(Hypertension. 1996;27:893-896.)
© 1996 American Heart Association, Inc.
Articles |
L-Arginine Restores Dilator Responses of the Basilar Artery to Acetylcholine During Chronic Hypertension
From the Departments of Internal Medicine and Pharmacology, Cardiovascular Center, University of Iowa College of Medicine, and Veterans Administration Medical Center, Iowa City.
Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242.
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Abstract |
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Abstract The objective of this study was to test the hypothesis that administration of L-arginine, a substrate for nitric oxide synthase, restores acetylcholine-induced dilatation of the basilar artery in chronically hypertensive rats. Basilar artery diameter was measured through a cranial window in anesthetized stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) aged 6 to 7 months (adult) and 12 months (older adult). Under control conditions, baseline basilar artery diameter was smaller in SHRSP (adult, 239±30 µm; older adult, 198±13 µm) (mean±SE) than in WKY (adult, 261±10 µm; older adult, 259±7 µm) (P<.05 versus SHRSP). Topical application of acetylcholine (10-5 mol/L) produced dilatation of the basilar artery in WKY, which was impaired in both adult and older SHRSP (P<.05). Topical L-arginine (10-3 mol/L for 30 minutes) did not affect responses to acetylcholine in adult SHRSP but enhanced vasodilatation in response to acetylcholine (10-5 mol/L) in older SHRSP without affecting responses to sodium nitroprusside. In contrast, D-arginine did not affect acetylcholine-induced vasodilatation in older SHRSP. These results suggest that impaired dilatation of the basilar artery in response to acetylcholine in older SHRSP is restored toward normal by L-arginine, a substrate for nitric oxide synthase.
Key Words: cerebral arteries • endothelium-derived factor • rats, stroke-prone SHR • acetylcholine • arginine • nitric oxide
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Introduction |
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Endothelium-dependent dilator responses of cerebral and noncerebral arteries are reduced during chronic hypertension.1 2 3 4 5 6 7 8 Dilatation of the basilar artery in response to acetylcholine and bradykinin is impaired in spontaneously hypertensive rats and SHRSP compared with normotensive WKY.2 5 6
Mechanisms that contribute to impaired dilator responses of the basilar artery in SHRSP are not known. Reduced production of EDRF or release of an endothelium-derived contracting factor8 9 could contribute to such impairment. However, indomethacin does not affect responses of the basilar artery to acetylcholine in spontaneously hypertensive rats.6 Thus, impaired responses of the basilar artery to acetylcholine do not appear to be due to release of a cyclooxygenase-dependent endothelium-derived contracting factor, as has been described in some noncerebral blood vessels and cerebral arterioles.3 4
Acute or chronic treatment with L-arginine, a substrate for NO synthase, restores impaired endothelium-dependent relaxation in hypercholesterolemic animals and humans toward normal.10 11 12 13 14 15 16 L-Arginine has also been reported to prevent the development of hypertension in Dahl/Rapp salt-sensitive rats.17 The goal of the present study was to test the hypothesis that acute treatment with L-arginine restores impaired dilatation of the basilar artery to acetylcholine in SHRSP.
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Methods |
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Animal Preparation
Male WKY and SHRSP aged 6 to 7 months (adult) and 11 to 13 months (older adult) were used in this study. The rats were anesthetized with pentobarbital sodium (50 mg/kg IP). Anesthesia was supplemented at approximately 20 to 25 mg/kg per hour IV. The trachea was cannulated, and the rats were mechanically ventilated with room air and supplemental oxygen. Skeletal muscle paralysis was produced with gallamine triethiodide (5 to 10 mg/kg). Depth of anesthesia was evaluated by applying pressure to a paw or the tail and observing changes in heart rate or blood pressure. When such changes occurred, additional anesthetic was administered. Catheters were placed in both femoral arteries to measure systemic arterial pressure and obtain arterial blood samples. A femoral vein was cannulated for drug infusion. Arterial blood gases were monitored and maintained within normal limits throughout the experiments in both strains.
A craniotomy was prepared over the ventral brain stem as previously described in detail.5 18 A portion of the dura mater was opened and the cranial window was suffused with artificial cerebrospinal fluid (temperature, 37°C; ionic composition [mmol/L]: NaCl 132, KCl 2.95, CaCl2 1.71, MgCl2 0.65, NaHCO3 24.6, D-glucose 3.69) that was bubbled continuously with appropriate gases to produce normal levels of pH and PCO2. Blood vessel diameter was measured with a microscope equipped with a television camera coupled to a video monitor and image-shearing device (model 908, Instrumentation for Physiology & Medicine). The images were recorded on videotape for later analysis.
Experimental Protocol
We examined responses of the basilar artery to topical
application of acetylcholine (10-6 and
10-5 mol/L) and sodium nitroprusside
(10-8 and
10-7 mol/L). Agonists were mixed in
artificial cerebrospinal fluid and suffused over the
craniotomy for 4 minutes. Basilar artery diameter was
measured immediately before and during the last minute of application
of each agonist. After application of each agonist, the vessel returned
to baseline diameter within 5 minutes. L-Arginine
(10-4 and
10-3 mol/L) and D-arginine
(10-3 mol/L) were then suffused for 30
minutes before and during application of acetylcholine and sodium
nitroprusside. Responses of the basilar artery to acetylcholine were
also examined before and after injection of 10 mg/kg
indomethacin IV. We have previously shown that this
indomethacin dose effectively inhibits dilatation of
cerebral vessels in response to arachidonic acid in
vivo.19
Statistical Analysis
All values are expressed as mean±SE. A Mann-Whitney
U test was used to compare responses in WKY with those in
SHRSP. ANOVA and Kruskal-Wallis test were used for multiple
comparisons. A value of P<.05 was considered significant.
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Results |
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Responses in Adult WKY and SHRSP
Under control conditions, basilar artery diameter was smaller in 6- to 7-month-old SHRSP (239±30 µm) than in age-matched WKY (261±10 µm) (P<.05, Table
). In adult WKY,
acetylcholine (10-5 mol/L) increased
basilar artery diameter by 28±4%. Vasodilator responses to
acetylcholine were markedly impaired in adult SHRSP. Acetylcholine
(10-5 mol/L) increased basilar artery
diameter by only 2±1% in SHRSP (P<.05 versus WKY). Sodium
nitroprusside (10-8 and
10-7 mol/L) produced similar dilatation
of the basilar artery in adult WKY and SHRSP (P>.05, data
not shown). Indomethacin (10 mg/kg IV) did not enhance
vasodilatation produced by acetylcholine in SHRSP (n=5,
P>.05; data not shown).
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Application of 10-3 mol/L L-arginine for 30 minutes did not affect baseline diameter (P>.05). L-Arginine did not affect dilator responses of the basilar artery to acetylcholine in 6- to 7-month-old SHRSP (P<.05, data not shown). These results suggest that L-arginine does not enhance impaired dilator responses of the basilar artery to acetylcholine in 6- to 7-month-old SHRSP.
Responses in 12-Month-Old WKY and SHRSP
Under control conditions, basilar artery diameter was smaller in
12-month-old SHRSP (198±13 µm) than in age-matched WKY
(259±7 µm, P<.05; Table
). In 12-month-old WKY,
topical application of acetylcholine increased basilar artery diameter
(Fig 1). Acetylcholine-induced dilatation of the
basilar artery was markedly impaired in SHRSP (P<.05 versus
WKY, Fig 1). In contrast to acetylcholine, sodium nitroprusside
(10-8 and
10-7 mol/L) produced similar dilatation
of the basilar artery in WKY and SHRSP (P>.05, Fig 2). Indomethacin (10 mg/kg IV) did not
enhance vasodilatation produced by acetylcholine in SHRSP (n=5,
P>.05; data not shown).
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Application of 10-3 mol/L
L-arginine for 30 minutes did not affect baseline basilar
artery diameter in 12-month-old SHRSP or WKY (P>.05).
Pretreatment of the basilar artery with L-arginine
(10-3 mol/L) enhanced dilator responses
to acetylcholine in 12-month-old SHRSP (P<.05, Fig 1).
In the presence of 10-3 mol/L
L-arginine, 10-5 mol/L
acetylcholine increased arterial diameter by 14±3% in
SHRSP compared with only 5±1% under control conditions in SHRSP (Fig 1). L-Arginine did not affect dilator responses of the
basilar artery in 12-month-old SHRSP to sodium nitroprusside
(P>.05, Fig 2). D-Arginine
(10-3 mol/L) did not affect vasodilator
responses to acetylcholine in SHRSP (P>.05, Fig 1). A lower
concentration of L-arginine
(10-4 mol/L) did not affect
acetylcholine-induced vasodilatation in SHRSP (n=4,
P>.05; data not shown). These results suggest that
L-arginine selectively enhances responses of the basilar
artery to acetylcholine in 12-month-old SHRSP. These findings are
in contrast to results obtained in 6- to 7-month-old SHRSP in which
L-arginine did not alter responses to acetylcholine.
In 12-month-old WKY, L-arginine
(10-3 mol/L) did not enhance responses of
the basilar artery to acetylcholine but caused some paradoxical
attenuation of acetylcholine-induced dilatation of the basilar
artery (P<.05, Fig 1). L-Arginine
(10-3 mol/L) did not affect dilatation of
the basilar artery in response to sodium nitroprusside in older WKY
(P>.05, Fig 2). D-Arginine
(10-3 mol/L) did not affect vasodilator
responses to acetylcholine in 12-month-old WKY (P>.05,
Fig 1). The lower concentration of L-arginine
(10-4 mol/L) did not affect
acetylcholine-induced vasodilatation in WKY (P>.05,
data not shown). Thus, L-arginine did not enhance dilator
responses of the basilar artery to acetylcholine in 12-month-old
WKY.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The major new finding in the present study is that treatment of the basilar artery with L-arginine restores impaired dilator responses to acetylcholine in 12-month-old SHRSP. The effect of L-arginine in older SHRSP appears to be specific because D-arginine did not alter responses to acetylcholine. In contrast to older SHRSP, L-arginine did not enhance dilatation of the basilar in response to acetylcholine in adult (6- to 7-month-old) SHRSP. Thus, impaired dilatation of the basilar artery in response to acetylcholine may be restored to normal by L-arginine in older but not in younger adult SHRSP.
Responses to Acetylcholine
We have previously shown that dilator responses of cerebral
arterioles to ADP, A23187, and bradykinin are impaired in
SHRSP,1 3 19 and dilatation of the basilar artery to
acetylcholine and bradykinin are also impaired in chronically
hypertensive rats.5 6 Thus,
endothelium-dependent dilator responses of cerebral
arterioles and the basilar artery are impaired during chronic
hypertension. In the present study, impaired responses to
acetylcholine were observed in both adult and older adult SHRSP.
Because sodium nitroprusside–induced vasodilatation was similar in
SHRSP and WKY, impaired dilatation of the basilar artery appears to be
specific for acetylcholine. These findings suggest that chronic
hypertension is associated with impaired
endothelium-dependent responses of cerebral
vessels.
Effects of L-Arginine in SHRSP
Mechanisms that contribute to impaired dilatation of cerebral
blood vessels in SHRSP do not appear to be uniform throughout the
cerebral circulation. Indomethacin, or an
antagonist of prostaglandin
H2/thromboxane A2 receptors,
restored impaired dilatation of cerebral arterioles in response to
endothelium-dependent agonists in chronically
hypertensive rats,3 4 suggesting that release of a
vasoconstrictor prostanoid (an endothelium-derived
contracting factor) may contribute to impaired vasodilator responses.
In the basilar artery, indomethacin did not enhance
acetylcholine-induced dilator responses in both SHRSP (present
study) and SHR.6 Thus, formation of prostanoids may not
contribute to impaired dilator responses of the basilar artery during
chronic hypertension.
Dilator responses of the basilar artery to acetylcholine were enhanced in the presence of L-arginine in older SHRSP. This effect of L-arginine on responses to acetylcholine in older SHRSP appears to be specific for several reasons. First, L-arginine did not affect dilatation of the basilar artery in response to sodium nitroprusside in SHRSP. Second, L-arginine did not enhance impaired dilator responses of the basilar artery to acetylcholine in younger SHRSP. Third, D-arginine did not affect acetylcholine-induced vasodilatation in older SHRSP. These results suggest that increased availability of L-arginine, a precursor of NO, enhances dilatation of the basilar artery in response to acetylcholine in older SHRSP. This enhancement by L-arginine may be due to increased production of EDRF (NO) in response to acetylcholine. Previous studies have suggested that metabolism of L-arginine in vascular endothelium is altered during chronic hypertension.20 21 22 It is possible that a deficiency in intracellular stores of L-arginine may contribute to the impaired dilator response of the basilar artery to acetylcholine in older SHRSP. We also cannot exclude the possibility that the presence of an inhibitor of NO synthase, such as NG,N'G-dimethyl-L-arginine,23 contributes to the impaired dilator responses of the basilar artery to acetylcholine.
In contrast to effects on the response to acetylcholine, application of L-arginine did not produce direct dilatation of the basilar artery in SHRSP. Thus, basal release of EDRF may not be increased by L-arginine. We have shown recently that application of NG-nitro-L-arginine methyl ester, an inhibitor of NO synthase, produces similar constriction of the basilar artery in WKY and SHRSP.5 Thus, the basilar artery in SHRSP may produce normal amounts of EDRF under control conditions. A recent study reported that inhibition of NO synthase produces similar reductions in cerebral blood flow in WKY and SHRSP.24 These findings suggest that chronic hypertension may not affect basal release of EDRF.
Because L-arginine did not restore acetylcholine-induced vasodilatation in younger adult SHRSP, mechanisms that contribute to impaired vasodilator responses appear to be different in young and old adult SHRSP.
Effects of L-Arginine in WKY
Pretreatment with a high concentration of L-arginine
produced modest and paradoxical attenuation of
acetylcholine-induced dilatation of the basilar artery in WKY. This
effect has been described previously in the basilar artery in
normotensive rats25 and appears to be specific for two
reasons. First, L-arginine did not affect vasodilatation in
response to sodium nitroprusside. Second, D-arginine did
not alter dilatation of the basilar artery in response to acetylcholine
in WKY. Thus, high concentrations of L-arginine may produce
modest but selective inhibition of acetylcholine-induced
vasodilatation in WKY. The mechanism that accounts for this effect is
not clear. Recent studies suggest that NO itself inhibits NO
synthase.26 It seems unlikely that L-arginine,
which is a substrate for NO synthase, can also inhibit NO synthase.
In summary, increased availability of L-arginine, a substrate for NO synthase, enhances the response of the basilar artery to acetylcholine in older adult SHRSP.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants NS-24621, AG-10269, HL-16066, HL-14388, and HL-38901; by research funds from the Veterans Administration; and by a Grant-in-Aid from the American Heart Association (95014510). F.M.F. is an Established Investigator of the American Heart Association.
Received December 13, 1995; first decision December 18, 1995; accepted December 18, 1995.
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