| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha, Nebraska 68198-4575
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
An
enhanced peripheral chemoreflex has been documented in patients with
chronic heart failure (CHF). This study aimed to examine the
characteristics of carotid body (CB) chemoreceptors in response to
isocapnic hypoxia in a rabbit model of pacing-induced CHF and to
evaluate the possible role that nitric oxide (NO) plays in the altered
characteristics. The chemosensitive characteristics of the CB were
evaluated by recording single-unit activity from the carotid sinus
nerve in both an intact and a vascularly isolated preparation. It was
found that the baseline discharge under normoxia (intact preparation:
arterial PO2 90-95 Torr;
isolated preparation: PO2
100-110 Torr) and the chemosensitivity in response to graded
hypoxia (PO2 40-70 Torr) were
enhanced in CHF vs. sham rabbits. These alterations were independent of the CB preparations (intact vs. isolated). NO synthase inhibition by
N
-nitro-L-arginine
increased the baseline discharge and the chemosensitivity in the intact
preparation, whereas L-arginine
(10
5 M) inhibited the
baseline discharge and the chemosensitivity in the isolated preparation
in sham but not in CHF rabbits.
S-nitroso-N-acetylpenicillamine, an NO donor, inhibited the baseline discharge and the chemosensitivity in both CB preparations in CHF rabbits but only in the isolated preparation in sham rabbits. The amount of NO produced in vitro by the
CB under normoxia was less in CHF rabbits than in sham rabbits
(P < 0.05). NO synthase-positive
varicosities of nerve fibers within the CB were less in CHF rabbits
than in sham rabbits (P < 0.05).
These data indicate that an enhanced input from CB occurs in the rabbit
model of pacing-induced CHF and that an impairment of NO production may
contribute to this alteration.
single-unit activity; carotid sinus nerve; N-nitro-L-arginine; S-nitroso-N-acetyl-penicillamine; hypoxia
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
ACTIVATION OF SYMPATHETIC NERVOUS SYSTEM is a prominent part of the pathophysiology of chronic heart failure (CHF) (8, 10, 32). Because the magnitude of sympathohumoral activation in CHF is a good predictor of mortality (22), understanding the mechanism(s) responsible for the increased sympathetic activity is of major importance. A popular hypothesis explaining the generalized sympathetic activation in CHF has been the impairment of inhibitory arterial and cardiac baroreflexes (15, 32). However, other evidence suggests that excitatory reflexes may also play a role (31). Peripheral chemoreceptor activation is an excitatory input that results in increased sympathetic outflow and blood pressure. Recent studies have shown that chemoreflex sensitivity is enhanced in patients with CHF (4-7). Studies from our laboratory in a rabbit model of pacing-induced CHF have also shown an enhanced sensitivity of the peripheral chemoreflex (25, 26). This enhanced sensitivity of the peripheral chemoreflex contributes, at least in part, to the sympathetic activation in this rabbit model of CHF, because inhibition of peripheral chemoreceptor activity decreased renal sympathetic nerve activity (RSNA) in CHF, but not in sham, rabbits (26). It is not known whether this enhanced reflex sensitivity results from an alteration in the chemosensitive characteristics of the peripheral chemoreceptors or from changes in the central integration of the chemoreceptor input. Therefore, the first goal of this study was to determine whether the chemosensitive characteristics of the peripheral chemoreceptors are altered in the CHF state. For this purpose, the chemosensitive characteristics of the carotid body (CB) chemoreceptors in response to isocapnic hypoxia were examined in sham and CHF rabbits by recording the single-unit activity from the carotid sinus nerve (CSN). It was found that the sensitivity of CB chemoreceptors in response to hypoxia was enhanced in CHF rabbits.
On the basis of these observations, the second goal of this study was to evaluate the mechanism that is responsible for the enhanced sensitivity of the CB chemoreceptors in CHF rabbit. There is evidence indicating that administration of exogenous nitric oxide (NO) inhibits the activity of the CB chemoreceptors in normal animals (19). Furthermore, it has been demonstrated that NO synthesis is suppressed in CHF (24). Therefore, it is reasonable to speculate that an attenuated NO synthesis in the CB during the development of CHF may account for the enhanced sensitivity of the CB chemoreceptors in this condition. To test our hypothesis, we examined the effect of inhibition of endogenous NO synthesis and of administration of exogenous NO on the chemosensitive characteristics of the CB chemoreceptors in sham and CHF rabbits. We also measured the basal NO production in vitro from the CB and examined the density of nerve fibers positive for NO synthase (NOS) within the CB in sham and CHF rabbits. We found that the basal NO production and the NOS-positive nerve fibers were less in CHF rabbits than in sham rabbits. Blockade of NO synthesis increased the discharge of the CB chemoreceptors in sham rabbits while restoration of NO by administration of an NO donor inhibited the discharge of the CB chemoreceptors in CHF rabbits.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
General Preparation
All experiments were carried out on male New Zealand White rabbits weighing 2.5-3.5 kg. Experiments were approved by the University of Nebraska Medical Center Institutional animal Care and Use Committee and were carried out in accordance with the National Institutes of Health and the American Physiological Society's Guides for the Care and Use of Laboratory Animals. Rabbits were anesthetized with an anesthetic mixture consisting of 1.2 mg/kg acepromazine, 5.9 mg/kg xylazine, and 58.8 mg/kg ketamine, given as an intramuscular injection. Supplemental anesthesia was provided by pentobarbital sodium (1.7 mg/kg iv) as needed.With the use of a sterile technique, a left thoracotomy was performed as previously described (18). Briefly, the pericardium was opened, and a pin electrode of our own design was implanted on the left ventricle for pacing. Two sonomicrometer crystals (Sonometrics) were attached to the epicardial surfaces at the base of the heart to measure external cardiac diameters. All leads exited the chest between the 3rd and 4th ribs. A ground lead was implanted on the left wall of the chest underneath the skin. The chest was then closed in layers and evacuated. Proper procedures were taken to ensure that atelectasis and pneumothorax were minimized. Rabbits were placed on an antibiotic regimen consisting of 2.3 mg/kg Baytril im for 5 days.
Production of Heart Failure
After the rabbits recovered from the thoracotomy (10-14 days), in the awake state, baseline cardiac end-systolic and end-diastolic external diameters, fractional shortening, and shortening velocity (dD/dtmax) were measured by sonomicrometry. An arterial blood sample (0.25 ml) was taken by needle puncture of an ear artery for measuring blood gas. After these prepacing measurements were completed, a pacing regimen was started. The pacing was started at a 320 beats/min rate, which was held for 7 days. Then the pacing rate was gradually increased to 380 beats/min, with an increment of 20 beats/min each week. The pacemaker was of our own design, with its output usually being set at 4-5 V and 0.5 ms. Sonograms were done and blood gases were measured weekly, with the rabbits sitting quietly in a Plexiglas box and with the pacemaker turned off for at least 30 min before recordings were made. Rabbits with >40% reductions in dD/dtmax and shortening fraction were considered in CHF (generally after 3-4 wk). Sham-operated rabbits underwent a similar period of sonographic measurements. Rabbits exhibiting abnormal arterial blood gases [either arterial PO2 (PaO2) <80 Torr, arterial PCO2 (PaCO2) >45 Torr, or PaCO2 <30 Torr] were excluded from the study.CB Preparations
On the day of the experiment, rabbits were anesthetized with urethan (0.8 g/kg iv) and
-chloralose (40 mg/kg iv). The trachea was
intubated, and the lungs mechanically ventilated (250-300 ml · kg1 · min1).
Tidal volume was adjusted to maintain
PaCO2 at 30-35 Torr. To prevent
spontaneous breathing, the rabbit was paralyzed with pancuronium
bromide (0.25 mg/kg iv). Supplemental doses were given as needed. Body
temperature was maintained at 37-38°C by a heating pad.
Polyethylene catheters were inserted into a femoral artery and a vein
for the measurement of, respectively, arterial pressure, blood gases,
and administration of drugs. Lactated Ringer solution was infused (3 ml · kg1 · h1)
through the venous line to avoid dehydration. Any metabolic acidosis
was corrected by and intravenous infusion of appropriate amounts (0.2 × body wt × base excess meq) of 1 M
NaHCO3.
Both carotid sinus regions were exposed gently. The vessels of one carotid sinus (randomly assigned) were left intact (intact preparation, perfused with animal's own arterial blood), and the CSN was exposed for recording chemoreceptor afferent activity (see Recording Techniques). An ultrasonic probe attached to a flowmeter (T106, Transonic Systems) was used to measure the blood flow of the common carotid artery, which supplied the carotid sinus and CB. After the protocol for afferent recordings was completed on the intact side, the opposite sinus region was vascularly isolated and perfused with Krebs-Henseleit solution (in mM: 120 NaCl, 4.8 KCl, 2.0 CaCl2, 2.5 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 5.5 dextrose). Briefly, the internal carotid artery and all branches of external carotid artery were ligated, and the common carotid and external maxillary arteries were cannulated to allow flow-through perfusion of the carotid sinus region with the buffer solution. Perfusate was bubbled with O2-CO2-N2 gas mixture at proper proportion (see Construction of Stimulus-Response Curves) to maintain PO2 at 100-110 Torr, PCO2 at 30-35 Torr, and pH at 7.35-7.45 as the normoxic condition. The temperature of the perfusate was maintained at 36-38°C by using a water bath. Pressure in the carotid sinus was maintained at 80 mmHg by adjusting the height of the perfusion reservoir, and the flow was kept constant (10 ml/min) by adjusting the outlet of the effluent cannula.
Autonomic innervation of both carotid sinus regions was eliminated by stripping all visible neural connections between the carotid sinus and the superior cervical and nodose ganglia. The CSN was totally transected near the petrosal ganglion, not only to facilitate dissection of afferent fibers but also to interrupt neural efferents to the CB.
Recording Techniques
The CSN was covered with mineral oil and fine slips of nerve filaments placed on a silver electrode. Impulses was amplified with a band width of 100 Hz-3 kHz (Grass P511), displayed on an oscilloscope (Gould 450) and fed into a rate meter (Frederick Haer), window discriminators of which were set to accept potentials of a particular amplitude. Impulses were counted by the ratemeter in 1-s bins. The action potentials and ratemeter signals were fed into an analog-to-digital converter (MacLab) attached to a Macintosh computer. Bundles that had one or, at most, two easily distinguishable active fibers were used. Chemoreceptor afferents were identified by their sparse and irregular discharge at normoxia and by their response to hypoxia.In Vitro NO Assay
NOS activity in the CB was estimated by measuring the rate of NO release from the CB with a chemiluminescence NO analyzer (Sievers 208). At the end of afferent recording experiments, the blood-perfused sinus was removed, and the CB was dissected within ice-cold Krebs-Henseleit solution bubbled with 100% O2. The CB was then allowed to equilibrate at room temperature for 30 min in 30 ml of Krebs-Henseleit solution containing 1 mM of L-arginine and bubbled with 100% O2. After this incubation, the CB was removed and placed into a small incubation vial containing 0.5 ml of Krebs-Henseleit solution with 1 mM of L-arginine at 90-95 Torr PO2/30-40 Torr PCO2 (normoxia) and allowed to incubate at 37-38°C for 30 min. At the end of the incubation period, the CB was removed, and the incubation vial was sealed immediately and stored in a freezer (
20°C) for later
measurement of nitrite (NO2) and
nitrate (NO3) concentration by the
chemiluminescence analyzer. The analyzer was calibrated by using
standard solutions of NaNO2. This
system regenerates all of the NO oxidized to
NO2 or
NO3. To control for background
NO2 and
NO3 signals, aliquots from
incubation vials with Krebs-Henseleit solution without CB were assayed
in parallel with those from vials containing CB, and the background
level was subtracted from assay measurements. The CB was weighed at the
end of the experiment, and NO release was expressed as picamoles per
milligram wet weight per 30 min. NO release from the common carotid
artery (CA) and the superior cervical ganglion (SCG) were also assayed
for comparison.
Histochemical Studies
The localization of NOS in the CB of sham and CHF rabbits was examined by using NADPH-diaphorase histochemistry, as described previously (27). At the end of the afferent-recording experiments, the blood-perfused carotid sinus was vascularly isolated and perfused with Krebs-Henseleit solution until free of blood. Then the carotid sinus was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 20 min at room temperature. The sinus was then infiltrated with 30% sucrose in 0.1 M phosphate buffer and stored frozen at70°C until sectioned.
Cryostat sections (15 µm) were cut and incubated in 0.1 M phosphate
buffer, pH 7.4, containing 0.3% Triton X-100, 0.1 mg/ml nitroblue
tetrazolium, and 1.0 mg/ml 
-NADPH at 37°C for 60 min. After the
reaction, the sections were rinsed in phosphate buffer, pH 7.4, and
mounted onto chrome-alum-coated slides. The slides were air-dried, then
rinsed in distilled water, and dried again. Coverslips were then
mounted directly with Entellan. As a control, some sections were
incubated with the oxidized form of NADP along with nitroblue
tetrazolium. Sections were examined under a microscope, and the density
of the varicosities of nerve fibers positive for NADPH-diaphorase was
counted within unit area (0.5 mm2). Five separate areas within
each section were counted, and the values were averaged for the
section. Three to five sections were counted from each CB. Values from
sections of the same CB were averaged again as the value of the density
of nerve fibers within this CB.
Protocols
Discharge characteristics of the carotid body chemoreceptors in sham and CHF rabbits. The stimulus-response curves of single-unit activity of CB chemoreceptors to alterations in PO2 were examined in eight sham and eight CHF rabbits. In the intact preparation, PaO2 was altered by ventilating the rabbits with 20% O2-80% N2, 15% O2-85% N2, 10% O2-90% N2, and 100% O2 sequentially. Each stimulus lasted until a steady response was reached (40-60 s). An arterial blood sample was obtained at the end of each stimulus for blood-gas measurement. Stimuli were separated by at least 10 min to allow complete recovery of the fiber. PaCO2 was controlled by adjusting the tidal volume, and pH was adjusted by intravenous NaHCO3. In the vascularly isolated preparation, PO2 was altered by bubbling the perfusate with gas mixtures of different O2 concentrations (50, 10, 5, 0%) to achieve a PO2 of 300-400, 100-110, 60-70, and 40-50 Torr, respectively. The gas mixtures contained a constant fraction of 5% CO2 and a balance of N2. pH was adjusted by adding NaHCO3 or HCl into the perfusate. The afferent responses of the CB chemoreceptors from the intact preparation and from the isolated preparation were compared between sham and CHF rabbits. The comparison allowed us to deduce whether changes in afferent sensitivity between sham and CHF rabbits were the consequence of either intrinsic differences within the CB (isolated sinus) or differences as a result of alterations in blood-borne substances that influenced chemoreceptor activity.Contribution of NO to the alteration of chemosensitive
characteristics of carotid body chemoreceptors in CHF rabbits.
GROUP A: EFFECT OF NOS INHIBITION IN THE INTACT PREPARATION.
The response of CB chemoreceptors to alterations in
PaO2 was examined before
and after treatment of the CB with a competetive NOS antagonist
N-nitro-L-arginine
(L-NNA) in both sham
(n = 7) and CHF
(n = 7) rabbits. After the control
response was obtained, a bolus injection of
L-NNA was given intravenously at
a dose of 30 mg/kg. Twenty minutes later, the response to the same
level of hypoxia was reexamined. Then a large dose (60 mg · kg1 · min1)
of L-arginine, the endogenous
substrate for NOS, was infused intravenously for 10 min. The response
to hypoxia was examined for the third time with the infusion of
L-arginine to determine whether
the effect of L-NNA was
reversible by its competitive agonist. Only the response to severe
hypoxia (PaO2 at 40-50 Torr) was
examined, since we had shown the graded characteristics of the CB
chemoreceptors from the previous protocol.
5-104
M) on the response of CB chemoreceptors to hypoxia either in sham or in
CHF rabbits. Because the doses of
L-NNA we used were high enough
to effectively block the NOS activity (17, 23), we speculated that the
ineffectiveness of L-NNA in the
isolated preparation may be due to absence of the substrate
(L-arginine) for NOS within the buffer
solution. Therefore, we switched the sequence of administration of
L-NNA and L-arginine in the
isolated preparation to examine the effect of
L-arginine on the response of CB
chemoreceptors in sham (n = 7) and CHF
(n = 7) rabbits. After the control
response was obtained,
L-arginine
(105 M) was added to the
perfusate, and the response to hypoxia
(PO2 at 40-50 Torr) was then
examined as explained above. We then added L-NNA
(103 M) to the
L-arginine-supplemented
perfusate to determine whether the effect of
L-arginine on the response of
the CB chemoreceptors could be blocked by its competitive antagonist.
GROUP C: EFFECT OF
N -NITRO-D-ARGININE
(D-NNA) AND
D-ARGININE IN SHAM RABBITS.
As a negative control, the effect of biologically inactive isomers of
L-NNA and
L-arginine, i.e.,
D-NNA and
D-arginine, on the response of
the CB chemoreceptors to isocapnic hypoxia
(PO2 at 40-50 Torr) was examined
in six sham rabbits. This was not done in CHF rabbits, because neither
L-NNA nor
L-arginine was found effective
in CHF rabbits (see RESULTS). The
effect of D-NNA was examined
only in the intact preparation, whereas the effect of
D-arginine was examined only in
the isolated preparation.
GROUP D: EFFECT OF EXOGENOUS NO ON THE RESPONSE OF CB
CHEMORECEPTORS.
To assess whether the effects of endogenous NO on the sensitivity of
the CB chemoreceptors were maximally expressed in each group of
animals, the effect of an NO donor,
S-nitroso-N-acetyl-penicillamine (SNAP), on the response of the CB chemoreceptors was examined in five
sham and six CHF rabbits. Unlike
L-arginine, which requires NOS
to produce NO, SNAP spontaneously releases NO under aqueous conditions
via homolytic cleavage of the S-N
bond. In the intact preparation, SNAP was infused intravenously at a
dose of 60 nmol · kg1 · min1
throughout the experiment. In the isolated preparation, SNAP was added
into the perfusate at a concentration of
104 M. The response of the
CB chemoreceptors to isocapnic hypoxia (PO2 at 40-50 Torr) was obtained
before and during administration of SNAP.
Data Analysis
Baseline discharge of the CB chemoreceptors and the discharge at different levels of PO2 were compared within group and between groups by using a two-way ANOVA for repeated measures. Post hoc comparison was done by using the Bonferroni procedure. To quantify the stimulus-response characteristics of the CB chemoreceptors, experimental data from each animal were also fitted to a hyperbolic function described previously (13) by using the following equation: y = a + b/(x c), where
y is the discharge frequency of the CB afferents and x is
PO2. The values
a, b,
and c are constant, a being the horizontal asymptote,
b a "shaping term," and
c the vertical asymptote. The values
of r2 were at
0.95-0.99 (P < 0.01) for all of
the curve fittings. The parameters derived from curve fittings and
those from NO measurement and histological study were compared between
sham and CHF rabbits by unpaired Student's
t-test. Statistical analyses were
performed by using commercial statistical software (GB-STAT, Dynamic
Microsystems). All data are expressed as means ± SE. Statistical
significance was accepted when P < 0.05.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
As described in our accompanying paper (26), paced rabbits exhibited overt evidence of heart failure, including an enlarged heart, an attenuated contractility, and an elevated central venous pressure. Out of a total of 61 rabbits (33 sham, 28 CHF) used in the present study, 46 rabbits (26 sham and 20 CHF) were also used in the reflex study reported in our accompanying paper. Because the alterations of hemodynamics and cardiac dimensions due to cardiac pacing in the present study were not significantly different from those described in the reflex study, the reader is referred to Tables 1 and 2 in our accompanying paper (26) for these data.
Discharge Characteristics of the CB Chemoreceptors in Sham and CHF Rabbits
Isocapnic hypoxia increased the discharge frequency of the CB chemoreceptors in both sham and CHF rabbits in a level-dependent manner (Fig. 1). The PCO2 and pH were maintained within the normal range during the hypoxic stimulation (Table 1). However, the baseline discharge in the normoxic state and the magnitude of the response to corresponding levels of hypoxia were greater in CHF than in sham rabbits (Fig. 2), suggesting an enhanced sensitivity of the CB chemoreceptors in CHF rabbits. These alterations were observed both in the intact and in the isolated CB preparations (Fig. 2). The discharge frequency of the CB chemoreceptors in the hyperoxic state (300-400 mmHg) was not different between sham and CHF rabbits in both preparations (Fig. 2). Comparisons of the parameters derived from the hyperbolic curve fitting showed that the shaping coefficient b was greater, whereas the vertical asymptote c was smaller in CHF than in sham rabbits (Table 2), again suggesting a left-upward shift of the PO2-discharge relationship or a sensitization of the CB chemoreceptors in CHF rabbits.
|
|
|
|
We found that blood flow decreased in the carotid artery during severe hypoxia (PaO2 at 40-50 Torr), when the afferent recording was performed in the intact preparation [sham: from 93 ± 19 ml/min (normoxia) to 75 ± 15 ml/min (hypoxia); CHF: from 91 ± 18 ml/min (normoxia) to 74 ± 17 ml/min (hypoxia)]. The magnitude of decrease in the carotid blood flow was not different between sham and CHF rabbits. Mean arterial blood pressure did not change significantly [sham: 75 ± 2 mmHg (normoxia) vs. 76 ± 2 mmHg (hypoxia), P > 0.05; CHF: 69 ± 2 mmHg (normoxia) vs. 70 ± 4 mmHg (hypoxia), P > 0.05] during the same interval. Neither carotid artery blood flow nor blood pressure changed in either group during mild hypoxia (PaO2 at 60-70 Torr).
Contribution of NO to the Alteration of Chemosensitive Characteristics of the CB Chemoreceptors in CHF Rabbits
Group A. In the intact preparation, administration of the NOS inhibitor L-NNA (30 mg/kg) increased the baseline discharge in the normoxic state and the response to hypoxia in sham, but not in CHF, rabbits (Fig. 3). The effect of L-NNA was reversed by the NOS substrate L-arginine (60 mg · kg1 · min1)
(Fig. 3).
|
The administration of L-NNA elevated arterial blood pressure in both groups. The elevation was 27 ± 2 mmHg (from 77 ± 5 to 104 ± 7 mmHg) in sham rabbits and 21 ± 1 mmHg (from 70 ± 5 to 92 ± 6 mmHg) in CHF rabbits. To assess the possibility that the elevation of the blood pressure contributed to the effect of L-NNA on the CB chemoreceptors, we increased blood pressure to an equivalent level by mechanical occlusion of the descending aorta in two sham and two CHF rabbits. One fiber was studied in each rabbit. It was found that the equivalent elevation of blood pressure induced mechanically did not increase the baseline discharge nor the response to hypoxia in these rabbits. Therefore, it seems unlikely that the difference in the effect of L-NNA on the chemoreceptor activity between sham and CHF rabbits was due to the changes in the arterial blood pressure.
Group B. In the isolated preparation,
addition of L-NNA
(105 and
104 M) to the buffer
solution did not affect the baseline discharge or the response to
hypoxia in either sham or CHF rabbits (data not shown). However, when
L-arginine
(104 M) alone was added
into the perfusate, the baseline discharge and the responses to hypoxia
were inhibited in sham but not in CHF rabbits (Fig.
4). The inhibitory effect of
L-arginine on the activity of
the CB chemoreceptors was reversed by the subsequent administration of
L-NNA (10 mM) (Fig. 4).
|
Group C. D-NNA and
D-arginine, when given in the
same doses as the L-isomers, did
not affect the activity of the CB chemoreceptors either in the intact
or in the isolated preparation in sham rabbits (Fig
5).
|
Group D. In the intact preparation,
administration of the NO donor SNAP (60 nmol · kg1 · min1
iv) inhibited the baseline discharge and the response to hypoxia in CHF
but not in sham rabbits (Fig. 6). In the
isolated preparation, SNAP
(104 M) inhibited the
baseline discharge and the response to hypoxia in both sham and CHF
rabbits (Fig. 7). Administration of SNAP in
the intact preparation decreased arterial blood pressure from 78 ± 3 to 68 ± 4 mmHg (P < 0.01) in
sham rabbits and from 71 ± 3 to 58 ± 2 mmHg in CHF rabbits
(P < 0.01). To assess the
possibility that the decrease in blood pressure might contribute to the
effect of SNAP on the CB chemoreceptors, experiments were conducted in one fiber from a sham and one fiber from a CHF rabbit, with the blood
pressure maintained at the equivalent level induced by SNAP by partial
occlusion of the inferior vena cava. It was found that an equivalent
decrease in blood pressure caused by the vena cava occlusion did not
alter the characteristics of either chemoreceptor in response to
hypoxia.
|
|
In Vitro NO Production from the Carotid Bodies of Sham and CHF Rabbits
Basal NO production from the CB, CA, and SCG in the normoxic state was measured in vitro in six sham and eight CHF rabbits. It was found that the CBs from CHF rabbits produced markedly less NO than those from sham rabbits (Table 3). The NO production from the CA was also less in CHF than in sham rabbits (Table 3). Minimal NO was produced from the SCG, and no difference was found between CHF and sham rabbits (Table 3).
|
Histochemical Staining of NADPH-diaphorase in the CB from Sham and CHF Rabbits
NADPH-diaphorase histochemistry was carried out in the CB from seven sham and nine CHF rabbits. The histochemistry showed that the density of NADPH-diaphorase-positive sites within the CB from CHF rabbits was significantly less than that from sham rabbits (21 ± 5 vs. 34 ± 5 sites/0.5 mm2, P < 0.05). Figure 8 illustrates the representative results from a CB of a sham rabbit and a CB of a CHF rabbit. No positive staining was found when the section was incubated with the oxidative form of NADP.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The major findings of the present study were as follows. 1) The baseline discharge in the normoxic state and the response to isocapnic hypoxia of the CB chemoreceptors were enhanced in CHF rabbits compared with sham rabbits. These alterations were independent of the CB preparations (intact vs. isolated). 2) NOS inhibition enhanced the sensitivity of CB chemoreceptors in sham but not in CHF rabbits. 3) Administration of NOS substrate, L-arginine, decreased the sensitivity of CB chemoreceptors in sham but not in CHF rabbits. 4) Increase in NO production independently of NOS activity by administration of SNAP inhibited the sensitivity of CB chemoreceptors in CHF rabbits. 5) The in vitro basal NO production from the CB and the varicosities of nerve fibers positive for NADPH-diaphorase within the CB were less in CHF rabbits than in sham rabbits. From these results, we conclude that input from CB chemoreceptors is enhanced in the rabbit model of pacing-induced CHF and that an attenuated NOS activity in the CB contributes to the enhanced activity of the CB chemoreceptors in this condition.
Previous studies have documented that an enhanced peripheral chemoreflex is a common finding in patients with increasing severity of heart failure (4-6). In our accompanying paper (26), we have reported similar results documenting that peripheral chemoreflex control of RSNA and ventilation is enhanced in conscious rabbits with pacing-induced CHF. The important question that arises from these studies is defining the mechanism(s) responsible for this altered chemoreflex function. Although we cannot tell from the present study whether the central gain of the peripheral chemoreflex is altered, our findings provide direct evidence for an enhanced afferent input from the CB chemoreceptors in the CHF state, which would provide a primary contribution to the augmentation of reflex function. Furthermore, the fact that the enhanced afferent sensitivity of the CB chemoreceptors is independent of the preparations (intact vs. isolated) suggests that an intrinsic alteration occurs within the CB in the CHF state.
Although there are numerous factors that may affect peripheral chemoreceptor function (1, 9), we were interested in the possible role that NO plays in this alteration. NO is of importance for several reasons. 1) NOS activity has been detected in the CB of rats (29), cats (11), and rabbits (19). 2) NO inhibits the activity of CB chemoreceptors in normal animals (19, 28). 3) NOS activity or NO production is decreased in the CHF state (16, 24, 30). These data imply that a reduction of NO release in the CHF state may lead to a disinhibition of the CB chemoreceptors, which could account for the enhanced afferent sensitivity of the CB chemoreceptors in the CHF condition.
The results of the present study provide direct evidence for the above hypothesis. We found that basal NO production from the CB and the density of NADPH-diaphorase-positive cells (a maker of NOS) (2, 27) within the CB were less in CHF rabbits than in sham rabbits. These results suggest an attenuated NOS activity and a lower content of NOS within the CB in CHF rabbits. Moreover, we found in the intact preparation that inhibition of NOS activity by administration of L-NNA enhanced the activity of the CB chemoreceptors in sham but not in CHF rabbits and that the administration of the NO donor SNAP inhibited the chemoreceptor activity in CHF but not in sham rabbits. These results indicate that a tonic inhibitory effect of NO on the activity of the CB chemoreceptors is fully expressed in the CB in sham rabbits but virtually absent in the CB in CHF rabbits. A possible nonspecific effect produced by L-NNA is unlikely, since the fact that the effect of L-NNA could be reversed by L-arginine and D-NNA was without affect. These data strongly support the idea that an attenuated NOS activity in the CB of CHF rabbits, rather than an inability to respond to NO, contributes to the enhanced activity of the CB chemoreceptors in this condition.
An augmented activity of the CB chemoreceptors was also identified in the isolated preparation in CHF rabbits. Unlike in the intact preparation, NOS inhibition produced by L-NNA (alone) did not affect the activity of the CB chemoreceptors in either sham or CHF rabbits. However, administration of the NOS substrate L-arginine (alone) to the isolated preparation attenuated the afferent activity of the CB chemoreceptors in sham but not in CHF rabbits. This result suggests that NO production in the isolated CB under our conditions was substrate limited in the sham group. By contrast, we found that SNAP (which produces NO independently of NOS activity) produced a similar inhibitory effect in the isolated preparation of both groups. This result further suggests that the enhanced sensitivity of the CB chemoreceptors in the isolated preparation from CHF animals could be linked to an attenuated NOS activity. These data support our hypothesis that the downregulation of NOS activity or a decrease in NO release in the carotid sinus region plays an important role in the alteration of the characteristics of the CB chemoreceptors in CHF rabbits.
Three isomers of NOS have been found, i.e., neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) (3). From the data presented here it is not possible to determine which isomer is altered in the CB of rabbits with CHF. However, because NADPH-diaphorase histochemistry is relatively specific for the neuronal isomer (2), we believe that a downregulation of nNOS in the CB cells contributes to the reduced capacity to synthesize NO in the CHF state. The results from previous studies (19, 28, 29) indicate that NADPH-diaphorase-containing elements within the CB are mainly composed of nerve fibers and scattered neurons. We found that the density of NADPH-diaphorase-positive fibers is decreased in the CB in CHF rabbits.
However, a participation of eNOS in the alteration of the characteristics of the CB chemoreceptors in CHF rabbits cannot be excluded. A recent study supports that eNOS is more important than nNOS in the regulation of respiration in response to the stimulation of peripheral chemoreceptors in the normal condition (12). Because it is known that eNOS exists in the endothelium of the CB vasculature (19) and that eNOS activity is decreased in the CHF state (16, 24), it is reasonable to speculate that downregulation of NOS activity during CHF may result in a vasoconstriction within the CB and, therefore, lead to a decrease in the blood supply to the CB. The decreased blood supply may, in turn, cause the activation of the CB chemoreceptors.
It is not clear how NO alters O2 sensitivity in the CB. The activation of chemoreceptors by hypoxia has been postulated to be due to decreased availability of an inhibitory chemical messenger (19, 20). It has been documented that 1) many nerve plexuses innervating the chemoreceptor tissue were positive for NOS (19); 2) inhibition of NOS activity augments CB chemoreceptor activity, indicating a tonic inhibitory effect of endogenous NO on the chemoreceptor activity (19, 28); and 3) the NOS activity is less at low PO2 compared with normoxic controls (21). Therefore, it is possible that low sensory activity during normoxia is due to a constant inhibitory action of NO, whereas increased sensory activity under hypoxia is due to disinhibition resulting from reduced production of NO. Because NOS activity or NO synthesis is attenuated in the CHF state (16, 24, 30, present study), less inhibition produced by NO and, therefore, an enhanced activity of the chemoreceptors should be expected in this condition.
Although one would expect little NO released from the CB in the isolated preparation from both sham and CHF rabbits because of the substrate limitation of the perfusate, an enhanced activity of the CB chemoreceptors (compared with shams) was still observed in the isolated preparation in CHF rabbits. This implies that, although NO is important in the alteration of the characteristics of the CB chemoreceptors in the CHF state, other factors besides NO may also play a role in this pathophysiological process. An alternative explanation is that the enhanced activity of the CB chemoreceptors results from an intrinsic alteration in the CB due to chronic downregulation of NO synthesis during the development of CHF, which cannot be mimicked by the acute depletion of NO as occurred during the preparation for the isolated carotid sinus.
We found that there was a reduction of blood flow in the carotid artery during severe hypoxia (PaO2 at 40-45 Torr) in both sham and CHF rabbits, when the afferent recording was performed in the intact preparation. Because the blood pressure did not change significantly during the same interval, the reduction of carotid blood flow was probably due to a vasoconstriction, which may have resulted from a sympathoadrenal activation in response to hypoxia and/or an attenuated cardiac function because of hypoxia. It is open to question whether the reduction of blood flow affected the sensitivity of the CB chemoreceptors to hypoxia in this instance. However, it seems unlikely that the reduction of blood flow to the carotid sinus area contributed to the difference in the sensitivity of the CB chemoreceptors between sham and CHF rabbits because the degree of reduction of blood flow was equivalent in both groups. Moreover, the enhanced sensitivity of the CB chemoreceptors to hypoxia in CHF was also observed in the isolated preparation where the flow was held constant.
Limitations
First, the present study was carried out in the anesthetized state. The anesthesia and the surgical procedure could directly or indirectly affect the characteristics of the CB chemoreceptor in response to hypoxia. Therefore, care should be taken when extrapolating the data obtained here to the clinical phenomena observed in patients. Second, we did not measure the conduction velocity of the afferent fibers of the CB. Therefore, it is not known what type of afferents from the CB was recruited in the present study. Third, a nonspecific NOS inhibitor L-NNA was employed in the present study. From the data presented here, it is impossible to determine which isomer is responsible for the alteration of the characteristics of the CB chemoreceptors in the CHF state. Further study is needed to evaluate the relative role of eNOS and nNOS.Significance
To our knowledge, this is the first time that the characteristics of the CB chemoreceptors have been examined in an experimental model of CHF by directly recording the single-unit activity from the CB afferents. The data shown in the present study provide direct evidence for an enhanced afferent input from the CB chemoreceptors in CHF rabbits, which suggests a possible mechanism for the augmentation of the peripheral chemoreflex function in the CHF state. Furthermore, our findings indicate that an attenuated NOS activity in the CB contributes to the enhanced activity of the CB chemoreceptors in this condition.| |
ACKNOWLEDGEMENTS |
|---|
The authors thank Dr. Kaushik P. Patel and Navasha McGlothan for generous help in the histochemical study and Johnnie F. Hackley, Pamela Curry, Phyllis Anding, and Lei Wang for excellent technical assistance.
| |
FOOTNOTES |
|---|
This study was supported by grants from the Nebraska Affiliate of the American Heart Association (Grant 9704521S) and by the National Heart, Lung, and Blood Institute Grants HL-52190, HL-54586, and HL-38690.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. D. Schultz, Dept. of Physiology and Biophysics, Univ. of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: hschultz{at}unmc.edu).
Received 30 September 1998; accepted in final form 8 December 1998.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Bee, D.,
and
P. Howard.
The carotid body: a review of its anatomy, physiology and clinical importance.
Monaldi Arch. Chest Dis.
48:
48-53,
1993[Medline].
2.
Beesley, J. E.
Histochemical methods for detecting nitric oxide synthase.
Histochem. J.
27:
757-769,
1995[Medline].
3.
Billiar, T. R.
Nitric oxide: novel biology with clinical relevance.
Ann. Surg.
221:
339-349,
1995[Medline].
4.
Chua, T. P., A. L. Clark, A. A. Amadi, and A. J. Coats. Increased chemoreceptor
sensitivity: a contributory cause of dyspnea in chronic heart failure
(Abstract). J. Am. Coll. Cardiol.
(special issue): 265A, 1995.
5.
Chua, T. P.,
A. L. Clark,
A. A. Amadi,
and
A. J. Coats.
Relation between chemosensitivity and the ventilatory response to exercise in chronic heart failure.
J. Am. Coll. Cardiol.
27:
650-657,
1996[Abstract].
6.
Chua, T. P.,
P. Ponikowski,
K. Webb-Peploe,
D. Harrington,
S. D. Anker,
M. Piepoli,
and
A. J. Coats.
Clinical characteristics of chronic heart failure patients with an augmented peripheral chemoreflex.
Eur. Heart J.
18:
480-486,
1997
7.
Chugh, S. S.,
T. P. Chua,
and
A. J. Coats.
Peripheral chemoreflex in chronic heart failure: friend and foe.
Am. Heart J.
132:
900-904,
1996[Medline].
8.
Esler, M.,
D. Kaye,
G. Lambert,
D. Esler,
and
G. Jennings.
Adrenergic nervous system in heart failure.
Am. J. Cardiol.
80:
7L-14L,
1997[Medline].
9.
Finley, J. C.,
J. Polak,
and
D. M. Katz.
Transmitter diversity in carotid body afferent neurons: dopaminergic and peptidergic phenotypes.
Neuroscience
51:
973-987,
1992[Medline].
10.
Floras, J. S. Clinical aspects of sympathetic
activation and parasympathetic withdrawal in heart failure.
J. Am. Coll. Cardiol. 22, Suppl. A: 72A-84A, 1993.
11.
Grimes, P. A.,
S. Lahiri,
R. Stone,
A. Mokashi,
and
D. Chug.
Nitric oxide synthase occurs in neurons and nerve fibers of the carotid body.
In: Arterial Chemoreceptors: Cell to System, edited by R. G. O'Regan,
D. S. McQueen,
and D.-J. Paterson. New York: Plenum, 1994, p. 221-224.
12.
Gozal, D.,
E. Gozal,
Y. M. Gozal,
and
J. E. Torres.
Nitric oxide synthase isoforms and peripheral chemoreceptor stimulation in conscious rats.
Neuroreport
7:
1145-1148,
1996[Medline].
13.
Kumar, P.,
and
M. A. Hanson.
Re-setting of the hypoxic sensitivity of aortic chemoreceptors in the new-born lamb.
J. Dev. Physiol. (Eynsham)
11:
199-206,
1989[Medline].
14.
Kummer, W.,
and
J. O. Habeck.
Substance P- and calcitonin gene-related peptide-like immunoreactivities in the human carotid body studied at light and electron microscopical level.
Brain Res.
554:
286-292,
1991[Medline].
15.
Mark, A. L. Sympathetic dysregulation in heart
failure: mechanisms and therapy. Clin.
Cardiol. 18, Suppl. I:
I-3-I-8, 1995.
16.
Mohri, M.,
K. Egashira,
T. Tagawa,
T. Kuga,
H. Tagawa,
Y. Harasawa,
H. Shimokawa,
and
A. Takeshita.
Basal release of nitric oxide is decreased in the coronary circulation in patients with heart failure.
Hypertension
30:
50-56,
1997
17.
Mülsch, A.,
and
R. Russe.
NG-nitro-L-arginine impairs endothelium-dependent dilatations by inhibiting cytosolic nitric oxide synthesis from L-arginine.
Naunyn Schmiedebergs Arch. Pharmacol.
341:
143-147,
1990[Medline].
18.
Murakami, H.,
J. L. Liu,
and
I. H. Zucker.
Blockade of AT1 receptors enhances baroreflex control of heart rate in conscious rabbits with heart failure.
Am. J. Physiol.
271 (Regulatory Integrative Comp. Physiol. 40):
R303-R309,
1996
19.
Prabhakar, N. R.,
G. K. Kumar,
C. H. Chang,
F. H. Agani,
and
M. A. Huxhiu.
Nitric oxide in the sensory function of the carotid body.
Brain Res.
625:
16-22,
1993[Medline].
20.
Prabhakar, N. R.
Significance of excitatory and inhibitory neurochemicals in hypoxic chemo-transmission of the carotid body.
In: Control of Breathing: Modelling and Perspectives, edited by Y. Honda,
and J. G. Widdicombe. New York: Plenum, 1993, p. 141-148.
21.
Prabhakar, N. R.
Neurotransmitters in the carotid body.
In: Arterial Chemoreceptors: Cell to System, edited by R. G. O'Regan,
D. S. McQueen,
and D.-J. Paterson. New York: Plenum, 1994, p. 57-69.
22.
Rector, T. S.,
M. T. Olivari,
T. B. Levine,
G. S. Francis,
and
J. N. Cohn.
Predicting survival for an individual with congestive heart failure using norepinephrine concentration.
Am. Heart J.
114:
148-152,
1987[Medline].
23.
Rees, D. D.,
R. M. J. Palmer,
R. Schulz,
H. F. Hodson,
and
S. Moncada.
Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo.
Br. J. Pharmacol.
101:
746-752,
1990[Medline].
24.
Smith, C. J.,
D. Sun,
C. Hoegler,
B. S. Roth,
X. Zhang,
G. Zhao,
X. B. Xu,
Y. Kobary,
K. Pritchard, Jr.,
W. C. Sessa,
and
T. H. Hintze.
Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure.
Circ. Res.
78:
58-65,
1996
25.
Sun, S.-Y., I. H. Zucker, and H. D. Schultz.
Enhanced chemoreflex function contributes to sympathetic
hyperactivity and baroreflex deterioration in heart failure rabbits
(Abstract). Circulation 96, Suppl. I: I-70, 1997.
26.
Sun, S.-Y.,
W. Wang,
I. H. Zucker,
and
H. D. Schultz.
Enhanced peripheral chemoreflex function in conscious rabbits with pacing-induced heart failure.
J. Appl. Physiol.
86:
1264-1272,
1999
27.
Vincent, S. R.,
and
H. Kimura.
Histochemical mapping of nitric oxide synthase in the rat brain.
Neuroscience
46:
755-784,
1992[Medline].
28.
Wang, Z.-Z.,
D. S. Bredt,
S. H. Snyder,
S. J. Fidone,
and
L. J. Stensass.
Nitric oxide synthase in the carotid body.
Soc. Neurosci. Abstr.
18:
1197,
1992.
29.
Wang, Z.-Z.,
D. S. Bredt,
S. J. Fidone,
and
L. J. Stensass.
Neurons synthesizing nitric oxide innervate the mammalian carotid body.
J. Comp. Neurol.
336:
419-432,
1993[Medline].
30.
Zhang, K.,
I. H. Zucker,
and
K. P. Patel.
Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure.
Brain Res.
786:
219-225,
1998[Medline].
31.
Zucker, I. H.,
W. Wang,
M. Brandle,
and
H. D. Schultz.
Baroreflex and cardiac reflex control of the circulation in pacing-induced heart failure.
In: Pathophysiology of Tachycardia-Induced Heart Failure, edited by F. G. Spinale. Mount Kisco, NY: Futura, 1996, p. 193-226.
32.
Zucker, I. H.,
W. Wang,
M. Brandle,
H. D. Schultz,
and
K. P. Patel.
Neural regulation of sympathetic nerve activity in heart failure.
Prog. Cardiovasc. Dis.
37:
397-414,
1995[Medline].
This article has been cited by other articles:
|
|
 |
|
  Y. Ding, Y.-L. Li, and H. D. Schultz Downregulation of carbon monoxide as well as nitric oxide contributes to peripheral chemoreflex hypersensitivity in heart failure rabbits J Appl Physiol, July 1, 2008; 105(1): 14 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kurzyna and A. Torbicki Neurohormonal modulation in right ventricular failure Eur. Heart J. Suppl., December 1, 2007; 9(suppl_H): H35 - H40. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Hansen, G. Ulubay, B. F. Chow, X.-G. Sun, and K. Wasserman Mixed-Expired and End-Tidal CO2 Distinguish Between Ventilation and Perfusion Defects During Exercise Testing in Patients With Lung and Heart Diseases Chest, September 1, 2007; 132(3): 977 - 983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Li, L. Gao, I. H. Zucker, and H. D. Schultz NADPH oxidase-derived superoxide anion mediates angiotensin II-enhanced carotid body chemoreceptor sensitivity in heart failure rabbits Cardiovasc Res, August 1, 2007; 75(3): 546 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Di Vanna, A. M. F. W. Braga, M. C. Laterza, L. M. Ueno, M. U. P. B. Rondon, A. C. P. Barretto, H. R. Middlekauff, and C. E. Negrao Blunted muscle vasodilatation during chemoreceptor stimulation in patients with heart failure Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H846 - H852. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. D. Schultz, Y. L. Li, and Y. Ding Arterial Chemoreceptors and Sympathetic Nerve Activity: Implications for Hypertension and Heart Failure Hypertension, July 1, 2007; 50(1): 6 - 13. [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Stickland, J. D. Miller, C. A. Smith, and J. A. Dempsey Carotid Chemoreceptor Modulation of Regional Blood Flow Distribution During Exercise in Health and Chronic Heart Failure Circ. Res., May 11, 2007; 100(9): 1371 - 1378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Gao, Y.-X. Pan, W.-Z. Wang, Y.-L. Li, H. D. Schultz, I. H. Zucker, and W. Wang Cardiac sympathetic afferent stimulation augments the arterial chemoreceptor reflex in anesthetized rats J Appl Physiol, January 1, 2007; 102(1): 37 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Weiss, M. D. Y. Liu, and J. Huang Sleep Apnoea & Hypertension: Physiological bases for a causal relation: Physiological basis for a causal relationship of obstructive sleep apnoea to hypertens |
P < 0.05, hypoxia vs.
normoxia.
