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Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Rats were fed a low-salt (LS;
0.4% NaCl) or high-salt (HS; 4.0% NaCl) diet for 3 days, and the
responses of isolated cerebral arteries to acetylcholine (ACh), the
nitric oxide (NO)-dependent dilator bradykinin, and the NO donor
6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex-anamine (NOC-9) were determined. ACh-induced vasodilation and NO release, assessed with the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2) diacetate, were eliminated with the HS diet. Inhibition of
cyclooxygenase, cytochrome P-450 epoxygenase, and
acetylcholinesterase did not alter ACh responses. Bradykinin and NOC-9
caused a similar dilation in cerebral arteries of all groups. Arteries
from animals on LS or HS diets exhibited similar levels of basal
superoxide (O
bradykinin; cyclooxygenase; endothelium; epoxyeicosatrienoic acids; nitric oxide; superoxide
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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RECENT STUDIES INDICATE that a high-salt (HS) diet can impair vessel responses to endothelium-dependent vasodilator stimuli, including acetylcholine (ACh) (4, 15-17, 34, 35, 47). Under normal conditions, ACh-induced dilation may involve the release of several different vasoactive compounds including nitric oxide (NO) (1), endothelium-derived hyperpolarizing factor (EDHF) (6, 28, 30), and/or cyclooxygenase metabolites of arachidonic acid (25), depending on the specific vessel studied. In cerebral arteries, ACh generally induces vasodilation through a NO-mediated process (9, 41), although there is also evidence suggesting a role for EDHF in ACh-induced dilation of the rabbit middle cerebral artery (49). The extent to which dietary salt may alter the relative contribution of the potential mediators of ACh-induced vasodilation in cerebral arteries remains unknown.
The reduced dilation in response to ACh in animals on a HS diet could
also result from the liberation of an endothelium-derived contracting
factor derived from cyclooxygenase metabolism, as has been reported in
some animal models of hypertension (13, 19, 24, 26). For
example, Luscher et al. (37) observed that attenuated
dilator responses to ACh in mesenteric resistance vessels of
spontaneously hypertensive rats could be partially restored by
indomethacin, suggesting that ACh stimulates the release of a
constrictor prostanoid, in addition to NO. Other studies have described
the effect of a HS diet on the response of arterioles and resistance
arteries to NO donors and concluded that NO sensitivity remains
unaltered in animals on a HS diet (4, 16, 17, 35, 47).
This suggests that changes in ACh signal transduction occur either at
the level of NO synthase (NOS) and/or upstream from the activation of
NOS. Finally, Lenda et al. (32) assessed the potential
role of O
The overall goal of the present study was to test the hypothesis that
short-term exposure to a HS diet impairs ACh-induced vasodilation
upstream from NOS. The specific aims of the study were to determine the
effect of a short-term HS diet on ACh-induced dilation of rat cerebral
arteries, to identify possible changes in the ACh signaling cascade
that may contribute to any impaired vascular reactivity observed with a
HS diet, and to evaluate the potential role of O
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and preparations. Age-matched male Sprague-Dawley rats (Harlan Teklad; Madison, WI) were maintained on a low-salt (LS; 0.4% NaCl) or HS (4.0% NaCl) diet (Dyets; Bethlehem, PA) with tap water ad libitum for 3 days. All rats were housed in an animal care facility at the Medical College of Wisconsin, which is approved by the American Association for the Accreditation of Laboratory Animal Care, and all protocols were approved by the Animal Care Committee at the Medical College of Wisconsin.
On the day of the experiment, rats were anesthetized with pentobarbital sodium (50 mg/kg ip, Abbott Laboratories; Chicago, IL), and a carotid artery was cannulated for determination of arterial pressure. After arterial pressure was measured, the brain was quickly removed and immersed in physiological salt solution (PSS) having the following composition (in mM): 119.0 NaCl, 4.7 KCl, 1.6 CaCl2, 1.18 NaH2PO4, 1.17 MgSO4, 24.0 NaHCO3, 5.5 dextrose, and 0.03 EDTA. Middle cerebral arteries were carefully isolated using a dissecting microscope (Leica; Buffalo, NY) and cannulated as described below.Isolated vessel protocol. The isolated vessels were transferred to a superfusion-perfusion chamber and doubly cannulated with glass micropipettes (100-150 µm), as described previously (14, 36). The vessels were continuously perfused and superfused with warmed PSS (37°C) equilibrated with a 21% O2-5% CO2-74% N2 gas mixture. Intralumenal pressure was set at 80 mmHg for the isolated vessel to approximate the in vivo pressure encountered by the vessel (31, 42). Internal diameter of the vessel was measured with a video micrometer (model IV-550, FOR.A; Tokyo, Japan). Vessel diameters were measured under resting conditions in PSS and after maximum relaxation of the artery induced by perfusion and superfusion of the vessel with Ca2+-free PSS having the following composition (in mM): 119.0 NaCl, 20.0 MgCl2, 4.7 KCl, 1.18 NaH2PO4, 1.17 MgSO4, 24.0 NaHCO3, 5.5 dextrose, and 2.0 EGTA. Any vessel that did not exhibit significant levels of active tone (as evidenced by a substantial increase in resting diameter upon exposure to Ca2+-free PSS) was not used in this study. Because larger arteries were required for the NO indicator assay (see below), responses to ACh were also evaluated in basilar arteries of rats on LS and HS diets.
Vascular response protocols.
To assess potential mediators and modulators of ACh-induced
responses in the two groups, ACh concentration-response experiments (10
7-104 M) were performed before and
after removal of the endothelium by air perfusion (14) and
in the presence and absence of inhibitors of several vasoactive
compounds that have been proposed to contribute to ACh-induced
dilation. These included 1) the NOS inhibitor
NG-monomethyl-L-arginine
(L-NMMA; 104 M); 2) the
cyclooxygenase inhibitor indomethacin (106 M); and
3)
N-methylsulfonyl-6-(2-propargyloxyphenyl)-hexanamide (MS-PPOH; 2 × 106 M), an irreversible inhibitor of
the formation of epoxyeicosatrienoic acids (EETs) (46),
which are putative EDHFs (5). ACh
concentration-response experiments were also performed before and after
the addition of the acetylcholinesterase inhibitor physostigmine
(105 M) to verify that differences in
acetylcholinesterase activity do not modify vascular responses to ACh
in middle cerebral arteries obtained from rats on LS or HS diets.
10-106 M) and
6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine
(NOC-9; a NO donor; 1011-105 M) were
also recorded in isolated middle cerebral arteries of rats on LS and HS
diets. The bradykinin concentration-response experiments were
determined before and after the addition of the NOS inhibitor
L-NMMA (104 M) to the tissue bath.
To evaluate the potential contribution of reactive oxygen species to
the impaired relaxation to ACh in animals on a HS diet, responses of
middle cerebral arteries to ACh were also recorded in the presence and
absence of either superoxide dismutase (SOD; 80 U/ml, Sigma; St. Louis,
MO) and catalase (120 U/ml, Sigma) or polyethylene glycol (PEG)-SOD
(250 U/ml, Sigma) and catalase in the perfusion solution. In those
experiments, SOD and catalase or PEG-SOD and catalase were added to the
perfusate reservoir, and the vessels were incubated for ~15 min.
NO indicator assay. ACh-induced NO release was assessed in basilar arteries of rats administered a LS or HS diet using the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2) diacetate (Calbiochem; La Jolla, CA), as described by Kojima et al. (29) and Zhang et al. (51). DAF-2 diacetate is cell permeable and is hydrolyzed by cytosolic esterases to DAF-2. The relatively nonfluorescent DAF-2 is converted to the highly fluorescent DAF-2 triazole in the presence of NO and oxygen, and the fluorescence intensity of DAF-2 is directly proportional to the NO concentration.
Basilar arteries were selected because the larger internal diameter of these vessels compared with that of middle cerebral arteries facilitates longitudinal sectioning of the vessel, which is necessary to view the endothelium. In these experiments, the arteries were doubly cannulated with glass micropipettes, as described in Isolated vessel protocol. Vessels were flushed with PSS to remove blood cells, sectioned longitudinally, and equilibrated with PSS (37°C) in the vessel chamber for 1 h. Care was taken to ensure that the endothelium remained intact during sectioning. The arteries were then pinned down on a Sylgard (Dow Corning; Midland, MI)-coated dissecting dish with the endothelial surface positioned upward and were submerged in HEPES-buffered PSS having the following composition (in mM): 140 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, and 10 glucose. Arteries were incubated with DAF-2 diacetate (105 M) for 30 min at room temperature and rinsed three
times with PSS before experimental observations with an epifluorescence
microscope (Nikon E600; Tokyo, Japan) equipped with a ×20 objective
and 490-nm excitation and 510- to 560-nm emission filters.
Digital images were captured using a PC-controlled charge-coupled
device camera (Roper Scientific RTE/CCD-1300-Y/HS; Trenton, NJ) and
Metamorph imaging and analysis software (Universal Imaging; Downington, PA).
ACh (105 M) was added to the basilar artery, and changes
in fluorescence were recorded at 5-min intervals for ~30 min. An
additional group of vessels obtained from rats on a LS diet was
incubated with the NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME; 104 M) for 15 min before
experimental observations and the addition of ACh. The response to the
NO donor 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NONOate; 5 × 104 M) was also evaluated in vessels of animals on
either LS or HS diets as a positive control to demonstrate the ability
of the DAF-2 assay to increase its fluorescence in response to NO.
Responses were expressed as the maximum change in fluorescence
intensity after normalization for differences in baseline parameters.
Dihydroethidine assay.
On the basis of the procedure described by Bindokas et al.
(2), a dihydroethidine (DHEt) assay was used to assess
basal O5 M
hydroethidine solution in PSS and incubated for 20 min. After incubation, the vessels were removed, rinsed with PSS, and placed on
microscope slides for subsequent observation utilizing a confocal microscope. O4 M, Sigma), a known stimulator
of O5 M
Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; Cayman
Chemical; Ann Arbor, MI), a membrane-permeable SOD mimetic (8), was added to middle cerebral arteries of animals on a LS or HS diet as a further verification of the effectiveness of the
DHEt assay.
Western blotting.
Muscarinic type 3 (M3) receptor protein expression was
evaluated utilizing a Western blotting protocol modified from Mattson and Higgins (38). Each experimental group consisted of
tissues pooled from four to eight Sprague-Dawley rats fed an acute LS or HS diet. Rats were anesthetized with pentobarbital sodium, and
aortas and cerebral vessels (including middle cerebral arteries, basilar arteries, and similar-sized arteries from the Circle of Willis)
were freshly dissected and cleared of adhering parenchymal tissue. The
vessels were snap-frozen in liquid nitrogen and stored in 0.5-ml
microcentrifuge tubes in a freezer (
85°C) until the time of
homogenization. Tissues were subsequently placed in homogenization buffer composed of (in mM) 100 K2HPO4, 100 KH2PO4, 475 sucrose, 100 EDTA, 1.0 pepstatin,
1.0 leupeptin, and 100 phenylmethylsulfonyl fluoride. Cerebral vessels
were hand homogenized in microcentrifuge tubes using a pestle (Kontes;
Vineland, NJ). Aortas were homogenized at 3,000 rpm with a
Potter-Elvehjem tissue grinder. The homogenates were centrifuged for 20 min at 14,000 g for protein isolation. The supernatant was
collected, and the protein content was estimated using a standard
protein determination assay (Coomassie Protein Assay, Pierce; Rockford,
IL). Fifty micrograms of protein from each sample were suspended in a
loading buffer, incubated (10 min at 37°C), and loaded into a single
lane of a 10% polyacrylamide gel (Zaxis; Hudson, OH) for
electrophoretic separation (200 V, 60 min, Owl Separation Systems;
Portsmouth, NH). Kaleidoscope prestained standards (Bio-Rad
Laboratories; Hercules, CA) were added into one lane to serve as size
standards. The protein was then transferred to a nitrocellulose
membrane (100 V, 60 min), washed in Tris-buffered saline (3 times for
10 min), and blocked with 10% nonfat dry milk in Tris-buffered saline
for 2 h. A polyclonal antibody specific for the M3
receptor (1:500 dilution in 4% nonfat dry milk in Tris-buffered
saline, Santa Cruz Biotechnology; Santa Cruz, CA) was applied to the
nitrocellulose membrane for 1 h at room temperature and
subsequently conjugated to anti-goat IgG horseradish peroxidase for
protein recognition (1:2,500 dilution, 1 h, Santa Cruz
Biotechnology). Antibodies were detected using enhanced
chemiluminescence (Amersham; Arlington Heights, IL) on X-ray film.
Protein expression was evaluated quantitatively using densitometric
analysis. After blotting for the M3 receptor, the blot was
stripped with a Tris-buffered solution containing 2% sodium dodecyl
sulfate and 100 mM 
-mercaptoethanol at 50°C. After the stripping
procedure, the blots were probed with a monoclonal mouse antibody that
recognizes -actin (1:1,000 dilution, Sigma) to obtain a loading control.
Data analysis. Vessel responses were described as the change in internal diameter and expressed as means ± SE. A Student's t-test for paired or unpaired data was used to evaluate results in experiments involving single comparisons. All other comparisons were made using ANOVA. The Student-Newman-Keuls test was used for post hoc analysis. A probability value of <0.05 was considered to be statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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General characteristics of the experimental groups and response to
ACh in animals on HS and LS diets.
Table 1 provides summary data describing
the rats from the different experimental groups employed in this study.
The HS diet had no effect on body weight, mean arterial pressure, or
vessel diameter.
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5 M). In those experiments, responses to ACh were
unaffected by physostigmine in vessels from animals on either LS or HS
diets (data not shown).
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Responses to bradykinin and NOC-9.
To determine whether the attenuated ACh-induced dilations in arteries
of animals on a HS diet are caused by specific changes in ACh
reactivity or whether the impaired relaxation to ACh is due to a
nonspecific effect of dietary salt intake to reduce the ability of the
vessel to relax in response to any vasodilator stimulus, we tested the
responses of middle cerebral arteries to bradykinin (an unrelated
receptor-mediated vasodilator agonist that releases NO). In those
experiments, bradykinin induced a significant, dose-dependent dilation
that was similar in magnitude in middle cerebral arteries of rats on
both LS and HS diets (Fig. 5). The
bradykinin-induced dilations of the vessels were significantly attenuated by the NOS inhibitor L-NMMA (Fig. 5,
inset).
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ACh-induced NO release.
The fluorescent NO indicator DAF-2 diacetate was used to evaluate the
level of ACh-induced NO release in basilar arteries from rats
administered LS or HS diets (Fig. 7).
Arteries from rats on a LS diet exhibited a significant increase in NO
levels upon stimulation with ACh, and this response was attenuated in vessels from rats on a HS diet. Vessels from rats on a LS diet that
were incubated with the NOS inhibitor L-NAME did not
exhibit an increase in NO concentration in response to ACh, whereas
treatment of the vessels with the NO donor DEA-NONOate caused a marked
increase in DAF-2 fluorescence.
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Role of O
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Effect of dietary salt intake on M3 receptor
expression.
We also evaluated M3 receptor expression using immunoblot
analysis (Fig. 10) to study the
potential effect of dietary salt intake on the expression of the ACh
receptor responsible for initiating ACh-induced dilation in cerebral
arteries. The M3 receptor is expressed in freshly isolated
endothelial cells but is not expressed in cultured endothelial cells
(44). The latter finding suggests that the M3
receptor is capable of undergoing dramatic changes in protein
expression under different experimental conditions. Of particular
relevance to this study, loss of the M3 receptors that are
necessary for ACh signal transduction may constitute a potential
mechanism for the impaired ACh reactivity observed in arteries of rats
on a HS diet. However, arteries from animals on LS and HS diets
expressed similar levels of M3 receptor in the present
study (Fig. 10).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cerebral arteries of rats on a short-term HS diet exhibited attenuated vasodilation to ACh (Figs. 1 and 2) despite having similar body weight, mean arterial pressure, and diameter as the control animals on a LS diet (Table 1). These results are consistent with those of earlier studies showing impaired ACh reactivity in arterioles and skeletal muscle resistance arteries of rats fed an acute HS diet (16, 47). Together, these findings demonstrate that elevated dietary salt intake leads to impaired ACh-induced dilation in resistance arteries of different vascular beds. Thus loss of vascular relaxation in response to ACh in animals on a HS diet is not restricted to the skeletal muscle circulation but is instead a more generalized phenomenon in the peripheral vasculature.
In the present studies, cerebral arteries of rats on LS and HS diets had identical responses to Ca2+-free PSS, demonstrating that short-term exposure to a HS diet does not alter resting tone or cause structural remodeling of the middle cerebral artery. This finding is consistent with the lack of structural remodeling of skeletal muscle resistance arteries after short-term exposure to a HS diet (16, 47) but contrasts with the effect of a chronic (4 wk) HS diet on skeletal muscle resistance arteries, where structural remodeling is evident as a decrease in the diameter of the maximally relaxed vessel (15, 47). Thus the observed alterations in ACh reactivity in cerebral arteries of animals on a HS diet are due to changes in vasodilator pathways rather than the inability of the vessel to increase its diameter due to structural remodeling.
Alterations in ACh reactivity in response to a HS diet could be due to a generalized effect of elevated salt intake to reduce the ability of vessels to relax in response to any vasodilator stimulus. This would presumably result in the attenuation of all receptor-mediated dilator pathways, including those mediating relaxation in response to ACh. However, bradykinin, an endothelium-dependent dilator that causes NO release through a receptor-mediated signal transduction pathway independent of ACh [bradykinin type 2 (B2) receptors (10, 22)], caused a similar dilation of middle cerebral arteries from animals on LS and HS diets (Fig. 5). Of particular interest in this regard is the observation that bradykinin relaxes middle cerebral arteries from animals on a HS diet even though the B2 receptor operates through a Gq protein-mediated pathway (33), similar to ACh. The demonstration of a normal relaxation in response to a receptor-mediated vasodilator agonist other than ACh in arteries from rats on a HS diet suggests that the attenuated ACh reactivity in these animals is due to intrinsic alterations in the ACh pathway and is not due to a nonspecific effect of HS diet to depress all receptor-mediated signaling pathways in the vasculature.
In the present study, ACh reactivity in rat middle cerebral arteries was mediated primarily or entirely by NO, as evidenced by the elimination of the dilator response to ACh in the presence of the NOS inhibitor L-NMMA. The lack of a significant effect of either the cyclooxygenase inhibitor indomethacin (Fig. 3) or MS-PPOH (Fig. 4) (eliminating the potential involvement of EETs in this vascular response) on the response of the vessels to ACh further supports the hypothesis that ACh-induced dilation of the middle cerebral artery is mediated via NO. However, indomethacin treatment also tended to decrease the vasodilator response to ACh in arteries from animals on a LS diet. The latter observation could be consistent with previous reports suggesting that a dilator compound derived from cyclooxygenase may contribute to the vascular response to ACh observed in rats on a standard salt diet (12, 50) but most likely reflects the tendency for resting tone to increase in response to indomethacin due to inhibition of basal prostaglandin production by the endothelium. However, the most important finding from the indomethacin experiments was that the addition of the cyclooxygenase inhibitor did not restore ACh-induced dilation in arteries from rats on a HS diet. The latter observation indicates that elevations in dietary salt intake do not eliminate ACh-induced relaxation by causing the release of a cyclooxygenase-dependent endothelium-derived contracting factor, as reported in certain models of hypertension (26, 27, 37, 40).
The specific component(s) of the ACh cascade that are altered by an acute HS diet remain unknown. In the present study, the dilation of the cerebral vessels to bradykinin was unaffected by dietary salt intake. Bradykinin-induced dilation was significantly attenuated by L-NMMA (Fig. 5, inset), demonstrating that NO release plays a major role in the relaxation of the vessels in response to the agonist. The absence of an impaired response to bradykinin in arteries of animals on a HS diet indicates that NOS function is intact in the vessels and that loss of the vasodilator response to ACh in arteries of animals on a HS diet is not due to an inability of the enzyme to catalyze the formation of NO in these animals.
In the present study, we observed that basilar arteries of rats on a HS diet, which exhibited an impaired dilation in response to ACh (Fig. 2), also exhibited an attenuated release of NO in response to ACh, as revealed by decreased DAF-2 fluorescence (Fig. 7). The latter observation strongly indicates that the impaired dilation that occurs in response to ACh in cerebral arteries of rats on a HS diet is due to attenuated NO release.
A reduced sensitivity of the vascular smooth muscle cells to the vasodilator effect of NO could also contribute to the impaired relaxation of the vessel in response to ACh in arteries of animals on a HS diet. To determine whether a decreased sensitivity of the vascular smooth muscle cells to NO could contribute to the reduced dilation in response to ACh in middle cerebral arteries of animals on a HS diet, we tested the effect of increasing concentrations of the NO donor NOC-9 on vessel diameter (Fig. 6). Consistent with the results of several previous studies (4, 16, 17, 35, 47), arteries from animals on LS and HS diets exhibited a similar dilation in response to the NO donor, suggesting that vascular sensitivity to NO is unaltered by elevated dietary salt intake.
Experimental evidence obtained from in vivo studies of skeletal muscle
arterioles suggests that the attenuated ACh reactivity observed in rats
on a chronic HS diet may be due to an elevated production of
O
Although the administration of O
On the basis of the findings of this study, we postulate that the impaired relaxation in response to ACh that occurs in middle cerebral arteries from animals on a HS diet is due to alterations in the ACh cascade upstream from NOS. One possible mechanism for a reduced relaxation to ACh would be the loss of the M3 receptor responsible for initiating the signaling cascade. The M3 receptor is the mediator of ACh-induced vasodilation (7, 21, 43), and its expression is downregulated in certain experimental models, including cultured endothelial cells (44). However, M3 receptors were still expressed in cerebral arteries of both groups of animals, and the level of expression was unaffected by dietary salt intake (Fig. 10).
The finding that M3 receptors are still expressed in cerebral arteries of animals on a HS diet indicates that the impaired ACh reactivity in the cerebral vasculature during elevated salt intake is not due to M3 receptor downregulation. However, it does not preclude changes in receptor affinity or receptor desensitization as possible mechanisms resulting in attenuated ACh-induced vasodilation. Wu et al. (48) reported that M3 receptors possess a phosphorylation site for G protein-coupled receptor kinase 2 that may be responsible for regulating M3 signal transduction, and such a mechanism could lead to receptor desensitization in animals on a HS diet. We believe that a more likely explanation for the altered response to ACh in vessels from animals on a HS diet may be an uncoupling of the muscarinic receptor from the Gq protein, precluding it from increasing intracellular Ca2+ concentration in the endothelial cells. The latter hypothesis is supported by recent studies suggesting that impaired coupling between membrane receptors and G proteins may be responsible for the reduced responses to some vasodilator agonists in skeletal muscle resistance arteries from rats administered acute or chronic HS diets (18, 47). However, further investigation is required to determine the role of these possible mechanisms in mediating impaired vascular relaxation during elevated dietary salt intake.
In conclusion, middle cerebral arteries from rats fed a short-term HS
diet exhibit attenuated dilator responses to ACh, and ACh-induced
dilation and NO release are also attenuated in basilar arteries of rats
on a HS diet. However, vascular responses to bradykinin, another
NO-dependent vasodilator agonist, and to the NO donor NOC-9 remain
intact, suggesting that the downstream components of the ACh cascade
and vascular sensitivity to NO are unaffected by a HS diet. In contrast
to the studies of Lenda et al. (32), short-term exposure
to elevated dietary salt intake does not appear to elevate
O
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ACKNOWLEDGEMENTS |
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The authors thank Drs. William Chilian and Kirkwood Pritchard for technical advice in performing the DHEt assay, Dr. Ai-Ping Zou and David Zhang for advice concerning the use of DAF-2 diacetate in the NO indicator assay, Dr. J. R. Falck for generously supplying MS-PPOH, and Dr. Richard Roman for insight concerning the use of MS-PPOH.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-29587, HL-37374, HL-65289, GM-31278, and F32-HL-09994.
Address for reprint requests and other correspondence: J. H. Lombard, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (E-mail: jlombard{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 28, 2002;10.1152/ajpheart.00127.2002
Received 21 February 2002; accepted in final form 25 March 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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