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(Circulation Research. 1996;79:802-811.)
© 1996 American Heart Association, Inc.

Articles

Oxygen-Derived Free Radicals Contribute to Baroreceptor Dysfunction in Atherosclerotic Rabbits

Zhi Li, Hui Z. Mao, Francois M. Abboud, Mark W. Chapleau

the Cardiovascular Center and the Departments of Internal Medicine (Z.L., H.Z.M., F.M.A., M.W.C.) and of Physiology and Biophysics (F.M.A.), University of Iowa College of Medicine, and the Department of Veterans Affairs Medical Center (M.W.C.), Iowa City, Iowa.

Correspondence to Mark W. Chapleau, PhD, Department of Internal Medicine, University of Iowa College of Medicine, 200 Hawkins Dr, Iowa City, IA 52242.

	Abstract

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The goal of the present study was to determine whether oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerosis. Baroreceptor activity was measured from the carotid sinus nerve during pressure ramps in isolated carotid sinuses of anesthetized rabbits. Rabbits fed a 0.5% to 1.0% cholesterol diet for 7.9±0.4 months (mean±SE; range, 5.5 to 10) developed atherosclerotic lesions in the carotid sinuses. Maximum baroreceptor activity measured at 140 mm Hg and the slope of the pressure-activity curve were reduced in atherosclerotic (n=15) compared with normal (n=13) rabbits (425±34 versus 721±30 spikes per second and 6.2±0.6 versus 10.8±0.8 spikes per second per mm Hg, respectively, P<.05). The level of activity was inversely related to plasma cholesterol concentration (r=.86, P<.001) and total cholesterol load (plasma concentrationxduration of diet, r=.92). Mean arterial pressure was normal in both groups. Exposure of the carotid sinus to the free-radical scavengers superoxide dismutase (SOD) and catalase significantly increased maximum baroreceptor activity by 25±4% in atherosclerotic rabbits (n=6) but caused only small and irreversible changes in activity in normal rabbits (n=8). Catalase alone but not SOD also increased baroreceptor activity in atherosclerotic rabbits (n=7). Exposure of the carotid sinus of normal rabbits to exogenous free radicals generated from the reaction between xanthine and xanthine oxidase inhibited baroreceptor activity in a dose-dependent and reversible manner (n=8, P<.05). The inhibition of activity was attenuated by SOD and catalase but was not attenuated by the inhibitor of hydroxyl radical formation, deferoxamine. Neither restoration of baroreceptor activity in atherosclerotic rabbits by catalase nor inhibition of activity by xanthine/xanthine oxidase could be explained by changes in the carotid pressure-diameter relation or prostacyclin formation. These results indicate that oxidant stress inhibits baroreceptor activity and that endogenous oxyradicals produced in atherosclerotic carotid sinuses contribute to baroreceptor dysfunction.

Key Words: baroreceptors • atherosclerosis • oxygen-derived free radicals • carotid sinus • catalase • superoxide dismutase

	Introduction

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Arterial baroreceptors are mechanosensitive nerve endings located in the vessel wall of the aortic arch and carotid sinuses that are activated by vascular stretch during increases in arterial pressure.^{1 2} It has been demonstrated in animal models of atherosclerosis that there is decreased sensitivity of arterial baroreceptors³ and impaired baroreflex control of the circulation.^{4 5 6} The decreased baroreceptor sensitivity in atherosclerosis has generally been ascribed to increased vascular stiffness, degeneration of baroreceptor endings, and increased arterial pressure.^{3 4 5 6} Numerous studies have indicated that baroreceptor sensitivity is not only determined by the level of arterial pressure and vascular stretch but also modulated by various neurohumoral substances and paracrine factors.^{7 8 9 10 11} We reasoned that altered formation of the paracrine factors that modulate baroreceptor sensitivity might contribute to baroreceptor dysfunction in atherosclerosis.

Oxygen-derived free radicals (oxyradicals) are produced in atherosclerotic lesions and are thought to contribute to the pathogenesis of atherosclerosis.^{12 13} Atherosclerotic lesions are particularly prominent in the carotid sinuses in humans.¹⁴ We hypothesized that oxyradicals produced in the atherosclerotic carotid sinus might contribute to baroreceptor dysfunction in atherosclerosis. Two major groups of experiments were performed. First, we investigated whether scavenging of endogenous oxyradicals in the isolated carotid sinus by SOD and catalase influences baroreceptor activity in normal rabbits and in rabbits fed a high-cholesterol diet. SOD reduces the steady-state concentration of superoxide anion by catalyzing the conversion of this molecule to H₂O₂. This reaction occurs spontaneously and rapidly even without SOD. Therefore, addition of SOD reduces the superoxide anion concentration without necessarily influencing the concentration of H₂O₂. Catalase reduces the concentration of H₂O₂ by increasing its conversion to water. Second, we determined whether oxyradicals generated by the reaction of xanthine and XO¹⁵ influence baroreceptor activity in normal rabbits. Because oxyradicals may alter vascular tone^{16 17} and PGI₂ formation,^{18 19 20 21 22} we also determined whether the effects of oxyradicals and free-radical scavengers on baroreceptor activity were related to changes in vascular distensibility or PGI₂ formation.

	Materials and Methods

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New Zealand White rabbits of either sex were fed standard rabbit chow or chow supplemented with 0.5% (n=9) or 1.0% (n=3) cholesterol for 5.5 to 10 months (mean±SE, 7.9±0.4).²³ The cholesterol diet was prepared by dissolving the cholesterol in warm vegetable oil and then mixing it with standard rabbit chow. The rabbits were 3 to 13 months old at the time of study and within this age range are considered to be juveniles and young adults.²⁴ The life span of rabbits may exceed 84 months.²⁴ At the time of the experiments the animals were anesthetized with sodium pentobarbital (30 to 35 mg/kg body weight) slowly injected through an ear vein over 3 to 5 minutes. The trachea was cannulated for artificial ventilation with room air supplemented with O₂. The pH (7.3 to 7.4), PCO₂ (30 to 45 mm Hg), and PO₂ (>100 mm Hg) of the arterial blood were maintained by adjusting the ventilation and by intravenous administration of 5% NaHCO₃ when necessary. The femoral artery and vein were catheterized for measurement of arterial pressure and administration of anesthetic, respectively. A blood sample of 5 mL was withdrawn into a tube containing EDTA at the beginning of each experiment for measurement of plasma lipoprotein cholesterol and triglyceride.²⁵ Body temperature was maintained at 37°C with a heating pad.

Isolation of the Carotid Sinus
The carotid sinus was isolated as described previously.^{9 10 11} All visible branches of the common and external carotid arteries in the region of the carotid sinus were ligated, and catheters were gently placed in the common, external, and internal carotid arteries. The sinus was filled with K-H physiological saline of the following composition (in mmol/L): NaCl 118.0, KCl 4.7, NaHCO₃ 24.0, MgSO₄ 1.2, CaCl₂ 2.5, KH₂PO₄ 1.1, and glucose 10.0. The K-H solution was bubbled beforehand with a 95% O₂/5% CO₂ gas mixture to adjust the pH to 7.3 to 7.4, the PO₂ to 150 to 200 mm Hg, and the PCO₂ to 30 to 40 mm Hg and then kept in a sealed glass container in a 37°C water bath. The common carotid artery catheter was connected to a pressure bottle filled with K-H solution, and the pressure in the carotid sinus was controlled by regulating the air flow to the pressure bottle from a pressurized air source. Thus, the pressure in the carotid sinus was precisely controlled to the desired levels in the absence of flow and was measured with a transducer (Statham model P23 XL) connected to the external carotid artery catheter. The carotid sinuses were successfully isolated, as indicated by the absence of blood leaking into the sinus at 0 mm Hg intrasinus pressure and the absence of K-H solution leaking from the sinus when pressure was held at 60 mm Hg. The cervical sympathetic nerve was cut below the level of the sinus to avoid any possible influence of sympathetic activity on baroreceptor discharge.^{7 8} Decamethonium bromide (0.3 mg/kg body weight IV) was administered before recording nerve activity to eliminate skeletal muscle contraction. Supplemental doses of anesthetic and decamethonium bromide were given during the experiment as needed.

Measurement of Baroreceptor Nerve Activity
The carotid sinus nerve was carefully isolated and sectioned at its junction with the glossopharyngeal nerve. The nerve was desheathed, placed on a unipolar platinum electrode, and encased in silicone gel for stable contact with the recording electrode. The in situ carotid sinus preparation was immersed in warm (37°C) paraffin oil to prevent drying and to stabilize the temperature. Nerve activity was recorded with a high-impedance probe (model HIP511J, Grass Instrument Co) and a Grass band-pass amplifier (model P511J; bandwidth, 100 Hz to 3 kHz). The electroneurogram was displayed on a Tektronix dual-beam storage oscilloscope (model 5113), and signal output was made audible through a loudspeaker. The discharge frequency of the action potentials that exceeded a selected threshold voltage set just above that of electrical noise was counted by a nerve traffic analyzer (model 706C, Department of Bioengineering, University of Iowa, Iowa City). The nerve activity was continuously averaged electronically over 0.5- to 1.0-second time bins. In a few experiments the nerve traffic signal was full-wave rectified and quantified by voltage integration in addition to spike counting. Quantified nerve activity, the raw neurogram, and the carotid sinus and systemic arterial pressures were recorded continuously on an electrostatic recorder (model ES1000, Gould Inc).

Measurement of Carotid Pressure-Diameter Relation
To record the carotid vessel diameter, the carotid sinus and arteries adjacent to the sinus were viewed under magnification (x16) through a stereomicroscope (model M3C, Wild) and the image was projected on a video monitor (model BWM15, Javelin Electronics).^{11 26} The images were recorded on videotape with a camera (model JE2362, Javelin Electronics) and a videocassette recorder (model SLV-585HF, Sony). A digital readout of carotid sinus pressure on the Gould recorder was filmed simultaneously (camera model JE 7542B, Javelin Electronics) and projected on the same monitor and recorded on the same videotape as the carotid artery image with the use of a beam splitter (model MPS-50, Image Labs). The diameter of the carotid artery just caudal to the carotid bifurcation was measured at 20–mm Hg increments in pressure (0, 20, 40, ... 140 mm Hg) with a videomicrometer (model VIA-100, Boeckeler Instruments). This system was able to detect changes of 12 µm in diameter over an absolute range of 0 to 4 mm.

Protocols
Baroreceptor activity was recorded during slow ramp increases (2 to 4 mm Hg/s) in nonpulsatile carotid sinus pressure from 0 to 150 mm Hg. The rate of pressure rise (dP/dt) during the ramps was kept the same within each experiment. Pressure was held constant at 60 mm Hg when ramps were not being applied. The isolated carotid sinus was refilled with fresh, oxygenated K-H buffer approximately every 15 minutes throughout the experiment. Pressure ramps were applied once every 5 minutes during a 15- to 30-minute period to ensure a stable preparation before exposure of the carotid sinus to any pharmacological intervention. A preparation was judged to be stable by visual inspection of the recording of mean discharge frequency when no discernible changes (<5%) in the baroreceptor response to repeated pressure ramps were observed.

Carotid arteries were videotaped simultaneously with the measurement of nerve activity during pressure ramps for later measurement of vessel diameter. The stable PGI₂ metabolite 6-keto-PGF₁ was analyzed by radioimmunoassay from 200-µL samples of K-H buffer withdrawn from the isolated sinus through the internal carotid artery catheter.^{10 26 27}

Protocol 1: Influence of SOD and Catalase on Baroreceptor Activity in Normal (n=8) and Atherosclerotic (n=6) Rabbits
After stable baroreceptor responses to pressure ramps were obtained, SOD (300 U/mL) and catalase (1200 U/mL) dissolved in K-H buffer were injected via the common carotid artery catheter. The isolated carotid sinus was completely refilled with fresh solution, thus replacing the prior solution, which exited the carotid sinus via the internal and external carotid artery catheters. Pressure ramps were applied after 5, 10, and 15 minutes of exposure to SOD and catalase. SOD and catalase were then washed out of the sinus with fresh K-H buffer, and additional pressure ramps were applied in an attempt to reverse the effect of SOD and catalase.

Protocol 2: Influence of SOD or Catalase Alone on Baroreceptor Activity in Atherosclerotic Rabbits (n=7)
To determine whether responses to SOD plus catalase observed in protocol 1 were caused by SOD, catalase, or both, an additional set of experiments in atherosclerotic rabbits was performed. The baroreceptor responses to pressure ramps were determined before and after 5, 10, and 15 minutes of exposure of the sinus to SOD alone (300 U/mL), catalase alone (1200 U/mL), and a combination of SOD and catalase. The carotid sinus was routinely refilled with fresh K-H buffer, and pressure ramps were repeated before and after injection of either catalase alone or SOD plus catalase to demonstrate response recovery. Since SOD alone did not alter baroreceptor activity, administration of SOD alone was followed immediately by injection of catalase alone in one of the seven experiments.

Protocol 3: Influence of Exogenous Oxyradicals on Baroreceptor Activity in Normal Rabbits
Oxyradicals were generated by the chemical reaction between xanthine and XO.^{15 16} Xanthine and XO were mixed in K-H buffer immediately before injection into the isolated carotid sinus (n=8). The baroreceptor responses to pressure ramps were obtained before and after 5, 10, and 15 minutes of exposure of the sinus to xanthine (0.1 mmol/L) and XO (20, 40, and 60 mU/mL). The xanthine/XO was flushed from the sinus and replaced with fresh K-H buffer, and additional pressure ramps were applied at the same intervals in an attempt to reverse the effect of each concentration of xanthine/XO. To demonstrate that the response to xanthine/XO was mediated by free radicals, the effect of xanthine and the high concentration of XO (60 mU/mL) was determined again in the presence of SOD (300 U/mL) and catalase (1200 U/mL) in the same experiments. Recovery responses were measured after flushing the drugs from the carotid sinus and refilling it with fresh K-H buffer. In four of eight experiments baroreceptor responses were also measured before and after injection of xanthine (0.1 mmol/L) alone into the isolated sinus.

In a separate group of experiments the carotid sinus was pretreated with the iron chelator deferoxamine (1 mmol/L) before xanthine/XO (60 mU/mL) was added (n=5). The baroreceptor responses to pressure ramps were measured before and during exposure to deferoxamine alone, during exposure to the combination deferoxamine/xanthine/XO, and again after the drug solution was replaced with fresh K-H buffer (recovery).

Histological Examination of the Carotid Sinus
At the end of each experiment the carotid sinuses and the adjacent common, external, and internal carotid arteries were removed, flushed with K-H buffer, and submerged in 10% formaldehyde. Sections of the preserved vessels were obtained from the carotid sinuses and the common, external, and internal carotid arteries and were stained with either Verhoeff–van Gieson's stain or oil red O.²⁸

Data Analysis
Baroreceptor discharge frequency in spikes per second was measured directly from the electrostatic recording at 20–mm Hg pressure increments from 0 to 140 mm Hg (0, 20, 40, . . . 140 mm Hg). The slope of the pressure-activity curve within the linear portion (40 to 80 mm Hg) was obtained by linear regression.^{11 26} Differences in overall pressure-activity curves between normal and atherosclerotic rabbits were determined by ANOVA from data measured at eight pressure levels (0, 20, 40, . . . 140 mm Hg) followed by contrast testing.²⁹ In addition, baroreceptor activity was normalized as a percentage of the maximum nerve activity recorded during the pressure ramp under control conditions in each experiment. The effects of SOD and catalase, deferoxamine, and xanthine/XO on the normalized pressure–baroreceptor activity curve, the pressure-diameter curve, and PGI₂ formation were analyzed by ANOVA followed by contrast testing.²⁹ Probability values were corrected by the Bonferroni method for multiple comparisons. Vascular distensibility was calculated as the slope of the pressure-diameter relation with measurements obtained at 20, 40, 60, and 80 mm Hg, which represented the linear range. Differences in maximum baroreceptor activity measured at 140 mm Hg, baroreceptor slope, and vascular distensibility in normal compared with atherosclerotic rabbits were determined by unpaired t test.²⁹ The relations between the absolute level of maximum baroreceptor activity (in spikes per second) in individual experiments and plasma cholesterol concentration, duration of the cholesterol diet, and age of the rabbits were analyzed by linear regression and correlation analysis. Since baroreceptor activity could be measured only at the time of the experiment, data for these analyses were taken from all rabbits measured at different time intervals, with each data point representing the value measured in a single rabbit. Data are expressed as the mean±SE. Differences were considered significant when P<.05.

	Results

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Baseline Plasma Lipids and Arterial Pressures in Normal and Atherosclerotic Rabbits
There were significant increases in total plasma cholesterol, LDL cholesterol, and triglycerides and decreases in HDL cholesterol in rabbits fed the cholesterol diet compared with rabbits that were fed the normal diet (Table). The mean arterial pressure measured under anesthesia before isolation of the carotid sinus was not significantly different between normal and cholesterol-fed rabbits (Table).

View this table: [in this window] [in a new window]   Table 1. Baseline Measurements of Mean Arterial Pressure (MAP), Plasma Cholesterol (Chol) Level, and Lipoprotein Concentrations in Rabbits Fed Normal and High-Cholesterol Diets

Prominent atherosclerotic lesions characterized by localized intimal thickening and extrusion into the lumen were consistently present in the carotid sinuses and to a lesser extent in the adjacent carotid arteries in cholesterol-fed rabbits but were not present in rabbits that were fed the normal diet (Fig 1). Oil red O staining indicated extensive lipid infiltration into the intimal lesions in the cholesterol-fed rabbits.

View larger version (136K): [in this window] [in a new window]   Figure 1. Light photomicrographs of cross sections of carotid sinuses from a normal rabbit (left) and a rabbit fed the 0.5% cholesterol diet for 7 months (right). Vessel sections were stained with Verhoeff–van Gieson's method.28 Intimal proliferation is evident in the carotid sinus of the cholesterol-fed rabbit.

Baroreceptor Activity in Atherosclerotic and Normal Rabbits
When measured under identical experimental conditions, baroreceptor activity in the atherosclerotic rabbits was markedly reduced compared with that in normal rabbits (Fig 2, left). Baroreceptor activity measured at 140 mm Hg averaged 721±30 spikes per second in normal (n=13) and 425±34 spikes per second in atherosclerotic (n=15, P<.05) rabbits. The slope of the pressure–nerve activity relation was 10.8±0.8 and 6.2±0.6 spikes per second per mm Hg in normal and atherosclerotic rabbits, respectively (P<.05). In three atherosclerotic rabbits both carotid sinuses were isolated, and baroreceptor activity on both sides was reduced to a similar extent.

View larger version (15K): [in this window] [in a new window]   Figure 2. Left, Relation between carotid sinus pressure and the absolute level of baroreceptor activity in normal (n=13) and cholesterol-fed (n=15) rabbits. Right, The corresponding relation between carotid sinus pressure and carotid diameter in normal (n=8) and cholesterol-fed (n=10) rabbits. Baroreceptor (BR) activity was significantly less over the entire pressure range in the atherosclerotic rabbits despite a significantly larger carotid artery diameter and wall strain (*P<.05, ANOVA followed by contrast testing). Data are mean±SEM.

The marked reduction in baroreceptor activity in the atherosclerotic rabbits could not be explained by a reduction in vascular distensibility. Carotid diameter was significantly higher over a wide pressure range in atherosclerotic than in normal rabbits (Fig 2, right). The slope of the pressure-diameter relation (distensibility) was not significantly different in the two groups of rabbits (8.2±0.7 and 9.8±0.6 µm/mm Hg in normal and atherosclerotic rabbits, respectively).

The level of maximum baroreceptor activity measured in individual normal and atherosclerotic rabbits was inversely correlated with both plasma cholesterol concentration (r=.86, P<.001; Fig 3) and duration of cholesterol feeding (r=.84, P<.001). The correlation was improved by calculating the total cholesterol load as the plasma cholesterol concentration times the duration of cholesterol feeding (r=.92, P<.001; Fig 3). When the regression analysis was performed with data from the cholesterol-fed rabbits only, the inverse correlation between baroreceptor activity and cholesterol load remained significant (r=.60, P<.05). Baroreceptor activity was not correlated with the age of the rabbits (r=.13).

View larger version (14K): [in this window] [in a new window]   Figure 3. Relation between maximum baroreceptor activity measured at 140 mm Hg and plasma cholesterol concentration (left) and cholesterol concentrationxduration of cholesterol feeding (right). Each point represents data from individual rabbits fed the normal diet (n=5) and the high-cholesterol diet (n=12). In 3 of 12 atherosclerotic rabbits baroreceptor activity was recorded from both carotid sinus nerves and the results averaged. Lines were obtained by linear regression and correlation analysis. Baroreceptor activity was inversely and significantly correlated with plasma cholesterol concentration and with cholesterol concentrationxduration of cholesterol feeding. BR indicates baroreceptor; Chol, cholesterol.

Responses to SOD and Catalase in Normal and Atherosclerotic Rabbits
In normal rabbits exposure of the isolated carotid sinus to SOD and catalase slightly but significantly increased baroreceptor activity at pressures of 60 and 80 mm Hg, but this increase was not reversed after washout of the SOD and catalase (Fig 4, left). Furthermore, SOD and catalase did not alter either maximum baroreceptor activity or the slope of the pressure-activity curve in normal rabbits (Fig 4). In contrast, SOD and catalase significantly increased baroreceptor activity in atherosclerotic rabbits over a wide pressure range, with the most prominent increases at higher pressure levels (Fig 4, middle, and Fig 5). At 140 mm Hg the increase in activity averaged 25±4% in atherosclerotic rabbits, in contrast to the lack of effect of SOD and catalase in normal rabbits (0±1%, Fig 4). The increase in baroreceptor activity in atherosclerotic rabbits in response to SOD and catalase was (1) evident when activity was quantified as either spikes per second or as integrated voltage (Fig 5), (2) not accompanied by any change in the pressure-diameter relation (n=6; Fig 4, right), and (3) reversed after washout of SOD and catalase (maximum activity, 105±7% versus 125±4% during recovery and during SOD plus catalase, respectively).

View larger version (14K): [in this window] [in a new window]   Figure 4. Effects of SOD and catalase on carotid sinus pressure–normalized baroreceptor activity relation in normal (left, n=8) and atherosclerotic (middle, n=6) rabbits and the pressure-diameter relation in atherosclerotic rabbits (right, n=6). SOD and catalase caused a slight but significant increase in baroreceptor (BR) activity at low pressures but did not influence activity at high pressures in rabbits fed a normal diet. In contrast, SOD and catalase caused a significant increase in activity over a wide range of pressures in the cholesterol-fed rabbits in the absence of any change in the pressure-diameter relation. *Significant increase in baroreceptor activity (BR) compared with control (P<.05, ANOVA followed by contrast testing).

View larger version (14K): [in this window] [in a new window]   Figure 5. Original recordings obtained from an atherosclerotic rabbit that demonstrate the increase in baroreceptor activity measured in spikes per second and as integrated voltage in response to SOD and catalase injected into the isolated carotid sinus. Recordings were obtained before and after 15-minute exposure to SOD and catalase. CSP indicates carotid sinus pressure.

In a separate series of experiments (n=7) administration of SOD alone into the isolated carotid sinus failed to influence baroreceptor activity, whereas catalase alone significantly increased activity (Fig 6, P<.05). SOD and catalase in combination again increased baroreceptor activity, but this increase was not significantly greater than that observed with catalase alone (Fig 6). The increases in activity during catalase alone and SOD plus catalase administration were reversed after the carotid sinus was refilled with K-H buffer (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 6. A, Effects of SOD alone (left), catalase alone (middle), and SOD plus catalase (right) on the carotid sinus pressure–baroreceptor (BR) activity relation in atherosclerotic rabbits (n=7). B, Percentage changes in baroreceptor activity measured at 140 mm Hg that occurred in response to SOD, catalase, and the SOD/catalase combination. *Significant increase in BR activity compared with control (ANOVA followed by contrast testing, P<.05). The magnitude of increase in activity was similar with catalase alone and with SOD plus catalase.

To determine whether the influence of SOD and catalase on baroreceptor activity might be caused by increased formation of PGI₂, we measured the stable PGI₂ metabolite 6-keto-PGF₁ in samples of K-H buffer obtained from the isolated carotid sinus.^{10 26 27} SOD and catalase did not significantly alter the formation of PGI₂ in either atherosclerotic or normal rabbits. 6-Keto-PGF₁ levels averaged 3.9±0.7 and 4.7±0.7 ng/mL before and after SOD and catalase in atherosclerotic rabbits (n=13) and 2.1±0.4 and 2.7±0.5 ng/mL before and after SOD and catalase in normal rabbits (n=6). The basal level of 6-keto-PGF₁ was significantly higher in atherosclerotic rabbits than in normal rabbits.

Responses to Xanthine/XO in Normal Rabbits
Effect on Baroreceptor Activity
Injection of the combination of xanthine and XO into the isolated carotid sinus significantly decreased baroreceptor activity in a reversible and concentration-dependent manner (Figs 7 through 10). Suppression of baroreceptor activity was most prominent at higher pressure levels. Xanthine alone had no effect on baroreceptor activity (Figs 7 and 9). Suppression of baroreceptor activity by xanthine and XO was significantly attenuated in the presence of SOD and catalase (Figs 7, 8, and 10, right). Suppression of baroreceptor activity by xanthine/XO was not accompanied by any significant change in the pressure-diameter relation (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 7. Original recordings of baroreceptor activity measured at a constant carotid sinus pressure of 60 mm Hg from a rabbit fed a normal diet. Injection of xanthine alone (X, 0.1 mmol/L) into the isolated carotid sinus failed to decrease baroreceptor activity (upper left), whereas X and XO (60 mU/mL) decreased baroreceptor activity (upper right). Baroreceptor activity was restored after flushing X/XO from the sinus and replacing it with fresh K-H buffer (lower left). The inhibitory effect of X/XO was attenuated by exposure of the carotid sinus to SOD and catalase (lower right).

View larger version (18K): [in this window] [in a new window]   Figure 8. Original recordings from a rabbit fed a normal diet that demonstrate the effect of xanthine (X, 0.1 mmol/L) plus XO (60 mU/mL) on baroreceptor activity measured during the pressure ramp before and in the presence of SOD and catalase. The pressure ramp was essentially superimposable under various conditions and is shown only once for clarity. X/XO suppressed baroreceptor activity and decreased the slope of the pressure-activity relation, and these effects were blunted by SOD and catalase. CSP indicates carotid sinus pressure.

View larger version (16K): [in this window] [in a new window]   Figure 9. Effects of xanthine (0.1 mmol/L) and varying concentrations of XO (20, 40, and 60 mU/mL) or xanthine alone on baroreceptor (BR) activity measured at a carotid sinus pressure of 140 mm Hg. Control (C) and recovery (R) data are shown by open bars. Measurements were taken after a 15-minute exposure of the carotid sinus to the various agents. *Significant decrease in baroreceptor activity compared with control (ANOVA followed by contrast testing, P<.05). The number of experiments (n) in each group is shown in each bar.

View larger version (16K): [in this window] [in a new window]   Figure 10. Effects of xanthine (X, 0.1 mmol/L) and XO (60 mU/mL) on the baroreceptor pressure-activity relation of normal rabbits without (left, n=8) or with (right, n=7) SOD and catalase. X/XO decreased baroreceptor (BR) activity significantly at pressures 60 mm Hg. Inhibition of activity by X/XO was significantly attenuated by SOD and catalase and was no longer statistically significant at high carotid sinus pressures. *Significant decrease in activity during exposure of the carotid sinus to X/XO compared with control (ANOVA followed by contrast testing, P<.05).

We considered the possibility that hydroxyl radical might have mediated the inhibition of baroreceptor activity during exposure of the carotid sinus to xanthine/XO. To confirm or refute this possibility, deferoxamine was administered into the isolated carotid sinus to inhibit hydroxyl radical formation. Deferoxamine did not influence baroreceptor activity and did not attenuate the xanthine/XO–induced inhibition of activity (Fig 11). In fact, the inhibitory response to xanthine/XO was significantly greater in the presence of deferoxamine (Fig 11).

View larger version (22K): [in this window] [in a new window]   Figure 11. Effect of xanthine (X, 0.1 mmol/L) and XO (60 mU/mL) on the baroreceptor pressure-activity relation in carotid sinuses pretreated with deferoxamine (Def, 1 mmol/L; n=5). Deferoxamine alone did not alter baroreceptor (BR) activity. X/XO in the continued presence of deferoxamine significantly decreased activity (ANOVA followed by contrast testing, P<.05), and the inhibition of activity was reversed after removal of the drug solution and replacement with fresh K-H buffer (recovery).

Effect on PGI₂ Formation
We also considered the possibility that oxyradicals generated by xanthine/XO might have suppressed baroreceptor activity indirectly as a result of inhibition of PGI₂ formation.^{18 19 20} We have previously shown that endogenous prostanoids contribute to baroreceptor activity.^{9 10 26} Xanthine/XO did not inhibit, but instead significantly increased, PGI₂ formation in a concentration-dependent and reversible manner (Fig 12). Xanthine alone did not alter PGI₂ production (Fig 12). The increase in PGI₂ formation caused by xanthine/XO was prevented by SOD and catalase (Fig 12).

View larger version (15K): [in this window] [in a new window]   Figure 12. Levels of PGI2 measured as the stable breakdown product 6-keto-PGF1 in the buffer sampled from the isolated carotid sinus under various conditions. Xanthine (X, 0.1 mmol/L) plus XO (20, 40, or 60 mU/mL) increased PGI2 formation in a dose-dependent and reversible manner. Neither xanthine alone nor SOD plus catalase influenced PGI2 formation. With SOD and catalase, X/XO failed to increase PGI2 formation. *Significant increase compared with control (C) (paired t test, P<.05). The number of experiments (n) performed for each condition is shown in each bar. R indicates recovery.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of the study are the following: (1) Rabbits fed a high-cholesterol diet for 5.5 to 10 months develop atherosclerotic lesions in the carotid sinuses, which are associated with decreased carotid baroreceptor sensitivity. (2) The decrease in baroreceptor activity is inversely correlated with plasma cholesterol concentration and duration of cholesterol feeding. (3) Short-term exposure of the isolated carotid sinuses to the oxyradical scavengers SOD and catalase or catalase alone increases maximum baroreceptor activity in atherosclerotic but not in normal rabbits. (4) The increase in baroreceptor activity caused by oxyradical scavengers is not accompanied by a significant change in the carotid pressure-diameter relation or PGI₂ formation. (5) In normal rabbits exogenous oxyradicals generated by the chemical reaction between xanthine and XO inhibit baroreceptor activity in a dose-dependent and reversible manner.

Influence of Atherosclerosis on Baroreceptor Sensitivity
Previous studies have demonstrated that baroreflex control of sympathetic nerve activity, heart rate, and vascular resistance is impaired in animal models of atherosclerosis.^{4 5 6} Angell-James³ demonstrated decreased aortic baroreceptor sensitivity in rabbits fed a high-cholesterol diet and attributed the impaired baroreceptor function to hypertension, decreased vascular distensibility, and degeneration of baroreceptor nerve terminals. In the present study the mean arterial pressure was similar in atherosclerotic and normal rabbits. Therefore, the decreased baroreceptor sensitivity in hypercholesterolemic rabbits cannot be attributed to hypertension. The question may arise as to why the decreased baroreceptor activity failed to reflexly increase arterial pressure in the cholesterol-fed rabbits. Adaptive processes in the central nervous system or peripheral blood pressure–regulating mechanisms may compensate for decreased baroreceptor activity, which likely occurs gradually in response to the cholesterol diet. It is not known whether hypercholesterolemia or atherosclerosis increases the resting level of sympathetic nerve activity in conscious animals or humans. Results from such an experiment would suggest whether central or peripheral mechanisms are responsible for maintaining normal arterial pressure. The lack of chronic hypertension in the cholesterol-fed rabbits does not preclude important functional implications of decreased baroreceptor sensitivity. Impaired short-term regulation of arterial pressure and associated fluctuations in pressure may contribute to orthostatic intolerance and occurrence of stroke or myocardial ischemia in patients with atherosclerosis.

In the present study the diameter of the carotid artery was significantly greater in the atherosclerotic rabbits (Fig 2). The increase in carotid artery diameter may reflect a compensatory remodeling of the vessel wall that has been described in atherosclerotic arteries, including the carotid sinus.^{30 31 32} This increased diameter and vessel wall strain would not be expected to reduce baroreceptor activity. Therefore, the decrease in baroreceptor activity observed in our experiments cannot be attributed to altered vascular distensibility or strain.

We recorded baroreceptor activity from multiple fibers in the whole carotid sinus nerve. The absolute value of baroreceptor activity measured by whole-nerve recording may be influenced by the proximity of active fibers to the recording electrode, electrical noise, or superimposition of spikes. The limitations in precisely quantifying absolute nerve activity should make it more difficult to show differences between separate groups of animals. Despite these limitations, we consistently found that baroreceptor activity was significantly less in cholesterol-fed rabbits than in rabbits fed the normal diet (Fig 2). Furthermore, the absolute level of activity was inversely correlated with plasma cholesterol concentration and duration of cholesterol feeding (Fig 3), suggesting that the decrease in baroreceptor activity was indeed related to the cholesterol diet. Our conclusion that baroreceptor sensitivity is decreased in hypercholesterolemic rabbits is reinforced by the study of Angell-James,³ in which the activity of single baroreceptor fibers and the slope of the pressure-activity relation were significantly decreased in the same animal model.

The carotid sinus nerve also contains chemoreceptor afferent fibers that may "contaminate" the recording of baroreceptor activity. Chemoreceptor activity was minimized in our experiments by periodically refilling the isolated carotid sinus with fresh, oxygenated buffer and appeared to be negligible, as judged by the absence of nerve activity when pressure was rapidly lowered to 0 mm Hg (Figs 5 and 8).

Oxyradicals as a Cause of Baroreceptor Dysfunction
The substantial evidence that oxyradicals are produced in atherosclerotic blood vessels^{12 13} and the fact that the carotid sinus is a prominent site of atherosclerosis¹⁴ led us to propose that oxyradicals produced in the atherosclerotic sinus may contribute to baroreceptor dysfunction. The finding that oxyradical scavengers significantly increased baroreceptor activity in atherosclerotic rabbits suggests that radicals that are generated endogenously tonically suppress baroreceptor activity (Figs 4 through 6). The response to free-radical scavengers was small and irreversible in normal rabbits, a finding consistent with the limited production of radicals in the normal arterial wall. As one would expect, the increase in baroreceptor activity in response to scavenging of radicals was greater in cholesterol-fed rabbits. To confirm that oxyradicals modulated baroreceptor activity, we tested whether radicals generated exogenously with the xanthine/XO reaction influenced baroreceptor activity in the carotid sinus of normal rabbits. We found that xanthine/XO inhibited baroreceptor activity and that the inhibitory effect was significantly attenuated by SOD and catalase (Figs 7 through 10). Xanthine alone (Figs 7 and 9) or XO alone (data not shown) failed to decrease baroreceptor activity, suggesting that the xanthine/XO chemical reaction was required to produce the oxyradicals that inhibited baroreceptor activity.

The question arises as to which reactive oxygen species is responsible for the suppression of baroreceptor activity. In atherosclerotic rabbits SOD, which reduces the concentration of superoxide anion, failed to increase baroreceptor activity, whereas catalase, which reduces the concentration of H₂O_2, increased baroreceptor activity. The increase in activity with catalase was similar in magnitude to that observed with SOD and catalase combined (Fig 6). These results suggest that H₂O₂ and/or radicals derived therefrom, but not superoxide anion, are in part responsible for the lower level of baroreceptor activity in atherosclerotic rabbits. Consistent with this hypothesis is our finding that exposure of the isolated carotid sinus to exogenous H₂O₂ significantly inhibited baroreceptor activity (n=5, data not shown). Our finding that deferoxamine did not blunt and in fact actually enhanced the magnitude of inhibition of baroreceptor activity during xanthine/XO administration (Fig 11) argues against an important role for hydroxyl radical in mediating the response. Others have also demonstrated that H₂O₂ can exert biological actions independent of hydroxyl radical generation. For example, H₂O₂ and not hydroxyl radical mediates the vasodilator responses to xanthine/XO or bradykinin in some blood vessels.^{33 34 35}

The source of endogenous oxyradicals responsible for inhibition of baroreceptor activity cannot be determined from our data. Endothelial cells,^{36 37} smooth muscle cells,³⁸ and monocytes/macrophages in atherosclerotic lesions^{13 39 40} are potential sources of oxyradicals.

We considered the possibility that oxyradicals may suppress baroreceptor activity indirectly, eg, by an influence on carotid vascular tone and distensibility^{16 17} or by altering PGI₂ formation.^{18 19 20 21 22} Neither oxyradical scavengers nor xanthine/XO altered the carotid pressure-diameter relation, suggesting that the changes in baroreceptor activity were not caused by a change in vascular tone or distensibility.

The effects of oxyradicals and atherosclerosis on PGI₂ formation are complex. Free radicals and lipid peroxides may inhibit cyclooxygenase to reduce PGI₂ formation.^{18 19 20} In contrast, oxidant stress may increase PGI₂ formation through activation of phospholipase A₂ and release of arachidonic acid, particularly during acute oxidant stress.^{21 22} The latter mechanism may account for the increased formation of PGI₂ that we observed during short-term exposure of the carotid sinus to xanthine/XO. We have previously shown that PGI₂ exerts an excitatory influence on baroreceptor activity.^{9 10} Therefore, the increased formation of PGI₂ cannot be responsible for and may actually oppose the suppression of baroreceptor activity by oxyradicals.

We observed an increase in the basal formation of PGI₂ in the carotid sinuses of atherosclerotic rabbits compared with those in normal rabbits. This increased basal production of PGI₂ does not appear to be the result of oxyradicals, since treatment of the atherosclerotic carotid sinus with SOD and catalase did not reduce PGI₂ formation. Enhanced PGI₂ formation in atherosclerosis has been demonstrated previously in vivo⁴¹ and in some in vitro studies.^{42 43} Since PGI₂ increases baroreceptor sensitivity,^{9 10} the increased basal level of PGI₂ may be a compensatory mechanism that attempts to preserve baroreceptor sensitivity in atherosclerosis.

These results suggest that free radical–mediated suppression of baroreceptor activity cannot be explained by changes in vascular distensibility or PGI₂ formation. Therefore, a possible direct effect on baroreceptor sensory nerve endings should be considered. Free radicals and oxidant stress have been shown to alter membrane excitability.^{44 45} These actions may be mediated through the effects of oxidants on the activity of ion pumps or channels.^{45 46 47 48} For example, oxidant stress may increase potassium channel activity,^{47 48} which would promote hyperpolarization and a decrease in the frequency of action potential discharge. Interestingly, recent studies have demonstrated that hydroxyl radicals generated from H₂O₂ activate chemosensitive afferent fibers in the heart and other visceral organs.^{49 50} Therefore, free radicals and oxidant stress may cause differential effects on the activity of different types of sensory nerves. The reversible nature of free radical–mediated suppression of baroreceptor activity and the opposite effect of radicals on the activity of chemosensitive afferents^{49 50} suggest that our results do not reflect nonspecific effects or irreversible damage to baroreceptor nerve endings.

Pathophysiological Implications
It is generally accepted that structural changes, such as decreased distensibility of large arteries, cause decreased baroreflex sensitivity in atherosclerosis.^{3 4 5 6} We propose that functional changes in paracrine systems, such as increased formation of oxyradicals, contribute significantly to baroreceptor dysfunction. Previous studies in our laboratory suggest that loss of the excitatory influence of PGI₂ on baroreceptor activity⁵¹ and release of factors from aggregating platelets^{11 26} may contribute to decreased baroreceptor sensitivity in atherosclerosis. Decreased formation of PGI₂, platelet activation, and increased formation of free radicals in atherosclerotic vessels may also facilitate each other's inhibitory influence to exacerbate baroreceptor dysfunction.^{13 52}

The finding that altered paracrine mechanisms contribute to baroreceptor dysfunction in atherosclerosis raises the possibility that therapies designed to correct the paracrine imbalances may rapidly restore baroreceptor sensitivity before structural vascular changes can be reversed. For example, the significant increase in baroreceptor activity after short-term exposure to catalase suggests that antioxidant therapies may exert beneficial effects on neural control of the circulation. The ability to restore baroreceptor sensitivity in atherosclerosis may have important implications, not only for improving the control of arterial pressure, but also for protecting against ventricular arrhythmias and sudden cardiac death that are associated with decreased baroreflex sensitivity in atherosclerosis.^{53 54}

	Selected Abbreviations and Acronyms

 

6-keto-PGF1 = 6-ketoprostaglandin F1 K-H = Krebs-Henseleit PGI2 = prostacyclin SOD = superoxide dismutase XO = xanthine oxidase

	Acknowledgments

 
The work was supported by research funds from the Department of Veterans Affairs, the National Institutes of Health (P01-HL 14388), and the Iowa Affiliate of the American Heart Association. Dr Zhi Li was a Research Fellow of the Iowa Affiliate of the American Heart Association when this study was performed. The authors thank Dr Donna Farley for the analysis of 6-keto-PGF₁ levels, Pamela K. Tompkins for performing the histology, Dr Bridget Zimmerman for the statistical analysis of the data, and Shawn M. Roach and Deb Schiek for preparation of the figures.

Received February 7, 1996; accepted July 1, 1996.

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