aortic rings either remained untreated or
were treated with indomethacin (105 mol/l)
or ifetroban (106 mol/l). Fifteen minutes
later, while monitoring isometric tension, each aortic ring was frozen
in liquid nitrogen and subsequently transferred to a solution of
ice-cold trichloroacetic acid (10%) and stored at 70°C. To assay
cGMP, each ring was homogenized in the trichloroacetic acid solution.
After centrifugation, the supernatant was extracted four times with 2 volumes of diethyl ether, and the aqueous phase was evaporated under
vacuum. After reconstitution with deionized water, cGMP was measured by
radioimmunoassay (Advanced Magnetics, Cambridge, MA). The tissue
content of cGMP is expressed as femtomoles per milligram of protein.
Protein was measured using the BioRad Protein Assay Kit (BioRad
Laboratories, Hercules, CA).
To evaluate the effect of indomethacin and ifetroban on the release of
nitrite by aortic tissue, the descending thoracic aortae of rats with
aortic coarctation-induced hypertension was excised and cut into rings
which were placed in 1.5-ml microcentrifuge tubes containing 250 µl
of Krebs' bicarbonate buffer. Aortic rings were incubated at 37°C in
an atmosphere of 95%O2-5%
CO2 for 20 min in the absence and presence of
indomethacin (105 mol/l) or ifetroban
(106 mol/l). At the conclusion of the
incubation, the aortic rings were dried overnight and the weight was
taken, whereas the concentration of nitrite in the incubation media was
measured according to a previously described fluorometric assay (Misko
et al., 1993). Nitrite release is expressed as picomoles of
nitrite released during a 20-min period per milligram of dry tissue.
Protocols to Investigate the Effect of L-NAME on Prostaglandin Synthesis in Aortic Rings
In one protocol, the effect of L-NAME on the release
of 6-keto-PGF1
, an index of
PGI2 release, was examined in rings of descending
thoracic aortae taken from rats with aortic coarctation-induced hypertension. To this end, paired aortic rings were preincubated for 20 min at 37°C in Krebs' bicarbonate buffer equilibrated in an
atmosphere of 95% O2-5%
CO2, with and without L-NAME (3 × 104 mol/l). Subsequently, each ring was
transferred to a new vial containing 1.0 ml of the corresponding fresh
media and was incubated for an additional 20-min period. The rings were
saved for measurement of dry weight and the media were stored at
20°C until assayed for
6-keto-PGF1 with an
enzyme-immunoassay kit obtained from Cayman Chemical (Ann Arbor, MI).
Release of 6-keto-PGF1 is expressed as
nanograms of 6-keto-PGF1 released during
a 20-min period per milligram of dry tissue.
In another protocol, the effect of L-NAME on the conversion
of PGH2 to PGI2 (measured
as 6-keto-PGF1) was studied in aortic
rings of rats with aortic coarctation-induced hypertension. To this
end, paired aortic rings from hypertensive rats were preincubated at
37°C for 20 min in Krebs' bicarbonate buffer containing indomethacin (105 mol/l), with and without
L-NAME (3 × 104 mol/l),
in an atmosphere of 95% O2-5%
CO2. Subsequently, each ring was then transferred
to a new vial containing 1.0 ml of the corresponding fresh media.
Authentic PGH2 (105
mol/l) was added to the media and the incubation was carried out under
identical conditions for 3 min. At the conclusion of the incubation
period, the rings were removed and saved for estimation of dry weight,
and the media were stored at 20°C until assayed for
6-keto-PGF1 as indicated above. The
concentration of 6-keto-PGF1 in media
derived from the incubation of aortic rings in indomethacin-containing
buffer without exogenous PGH2 was <6% of the
concentration in media derived from incubations carried out in the
presence of exogenous PGH2. Hence, when
cyclooxygenase is inhibited by indomethacin, the conversion of
exogenous PGH2 to PGI2 by
aortic rings reflects the tissue activity of prostacyclin synthase (Lin
et al., 1994). The conversion of PGH2
to PGI2 is expressed as nanograms of
6-keto-PGF1 formed during the 3-min incubation period per milligram of dry tissue.
Statistics
Results are expressed as mean ± S.E.M. Data were analyzed as appropriate by Student's t test. The null hypothesis was rejected at P < .05.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Figure 1 shows representative
tracings of experiments which examined the effects of successive
exposure to calcium-free Krebs' bicarbonate buffer, regular Krebs'
bicarbonate buffer and either the cyclooxygenase inhibitor,
indomethacin (105 mol/l), or the
TxA2/PGH2 receptor
antagonist, ifetroban (106 mol/l), on
isometric tension developed by rings of thoracic aortae. In rings of
thoracic aortae taken from normotensive rats, isometric tension did not
change during exposure to calcium-free media or during subsequent
reexposure to calcium-containing media. On the other hand, in rings of
thoracic aortae taken from hypertensive rats, isometric tension fell
progressively during exposure to calcium-free media, and this change
was readily reversed upon reexposure to calcium-containing media. The
increase in tension brought about by reexposure of the aortic rings to
calcium-containing media was presumed to reflect the calcium-dependent
active tone of the rings. This tone was virtually undetectable in
aortic rings taken from normotensive rats (n = 8) and
averaged 1.16 ± 0.09 g in aortic rings taken from rats with
aortic coarctation-induced hypertension of 7 to 14 days duration
(n = 36). In experiments with hypertensive rats, these
estimates of calcium-dependent tone in aortic smooth muscle matched
well with the relaxing responses to a maximally effective concentration
of the calcium channel blocker nifedipine
(106 mol/l) in aortic rings never before
exposed to calcium-free media (0.95 ± 0.18 g,
n = 4).
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Indomethacin did not affect isometric tension development in aortic
rings taken from normotensive rats (n = 4), but
elicited a 0.60 ± 0.09 g reduction of tension in aortic
rings from hypertensive rats (n = 18), which
corresponds to a 69 ± 11% reduction in the prevailing
calcium-dependent active tone of the rings. Similarly, ifetroban did
not affect development of isometric tension in aortic rings from
normotensive rats (n = 4), but caused a 0.61 ± 0.08 g reduction of tension in aortic rings from hypertensive rats (n = 18), which corresponds to a 68 ± 11%
decrease in the calcium-dependent active tone displayed by the rings.
In experiments with hypertensive rats, indomethacin and ifetroban also
elicited reductions of basal tone in aortic rings never before exposed
to calcium-free media (0.53 ± 0.09 g, n = 16; and 0.64 ± 0.12 g, n = 16, respectively).
Figure 2 contrasts the effects of
indomethacin (105 mol/l), ifetroban
(106 mol/l) and the thromboxane synthase
inhibitor CGS13080 (105 mol/l) on the
calcium-dependent active tone displayed by endothelium-intact and
endothelium-denuded rings of descending thoracic aortae taken from
hypertensive rats. The calcium-dependent active tone of aortic rings
denuded of endothelium by rubbing (1.73 ± 0.18 g) surpassed (P < .01) that of unrubbed aortic rings (1.16 ± 0.09 g). The calcium-dependent active tone of unrubbed rings of aortae was
decreased (P < .01) both by indomethacin and ifetroban. However,
the calcium-dependent active tone of rubbed aortic rings was virtually
unaffected by indomethacin or ifetroban. The thromboxane synthase
inhibitor CGS13080 had minimal effect on the calcium-dependent active
tone displayed either by rubbed or unrubbed aortic rings.
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Figures 3, 4, 5, 6, 7 show the results of experiments aimed at investigating
the relationship between vascular nitric oxide synthesis and the
ability of indomethacin and ifetroban to produce endothelium-dependent
reductions of calcium-dependent active tone in aortic rings of
hypertensive rats. Figure 3 depicts the
effects of indomethacin (105 mol/l) and
ifetroban (106 mol/l) on the
calcium-dependent active tone displayed by aortic rings from
hypertensive rats while bathed in Krebs' bicarbonate buffer with and
without the nitric oxide synthesis inhibitor L-NAME (3 × 104 mol/l) or L-NA (3 × 104 mol/l). The calcium-dependent
active tone displayed by rings of descending thoracic aortae in media
without inhibitors of nitric oxide synthase (1.21 ± 0.07 g)
was exceeded (P < .05) by that displayed by aortic rings in media
containing L-NAME (1.63 ± 0.08 g) or
L-NA (1.78 ± 0.13 g). Indomethacin elicited more
prominent (P < .01) reductions of calcium-dependent active tone
in aortic rings bathed in control media without inhibitors of nitric
oxide synthesis than in aortic rings bathed in media containing
L-NAME or L-NA. Likewise, ifetroban produced
more prominent reductions (P < .01) of calcium-dependent active
tone in aortic rings bathed in control media than in rings bathed in
media containing L-NAME or L-NA.
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Figure 4 contrasts the effects of
indomethacin (105 mol/l) and ifetroban
(106 mol/l) on the calcium-dependent
active tone displayed by aortic rings from hypertensive rats in Krebs'
bicarbonate buffer with and without ODQ
(105 mol/l), an inhibitor of soluble
guanylate cyclase. The calcium-dependent active tone displayed by
aortic rings in control media (1.38 ± 0.33 g) did not differ
significantly from that in aortic rings bathed in media containing ODQ
(1.54 ± 0.37 g). Indomethacin elicited more prominent
reductions of calcium-dependent active tone (P < .01) in aortic
rings bathed in control media than in aortic rings bathed in media
containing ODQ. Likewise, ifetroban produced more prominent reductions
of calcium-dependent tone (P < .01) in aortic rings bathed in
control media than in aortic rings bathed in media containing ODQ.
Figure 5 shows the effects of
indomethacin (105 mol/l) and ifetroban
(106 mol/l) on calcium-dependent tone in
aortic rings taken from hypertensive rats bathed in Krebs' bicarbonate
buffer containing L-NAME (3 × 104 mol/l) either with or without enough
sodium nitroprusside to replace L-NAME-induced losses of
endogenous nitric oxide. Sodium nitroprusside (5.84 ± 0.92 nmol/l) caused a small reduction in calcium-dependent tone (18 ± 2%). Moreover, the reductions in calcium-dependent tone elicited by
indomethacin or ifetroban in aortic rings bathed in media containing
L-NAME and sodium nitroprusside were more prominent than
those obtained in rings bathed in media with L-NAME alone.
Figure 6 shows data on cGMP content and
nitrite release in aortic rings from hypertensive rats during
incubation in Krebs' bicarbonate buffer with and without indomethacin
(105 mol/l) or ifetroban
(106 mol/l). Neither indomethacin nor
ifetroban modified the cGMP content or nitrite release of aortic rings.
Figure 7 (upper panel) shows data on
PGI2 release (measured as
6-keto-PGF1) from aortic rings of
hypertensive rats during incubation in Krebs' bicarbonate buffer with
and without L-NAME (3 × 104 mol/l). L-NAME caused a
small but significant (P < .05) increase in the release of
PGI2 from aortic rings taken from hypertensive rats. As shown in figure 7 (lower panel), the conversion of exogenous PGH2 to PGI2 by aortic
rings from hypertensive rats, during short-term incubation in media
containing indomethacin to suppress cyclooxygenase, was not affected by
L-NAME.
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Discussion |
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|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Rings of thoracic aortae taken from rats with aortic
coarctation-induced hypertension, but not from normotensive rats,
display a calcium-dependent basal tone (Pucci et al., 1995).
These studies demonstrate that this calcium-dependent tone displayed in
rings from hypertensive rats can be attenuated 65 to 75% by both
indomethacin and ifetroban, which suggests the contribution of a
constrictor prostanoid that binds to the
TxA2/PGH2 receptor. This
constrictor prostanoid is presumed to be PGH2,
because CGS13080, an inhibitor of thromboxane synthase, did not reduce
the basal tone. Previous studies have implicated
PGH2 in the constrictor responses of aortic rings
to acetylcholine (Ito et al., 1991; Luscher and Vanhoutte, 1986) and arachidonic acid (Ge et al., 1995; Boulanger and
Vanhoutte, 1993) in the spontaneously hypertensive rat.
PGH2 also was proposed to contribute to the
constrictor effect of AngII, arachidonic acid and calcium ionophore in
aortic rings of rats with aortic coarctation-induced hypertension (Lin
et al., 1994; Lin and Nasjletti, 1992, 1991). The present
study did not investigate the contribution of
PGH2-mediated mechanisms of vascular contraction
to the tone of resistance vessels in rats with aortic
coarctation-induced hypertension. However, such a contribution is
likely in view of a report which showed that treatment with
TxA2/PGH2 receptor
antagonists lower blood pressure in rats with aortic
coarctation-induced hypertension (Lin et al., 1991).
Confirming a previous report (Pucci et al., 1995), removal
of the endothelium results in an augmentation of basal tone in aortic
rings of rats with aortic coarctation-induced hypertension. However,
neither indomethacin nor ifetroban decreased the basal tone of rings
without endothelium. These findings suggest that although the tone of
aortic rings with endothelium is mediated by both prostanoid-dependent
and -independent mechanisms, the tone of rings without endothelium is
mediated by a prostanoid-independent mechanism. Moreover, our findings
indicate that removal of the endothelium from aortic rings of rats with
aortic coarctation-induced hypertension blunts the prostanoid-dependent
component of the basal tone and magnifies the prostanoid-independent
component.
One possible explanation to account for the finding that indomethacin and ifetroban decrease the basal tone of unrubbed aortic rings only is that the constrictor prostanoid contributing to the tone originates in the endothelium. An alternative explanation is that blockade of either PGH2 formation or its receptor unmasks a vasodilator mechanism linked to production of endothelium-derived nitric oxide. In this regard, when the rings of thoracic aortae taken from hypertensive rats were pretreated with L-NAME or L-NA, both inhibitors of nitric oxide synthase, the relaxant responses to indomethacin and ifetroban were attenuated. Furthermore, when the rings were pretreated with ODQ, an inhibitor of soluble guanylate cyclase, the relaxant responses to indomethacin and ifetroban also were attenuated. These data support the notion that the responses to indomethacin and ifetroban are partly linked to a nitric oxide-dependent mechanism.
Recent reports of interactions between nitric oxide and constrictor
prostanoids support the possibility that PGH2
either decreases the formation or increases the inactivation of nitric
oxide. For example, PGH2 was shown to attenuate
the relaxing effect of acetylcholine in aortic rings from rats
(Tesfamariam, 1994), whereas cyclooxygenase inhibitors and
TxA2/PGH2 receptor
antagonists were found to increase the relaxing effect of acetylcholine
in aortic rings of spontaneously hypertensive rats (Kato et
al., 1990; Ito et al., 1991; Luscher and Vanhoutte,
1986). If PGH2 does interfere with vasorelaxing mechanisms mediated by nitric oxide, pharmacological blockade of
PGH2 formation or actions may be expected to
either increase the formation or decrease the inactivation of nitric
oxide. However, neither indomethacin nor ifetroban increased nitrite
release or cGMP content of aortic rings from hypertensive rats.
Together, these data argue against the idea that the reduction in tone
elicited by indomethacin and ifetroban is mediated by increased aortic levels of nitric oxide and/or cGMP.
Another possibility to explain the finding that nitric oxide synthase
inhibitors attenuate the relaxant responses to indomethacin and
ifetroban is that nitric oxide fosters the activity of a
PGH2-mediated mechanism of vascular contraction.
Previous studies have shown that nitric oxide can stimulate
heme-containing enzymes, such as cyclooxygenase (Davidge et
al., 1995; Salvemini et al., 1993, 1994). If nitric
oxide were stimulating cyclooxygenase, we would expect that inhibition
of nitric oxide synthase would decrease the release of
6-keto-PGF1, an index of
PGI2 production. However, our data argue against
this hypothesis, because L-NAME caused an increase in
PGI2 release from rings of hypertensive rats.
This observation calls attention to the possibility that endogenous
nitric oxide inhibits the conversion of PGH2 to
PGI2 by prostacyclin synthase, thus favoring
elevation of PGH2 levels. However, our findings
are inconsistent with this hypothesis, because the conversion of
PGH2 to PGI2, an index of
prostacyclin synthase activity, was unaffected by L-NAME in
aortic rings from hypertensive rats.
An alternative explanation to account for the finding that inhibition of nitric oxide synthase attenuates the relaxant responses to indomethacin and ifetroban in aortic rings of hypertensive rats is that nitric oxide subserves a permissive role, which facilitates the deactivation of a prostanoid-mediated mechanism of vascular contraction. This possibility agrees with our finding that relaxant responses to indomethacin or ifetroban are unimpeded in aortic rings pretreated with L-NAME and concurrently exposed to sodium nitroprusside to replace the loss of endogenous nitric oxide. However, the present study offers no information on the precise mechanism underlying the proposed permissive role of nitric oxide.
In conclusion, we have shown that the basal calcium-dependent tone that is expressed in rings of thoracic aorta taken from rats with aortic coarctation-induced hypertension is partially attenuated by indomethacin and ifetroban, but not by an inhibitor of thromboxane synthase. The reductions in basal tone elicited by indomethacin and ifetroban were attenuated by nitric oxide synthase inhibitors as well as by an inhibitor of soluble guanylate cyclase. These data suggest that a constrictor prostanoid other than TxA2, presumably PGH2, contributes to the implementation of the basal tone in rings from hypertensive rats and that part of the response to indomethacin and ifetroban is linked to nitric oxide.
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Acknowledgments |
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The authors thank Lucas Guideri for technical assistance.
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Footnotes |
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Accepted for publication June 10, 1997.
Received for publication February 5, 1997.
1 This work was supported by Grants HL-18579 and 5PO1 HL-34300 from the US Public Health Service.
Send reprint requests to: AnnMarie DelliPizzi, Department of Pharmacology, New York Medical College, Valhalla, NY 10595.
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Abbreviations |
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AngII, angiotensin II;
TxA2, thromboxane A2;
PGH2, prostaglandin
endoperoxide H2;
L-NA, N-nitro-L-arginine;
L-NAME, N-nitro-L-arginine methyl ester;
CGS13080, imidazol
[1,5-
]pyrine 5-hexanoic acid;
ifetroban, [1S-(1,2,3,4)]-2-[(3-{4-[(pentylamino)carbonyl]-2-oxazolyl}-7-oxabicyclo[2.2.1]hept-2-yl)methyl]benzene-propanoic
acid ;
DAN reagent, 2,3-diaminonaphthalene;
ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one;
PGI2, prostaglandin I2;
6-keto-PGF1, 6-keto-prostaglandin F1.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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