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1 Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES, Brazil 20042-755; 2 The Cardiovascular Center and the Departments of 3 Internal Medicine, 4 Anatomy and Cell Biology, and 5 Physiology and Biophysics, The University of Iowa, Iowa City 52242; and the 6 Veterans Affairs Medical Center, Iowa City, Iowa 52246
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Administration of nitric oxide (NO) or
NO donors to isolated carotid sinus and carotid bodies inhibits the
activity of baroreceptor and chemoreceptor afferent nerves.
Furthermore, NO synthase (NOS) is present in endothelial cells and in
sensory nerves innervating the carotid sinus region. The major goal of
this study was to determine whether overexpression of NOS in carotid
sinus modulates baroreceptor activity. Rabbits were anesthetized, and
adenoviral vectors (5 × 108 plaque-forming units)
encoding genes for either 
-galactosidase (-Gal) or endothelial
type III NOS (eNOS) were applied topically to the adventitial surface
of one carotid sinus. In some experiments, the NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME) was applied to the carotid sinus immediately after
the vector. Four to five days later, baroreceptor activity and carotid
sinus diameter were measured from the vascularly isolated carotid sinus
of the anesthetized rabbits. Transgene expression was confirmed by
X-Gal staining of -Gal and measurement of NOS activity by citrulline
assay. The expression was restricted to the carotid sinus adventitia.
Baroreceptor activity was decreased significantly, and the
pressure-activity curve was shifted to higher pressures in
eNOS-transduced (n = 5) compared with
-Gal-transduced (n = 5) carotid sinuses. The
pressure corresponding to 50% of maximum activity averaged 55 ± 6 and 76 ± 7 mmHg in -Gal- and eNOS-transduced carotid
sinuses, respectively (P < 0.05). Decreased baroreceptor activity was accompanied by a significant increase in
carotid diameter in the eNOS-transduced carotid sinuses
(n = 5). L-NAME prevented the inhibition of
baroreceptor activity and the increase in carotid diameter in
eNOS-transduced carotid sinuses (n = 5). We conclude
that adenoviral-mediated gene transfer of eNOS to carotid sinus
adventitia causes sustained, NO-dependent inhibition of
baroreceptor activity and resetting of the baroreceptor function curve
to higher pressures.
baroreceptor resetting; adenoviral vectors; nitric oxide
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE (NO) is an important autocrine/paracrine factor that mediates vascular relaxation, signaling within the nervous system, and immunological responses (28). Type III endothelial NO synthase (eNOS) and type I neuronal NOS (nNOS) isoforms are present in vascular endothelial cells and sensory nerves innervating the carotid sinus region, respectively (9, 14, 41). Furthermore, NO inhibits chemoreceptor and baroreceptor afferent nerve activity acutely (25, 33, 38). The functional role of endogenous NO and its potential to chronically modulate baroreceptor sensitivity are unknown.
The major goal of the present study was to determine whether sustained overexpression of NOS in the carotid sinus modulates carotid sinus baroreceptor activity. Localized overexpression of NOS was accomplished by adenoviral-mediated gene transfer of eNOS to the carotid sinus (5, 26, 34).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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A total of 32 New Zealand White rabbits of either sex were
studied. Experimental procedures were carried out in accordance with
the American Physiological Society's "Guiding Principles for
Research Involving Animals and Human Beings" and institutional guidelines. One carotid sinus from each rabbit was subjected to gene
transfer of either the reporter gene -galactosidase (-Gal) or
bovine type III eNOS. In some experiments the NOS inhibitor NG-nitro-L-arginine methyl ester
(L-NAME) was applied to the carotid sinus immediately after
application of the viral vector suspension. The contralateral carotid
artery and carotid sinus region (nontransfected) provided additional
controls for some of the protocols.
Adenoviral Vectors
Adenoviral vectors were constructed in the University of Iowa Gene Transfer Vector Core Laboratory (7, 8, 12, 26, 31, 34). The cDNAs for the reporter gene-galactosidase (-Gal) and type III eNOS were cloned into shuttle vectors containing sequences
from serotype 5 human adenovirus, and the Rous sarcoma virus (RSV)
promoter was used to drive transcription. The transgene plasmid was
transfected into human embryonic kidney 293 cells along with a plasmid
containing the serotype 5 human adenovirus genome with deletions in the
E1A, E1B, and E3 regions. Replication-deficient vectors were produced
by homologous recombination within the 293 cells. Each vector was
prepared by double-cesium gradient purification, suspended in 3%
sucrose solution [~1010 plaque-forming units (pfu)/ml],
and stored at 
70°C.
Topical Application of Vectors to Carotid Sinus
Rabbits were anesthetized with intramuscular injection of xylazine (20 mg/kg) and ketamine (55 mg/kg). Using sterile surgical technique, one carotid sinus was exposed via a small incision in the cervical region. Approximately 50 µl of adenoviral vector suspension (5 × 108 pfu) was applied topically to the carotid sinus region through an opening in the carotid sheath using a pipetter. In subsets of rabbits, 50 µl of poloxamer gel (Pluronic F-127, Molecular Probes, Eugene, OR) containing L-NAME (1 mM) was topically applied to the carotid sinus immediately after applying the viral vector. The incisions were closed, and the rabbits were observed until they regained consciousness. To minimize risk of infection, penicillin (60,000 U) was administered after performing the surgical procedures.Analysis of Transgene Expression
-Gal transgene expression was confirmed 4-5 days after
application of the -Gal vector to the carotid sinus by histochemical analysis (8, 26, 34). The carotid sinus region was
removed, rinsed with phosphate-buffered saline, and fixed in
glutaraldehyde (0.5%) for 30 min at room temperature. The carotid
sinuses were then washed with phosphate-buffered saline containing
MgCl2 (1 mM) and exposed to
K3Fe(CN)6 (4.9 mM),
K4Fe(CN)6 (4.7 mM), and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal,
2.4 mM) in saline containing MgCl2 for 1 h at 37°C.
The X-Gal solution was then replaced with saline, and the blood vessels
were stored at 4°C. The same procedure was performed on the
contralateral, nontransfected carotid sinuses.
The citrulline assay (3, 8, 12) was used to quantify NOS
activity in four NOS-transduced and four contralateral control carotid
sinuses removed from four rabbits 4-5 days after application of
the NOS vector to one carotid sinus. The carotid sinuses were homogenized on ice using a glass homogenizer fitted with a ground glass
pestle in 50 mM Tris · HCl (pH 7.4) containing
0.1 mM EDTA, 0.1 mM EGTA, 12 mM 2-mercaptoethanol, 1 mM
phenylmethysulfonyl fluoride, 3 µM leupeptin, 1 µM aprotinin, 1 µM pepstatin, and 1 µM soybean trypsin inhibitor. The assay mixture
contained 50 mM Tris · HCl (pH 7.4), 5 µM
L-arginine, 0.25 µCi
L-[3H]arginine, 0.5 mM 
-NADPH, 10 µM
tetrahydrobiopterin, 4 µM flavin mononucleotide, 4 µM FAD, 1 µg
calmodulin, 1 mM calcium, and 40-80 µg cell homogenate protein
in 200 µl volume. To determine calcium-independent NOS activity,
calcium was replaced by 1 mM EGTA. To confirm that the activity
measured reflected NOS activity, the NOS inhibitor NG-nitro-L-arginine
(L-NNA, 1 mM) was added to the assay. Enzyme assays were
carried out at 37°C for 15 min and terminated by adding 5.5 ml of
Dowex slurry (Dowex AG50W-X8, 100- to 200-mesh sodium form).
L-[3H]citrulline production was measured by
using a liquid scintillation spectrometer (Beckman-Coulter, model DU
640, Brea, CA).
Measurement and Analysis of Baroreceptor Pressure-Activity Relation
Baroreceptor activity was measured from isolated carotid sinuses subjected to-Gal gene transfer (n = 5), NOS
gene transfer (n = 5), NOS gene transfer plus
L-NAME treatment (n = 5), and -Gal gene
transfer plus L-NAME treatment (n = 5).
Four to five days after topical application of vector to the carotid
sinus, rabbits were anesthetized with pentobarbital sodium (30-35
mg/kg iv) and mechanically ventilated with room air supplemented with oxygen. Ventilation was adjusted to maintain arterial blood gases and
pH within normal values. Polyethylene catheters were placed into the
femoral artery and vein for measurement of arterial pressure and
administration of anesthetic, respectively.
The carotid sinus subjected to gene transfer 4-5 days beforehand was vascularly isolated as described previously (24-26). Briefly, the common, external, and internal carotid arteries were isolated and ligated along with other arterial branches, and catheters were placed in the common carotid and lingual arteries, thereby isolating the carotid sinus lumen from the adjacent circulation. The isolated carotid sinus was filled with an oxygenated Krebs-Henseleit buffer. Carotid sinus pressure was measured with a transducer (model P23ID, Statham) connected to the lingual artery catheter and was varied in a controlled manner by altering the inflow of air from a pressurized air source into a glass fluid reservoir attached to the common carotid artery catheter. The cervical sympathetic, aortic depressor, and vagus nerves were sectioned on the side of the isolated carotid sinus. Skeletal muscle contractions were prevented during periods of nerve recording by administration of the neuromuscular blocker decamethonium bromide (0.3 mg/kg iv).
The carotid sinus nerve was identified, sectioned, and draped over a unipolar electrode. The electrode and nerve were insulated by covering the area with warm paraffin oil (37°C) and/or by encasing the nerve in Wackers silicone gel (24-26). Nerve activity was recorded by using a high-impedance probe (model HIP511J, Grass Instrument) and a Grass band-pass amplifier (model P511J, 100- to 3,000-Hz bandwidth). The nerve recording was displayed on a dual-beam storage oscilloscope (model 5113, Tektronix). A nerve traffic analyzer (model 706C, Dept. of Bioengineering, Univ. of Iowa, Iowa City, IA) was used to count the frequency of action potentials with amplitude exceeding a selected voltage level set just above the electrical noise. Systemic arterial pressure, carotid sinus pressure, and the output of the nerve traffic analyzer were continuously recorded on a chart recorder (model 11-1202-25, Gould).
Baroreceptor activity was recorded during slow ramp increases in nonpulsatile carotid sinus pressure from 0 to 160 mmHg. Pressure ramps were repeated approximately once every 5 min until baroreceptor responses were consistent. Carotid sinus pressure was held at 80 mmHg in between pressure ramps. The rate of increase in carotid sinus pressure during the ramps was similar in all experiments and averaged approximately 2.5-3.0 mmHg/s.
Baroreceptor activity was measured at 20-mmHg increments over a range of 20-160 mmHg. The data from three or four pressure ramps were analyzed, and the average responses were calculated for each experiment. Because the absolute level of multifiber baroreceptor activity is dependent on the recording conditions and varies from preparation to preparation, activity is expressed as a percentage of the maximum activity recorded at high carotid sinus pressure up to 160 mmHg. The position of the baroreceptor function curve along the pressure axis was determined directly from the experimental traces in individual experiments by measuring the carotid sinus pressure corresponding to 50% of the maximum baroreceptor activity (EP50).
Measurement and Analysis of Carotid Pressure- Diameter Relation
The diameter of the carotid artery at the origin of the carotid sinus bifurcation was measured during ramp increases in carotid sinus pressure from 0 to 160 mmHg using a video micrometer (model VIA-100, Boeckeler Instruments, Tucson, AZ) (24, 25). Carotid sinuses exposed to the-Gal vector (n = 4), the NOS
vector (n = 4), and the NOS vector plus
L-NAME (n = 5) were studied 4-5 days
after topical application of the vectors to the carotid sinus. The
carotid sinus region was viewed under magnification (×16) through a
stereomicroscope (model M3C, Wild, Heerbrugg, Switzerland) during slow
ramp increases in carotid sinus pressure. The image was recorded on
videotape using a camera (model JE7362, Javelin Electronics, Torrance,
CA), videocassette recorder (model SLV-585HF, Sony, Japan), and video
monitor (model BWM12, Javelin Electronics). A digital readout of the
carotid sinus pressure was simultaneously filmed using a second camera
(model JE7542B, Javelin Electronics) and was projected on the same
video monitor as the carotid sinus image with the use of a beam
splitter (model MPS-50, Image Labs, Pearl River, NY). The video
micrometer system enabled detection of 12-µm changes in diameter over
an absolute range of 0-5 mm. The compliance of the carotid sinus
was estimated by calculating the slope of the pressure-diameter
relation over its steepest portion between 20 and 80 mmHg using linear regression.
Carotid Sinus Histology
Both common carotid arteries and the carotid sinus regions were removed bilaterally from rabbits subjected to-Gal or eNOS gene
transfer to one carotid sinus. The unpressurized arteries were covered
in OCT compound (Miles Scientific, Elkhart, IN) and frozen in a
cryostat. Cross-sections (20-µm thickness) through the common carotid
artery just below the bifurcation and through the carotid sinus and
external carotid artery were placed on polylysine-coated slides and
either processed for X-Gal staining (see Analysis of Transgene
Expression) or stained with hematoxylin or eosin and photographed
(Diaphot 300, Nikon, Japan).
Statistical Analysis
All data are expressed as means ± SE. Effects of carotid sinus pressure, treatment group (eNOS vs.-Gal gene transfer and effect of L-NAME), and the interaction between pressure and
treatment on baroreceptor activity and carotid diameter were analyzed
by two-factor ANOVA (GB-Stat 6.0 software). When the ANOVA was
significant, differences between two sets of data points were
determined by Fisher's least significant difference post hoc test
(GB-Stat 6.0 software). Effects of NOS gene transfer and administration
of L-NAME on baroreceptor EP50 were analyzed by
unpaired t-test. Differences were considered significant
when P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transgene Expression in Carotid Sinus
Topical perivascular application of the adenoviral vectors Ad-Gal and AdeNOS to carotid sinus led to significant expression of
the transgenes in carotid sinus measured 4-5 days later. The gene
transfer, visualized by X-Gal staining of -Gal, was localized to the
region of the carotid sinus and was selective to cells in the
adventitia (Fig. 1). Transgene expression
was not observed in the media layer of carotid artery walls or within
carotid sinus nerve fibers. NOS enzymatic activity was ~100 fold
higher in carotid sinuses exposed to AdeNOS (n = 4)
compared with the contralateral control carotid sinuses
(n = 4) (Fig. 2). The NOS
activity was essentially abolished by either replacement of calcium
with the calcium chelator EGTA or addition of the NOS inhibitor
L-NNA to the assay, confirming that the measured activity
was indeed calcium-dependent NOS activity (Fig. 2).
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Gene Transfer and L-NAME Effects on Baroreceptor Pressure-Activity Relation
Baroreceptor activity was inhibited significantly, and the pressure-activity curve was shifted to higher pressures in eNOS compared with-Gal-transduced carotid
sinuses (Figs. 3 and 4A). Consequently, the carotid sinus pressure corresponding to
EP50 was significantly higher in the
eNOS-transduced carotid sinuses (Figs. 3 and 4B). In a third
group of rabbits, local application of the NOS inhibitor
L-NAME to carotid sinus adventitia immediately after
application of the AdeNOS vector completely prevented the eNOS-induced
inhibition of baroreceptor activity and the increase in
EP50 observed 4-5 days later (Fig. 4).
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A possible role of endogenous NO in modulation of baroreceptor activity
was investigated in a fourth group of rabbits that underwent -Gal
gene transfer and L-NAME treatment of one carotid sinus.
Baroreceptor activity tended to be higher in the -Gal-transduced sinuses treated with L-NAME (n = 5)
compared with untreated sinuses (n = 5), but the
difference did not reach statistical significance (Fig.
5). The baroreceptor function curves for
L-NAME-treated -Gal and L-NAME-treated
eNOS-transduced carotid sinuses were essentially superimposable and not
significantly different from each other (Fig. 5). Combining the data
from these two groups enabled demonstration of a significant increase
in baroreceptor activity at low levels of carotid sinus pressure
(20-60 mmHg) in L-NAME-treated carotid sinuses
(n = 10) compared with untreated -Gal-transduced carotid sinuses (n = 5).
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Gene Transfer and L-NAME Effects on Carotid Pressure-Diameter Relation
The carotid pressure-diameter relation was measured to evaluate possible effects of eNOS gene transfer on vascular tone and structure that might indirectly influence baroreceptor activity. The diameter of eNOS-transduced carotid sinuses was increased significantly over a wide range of pressure compared with the diameter of-Gal-transduced
sinuses (Fig. 6). Treatment of
eNOS-transduced sinuses with L-NAME prevented the increase
in diameter (Fig. 6). The compliance of the carotid sinus (change in
diameter/change in pressure) was not significantly different in
-Gal-transduced (14.5 ± 0.7 µm/mmHg, r = 0.99) and eNOS-transduced (18.5 ± 2.3 µm/mmHg,
r = 0.99) carotid sinuses.
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To visually confirm the increased diameter of eNOS-transduced carotid
arteries, we examined cross sections of unpressurized common carotid
arteries and carotid sinuses from one rabbit. The diameter of carotid
arteries subjected to eNOS gene transfer was larger than the diameter
of the contralateral control arteries (Fig.
7).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The major findings of the present study are that adenoviral-mediated gene transfer of NOS to carotid sinus adventitia causes sustained NO-dependent inhibition of baroreceptor activity, a resetting of the baroreceptor pressure-activity curve to higher pressures, and an increase in carotid sinus diameter. The discussion addresses the possible mechanisms that may contribute to NOS-induced baroreceptor resetting, other factors to consider in interpreting the results, and the physiological and investigative implications of the findings.
Possible Mechanisms of Baroreceptor Resetting Induced by NOS Gene Transfer
Topical application of L-NAME to the carotid sinus prevented NOS-induced baroreceptor resetting (Fig. 4), strongly suggesting that the inhibition of baroreceptor activity was indeed dependent on NO production. The finding that topical application of adenoviral vectors to the carotid sinus led to localized transgene expression restricted to the adventitia layer of the vessel wall (Fig. 1) is in agreement with previous results (5, 26, 34).Cell source of transgene generated NO.
The predominant cells transduced by topical application of the AdeNOS
vector to large arteries are adventitial fibroblasts (5).
Previous studies have shown that adventitial cells expressing the NOS
transgene, when stimulated by bradykinin or calcium ionophore, produce
sufficient NO to provoke NO-dependent changes in vascular tone and
structure in both endothelium-intact and endothelium-denuded arteries
(5, 6, 31). Taken together, these results suggest that
adventitial fibroblasts were the likely source of NO responsible for
baroreceptor resetting in the NOS-transduced carotid sinuses. Generation of NO by eNOS is calcium dependent (9, 28). We speculate that calcium influx through mechanosensitive ion channels on
fibroblasts (10, 37) may have activated eNOS and increased NO production in our experiments. We did not detect -Gal transgene expression in the carotid sinus nerve (data not shown) consistent with
the significant diffusion barrier presented by the perineurium surrounding nerve terminals in mature animals (21).
Direct action of NO on baroreceptors. NO may inhibit baroreceptor activity directly by an effect on baroreceptor nerve terminals. A previous study in our laboratory demonstrated that acute exposure of the isolated rabbit carotid sinus to NO or NO donors inhibits baroreceptor activity (25). The inhibition of nerve activity appeared to be mediated by a cGMP-independent mechanism, could not be explained by NO-induced vasodilation, and was transient, consistent with the short half-life of NO (25). In subsequent studies, we demonstrated that NO acutely inhibits voltage-dependent Na+ currents in isolated baroreceptor neurons in culture (1, 23). The NO-induced inhibition of Na+ current involved nitrosylation of thiol residues within Na+ channels or channel-associated proteins (23). Thus NO may decrease baroreceptor activity by inhibiting Na+ channels that are essential for generation of action potentials. NO has also been shown to enhance activity of various K+ channels (2, 19) that conceivably could contribute to inhibition of baroreceptor activity. It is also possible that NOS-generated reactive oxygen species or peroxynitrite may inhibit baroreceptor activity (24).
Indirect action of NO via change in vascular compliance/structure.
Alternatively, NO may decrease baroreceptor activity indirectly
through changes in the compliance or structure of the blood vessel wall
or by altering the coupling of baroreceptor terminals to the vessel
wall. Arterial diameter of eNOS-transduced carotid sinuses was
significantly larger than the diameter of -Gal-transduced or control
sinuses not exposed to adenovirus (Figs. 6 and 7). The increased
diameter cannot be explained solely as a result of decreased vascular
tone. Baseline tone is low in this preparation; acute administration of
NO donors into the isolated carotid sinus causes only a small increase
in carotid sinus diameter (25). Furthermore, the increase
in diameter of eNOS-transduced carotid sinuses was evident in
unpressurized carotid arteries (Fig. 7), suggesting a structural
enlargement that may be analogous to the structural remodeling that
occurs in large arteries subjected to chronic increases in blood flow
(17, 22, 36). Sustained increases in arterial flow
increase vessel size (17, 22, 36), and increased flow or
shear stress increases vascular eNOS expression (30, 35).
The chronic flow-induced arterial enlargement is attenuated by NOS
inhibitors, suggesting that NO is a key mediator of the response
(39). Enhanced NOS expression and NO production also
mediate, at least in part, poststenotic dilatation of large arteries
(4). Flow-induced changes in arterial structure (increased diameter) occur over a period of weeks to months (17, 22, 36). Our results suggest that NO-dependent arterial enlargement may occur as soon as 4-5 days after eNOS expression is enhanced. The mechanism may involve changes in the extracellular matrix (11, 12, 32, 36, 42). Although one might expect increased vessel diameter to increase baroreceptor activity, changes in the
extracellular matrix may alter the mechanical coupling of the
baroreceptor terminals to the vessel wall, leading to inhibition of
activity despite the increase in carotid sinus diameter.
Possible role of arterial pressure in eNOS-induced baroreceptor
resetting.
The baroreceptor pressure-activity curve is reset to higher pressures
in hypertensive states (20). Mean arterial pressure was
not significantly different in rabbits subjected to -Gal gene
transfer (95 ± 2 mmHg) and eNOS gene transfer (93 ± 2 mmHg). Therefore, the resetting of the baroreceptor function curve in eNOS-transduced carotid sinuses cannot be explained by a difference in
arterial pressure.
Other Factors for Consideration
The goal of the present study was to determine the effect of sustained overexpression of eNOS over a period of days on baroreceptor activity, thereby necessitating a comparison of activity measured in separate groups of control (-Gal) and eNOS-transduced rabbits. The
absolute level of recorded multifiber nerve activity is dependent on
the recording conditions. Therefore, as is routinely done in these
types of studies (24-26), we expressed baroreceptor
activity as a percentage of maximum activity. This analysis allowed us to demonstrate a significant rightward shift of the pressure-activity relation to higher pressures for the eNOS-transduced carotid sinuses. The results do not enable an evaluation of a possible effect of NOS
expression on absolute or maximum baroreceptor activity. Furthermore, we cannot rule out the possibility that selective damage to
low-threshold baroreceptor fibers innervating the eNOS-transduced
carotid sinus might have contributed to the shift in the baroreceptor
function curve to higher pressures.
Adenovirus may elicit an inflammatory response, particularly after
intraluminal administration of adenoviral vectors to arteries that can
limit the duration of transgene expression and alter physiological
functions (29). In contrast, inflammation appears to be
less of a problem after topical application of adenoviral vectors to
adventitia of arteries (5, 26, 34). We have demonstrated
previously that adenoviral-mediated gene transfer of -Gal to carotid
sinus adventitia did not significantly alter the baroreceptor
pressure-activity relation (26), suggesting that
adenovirus itself, -Gal expression, and/or the potential inflammatory response did not alter baroreceptor sensitivity to a
significant extent. The finding that the baroreceptor pressure-activity curve is shifted to higher pressures in eNOS-transduced vs.
-Gal-transduced carotid sinuses and the prevention of resetting by
L-NAME suggest that the resetting was indeed caused by NOS
overexpression and not by adenovirus or inflammation.
We evaluated the effect of eNOS gene transfer on baroreceptor activity 4-5 days after application of the viral vector to the carotid sinus. We chose this time point based on our previous study showing that transgene expression driven by the RSV promoter increased markedly between 1 and 4 days after application of the vector to carotid sinus adventitia (26). We observed that transgene expression is essentially absent ~1 mo after vector application (unpublished results). The loss of transgene expression over time may limit use of this technique to alter baroreflex sensitivity chronically over longer periods. Nevertheless, the results provide proof of principle that gene transfer can be used to chronically modulate baroreceptor function. Development of new vectors for gene transfer may extend the duration of transgene expression.
Perspectives
Endothelium, carotid sinus nerves, and fibroblasts contain eNOS, nNOS, and inducible NOS, respectively (9, 14, 28, 33, 40, 41), all of which can be upregulated by specific stimuli (9, 28). Thus there are several potential sources of NO located near, and possibly in, baroreceptor terminals. An inhibitory influence of endogenous NO on carotid chemoreceptor afferent activity has been demonstrated (33, 38). The results of this study and previous studies in our laboratory (1, 23, 25) demonstrate an inhibitory influence of NO on baroreceptor activity. The physiological role of endogenous NO in modulation of baroreceptor activity remains to be demonstrated. We speculate that if NO is produced during increases in arterial pressure, it may function as a negative-feedback inhibitor of activity and contribute to hypertensive baroreceptor resetting (20). Our finding of increased baroreceptor activity at low carotid sinus pressure in L-NAME-treated carotid sinuses compared with untreated sinuses (Fig. 5) supports a possible role of endogenous NO in modulation of baroreceptor activity. Interestingly, in addition to its effect on baroreceptor afferents, NO may also modulate the baroreflex by acting at other sites, including nucleus tractus solitarius, rostral ventrolateral medulla, paraventricular nucleus, and cardiac parasympathetic nerves (13, 15, 16, 18, 27, 43).Gene transfer to sites of baroreceptor innervation potentially provides a method to address difficult, longstanding questions related to baroreceptor function. For example, by producing sustained changes in baroreceptor sensitivity, gene transfer experiments may provide new insight into the role of the baroreceptor reflex in long-term control of arterial pressure. In addition, gene transfer to carotid sinus and/or aortic arch may provide a means to restore baroreceptor sensitivity in pathological states and examine the cardiovascular consequences of the improved reflex sensitivity. The approach may also provide a tool to explore the relation between chronic changes in vascular structure and changes in baroreceptor sensitivity.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Harrison at Emory Univ. for supplying the cDNA for type III eNOS, and C. Whiteis and A. Holley at the Univ. of Iowa for preparation of figures and assistance with histological procedures, respectively. We also thank Drs. B. L. Davidson and R. D. Anderson and the Gene Transfer Vector Core at the Univ. of Iowa for providing the adenoviral vectors. The Vector Core is supported in part by funds from the University of Iowa Carver Trust.
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FOOTNOTES |
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This publication was made possible by National Heart, Lung, and Blood Institute Grant HL-14388 and a Dept. of Veterans Affairs Merit Review Award to M. W. Chapleau. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or the Dept. of Veterans Affairs.
S. S. Meyrelles was supported by funds from National Council for Science and Technology Development at the time the studies took place.
Present address for H. Z. Mao: Dept. of Medicine, Univ. of California-Los Angeles, Los Angeles, CA 90073.
Address for reprint requests and other correspondence: M. W. Chapleau, Dept. of Internal Medicine, E327-1 GH, The Univ. of Iowa, 200 Hawkins Dr., Iowa City, IA 52242 (E-mail: mark-chapleau{at}uiowa.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.
10.1152/ajpregu.00735.2002
Received 4 December 2002; accepted in final form 10 January 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Significant difference
between L-NAME-treated and untreated
eNOS-transduced carotid sinuses, P < 0.05.
