Activation of cardiac vagal afferents by oxygen-derived free radicals in rats. E E Ustinova and H D Schultz. doi: /01.RES.74.5.

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1 Activation of cardiac vagal afferents by oxygen-derived free radicals in rats. E E Ustinova and H D Schultz Circ Res. 1994;74: doi: /1.RES Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 1994 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation Research is online at: Downloaded from by guest on March 3, 214

2 895 Activation of Cardiac Vagal Afferents by Oxygen-Derived Free Radicals in Rats Elena E. Ustinova, Harold D. Schultz Abstract Myocardial ischemia and reperfusion can evoke excitation of cardiac vagal afferent nerve endings and activation of a cardiogenic depressor reflex (Bezold-Jarisch effect). We postulate that oxygen-derived free radicals, which are well known to be produced during prolonged ischemia and reperfusion, contribute to this excitation. Hydroxyl radicals derived from hydrogen peroxide (H22) activate abdominal sympathetic afferents and produce reflex excitation of the cardiovascular system. However, it is not known whether inhibitory vagal cardiac afferents are activated by oxygen-derived free radicals. We recorded activity from 52 single vagal afferent fibers in 29 rats; the endings of these fibers were located in the walls of all four chambers of the heart. Thirty-three (63%) of these fibers were classified as chemosensitive C-fiber endings because of their irregular discharge under resting conditions, their activation in response to the topical application of capsaicin (1 to 1 gg) to the surface of the heart encompassing the receptive field, and their conduction velocities. Fourteen (27%) of the remaining fibers were found to be mechanoreceptors. Topical application of H22 to the heart activated 5% of the chemosensitive endings and did not directly affect cardiac mechanoreceptors. Activity increased by 498% at a dose of 3,umol (P<.1). This effect was reproducible and dose dependent and was not due to [H']. Topical application A ctivation of cardiac vagal sensory endings during ischemia and reperfusion may be an important mechanism by which cardiovascular function is regulated in these pathological states. Several studies have attempted to define the nature of the stimuli that activate cardiac vagal afferents in ischemia and reperfusion. The first direct neural recordings from vagal afferents during myocardial ischemia were carried out by Thoren.12 These studies in cats indicated that the firing rate of nonmyelinated (C-fiber) mechanosensitive ventricular fibers increased 1 to 2 minutes after the onset of ischemia and returned to the basal level after 1 to 2 minutes of occlusion. Reperfusion after 2 to 5 minutes of ischemia caused a second elevation of the neural activity. They attributed the increased firing of these fibers to systolic bulging of the ischemic myocardium. In addition to mechanosensitive C fibers, there exists a separate population of C-fiber endings in the heart known as chemosensitive endings. These endings are activated by exposure to irritant chemicals such as capsaicin and phenyl diguanide, but they do not Received September 8, 1993; accepted February 2, From the Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha. Correspondence to Harold D. Schultz, PhD, Department of Physiology and Biophysics, University of Nebraska Medical Center, 6 S 42nd St, Omaha, NE of xanthine/xanthine oxidase (2 mmol/.3 mu) activated 8 of the 12 chemosensitive fibers tested and had no direct effect on mechanosensitive fibers. Activity increased by 287% (P<.1). Administration of the superoxide radical-scavenging enzyme superoxide dismutase (2 U/kg IV) significantly decreased the response of the fibers to xanthine/xanthine oxidase but had no effect on the activation caused by H22. The antioxidant deferoxamine (2 mg/kg IV), which prevents the formation of hydroxyl radical, abolished the responses to xanthine/xanthine oxidase and H22. Dimethylthiourea (1 mg/kg IV), which scavenges the hydroxyl radical, also abolished afferent responses to H22. Administration of indomethacin (5 mg/kg IV) had no effect on the afferent response to H22. These results indicate that (1) the rat heart possesses a notable innervation by chemosensitive afferent vagal C fibers, (2) these cardiac chemosensitive afferents can be activated by reactive oxygen species, (3) the hydroxyl radical appears to be more important than the superoxide anion for this activation, and (4) this activation is not mediated by the cyclooxygenase system. (Circ Res. 1994;74: ) Key Words * cardiac chemosensitive endings * ventricular C fibers * oxygen radicals * prostaglandins * antioxidants * ventricular receptors readily respond to changes in cardiac pressures or volume.34 Coleridge et a15 showed that two thirds of the chemosensitive C-fiber endings in the left heart of dogs were stimulated within 1 to 3 seconds of coronary occlusion and that activation of these cardiac endings was unrelated to changes in cardiac pressures. In other studies, these investigators have also shown that ventricular chemosensitive afferents can be stimulated directly by bradykinin and prostaglandins,6'7 metabolic factors known to exist in the myocardium under ischemic conditions. An important metabolic event that occurs in ischemia, and especially in reperfusion, is oxygen-derived free radical activation.8 Investigations of the various factors that could affect cardiac vagal afferents during myocardial ischemia and reperfusion have not yet addressed the possibility that these radicals can affect cardiac endings. However, it was recently shown that during mesenteric ischemia, free oxygen radicals can activate ischemia-sensitive sympathetic afferents within abdominal organs and thus elicit cardiovascular reflexes mediated by these afferents.9 The same authors showed that direct application of hydrogen peroxide (H22) to the gallbladder, duodenum, and stomach activated abdominal visceral afferents, which reflexly stimulated the cardiovascular system by a mechanism involving hydroxyl radicals. This effect of H22 was abolished by the antioxidants dimethylthiourea (DMTU) and deferoxamine.1

3 896 Circulation Research Vol 74, No 5 May 1994 In the present study in rats, we sought to investigate the possibility that oxygen-derived free radicals can directly activate cardiac vagal afferent endings. We included in our investigation both mechanosensitive and chemosensitive endings. The specific questions that we addressed were as follows: (1) Do the oxygen-derived radical species hydroxyl radical and superoxide anion have the same effect on the cardiac afferents? To address this question, we studied the effect of two different free radical-generating systems: H22, which produces the hydroxyl radical via the Haber-Weiss reaction, and xanthine/xanthine oxidase, which generates superoxide anion.8 (2) Are the effects of free radicals on vagal cardiac afferent endings decreased or abolished with antioxidants? To address this question, we studied the effect of free radicals before and after the administration of deferoxamine, which prevents hydroxyl radical formation in the Haber-Weiss reaction,11-13 DMTU, which is a direct scavenger of hydroxyl radical,8 and superoxide dismutase, which is a scavenger of superoxide anion.1415 (3) Are the effects of free radicals on cardiac vagal sensory endings mediated by the cyclooxygenase system and prostaglandin synthesis? To address this question, we studied the effect of free radicals on cardiac vagal afferent discharge before and after the administration of indomethacin, which blocks cyclooxygenase. Materials and Methods Young adult Sprague-Dawley rats (25 to 32 g, either sex) were anesthetized intraperitoneally with a mixture of 2% a-chloralose and 25% urethane in saline (5 ml/kg). The trachea was cannulated low in the neck, and the lungs were ventilated by a Harvard rat respirator (6 breaths per minute) with air supplemented with 2. Body temperature was maintained at 37 C by a heating pad. Polyethylene catheters were inserted in a carotid artery and jugular vein for measurement of arterial pressure and administration of drugs, respectively. The chest was opened via a sternotomy, and a 2F microcatheter pressure transducer (Millar) was inserted into the left ventricle via a needle puncture through the apex for measurement of left ventricular pressure. Heart rate was measured by a cardiotachometer triggered by the arterial pressure pulse. Arterial and tracheal pressures were measured by strain gauges (Hewlett-Packard 127A). Heart rate, arterial, ventricular, and tracheal pressures, and nerve activities (see below) were recorded by a thermal recorder (Astro-Med MT95). Silk snares were looped around the ascending aorta and posterior vena cava to produce occlusion when necessary to ascertain the mechanosensitivity of the endings (see below). Estimated fluid loss was replaced by intravenous administration of physiological saline at a rate of 4 to 6 ml/kg per hour. Recording of Afferent Vagal Impulses Fine slips of the left cervical vagus were covered with mineral oil and placed on a silver electrode. Impulses were amplified (Grass P511 amplifier), displayed on an oscilloscope (Gould 45), and fed into a rate meter (Frederick Haer) whose window discriminators were set to accept potentials of a particular amplitude. Impulses were counted by rate meter in 1-second bins. Bundles that had one, or at most two, easily distinguishable active fibers were used. Identification of Cardiac Fibers We studied only spontaneously active fibers that had receptive fields in the heart that could be located precisely. Conduction velocity was determined by measuring the latency between electrical stimulation of the receptive field and recording of the evoked potential and measuring the conduction distance between the receptive field and recording electrode. C fibers and AS fibers were classified as those with a conduction velocity of <2.5 and >2.5 m/s, respectively. Chemosensitivity of an ending was tested by intravenous injection and topical application of capsaicin to the surface of the heart. Capsaicin was chosen as the test chemical because it is known to directly stimulate only chemosensitive C-fiber endings and not cardiovascular mechanoreceptors Capsaicin was injected intravenously in doses of.5 to 1. gg, and then if the fiber was activated, capsaicin was applied directly to the surface of the heart with a small patch to locate the receptive field (see below). Some chemosensitive endings could be stimulated by probing the heart (eg, see Fig 3). Mechanosensitivity of an ending was tested by aortic and vena caval occlusions. Endings that readily responded to left ventricular diastolic pressures of < 15 mm Hg or left ventricular systolic pressures of < mm Hg were considered mechanoreceptors. Cardiac diastolic and systolic pressures were manipulated by an aortic and vena caval snare. Receptive fields were located by gently probing the surface of the heart with a fine-tipped probe. Drug Administration A small circle of filter paper 3 mm in diameter was placed on the surface of the heart above the receptive field to which 1,L of test solutions was applied for 2 seconds. Capsaicin was applied to the filter paper in concentrations of.1 to 1 mg/ml; capsaicin solutions were made from a stock solution (1 mg/ml dissolved in 9% saline/1% ethanol), with final dilutions made with saline. H22 was applied to the filter papers in concentrations of 3%, 1%, and 3% in distilled H2; 1,uL of each solution was equivalent to doses of 1, 3, and 9,umol, respectively. The doses of xanthine and xanthine oxidase were 2 mmol and.3 mu, respectively, in 1,L of distilled H2. Applications of capsaicin vehicle or distilled H2 alone had no effect on fiber activity. After each application, the paper was removed, and the surface of the heart was washed with warm saline. All drugs were obtained from Sigma Chemical Co. Protocol The receptive field of each fiber was identified, and mechanosensitivity of the ending was tested with aortic and posterior vena caval occlusions. Then the fiber was tested with applications of capsaicin (1 and 1,ug in 1,uL), H22 (1 to 9,umol in 1,uL), and/or xanthine/xanthine oxidase (2 mmol/.3 mu in 1,uL distilled H2). Intervals of 1 to 15 minutes were allowed between applications. If the fiber was activated with H22 and/or xanthine/xanthine oxidase, applications of the effective doses were repeated before and 1 to 2 minutes after intravenous administration of either deferoxamine (2 mg/kg), DMTU (1 mg/kg), superoxide dismutase (2 U/kg), or indomethacin (5 mg/kg). In some experiments, we studied the effect of H22 application before and after administration of iron-saturated deferoxamine. Iron-saturated deferoxamine was prepared by incubating 1 ml deferoxamine solution (1 mg/ml) with 5 mg FeCl3 for 1 hour at room temperature. Analysis of Data Reported values are mean+sem. The firing rate of vagal fibers was calculated as the average number of impulses per second (imp/s) over a period of 2 seconds. Fiber activity was calculated over the 2 seconds of maximal activity during a 1-minute control period and during the first minute of the experimental intervention. Because of the individual variability in the control activity of different fibers, the neural responses were also expressed as percent change from the baseline. Differences among groups were determined by ANOVA for repeated measures, and differences between means were isolated by the Bonferroni correction for multiple

4 Ustinova and Schultz Cardiac Afferents and Oxygen Radicals 897 TABLE 1. Characteristics of Cardiac Vagal Afferents in the Rat Activated With Recorded Fibers Capsaicin Activated With H22 Cardiac Cardiac Cardiac Rhythm* Rhythm Rhythm Total + - Total + - Total + Left ventricle Left atrium Right ventricle Right atrium Total indicates discharge pattern synchronized to cardiac cycle; -, discharge pattern unrelated to cardiac cycle. *Afferent endings with a positive cardiac rhythm (+) were found to be mechanosensitive endings. t tests. Student's paired and unpaired t tests were used for single comparisons. Statistical significance was accepted at P<.5. Results Chemosensitivity of Cardiac Vagal Afferents Fifty-two cardiac vagal fibers were recorded in 29 rats. Twelve of the recordings were obtained from filaments with only one active fiber. In the other 2 recordings, two active fibers were present, but differences in spike heights permitted activity in the individual fibers to be determined separately. Table 1 and Fig 1 describe the distribution of the receptive fields, type of firing pattern, and chemosensitivity of the 52 fibers tested with H22. Thirty-eight (73%) of the 52 fibers exhibited an irregular pattern of discharge at rest, which was unrelated to the cardiac cycle. The maximal resting activity (averaged over 2 seconds of maximal activity during the control period) of the irregularly firing fibers was imp/s (from.2 to 1 imp/s). Thirty-three (89%) of the 38 irregularly firing endings were activated in response to capsaicin applied to their receptive field (1 to 1,ug in 1 pl) for 2 seconds (eg, see Figs 3 through 5). These endings responded to capsaicin with a volley of impulses that subsided quickly over the course of 1 to 2 seconds. Activation of these endings by capsaicin consistently occurred with no relation to the cardiac cycle or to pressure. The latency of activa- Anterior wall ~~~~~~ Posterior wall RV L9 V FIG 1. Drawings showing the location of the receptive fields of 52 cardiac vagal afferent fibers that were tested with the topical applications of capsaicin and H22 in series 1. The left cranial (superior) vena cava has been omitted for clarity. Ao indicates aorta; PA, pulmonary artery; RV, right ventricle; RA, right atrium; LV, left ventricle; LA, left atrium; *, fibers exhibiting irregular discharge; and A, fibers exhibiting a cardiac rhythm when active. tion was seconds, and the duration of response was 15 to 2 seconds. The activation lasted 3 to 5 seconds after the removal of capsaicin from the receptive field. Activity increased from a maximum of imp/s during the control period to 7.±.6 imp/s (147+16% increase, P<.1) at a dose of 1,ug and from to imp/s (53+13% increase, P<.1) at a dose of 1,ug. Each of these afferent endings also responded to systemic administration of capsaicin. We did not test the effect of endogenous substances such as bradykinin or prostaglandins on these afferents; however, with respect to capsaicin, these chemosensitive cardiac vagal endings are similar to those described previously by Coleridge and coworkers in dogs.3-7 Five irregularly firing fibers failed to respond to capsaicin, even though their receptive field could be located by probing the surface of the heart. None of the 38 irregularly firing endings responded to small changes in cardiac pressure, nor did they show evidence of cardiac or respiratory modulation. All irregularly firing fibers tested had conduction velocities of <2.5 m/s and were classified as C fibers. Mechanosensitive endings (described below) rarely responded to capsaicin application. Nevertheless, five mechanoreceptors responded with an increase in activity to capsaicin application (1,ug in 1 4L) to their receptive field. In each of these cases, the pattern of activation was distinctly different than that observed in chemosensitive afferents. The mechanosensitive fibers responded in a pulse-related pattern that gradually increased by 1 to 2 impulses in parallel with an increase in aortic pressure. These changes in activity could be mimicked by gradually occluding the ascending aorta to produce similar changes in blood pressure. Thus, in these five mechanoreceptors, activation in response to capsaicin appeared to be secondary to changes in cardiac hemodynamics. Mechanosensitivity of Cardiac Vagal Afferents Fourteen of the 52 fibers exhibited a cardiac rhythm and readily responded to vascular occlusions or intravenous bolus injection of saline (2 ml) and gentle probing of the surface of the heart. Thus, these fibers were considered to arise from cardiac mechanoreceptors.

5 898 Circulation Research Vol 74, No 5 May (.)..5._. E - in 15* 5- - D v A B n.g:f O LVEDP (mm Hg) FIG 2. Graphs showing the effect of ascending aortic occlusion on the activity of vagal afferent C fibers in the left ventricle. A, Activity of a total of five fibers that exhibited a cardiac rhythm and were not activated with capsaicin. Activity was correlated with the increase in left ventricular end-diastolic pressure (LVEDP); the threshold of activation was 4 to 8 mm Hg. These endings were classified as mechanosensitive. imp/s indicates impulses per second. B, Activity of five representative fibers that exhibited irregular discharge and were capsaicin sensitive. These endings were classified as chemosensitive. Chemosensitive fibers did not increase their activity with an increase in LVEDP below the range of 24 to 28 mm Hg. A total of 12 chemosensitive endings were found in the left ventricle. These fibers generally discharged with 1 to 2 impulses per cardiac cycle at rest. Those in the atria sometimes exhibited a respiratory modulation as well. Over the course of the experiment, two mechanosensitive endings in the right atrium and one in the left atrium became silent during part or all of the respiratory cycle but could be readily activated by aortic occlusion or bolus intravenous saline injections. Mechanosensitive fibers in which conduction velocities were obtained were classified as C fibers. Chemosensitive endings never exhibited a cardiac or respiratory rhythm and failed to respond to changes in cardiac hemodynamics within a normal range (diastolic pressure, 15 mm Hg; systolic pressure, mm Hg). However, many chemosensitive endings could be activated by probing cardiac tissue. Only chemosensitive endings located in the left heart responded to aortic occlusion (see below). Vagal Afferents From the Left Ventricle Fig 2 illustrates the response of 1 representative fibers from the left ventricle to occlusion of the ascending aorta. Five fibers illustrated in Fig 2A exhibited a cardiac rhythm. The activity of each of these fibers increased during aortic occlusion, which produced increases in both systolic and diastolic pressures. Average activity of these five fibers increased from 5.9±.4 imp/s at a left ventricular end-diastolic pressure (LVEDP) of 4 mm Hg to 21.6±1.4 imp/s at an LVEDP of 32 mm Hg. Because the activity of these fibers was synchronized to the cardiac cycle and because they responded to changes in ventricular pressure within a normal range, we characterized these endings as ventricular mechanoreceptors. The five fibers illustrated in Fig 2A were the only left ventricular mechanoreceptors that we observed. Another five fibers illustrated in Fig 2B exhibited an irregular pattern of discharge at rest, unrelated to the cardiac cycle, and were capsaicin sensitive, ie, chemosensitive endings. These fibers are representative of 12 chemosensitive endings recorded from the left ventricle. Their activity did not change with an increase in the distension of the left ventricle in response to aortic occlusion at LVEDP of <24 to 28 mm Hg. However, all chemosensitive endings in the left ventricle and left atrium were activated by prolonged aortic occlusion such that LVEDP increased above 28 mm Hg. Nevertheless, even with activation at very high pressure, these endings never exhibited a cardiac rhythm. Average activity of the 12 fibers increased from 2.6±.7 imp/s at an LVEDP of 4 mm Hg to 11.2±2.8 at an LVEDP of 32 mm Hg. Thus, although the chemosensitive endings were not totally unresponsive to cardiac distension, a fairly high level of gross distension of the heart was necessary. None of the left ventricular endings was affected by inferior vena caval occlusion, which caused a decrease in left ventricular systolic pressure from 1 to 12 mm Hg to between 2 and 4 mm Hg. Response to H22 Topical application of H22 (1 to 9,mol) to the receptive field for 2 seconds increased the activity of 19 (36%) of the 52 fibers tested (Table 1). Fifteen of the H22-sensitive fibers exhibited irregularly firing discharge at rest and were also capsaicin sensitive; thus, these endings were considered chemosensitive afferents (eg, see Figs 3 and 4). However, among the total 33 irregularly firing capsaicin-sensitive (ie, chemosensitive) fibers recorded, only 45% were activated by H22. Six of the 12 chemosensitive endings in the left ventricle were activated by H22. Irregularly firing fibers that were not activated with capsaicin were not activated by H22. The response of the 15 chemosensitive endings to H22 was dose dependent. The firing rate of the fibers increased from 2.9±.3 to imp/s (125±17% increase, P<.1) at a dose of 1 gmol, from 2.7±.3 to 1.9±1. imp/s (498±12% increase, P<.1) at a dose of 3,umol, and from 2.8±.4 to 14.2± 1.1 imp/s (534±17% increase, P<.1) at a dose of 9 gmol. The latency of activation at a dose of 3,umol was 8.6±1.4 seconds; ie, it was the same as with capsaicin application and did not change significantly with the increase in dose. The duration of the response varied from 8 seconds to 2 minutes after the removal of filter paper from the heart. In 9 of 15 chemosensitive fibers that were activated by H22, their increased activity was accompanied by changes in left ventricular pressure and arrhythmias that appeared 2 to 4 seconds after the application (eg, see Fig 3). In 7 fibers, systolic pressure decreased by 24% in response to H22 application (Table 2). Arrhythmias were evident in 5 of these 7 fibers. These cardiac effects lasted 3 to 6 seconds, depending on the dose of H22, and then returned to normal. In 2 of the 15 chemosensitive fibers, a transient (2- to 5-second) increase in systolic and diastolic pressure occurred after

6 Ustinova and Schultz Cardiac Afferents and Oxygen Radicals FiG 3. Effects of capsaicin, H22, and deferoxamine on two types of cardiac vagal afferent C fibers. Recordings of two cardiac afferent C fibers with concentric receptive fields in the right atrium are shown. The fiber with a highamplitude action potential (AP) (fiber 1, upper rate meter) had a spontaneous and irregular discharge. The fiber with a low-amplitude AP (fiber 2, lower rate meter) initially possessed a cardiac rhythm but was silent at rest at the time of recording. A, Both fibers discharged in response to touching the right atrium (marked with a bar) with a glass probe. B, Effects of topical application of capsaicin (1,ug in 1 gl) to the receptive fields are shown. The arrow indicates the moment of topical application. Fiber 1 responded to capsaicin with a latency of 9 seconds. Fiber 2 did not respond to capsaicin. C, Effects of topical application of H22 (3 gg in 1 1gL) to the receptive fields are shown. H22 induced transient disturbances in contractions. Fiber 1, which was capsaicin sensitive, was activated by H22, whereas fiber 2 was ; only slightly activated at the moment of contraction disturbances. D, Effects of topical application of the same dose of H22 to the receptive fields 15 minutes after deferoxamine (2 mg/kg IV) are shown. Deferoxamine virtually abolished ~~~~~~~~~~~~~~~~~5$ the afferent response of the chemosensitive fiber (fiber 1) to H22. The disturbances of contraction and activation of fiber 2 were the same as before deferoxamine administration. IF indicates impulse frequency (impulses per second [imp/s]); LVP, left ventricular pressure. 25 A Af (imp/s) O 25 IF (imp/s) AP 1 1:... op I lp,iih IJ (mmhg ::- ]. 25 ~ IF (imp/s) OP 1 LVP (mm Hg) --I #I VLI> } 4;E e~~~~~~~~~~~~~~~~~~~~~~~~.. '....E IE 12!1 I in left ventricular pressure did not correlate with the pattern of activation of the chemosensitive fibers. The remaining 6 chemosensitive fibers, one of which is shown in Fig 4, were activated by H22 without any changes in left ventricular pressure or cardiac rhythm. Neither resting activities of the chemosensitive afferents nor their response to capsaicin was affected after removal of H22 from the receptive field. The second application of the same dose of capsaicin (1 gg), 15 to 2 minutes after H22, induced an increase of firing rate.k LVP (mm Hg). -a AP.1.: ;:.1` :: -; "ps kw ~ n B IF J (imp/s) 25 AP LVP i,1. (mm Hg) (imp/s) from 2.9±..4 to 12.2±.9 imp/s (52±116% increase, P<.1). TI[he response of the endings to repeated applications ; of H22 was reproducible as well. The second appl lication of H22 (3 gmol), 3 minutes after the first app:lication, caused an increase of activity from 2.8±.4 to 11.±.7 imp/s; ie, activity increased by 51±121% (P<.1) (Fig 6). Four mecchanosensitive endings were activated by H22. In eaceh of these fibers, their increased activity in response to ]H2O2was accompanied by an increase in left ventricular ppressure. The time course of the changes in FIG 4. Recordings showing the effects of capsaicin and H22 on a chemosensitive vagal Ei 1; 18afferent C fiber in the left atrium before and after administration of deferoxamine. Arrows indicate L the moment of application. A, Activation of the fiber in response to topical application of capi li-_ saicin (1 9g in 1 gl) to the receptive field is shown. B, Activation of the same fiber with.., topical H22 (3 gmol) is shown. Note that H22 application stimulated the afferent ending without appreciable changes in left ventricular pressures (LVPs). C, Application of the same dose of H22 1 minutes after administration of deferoxamine (2 mg/kg IV) is shown. Deferoxamine abolished the afferent response to H22. IF indicates impulse frequency (impulses per a~ m -ssnuamn second [imp/s]); AP, action potentials. ia.fj AP LVP, ; Ii:-I:I H22, as shown in Fig 3. The time course of the changes (imp/s) [1.t ::: 1 7: l 1.1 ijl MEDW REL _9~ IU c, _= usa-rn 899 Uhf. L t (mm Hg) 5S

7 9 Circulation Research Vol 74, No S May 1994 (imp/s) 2[ LV if -A iii _ ~~~~~~~(imp(s)l ;i ;Ei i;::e a i; Ii= > : I ha a a.i: iaa. * C..l..._.. (imp/s)s AP v,itimit T77 TI: r LVP F E j IF (im p/s) (imp/s) AP~ ~ ~~~1 77. t1i1tuhlt :1 ;1,!LAJ... il.,! 55S left ventricular pressure closely correlated with the pattern of activation of the mechanosensitive endings, unlike the chemosensitive endings (eg, see Fig 3). The remaining mechanosensitive fibers were not activated by H22. In these cases, left ventricular pressure decreased or remained unaltered, and cardiac rhythm was constant. Response to Xanthine/Xanthine Oxidase The effect of the application of the xanthine/xanthine oxidase mixture to the receptive fields of cardiac fibers varied among different fibers. Of 12 chemosensitive fibers tested with xanthine/xanthine oxidase. 8 were activated in about the same manner as with H22 (Figs 5 and 6). In these fibers, the second application of xanthine/xanthine oxidase caused a repeatable effect (Fig 6). Mechanosensitive fibers were not directly activated by xanthine/xanthine oxidase. Application of xanthine/xanthine oxidase to the heart produced a minor transient decrease in the left ventricular systolic pressure, but this effect was not statistically significant. Effects of Antioxidants The afferent response of cardiac chemosensitive afferents to H22 was abolished when the second application was preceded by injection of the antioxidant deferoxamine (2 mg/kg IV). After administration of deferoxamine, maximal activity was imp/s before application of H22 (3 gmol) and 2.4±.4 imp/s after H22 application (n=9, P= NS). At the same time, systemic administration of deferoxamine did not always completely prevent the disturbances of left ventricular pressure and arrhythmias caused by H22, although the decrease of systolic pressure was less and the difference compared with control was not significant (Table 2). Administration of superoxide dismutase (2 U/kg IV) did not prevent either activation of cardiac fibers or disturbances of contractile function in response to H22. 17U;l& Deferoxamine also decreased the response of the chemosensitive fibers to capsaicin (Fig 6). Application of the same dose of capsaicin (1 gg), 1 minutes after FIG 5. Recordings showing the effects of deferoxamine and superoxide dismutase on the responses of a chemosensitive vagal afferent fiber in the left ventricle to capsaicin and xanthine/xanthine oxidase. Arrows indicate the moment of topical applications to the receptive field. A, Activation of the fiber in response to capsaicin (1 Mg in 1 gl) is shown. B, Activation of the same fiber in response to xanthine/ xanthine oxidase (2 mmol/.3 mu) is shown. C, Application of capsaicin 1 minutes after administration of superoxide dismutase (2 U/kg IV) is shown. D, Application of xanthine/ xanthine oxidase 15 minutes after administration of superoxide dismutase is shown. The response of the fiber was decreased. E, Application of capsaicin 1 minutes after administration of deferoxamine (2 mg/kg IV) is shown. F, Application of xanthine/xanthine oxidase 15 minutes after administration of deferoxamine is shown. The response of the fiber was almost completely abolished. IF indicates impulse frequency (impulses per second [imp/s]); AP, action potentials; and LVP, left ventricular pressure. administratio in of deferoxamine, caused an increase in fiber activity: from 2.4±.3 to 8.5± 1. imp/s. This response was significajlntly less than the response before deferoxamine admir nistration ( % versus 271±5% increase; P<. )5). Resting activity of these fibers was not affected by d leferoxamine. In experir nents in which fibers were activated with xanthine/xan thine oxidase application, we examined the effects of delferoxamine or superoxide dismutase on this activation. A.pplication of xanthine/xanthine oxidase (2 mmol/.3 n nu) increased the activity of these fibers from to 7.7±.7 imp/s, ie, by 287±43% (n=8, P<.1). Th his response to xanthine/xanthine oxidase 15 ] control * capsaicin 3 H22 / X+XO u a 1 5 Q Li.. _ F1~ ~ ~ ~ ~ ~ ~ ~* l second after after rst application SOD deferoxamine applic qation FIG 6. Bar gr raph showing the effects of antioxidants deferoxamine (2 mg /kg IV) and superoxide dismutase (SOD, 2 responses of cardiac chemosensitive vagal U/kg IV) on t the Dpical cardiac applications of capsaicin (1 Mg), H2a (3 Msmol) I,and xanthine/xanthine oxidase (X+XO, 2 mmol/.3 mu). imp)/s indicates impulses per second. SOD significantly attenual Lted afferent responses to capsaicin and X+XO but not H22 (CPc t.5 compared with second application). Deferoxamine signifiecrantly attenuated afferent responses to capsaicin, (**P<.1 compared with second application). X+XO, and H2io 2 statistical differences between the first and seeond applic ations. All responses to chemicals were significantly greaterrthan their respective controls (P.c.5 to.1) except for thosseto H22 and X+XO after deferoxamine (P=NS).

8 Ustinova and Schultz Cardiac Afferents and Oxygen Radicals 91 TABLE 2. Effect of H22 and Deferoxamine on Cardiac Contractility and Arrhythmias Control Before deferoxamine LV systolic pressure, mm Hg t LV diastolic pressure, mm Hg ±.7 Number of premature beats for 1 seconds after application 8.3±1.9 After deferoxamine LV systolic pressure, mm Hg 125.5± ±9.1 LV diastolic pressure, mm Hg 3.1 ± ±.8 Number of premature beats for 1 seconds after application Values are mean +±SEM. *Values for ventricular pressures taken at the maximal response. tp<.5 compared with control. H22* (3,gmol) Q.-_ 15 1 Q -. 5 LL control * capsaicin El H22 T before DMTU after DMTU FIG 7. Bar graph showing the effects of dimethylthiourea (DMTU, 5 mg/kg IV) on the responses of cardiac chemosensitive vagal afferent fibers to topical cardiac applications of capsaicin (1 /gg) and H22 (3,umol). imp/s indicates impulses per second. DMTU decreased the afferent response to capsaicin and abolished the response to H22. *P<.5 compared with activity before DMTU. was significantly less after the administration of superoxide dismutase (88±16% increase, P<.5). Deferoxamine completely abolished responses of cardiac afferents to xanthine/xanthine oxidase (Figs 5 and 6). In additional experiments in six rats, we recorded action potentials of nine cardiac chemosensitive fibers in response to the application of H22 before and after the administration of a hydroxyl radical scavenger, DMTU (1 mg/kg IV). DMTU completely abolished the response of the chemosensitive cardiac fibers to H22 in the same manner as deferoxamine in the previous experiments. Similarly, DMTU had a tendency to diminish the response of the fibers to capsaicin, but this effect was not statistically significant (Fig 7). Administration of iron-saturated deferoxamine (2 mg/kg IV) in six rats did not change the response of the chemosensitive fibers to H22 application (3,umol). The increase in fiber activity was 328±77% before and 32±82% after systemic administration of iron-saturated deferoxamine (P=NS). Effects of Indomethacin Fig 8 shows the effects of capsaicin and H22 on eight cardiac chemosensitive afferents before and after administration of indomethacin to the animals. Indomethacin decreased activation of cardiac fibers in response to capsaicin: application of capsaicin (1,gg) increased fiber activity by 51 ± 12% before and by 26± 6% after the administration of indomethacin (P<.5). The effect of the application of H22 (3,umol) after indomethacin administration was not altered: activity increased by 445±1% before and 428±121% after indomethacin (P=NS). Discussion There are few studies of cardiac vagal afferents in rats. Thoren et al17 suggested that these afferents originate only from atria and cannot be found in the ventricles of the rat heart. In their study, the screening procedure for finding receptors in the heart was a short-lasting aortic occlusion that induced an increase in left atrial pressure from 2-5 to 2 mm Hg. In our experiments, we found that the threshold LVEDP for activation of the left ventricular chemosensitive fibers was between 24 and 28 mm Hg. This gross distension of the heart most likely produced a nociceptive stimulus capable of activating the chemosensitive endings. Thus, it is possible that the distension of the heart produced in Thoren's study activated mechanosensitive endings but was not enough to reveal the chemosensitive endings. Nevertheless, in the present experiments, we found the presence of both mechanosensitive and chemosensitive vagal afferents in both atria and ventricles of the rat heart and demonstrated that 63% of the left ventricular afferents are chemosensitive C fibers that are capable of responding to capsaicin. Although changes in cardiac pressures and cardiac rhythm sometimes accompanied activation of the chemosensitive endings in response to H22, it is unlikely that these mechanical changes are responsible for the increased activity. First, H22 generally decreased left ventricular systolic pressure, whereas experiments with aortic and inferior vena caval occlusion showed that these endings responded only to very large increases in left ventricular systolic and diastolic pressure. Second, deferoxamine administration abolished the effect of H22 on the afferent endings regardless of a - E 2 > 1- CD a). LL [] control * capsaicin E H22 IT before after indomethacin indomethacin FIG 8. Bar graph showing the effects of indomethacin (5 mg/kg IV) on the responses of cardiac chemosensitive vagal afferent fibers to topical cardiac applications of capsaicin (1,ug) and H22 (3 gmol). imp/s indicates impulses per second. Indomethacin decreased the afferent response to capsaicin but not to H22. *P<.5 compared with activity before indomethacin. T

9 92 Circulation Research Vol 74, No 5 May 1994 whether a decrease in left ventricular systolic pressure and arrhythmias occurred. Another possible mechanism involved in the activation of cardiac fibers by H22 could be its low ph, which is =5.9 at a concentration of 1 mol/l. We examined this possibility in several experiments (data not shown) by applying 1,tL of HCI solutions with ph in the range of 6.1 to 5.4 to the receptive fields of chemosensitive C fibers. HCl solutions with ph in this range did not cause activation of cardiac afferents. These findings are in accordance with the data of Uchida and Murao,18 which showed that the threshold ph for activation of cardiac sympathetic fibers with application of HCl is 3.85 to 3.2. Apparently, cardiac afferent endings, both vagal and sympathetic, are less sensitive to the changes of ph than are carotid body chemoreceptors, which can be activated at ph The results of the present study suggest that activation of cardiac vagal afferents sensitive to H22 is caused by hydroxyl radicals that are formed from H22.2,21 The antioxidant deferoxamine, which prevents the formation of hydroxyl radicals by its iron-chelating properties,22'23 and a scavenger of hydroxyl radicals, DMTU, both completely abolished the activation of chemosensitive fibers in response to H22 application. These results strongly suggest that activation of the fibers was mediated by the hydroxyl radical. At the same time, superoxide dismutase at a dose of U/kg, which has been shown to effectively metabolize the superoxide anion in the rat heart,23 did not prevent C-fiber activation in response to H22. Application of xanthine/xanthine oxidase to the heart results in formation of superoxide radicals. Both superoxide dismutase and deferoxamine decreased the effect of xanthine/xanthine oxidase on cardiac afferent fibers, but only deferoxamine abolished this effect. Deferoxamine chelates iron ions and limits the Haber-Weiss reaction, thus preventing the formation of hydroxyl radicals in the pathway. Superoxide dismutase scavenges the superoxide anion by transforming the superoxide anion into H22. However, H22 produced in this enzymatic reaction can, in turn, enter the Haber-Weiss reaction with the formation of hydroxyl radicals. The inability of superoxide dismutase to prevent the formation of hydroxyl radicals may explain why superoxide dismutase inhibited the effect of xanthine/xanthine oxidase less effectively than did deferoxamine. Because deferoxamine blocks production of the hydroxyl radical but not the superoxide anion, its ability to abolish the afferent response to xanthine/xanthine oxidase strongly suggests that the hydroxyl radical is a more important factor in the activation of cardiac afferents than is the superoxide anion. In the present experiments, neither deferoxamine nor DMTU totally abolished the alterations in left ventricular pressure or the arrhythmias produced by H22 application, even though the antioxidants effectively abolished afferent responses to H22. We did not specifically address the nature of the effects of the prooxidants on cardiac function in the present study; however, we observed in preliminary experiments (data not shown) that the alterations in cardiac function induced by H22, particularly arrhythmias, were not abolished by vagotomy and thus were not mediated to free radical-generating systems is known to impair contractile function and induce cardiac arrhythmias.21'23 Because H22 is readily metabolized by catalase and glutathione enzymes found in cardiac tissue,24 it is likely that much of the H22 applied to the epicardial surface was degraded considerably by the time H22 diffused into the subepicardium. Thus, the concentration of H22 diffusing to afferent endings located within the myocardium would have been far less than that reaching cardiac myocytes at the surface of the heart. These differences in the effective concentrations of H22 could explain the fact that deferoxamine and DMTU abolished H22-induced activation of nerve endings and had much less effect on H22-induced disturbances of cardiac contractility and rhythm. Only about half of the capsaicin-sensitive fibers could be activated by H22 application, which may suggest that the chemosensitive C-fiber afferents as a population are not uniform in chemical sensitivities. In addition, we could find no evidence that oxygen-derived free radicals directly affect either atrial or ventricular endings in the heart that did not respond to capsaicin. Changes in activity that did occur with the mechanosensitive afferents in response to H22 were always associated with changes in cardiac mechanics. One could postulate, however, that endings unresponsive to H22 were located much deeper in the myocardium and were not accessible to topical application of the chemical. Because H22 could not be administered into the systemic circulation, we were limited to topical administration of the pro-oxidant to the heart. However, mechanosensitive endings were unresponsive to capsaicin regardless of whether capsaicin was applied topically over the receptive field or injected into the systemic circulation. Chemosensitive afferent endings always responded with a volley of impulses to either route of administration of capsaicin. In a few experiments, we attempted systemic injections of xanthine/xanthine oxidase, but the cardiac effects of this pro-oxidant mixture were always accompanied by marked arrhythmias and changes in cardiac pressures, which made interpretation of the afferent recordings difficult. Nevertheless, even with systemic administration of the pro-oxidant, we found no evidence of a direct chemical stimulation of mechanosensitive endings. Our observation that the pro-oxidants stimulated some chemosensitive and no mechanosensitive afferents would argue against the notion that the effect of oxygen-derived free radicals on nerve endings is a nonspecific effect on cellular membrane integrity. The mechanism by which oxygen-derived free radicals activate chemosensitive endings remains to be elucidated. A variety of possible mechanisms could be proposed that include interaction with ligand-gated ion channels, inactivation of Na,K- ATPase,25 or release of paracrine mediators (eg, histamine, bradykinin, and leukotrienes). Inhibition of prostaglandin synthesis is known to abolish cardioinhibitory and vasodepressant reflexes mediated by activation of vagal afferent C fibers in ischemia.26 One of the questions that we addressed was whether the effect of free radicals on cardiac afferents could be mediated by stimulating prostaglandin synthesis. We found that inhibition of cyclooxygenase activity by indomethacin did not affect the activation of cardiac afferents in response to solely by a vagal reflex. Exposure of isolated rat hearts H22. This result indicated that the effect of hydroxyl

10 Ustinova and Schultz Cardiac Afferents and Oxygen Radicals 93 radicals to stimulate chemosensitive endings in the heart is not mediated by an effect on prostaglandin synthesis. A unique secondary finding in these experiments was the fact that antioxidants decreased the response of the chemosensitive endings to capsaicin, although the effect of DMTU was not statistically significant. This phenomenon may indicate that the endogenous production of free radicals acts to sensitize cardiac vagal chemosensitive afferent endings to chemical stimuli. The same assumption was made by Lai,27 who found that the administration of DMTU ameliorated capsaicin-induced bronchoconstriction in rats. In a similar manner, results from our experiments indicated that endogenous prostaglandins may sensitize chemosensitive C fibers in the heart to capsaicin, since the afferent response to capsaicin also was attenuated by indomethacin. These results raise the question whether prostaglandin production influences afferent C-fiber sensitivity to capsaicin through the generation of free radical intermediates such as those involved in the formation of prostaglandin G2. The functional implications of the effect of oxygenderived free radicals on cardiac vagal chemosensitive endings remain to be fully understood. However, it is likely that these metabolites play an important role in the activation of cardiac chemosensitive endings in response to oxidative stress to the myocardium. In the accompanying article in this journal,28 we describe the role of oxygen-derived free radicals in mediating the afferent response of vagal cardiac chemosensitive endings to myocardial ischemia and reperfusion. Such an effect of these oxygen-derived free radicals on cardiac chemosensitive afferents could initiate the vasodepressor and cardioinhibitory reflexes that are known to occur at the onset of myocardial reperfusion.29'3 In summary, the results of the present study indicate that the rat heart possesses a notable innervation of vagal chemosensitive afferent nerve endings and that many of these endings respond to the chemical generation of oxygen-derived free radicals. Formation of the hydroxyl radical appears to play a major role in this response, since the effects of both xanthine/xanthine oxidase and H22 can be totally abolished by either deferoxamine or DMTU. Activation of cardiac chemosensitive vagal afferents by free oxygen radicals is not mediated by the cyclooxygenase system. The mechanism by which these oxygen-derived free radicals stimulate chemosensitive endings in the heart remains an important question for further study. Acknowledgments This study was supported by grant HL from the National Heart, Lung, and Blood Institute and Grants-in-Aid from the American Heart Association. Dr Ustinova is a postdoctoral fellow of the American Heart Association, Nebraska Affiliate, Inc. Dr Schultz is an Established Investigator of the American Heart Association. References 1. Thoren P. Left ventricular receptors activated by severe asphyxia and by coronary artery occlusion. Acta Physiol Scand. 1972;85: Thoren P. Activation of left ventricular receptors with nonmedullated vagal afferent fibers during occlusion of a coronary artery in cat. Acta Physiol Scand. 1976;37: Coleridge HM, Coleridge JCG. Cardiovascular afferents involved in regulation of peripheral vessels. Annu Rev Physiol. 198;42: Coleridge HM, Coleridge JCG, Kidd C. Cardiac receptors in the dog with particular reference to two types of afferent ending in the ventricular wall. J Physiol (Lond). 1964;174: Coleridge JC, Coleridge HM, Pissari TE, Schultz HD. Stimulation of cardiac vagal chemosensitive C-fibers by coronary occlusion in dogs. FASEB J. 199;4:A77. Abstract. 6. Baker DG, Coleridge HM, Coleridge JCG. Vagal afferent C-fibers from the ventricle. In: Hainsworth R, Kidd C, Linden RJ, eds. Cardiac Receptors. London, England: Cambridge University Press; 1979: Kaufman MP, Baker HM, Coleridge HM, Coleridge JCG. Stimulation by bradykinin of afferent vagal C-fibers with chemosensitive endings in the heart and aorta of the dog. Circ Res. 198;46: Grisam MB, McCord JM. Chemistry and cytotoxicity of reactive oxygen metabolites. In: Taylor AE, Matalon S, Ward P, eds. Physiology of Oxygen Radicals. Bethesda, Md: American Physiological Society; 1985: Stahl GL, Pan H-L, Longhurst JC. Activation of ischemia- and reperfusion-sensitive abdominal visceral C fiber afferents: role of hydrogen peroxide and hydroxyl radicals. Circ Res. 1993;72: Stahl GL, Halliwell B, Longhurst JC. 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