© 1995 American Heart Association, Inc.
Articles |
Modulation of Baroreceptor Activity by Nitric Oxide and S-Nitrosocysteine
From the Cardiovascular Center and the Departments of Internal Medicine, Physiology and Biophysics, Anesthesiology, and Pharmacology, University of Iowa, and the Department of Veterans Affairs Medical Center, Iowa City, Iowa.
Correspondence to Mark W. Chapleau, PhD, Assistant Professor, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242.
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Abstract The goal of this study was to determine whether nitric oxide (NO) and the NO donor, S-nitrosocysteine (cysNO), modulate the activity of carotid sinus baroreceptors. Baroreceptor activity was recorded from the vascularly isolated carotid sinus in anesthetized rabbits. Baroreceptor activity decreased in a dose-dependent manner after injection of either NO or cysNO as constant pressure was maintained, and activity recovered spontaneously over time, within seconds to minutes. The baroreceptor pressure-activity relation was shifted significantly to the right by cysNO, with a profound suppression of activity at high pressure. Baroreceptor activity at 160 mm Hg averaged 76±8%, 60±6%, and 36±5% of the control maximum during exposure to 10-4, 2 to 3x10-4, and 10-3 mol/L cysNO, respectively. The inhibition of activity by the L and D isomers of cysNO was equivalent and was blocked by reduced hemoglobin, suggesting that the effect was mediated by NO. The suppression of baroreceptor activity by cysNO was not related to vascular relaxation as measured by videomicrometer. Inhibition of soluble guanylate cyclase with methylene blue or 6-anilinoquinoline-5,8-quinone (LY83583, 10-5 mol/L) did not attenuate and dibutyryl cGMP (10-3 mol/L) did not mimic the suppression of baroreceptor activity by cysNO, suggesting a cGMP-independent mechanism. Activation of endogenous NO formation with thimerosal (10-5 to 10-4 mol/L) reduced maximum baroreceptor activity in five of eight experiments to 59±7% of the control maximum. The NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME, 10-4 mol/L) by itself failed to influence baroreceptor activity but prevented thimerosal-induced suppression of activity. Addition of L-arginine (10-3 mol/L) after L-NAME restored the inhibitory influence of thimerosal. The results indicate that NO and cysNO suppress baroreceptor activity through a mechanism independent of guanylate cyclase activation and vascular relaxation and that endogenous NO released by chemical activation suppresses baroreceptor activity.
Key Words: carotid sinus • endothelium-derived relaxing factor • pressoreceptors • rabbits
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Introduction |
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Arterial baroreceptors are mechanically sensitive nerve endings innervating carotid sinuses and the aortic arch.1 The increased activity of baroreceptors during increases in arterial pressure triggers reflex circulatory adjustments that buffer or oppose the rise in pressure. The level of baroreceptor activity is not determined solely by the level of arterial pressure and vascular stretch but is modulated by neurohumoral substances and paracrine factors released from endothelial cells and platelets.2 3 4 5 6 7 8
Numerous studies have focused recently on endothelium-derived relaxing factors (EDRFs) and their importance in the regulation of vascular tone in both physiological and pathological states.9 10 11 12 13 One of the major EDRFs has been identified as nitric oxide (NO) or a related nitrosothiol compound, such as S-nitrosocysteine (cysNO).12 13 14 15 16 Recent studies suggest that NO not only plays an important role in vascular regulation but is also a key regulator of platelet adhesion and activation, host defense reactions, and neurotransmission.12 13 17 18 19 20
The major goal of the present study was to determine whether NO and cysNO modulate baroreceptor sensitivity. Such an influence would represent an important additional mechanism by which these factors may influence arterial pressure. In addition, we sought to determine whether an influence on baroreceptor activity might be related to vascular relaxation and/or activation of the soluble guanylate cyclase, the enzyme that mediates most of the actions of NO in other tissues.11 To evaluate a potential role of endogenous NO in the modulation of baroreceptor activity, we activated endogenous NO formation in the carotid sinus with thimerosal21 and blocked endogenous formation with nitro-L-arginine methyl ester (L-NAME).22
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New Zealand White rabbits of either sex were anesthetized with sodium pentobarbital (30 to 35 mg/kg) injected through the marginal ear vein. The trachea was cannulated to provide mechanical ventilation with room air supplemented with oxygen. The femoral artery and vein were catheterized for measurement of arterial pressure and administration of anesthetic, respectively.
Isolation of the Carotid Sinus
One carotid sinus was vascularly isolated as described
previously.5 7 8 All visible branches of the common and
external carotid arteries were ligated in the region of the carotid
sinus. Catheters were placed in the common carotid and lingual
arteries, and the sinus was filled with Krebs-Henseleit physiological
saline solution of the following composition (mmol/L): NaCl 118, KCl
4.7, NaHCO3 24, MgSO4 1.2, CaCl2
2.5, KH2PO4 1.1, and glucose 10. Before placing
the Krebs' buffer in the carotid sinus, it was warmed to 37°C and
was saturated with 95% O2/5% CO2. A
pressure reservoir was attached to the isolated carotid sinus via the
common carotid catheter. Carotid sinus pressure was controlled by
regulating the air inflow to the reservoir from a pressurized air
source. Pressure was measured with a transducer (Statham model P23XL)
connected to the lingual artery catheter and a pressure processor
amplifier (model 13-4615-52, Gould Inc). The carotid sinus was
pressurized as a closed blind sac in the absence of flow through the
sinus. The vagus, aortic, and cervical sympathetic nerves were
transected to eliminate a possible influence of sympathetic activity on
baroreceptor discharge. Decamethonium bromide (0.3 mg/kg) was injected
intravenously to eliminate skeletal muscle contraction before recording
nerve activity. Supplemental doses of pentobarbital and decamethonium
were given as needed.
Measurement of Baroreceptor Activity
The carotid sinus nerve was carefully isolated and sectioned.
The nerve was desheathed, placed on a platinum electrode, and encased
in silicone gel. The isolated carotid sinus and surrounding structures
were submerged in warm (37°C) paraffin oil to avoid drying of the
tissues. Multifiber activity was recorded from the carotid sinus nerve
with a high-impedance probe (model HIP511J, Grass Instrument Co) and a
Grass bandpass amplifier (model P511G; low-frequency to high-frequency
cutoff, 100 to 300 Hz to 3 to 10 kHz). The electroneurogram was
displayed on a dual-beam storage oscilloscope (model 5113, Tektronix),
and the nerve discharge was heard through a loudspeaker.
In the majority of experiments, baroreceptor activity was quantified by integrating the voltage of the neurogram with an integrator amplifier (model 13-4615-70, Gould Inc). The integrated voltage, the carotid sinus pressure, and the systemic arterial pressure of the rabbit were recorded on a pen recorder (model 11-1202-25, Gould Inc). The integrated voltage that remained during a rapid decrease in carotid sinus pressure to 0 mm Hg (electrical noise) was subtracted from all measurements. In some experiments, baroreceptor activity was quantified by counting the frequency of action potentials that exceeded a selected voltage threshold level set just above the electrical noise with a nerve-traffic analyzer (model 706-C, Department of Bioengineering, University of Iowa, Iowa City). The electroneurogram, the output of the spike counter, and carotid sinus pressure were recorded on an electrostatic recorder (model ES-1000, Gould Inc).
Measurement of Carotid Pressure- Diameter Relation
The carotid sinus region was videorecorded through a microscope,
and the diameter of the carotid artery at the origin of the carotid
sinus was measured at a later time with a videomicrometer as described
previously.7 In addition to the analog tracing of carotid
sinus pressure, a digital readout of the pressure was videotaped to
facilitate matching of diameter measurements with precise pressure
levels when the pressure-diameter relation was analyzed at a later
time.
Preparation of NO and cysNO
A saturated solution of NO was prepared by equilibrating
deoxygenated water and NO in a sealed bottle at a pressure slightly
above atmospheric.14 23 The concentration of NO measured
by chemiluminescence was 2.4±0.1 mmol/L.23 The
NO-containing solution was freshly made before each experiment,
although it has been demonstrated to remain stable for more than 6
months in the absence of oxygen.24
cysNO was synthesized from the reaction of 1 mmol cysteine and 2 mmol
nitrogen dioxide gas in 1 mL of cold methanol.14 The
resulting 1-mol/L solution of cysNO was stable in methanol at -20°C
for 
2 months when protected from light. Quantitative conversion of
cysteine to cysNO was confirmed by high-pressure liquid
chromatography.14 New solutions of cysNO were prepared
each week. To examine responses to NO and cysNO in the isolated sinus,
the stock solutions were dissolved into Krebs' buffer and injected as
rapidly as possible into the isolated carotid sinus. Injections of the
methanol vehicle solution did not influence baroreceptor activity.
Protocols
Influence of NO and cysNO on Baroreceptor Activity
Baroreceptor activity was recorded as carotid sinus pressure was
maintained constant, usually at 80 mm Hg, before and after
injection of oxygenated warmed (37°C) Krebs' buffer into the
isolated sinus. Within 1 to 3 minutes after the injection, the
baroreceptor pressure-activity relation was determined with a slow ramp
increase in nonpulsatile carotid sinus pressure from 0 to 160 mm Hg.
The rate of rise of pressure (dP/dt) was 2 to 4 mm Hg/s and was
equivalent during all pressure ramps within each experiment. Three to
four consecutive pressure ramps were applied at 3-minute intervals to
ensure reproducible control responses. When pressure ramps were not
being applied, carotid sinus pressure was held constant, again usually
at 80 mm Hg.
Baroreceptor activity was recorded continuously before and after injections of NO (10-5 to 10-3 mol/L) or cysNO (10-5 to 10-3 mol/L) as pressure was held constant. To determine the time course and maximum response to NO and cysNO, pressure was often maintained at a constant level until the maximum change in nerve activity was noted. Pressure ramps were then applied repeatedly, as described above, at intervals of 1 to 3 minutes, usually until spontaneous recovery of baroreceptor sensitivity was evident. In some experiments, pressure ramps were applied soon after the injection of NO or cysNO. The carotid sinus was usually flushed and refilled with fresh Krebs' buffer, and pressure ramps were repeated before injection of the next dose of NO or cysNO. The effects of the D and L isomers of cysNO on baroreceptor sensitivity were tested to investigate a possible stereoselective receptor-mediated action.25 In three experiments, the influence of cysNO on baroreceptor activity was determined before and after addition of reduced hemoglobin (10 µmol/L) to the Krebs' buffer.
In some experiments, carotid diameter was measured simultaneously with baroreceptor activity during pressure ramps by using the same protocol as described above. cysNO was injected both in the presence of relatively low vascular tone (n=4) and after increasing vascular tone with the injection of phenylephrine (10-6 mol/L) to enhance cysNO-induced vasodilatation (n=4).
Role of Guanylate Cyclase Activation and cGMP in cysNO-Mediated
Suppression of Baroreceptor Activity
Baroreceptor responses to cysNO were tested before and after 20
to 30 minutes of exposure of the isolated sinus to methylene blue or
LY83583 (10-5 mol/L, n=6), which are established
inhibitors of guanylate cyclase. In addition, the membrane-permeant
analogue of cGMP, dibutyryl cGMP (10-3 mol/L, n=5), was
injected into the carotid sinus, and pressure ramps were applied as
described above.
Role of Endogenous NO in Modulation of Baroreceptor Activity
Baroreceptor pressure-activity curves were generated before and
after injection of thimerosal (10-5 to 10-4
mol/L, n=8), a potent stimulator of endogenous NO
formation,21 in the isolated carotid sinus. Pressure ramps
were applied before and after injection of thimerosal at 3-minute
intervals, and data were analyzed when maximum inhibition of activity
occurred. To inhibit the endogenous formation of NO, the NO synthase
inhibitor L-NAME (10-4, n=8) was injected into the
isolated carotid sinus.22 After an 8- to 10-minute
exposure to L-NAME, three pressure ramps were applied at 3-minute
intervals. In the continued presence of L-NAME, thimerosal (n=8) was
then injected into the carotid sinus, and additional ramps were applied
at 3-minute intervals. To demonstrate that the influence of L-NAME on
the responses to thimerosal was a result of the inhibition of NO
synthase, we also tested responses to injection of thimerosal plus
L-arginine (10-3 mol/L, n=7) in the
continued presence of L-NAME, and pressure ramps were again applied at
3-minute intervals. Addition of excess L-arginine, the
substrate for NO formation, has been shown to overcome competitive
inhibition of NO synthase by arginine analogues.15 22
Drugs
LY83583 was obtained from Cal Biochem Co, and dibutyryl
cGMP, thimerosal, L-NAME, L-arginine, cysteine,
hemoglobin, and methylene blue were obtained from Sigma Chemical
Co.
Data Analysis
The absolute amount of baroreceptor activity recorded depends on
the number of baroreceptor fibers in contact with the electrode and
varies among preparations. Therefore, drug-induced changes in
baroreceptor activity that occurred as carotid sinus pressure was
maintained constant are expressed as the percent change from baseline,
and the pressure-activity curves were constructed after normalizing
baroreceptor activity as a percentage of the maximum activity recorded
during the control ramp.
The maximum inhibition of baroreceptor activity at constant pressure and the time required to reach maximum inhibition after injections of NO and cysNO were determined. The influence of various concentrations of NO and cysNO on baroreceptor activity was analyzed by repeated-measures ANOVA followed by contrast testing.26 The magnitude and time course of responses to NO were compared with responses to equivalent concentrations of cysNO by unpaired t test.
The effects of cysNO on the baroreceptor pressure-activity and pressure-diameter relations were analyzed by ANOVA. In addition, baroreceptor pressure threshold (Pth), the slope of the pressure-activity curve over the linear range (gain), maximum baroreceptor activity, and the pressure at which activity was maximal were derived from the pressure-activity relations. The effects of cysNO on these parameters were analyzed by ANOVA, followed by contrast testing.26 Baroreceptor Pth was defined as the pressure at which nerve activity began to increase in a continuous manner with increases in carotid sinus pressure. All data are presented as mean±SEM. Differences were considered significant at P<.05.
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Influence of NO and cysNO on Baroreceptor Activity
Injections of either NO or cysNO into the isolated carotid sinus decreased baroreceptor activity significantly over time as pressure was maintained constant (Figs 1 through 3
). The threshold concentration
required for inhibition varied among experiments. Baroreceptor activity
was clearly decreased in a reversible manner by cysNO in three of eight
experiments and by NO in two of three experiments at concentrations of
1 to 5x10-5 mol/L (see Figs 1 and 2), but the decrease in activity at this dose was not
statistically significant for the group. The inhibition of baroreceptor
activity was consistent and dose dependent at higher concentrations and
was equivalent in magnitude with NO and cysNO (Fig 3).
The inhibition of baroreceptor activity occurred more rapidly with
authentic NO than with cysNO, with the maximum inhibition occurring
40±5 and 81±6 seconds after injection of the two compounds,
respectively (P<.05). Baroreceptor activity recovered
spontaneously within seconds to minutes, reflecting the short
biological half-life of the compounds. The inhibitory influence of NO
on baroreceptor activity dissipated more rapidly than that of cysNO,
which prevented determination of its effect on the pressure-activity
relation.
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Significantly greater magnitude of inhibition with the
higher doses compared with the inhibition caused by 10-4
mol/L S-nitrosocysteine (P<.05). The values
represent mean±SEM. The number of experiments are indicated
within each bar. Carotid sinus pressure averaged 82±3 and 82±2 mm Hg
during exposure to nitric oxide and S-nitrosocysteine,
respectively.
The longer duration of action of cysNO enabled determination of its
effect on the pressure-activity relation. cysNO shifted the
pressure-activity curve to the right with pronounced suppression of
nerve activity at high carotid sinus pressure (Figs 1
, 2, and 4). Baroreceptor Pth was increased
significantly, and maximum baroreceptor activity and gain were
decreased significantly by cysNO (Table). The decrease
in maximum activity was significant at 10-4 mol/L cysNO,
whereas higher concentrations (2 to 3x10-4 and
10-3 mol/L) were required to significantly decrease the
slope of the pressure-activity curve and increase
Pth, respectively (Table). In contrast to the
control response, where baroreceptor activity continued to increase and
plateau at high pressures, nerve activity reached a maximum at lower
pressures during exposure to cysNO with a paradoxical decline in
activity as pressure continued to rise (Figs 2 and 4). The
D and L isomers of cysNO (10-3
mol/L in four experiments, 10-4 mol/L in one experiment,
and 5x10-4 mol/L in one experiment) suppressed
baroreceptor activity to an equivalent extent. Baroreceptor activity
recorded at 160 mm Hg averaged 47±7% and 46±8% of the control
maximum during exposure to the D and L
isomers, respectively (n=6).
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The suppression of baroreceptor activity by cysNO was not related to
vascular relaxation. The magnitude of baroreceptor inhibition was
similar after injection of cysNO into the carotid sinus with low and
high (precontracted with phenylephrine) vascular tone, despite a marked
difference in the vasodilator response to cysNO (Fig 5).
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Hemoglobin failed to influence baroreceptor activity by itself but prevented the inhibition of activity by cysNO. Baroreceptor activity at 160 mm Hg was decreased by cysNO to 63±5% of control before hemoglobin (n=3). In the presence of hemoglobin, nerve activity averaged 103±5% and 106±5% before and after injection of cysNO, respectively (n=3).
Role of Guanylate Cyclase Activation in cysNO-Mediated Suppression
of Baroreceptor Activity
Exposure of the isolated carotid sinus to two different inhibitors
of the soluble guanylate cyclase, methylene blue and LY83583, did not
alter the response to cysNO (Fig 6). Injection of the
membrane-permanent analogue of cGMP, dibutyryl cGMP, into the isolated
carotid sinus also did not influence baroreceptor activity (Fig 6).
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Role of Endogenous NO in Modulation of Baroreceptor Activity
Injection of thimerosal, an activator of endogenous NO
formation, caused a pronounced and sustained decrease in baroreceptor
activity in five of eight experiments (Fig 7). Treatment
of the isolated carotid sinus with the NO synthase inhibitor L-NAME had
no effect by itself on baroreceptor activity but prevented the
suppression of baroreceptor activity by thimerosal (Fig 7), suggesting
that the inhibitory effect of thimerosal was mediated through formation
of NO. Injection of the excess substrate for NO synthase,
L-arginine (10-3 mol/L), restored the
inhibitory influence of thimerosal on baroreceptor activity in the
presence of L-NAME (Fig 7). L-Arginine alone did not
influence baroreceptor activity.
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The major findings of the present study are as follows: (1) NO and cysNO suppress baroreceptor activity through a mechanism independent of vascular relaxation or guanylate cyclase activation. (2) The basal release of NO at least under the conditions of these experiments is insufficient to modulate baroreceptor activity. (3) Endogenous NO released by chemical activation suppresses baroreceptor activity.
NO as the Inhibitory Mediator
We propose that NO is responsible for the inhibition of
baroreceptor activity during exposure to both NO and cysNO. The
magnitude of the inhibition caused by the two compounds was similar
(Fig 3), although the effect of NO was more rapid in onset and
dissipated much more rapidly. These results are consistent with the
known ability of cysNO to spontaneously generate NO and the fact that
cysNO is the more stable of the two compounds.14 24 The
delay in the maximum response to cysNO and the slower recovery of nerve
activity may reflect the time needed to generate NO from cysNO and the
greater stability of cysNO, respectively. Reduced hemoglobin binds to
and neutralizes extracellular NO but does not penetrate cells. Our
finding that hemoglobin prevented the suppression of baroreceptor
activity by cysNO suggests that NO was generated from cysNO
extracellularly and was responsible for the inhibition of nerve
activity after injection of cysNO into the carotid sinus. The amino
acid cysteine alone (10-8 to 10-3 mol/L)
failed to influence baroreceptor activity (n=6, data not shown).
We had originally considered the possibility that nitrosothiols such as cysNO may trigger receptor-mediated actions that are not dependent on extracellular generation of NO.25 For example, the L and D isomers of cysNO injected into a cerebral ventricle produce markedly different neurally mediated responses despite equivalent formation of NO.25 We have found that the L and D isomers of cysNO suppressed baroreceptor activity to an equivalent extent, again suggesting that NO was responsible for the inhibition of baroreceptor activity. We have also found that other structurally different NO donors, including S-nitroso-mercaptoproprionic acid (n=3), S-nitroso-ß-mercaptoethanol (n=3), S-nitroso-cysteamine (n=2), and nitroglycerin (n=6) at concentrations of 10-4 to 10-3 mol/L suppress baroreceptor activity. Interestingly, sodium nitroprusside failed to suppress activity (n=6), perhaps because of its polarity and relatively poor penetration through the vessel wall.
NO decomposes to nitrate and nitrite. These compounds are relatively stable, suggesting that they are not responsible for the transient inhibition of baroreceptor activity observed after injections of NO or cysNO. Furthermore, we have found that injection of sodium nitrite (10-3 mol/L) into the carotid sinus failed to influence baroreceptor activity.
Mechanism of Suppression of Baroreceptor Activity
NO and cysNO are potent vasodilators.14 16 We
considered the possibility that vascular relaxation may have been
responsible for the suppression of baroreceptor activity by NO and
cysNO. To test this possibility, we enhanced the vasodilator response
to cysNO by first increasing the basal tone of the carotid sinus with
an injection of phenylephrine, reasoning that if the inhibitory
influence of cysNO on baroreceptor activity was secondary to the
vasodilation, then the enhancement of dilation should enhance the
suppression of baroreceptor discharge. Under conditions of low basal
tone, cysNO caused minimal vasodilation but pronounced suppression of
baroreceptor activity (Fig 5). After preconstriction of the carotid
sinus with phenylephrine, injection of cysNO caused marked vasodilation
but suppressed nerve activity to an extent similar to that found under
conditions of low tone (Fig 5). Thus, the suppression of baroreceptor
activity by cysNO was not related to vascular relaxation, suggesting a
direct effect on neuronal excitability of baroreceptors.
The actions of NO and cysNO on various target cells are often mediated
through activation of soluble guanylate cyclase and increased
formation of cGMP.11 Our results suggest that NO-mediated
suppression of baroreceptor activity may not occur through this
mechanism. Inhibitors of the soluble guanylate cyclase (methylene blue
and LY83583) did not attenuate the baroreceptor response to cysNO (Fig 6). Furthermore, the membrane-permeant analogue of cGMP, dibutyryl
cGMP, failed to mimic the effect of cysNO on baroreceptor discharge
(Fig 6). We considered the possibility that these compounds
administered into the carotid sinus lumen may not have reached the
nerve endings in the adventitia. Flooding the outside of the carotid
sinus with dibutyryl cGMP also failed to suppress baroreceptor activity
(n=2).
The mechanism responsible for NO-mediated suppression of baroreceptor activity is unclear. In addition to the activation of guanylate cyclase, NO has been reported to activate ADP-ribosyltransferase in platelets27 and to lower the intracellular concentration of calcium in BALB/c 3T3 fibroblasts through a cGMP-independent mechanism.28 NO may also interact with other iron-containing enzymes or proteins, such as ferritin,13 with the potential to release free iron, which may promote lipid peroxidation. NO may also interact with superoxide anion released from endothelial cells or leukocytes to form the potent cytotoxic compounds peroxynitrite and hydroxyl radical.29 Further studies are needed to explore the potential role of these mechanisms in NO-mediated suppression of baroreceptor activity. NO released from macrophages has cytotoxic activity.20 The suppression of baroreceptor activity by NO and cysNO was completely and rapidly reversible, demonstrating that the decrease in nerve activity was not caused by irreversible damage of baroreceptor neurons.
An attractive hypothesis is that NO may function as a hyperpolarizing
factor. NO has been shown to hyperpolarize vascular and esophageal
smooth muscle,19 30 31 possibly in part through a
cGMP-independent mechanism.32 33 Recently, Bolotina et
al33 have reported that NO can directly activate
calcium-dependent K+ channels in isolated membrane patches
from vascular smooth muscle in the absence of intracellular messengers,
including cGMP. We speculate that the activation of K+
channels by NO may hyperpolarize baroreceptors, thereby decreasing
their activity. The suppression of baroreceptor activity by cysNO is
particularly pronounced at high levels of carotid sinus pressure, when
nerve activity actually declines as pressure continues to rise (Figs 2 and 4). This unusual effect also occurs when baroreceptors are exposed
to endothelin.3 Interestingly, NO-mediated
hyperpolarization of vascular muscle is also dependent on the level of
stretch; hyperpolarization occurs only when the muscle is stretched and
not when it is relaxed.31
Functional Significance
Inhibition of formation of endogenous NO with L-NAME or
scavenging NO with hemoglobin failed to influence baroreceptor activity
in our experiments, suggesting that the basal release of NO under the
conditions of our experiments does not modulate baroreceptor activity.
It is possible that the endogenous formation of NO was low in our
preparation because of the absence of flow and/or pulsatile pressure.
We have performed experiments in which the buffer was perfused through
the common carotid and lingual arteries at constant flow, and NO
synthesis inhibitors still failed to alter baroreceptor activity (n=6).
Relatively high concentrations of exogenous NO or cysNO
(10-5 to 10-4 mol/L) were
required to suppress baroreceptor activity. Because of the short
half-life of NO and cysNO (<30 seconds),12 13 14 15 16 we believe
that the actual concentration of NO and cysNO at the baroreceptor
endings may have been considerably less than the calculated
concentration in the solution before injection.
The potential for chemically activated endothelium to produce endogenous levels of NO capable of decreasing baroreceptor activity is supported by our results using thimerosal. Thimerosal, an inhibitor of the enzyme acyl coenzyme A/lysolecithin acyltransferase, is a powerful stimulator of EDRF release from endothelial cells21 and hyperpolarizes vascular smooth muscle in an endothelium-dependent manner.34 Thimerosal also exerts other actions, including stimulation of prostaglandin formation. The inhibitory influence of thimerosal on baroreceptor activity was blocked by L-NAME and restored by excess L-arginine, strongly suggesting that the inhibitory effect was mediated by NO. We have demonstrated previously that stimulation of cultured bovine aortic endothelial cells with the calcium ionophore A23187 or bradykinin, which are also known to stimulate formation of NO, releases a factor that suppresses the activity recorded from single carotid sinus baroreceptor fibers of dogs.4 The factor released from cultured endothelium was not identified but may have been NO.
The cellular source of NO in the isolated carotid sinus during chemical activation with thimerosal is unknown. It may have been released from the endothelium but may have also been formed in the baroreceptor neurons themselves. NO synthase is present in neurons,17 including sensory neurons innervating the carotid sinus region,35 and NO has been implicated as an endogenous inhibitory modulator of carotid chemoreceptor sensory discharge.36 Thus, NO may exert an inhibitory influence on the sensory activity of both chemoreceptors and baroreceptors. Recent studies also suggest that NO may modulate the activity of nociceptive sensory receptors.37
An inducible NO synthase activated by cytokines produced in pathological inflammatory states has been demonstrated in macrophages, neutrophils, vascular muscle, and endothelium.12 13 The inducible NO synthase generates sustained release of very large quantities of NO.12 13 Thus, NO-mediated suppression of baroreceptor activity may be particularly relevant in pathological states associated with neutrophil and macrophage infiltration into the blood vessel wall and cytokine-induced NO formation, as occurs in atherosclerosis or sepsis.
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Acknowledgments |
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This study was supported by research funds from the Department of Veterans Affairs, grant HL-14388 from the National Institutes of Health, and a grant from the American Heart Association, Iowa Affiliate, Inc. The authors thank Shawn M. Roach and Deb Shiek for preparation of the figures, Maureen Kent and Kathleen Romero for typing the manuscript, and Dr Bridget Zimmerman for assistance with the statistical analysis. The authors also thank Drs Donald D. Heistad, Kathryn G. Lamping, and Frank M. Faraci for helpful suggestions concerning the manuscript and Laurie Waite for technical assistance.
Received September 6, 1994; accepted November 21, 1994.
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