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Am J Physiol Heart Circ Physiol 281: H93-H104, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 1, H93-H104, July 2001

Activation of cardiac afferents by arachidonic acid: relative contributions of metabolic pathways

Shu-Yu Sun, Wei Wang, and Harold D. Schultz

Department of Physiology and Biophysics, University of Nebraska College of Medicine, Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arachidonic acid (AA) is metabolized via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P-450 (CP450) pathways to a variety of bioactive products. The sensitivity of cardiac afferent endings to AA and its metabolites, especially those derived from LOX and CP450 pathways, is currently unclear. We examined AA-induced activation of cardiac vagal chemosensitive afferents in non- and postischemic hearts in rats and evaluated the relative contributions of the three metabolic pathways to the effects. Epicardial application of AA activated the cardiac afferents dose dependently in both nonischemic and postischemic hearts, with afferent responses greater in the latter condition. In nonischemic hearts, the afferent response to AA was abolished only after simultaneous administration of indomethacin and 17-octadecynoic acid (COX and CP450 inhibitors, respectively). Nordihydroguaiaretic acid (a LOX inhibitor) had no effect on the afferent response to AA. In postischemic hearts, abolition of the afferent response to AA required simultaneous blockade of all three pathways. None of the AA metabolic inhibitors affected resting activity of cardiac afferents in nonischemic hearts, but each suppressed afferent activity during ischemia-reperfusion. Most COX metabolites, CP450 metabolites, and 5-LOX metabolites tested were capable of activating cardiac afferents. The 12-LOX metabolites and 15-LOX metabolites had no effect on afferent activity. These data indicate that in the nonischemic heart, basal AA metabolism does not contribute to resting afferent activity, but AA is capable of activating cardiac afferents via COX and CP450 but not LOX pathways. During ischemia-reperfusion, all three metabolic pathways contribute to activation of cardiac vagal afferents with an enhanced responsiveness to AA. Our results suggest that induction of the 5-LOX pathway contributes to the enhanced sensitivity of cardiac vagal afferents to AA in the ischemic condition.

ventricular receptors; vagus; cyclooxygenase; lipoxygenase; cytochrome P-450; ischemia-reperfusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ARACHIDONIC ACID (AA), one of the principal fatty acids, is liberated from membrane lipids in response to physiological, pharmacological, and pathological stimuli. Free AA is metabolized by three enzyme systems: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P-450 (CP450), which produce prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs) (9). During the past two decades, studies of the biological significance of AA and its metabolites focused on their role in regulation of smooth muscle tone (respiratory, vascular, and intestinal), and their participation in inflammatory response and tissue injury under a variety of disease conditions (23). However, there has been evidence indicating that PGs, the COX metabolites of AA, may play an important role in the regulation of cardiovascular function via modulating cardiac afferent neural activity (11). For instance, intracoronary administration of PGI2 evokes a Bezold-Jarisch-like reflex in dogs (15). Moreover, brief occlusion of the circumflex coronary artery in dogs results in a decrease in arterial pressure and renal sympathetic nerve activity (31) and an inhibition of the arterial baroreflex (38). These effects can be reversed by blockade of PG synthesis with indomethacin. Recent evidence (32) has shown that indomethacin effectively prevents the activation of cardiac vagal chemosensitive afferent endings at an early stage of coronary occlusion. These data indicate that during myocardial ischemia, PGs serve as a major stimulus to cardiac chemosensitive endings with sympathoinhibitory vagal afferent fibers.

AA metabolism is enhanced not only during myocardial ischemia but also, even to a greater degree, during post-ischemia-reperfusion (10, 33). In addition to the more intensively studied COX pathway (4), AA metabolism through LOX and CP450 pathways is also augmented during ischemia, as manifested by increased accumulation of LOX and CP450 metabolites within cardiac tissue following myocardial ischemia-reperfusion (16, 20, 27). However, little is known regarding the role of AA metabolites on cardiac afferent function in the postischemic heart. Therefore, this study was designed to evaluate the relative contributions of the three AA metabolic pathways to activation of cardiac vagal afferent endings in normal (nonischemic) hearts and hearts subjected to myocardial ischemia-reperfusion. To do this, afferent responses to epicardial application of AA and to coronary occlusion and reperfusion were documented in the rat heart, and the effects of selective antagonists for COX, CP450, and LOX on the afferent responses were examined.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General Preparation

All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and were carried out in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the American Physiological Society.

Young adult Sprague-Dawley rats (250-320 g, male) were anesthetized intraperitoneally with combination of alpha -chloralose (40 mg/kg) and urethan (800 mg/kg). The trachea was cannulated and the lungs ventilated with a Harvard rat respirator (65 strokes/min × 1 ml/100 g) with air supplemented with O2. Body temperature was maintained at 37 ± 1°C by a heating pad. Polyethylene-50 catheters were inserted into a femoral artery and vein for the measurement of arterial pressure and administration of drugs, respectively. The chest was opened via a sternotomy, and the heart was exposed for epicardial application of drugs and for exploration of a receptive field for the afferent endings. Arterial pressure was measured with the use of a pressure transducer (model 1270A, Hewlett-Packard) connected to the arterial catheter. Heart rate was measured with the use of a cardiotachometer (model S77-26, Coulbourn Instruments) triggered by the differential signal of the pulsatile arterial pressure. Heart rate, arterial pressure, and nerve activity (see below) were monitored and recorded with the use of a MacLab System (ADInstruments) as well as by a thermal chart recorder (DASH IV, Astro-Med). Silk snares were looped around the ascending aorta and inferior vena cava to produce occlusion when necessary to ascertain the mechanosensitivity of the endings. Estimated fluid loss was replaced by intravenous infusion of physiological saline at 2-3 ml · kg-1 · h-1.

Nerve Recordings

The cervical vagus of either side was dissected free from the surrounding connective tissues and was then covered with mineral oil. Vagal afferent impulses were recorded from "single fibers" split from fine slips that were dissected from the distal cut end of the vagus. The nerve fibers were gently placed on a silver electrode. Impulses were amplified with a preamplifier (model P511J, Grass Instruments) with a filter width of 100 Hz-3 kHz, displayed on an oscilloscope, and fed into a rate meter with window discriminators set to accept potentials of a particular amplitude. The rate meter counted the impulses by 1-s bins.

Identification of Cardiac Afferent Axons

Only fibers with spontaneous discharge, and with their receptive fields located in either the left or right ventricle, were studied. The receptive field of a fiber was located first by gently probing the heart surface with a fine cotton-tipped applicator. Fibers that exhibited a volley of discharges in response to the probing were then tested for chemo- or mechanosensitivity. Fibers with receptive fields that could not be located were discarded.

The chemosensitivity of a sensory field associated with an afferent fiber was tested by epicardial application of capsaicin over its receptive field. Capsaicin was chosen as the test chemical because it is known to directly stimulate only chemosensitive endings, not cardiovascular mechanoreceptors (6). In addition, capsaicin is known to selectively stimulate afferent endings with slowly conducting unmyelinated and finely myelinated (C and Adelta ) fibers (6). However, because we did not measure conduction velocities in our afferent recordings, we did not attempt to categorize afferent responses with respect to fiber type. In preliminary experiments, we verified that epicardial capsaicin did not affect afferent responses to AA. Capsaicin was applied via a circular patch of filter paper with diameter of ~3 mm. The doses of capsaicin used were 0.1 and 1.0 µg in a volume of 10 µl.

Mechanosensitivity of a sensory field associated with an afferent fiber was identified by the timing and pattern of its discharge related to the cardiac rhythm and was further tested by aortic and inferior vena caval occlusions. Endings that exhibited rhythmic discharges and readily respond to changes in the intracardiac pressures were defined as mechanoreceptors. Mechanosensitive endings were not investigated in this study. Endings responding to both capsaicin and changes in intracardiac pressure were also excluded to avoid confusion of the categorization. Therefore, all of the studied afferent fibers were chemosensitive.

Drug Administration

AA and its metabolites were applied to the epicardium in the same manner as that of capsaicin (see Identification of Cardiac Afferent Axons). Each chemical was applied for 1 min. After the stimulus was removed, the epicardial field was washed with warm saline for 1 min. An interval of >= 10 min was allowed between two consecutive applications. In our preliminary experiments, it was determined that an interval of >= 5 min between two consecutive applications of AA was required to prevent tachyphylaxis. Three doses of AA (0.3, 3, and 30 nmol) were applied randomly. The AA metabolites tested in this study were PGI2, PGE2, PGF2alpha , thromboxane (Tx) A2 (TxA2) and TxB2 (COX metabolites); 14,15-EET (a CP450 epoxygenase metabolite) and 20-HETE (a CP450 hydroxylase metabolite); leukotriene (LT) B4, C4, D4, and E4 (5-LOX metabolites); 12-hydroperoxyeicosatetraenoic acid (HpETE) and 12-HETE (12-LOX metabolites); 15-HpETE, 15-HETE, 8(S), 15(S)-dihydroxyeicosatrienoic acid (DiHETE), and 8(R), 15(S)-DiHETE (15-LOX metabolites). The doses for each metabolite were 0.3 and 3 nmol. For the purpose of a negative control, three nonsubstrate forms of fatty acids were also tested for their effect on the activity of cardiac afferent axons. The forms were: palmitic acid (16:0), a saturated fatty acid; linoelaidic acid (18:2), a trans-polyunsaturated fatty acid; and oleic acid (18:1), a cis-monounsaturated acid.

The fatty acids and AA metabolites were aliquoted and diluted in 95% ethanol to appropriate stock concentrations in airtight microvials that had been purged with N2 and stored at -20°C. On a daily basis, the fatty acids and AA metabolites were removed from the vial and diluted with saline under N2 to appropriate concentration before each experiment. The final concentration of ethanol as a vehicle was not >1%. In our preliminary experiments, 1% ethanol was found to have no effects on the activity of cardiac afferent axons.

The inhibitors of COX, LOX, and CP450 used in the present study were indomethacin (INDO), nordihydroguaiaretic acid (NDGA), and 17-octadecynoic acid (17-ODYA), respectively. The inhibitors were dissolved in 95% ethanol to appropriate stock concentration and stored at -20°C. The final concentration was diluted with saline under N2 before each experiment.

The fatty acids, AA metabolites, and 17-ODYA were purchased from Cayman (Ann Arbor, MI). INDO and NDGA were purchased from Sigma (St. Louis, MO).

Protocols

Reproducibility of the AA-induced activation of cardiac afferent axons. First, we tested whether the AA-induced activation of cardiac afferents was reproducible over the course of our experimental design in 10 afferent fibers obtained from 6 rats. We then examined single-unit activity of the cardiac afferents in response to epicardial application of graded doses of AA (0.3, 3, and 30 nmol) before and 20 min after an intravenous injection of vehicle (1% ethanol) for AA metabolic inhibitors. The second test served as the time control for the effects of AA metabolic inhibition. The doses of AA were applied randomly to the sensory field.

Effects of COX, LOX, and CP450 inhibition on AA-induced activation of cardiac afferent axons in nonischemic hearts. We examined the effects of COX, LOX, and CP450 inhibition in three separated groups of normal hearts treated with either INDO (5 mg/kg), NDGA (5 mg/kg), or 17-ODYA (1 mg/kg), respectively. These doses have been shown to effectively block their corresponding enzymes in previous studies (2, 30, 37). All inhibitors were administered intravenously in a volume of 0.3-0.5 ml >1 min. We examined the responses of cardiac afferents to epicardial application of graded doses of AA before and 20 min after administration of the AA metabolic inhibitors. We used nine rats in the INDO group, eight in the NDGA group, and nine in the 17-ODYA group. We studied one fiber from each rat.

Because we found that INDO and 17-ODYA attenuated but did not abolish AA-induced activation of the cardiac afferents, we examined the effect of combined blockade of COX and CP450 by coadministration of INDO (5 mg/kg) and 17-ODYA (1 mg/kg) in an additional group of rats (n = 7).

Effects of COX, LOX, and CP450 inhibition on AA-induced activation of cardiac afferent axons in postischemic hearts. AA metabolism within cardiac tissue ensues at a low level under normal conditions but is activated during myocardial ischemia-reperfusion (23). Therefore, we conducted a series of experiments to evaluate whether the enhanced AA metabolism via the various potential pathways due to myocardial ischemia-reperfusion has any impact on the activity of cardiac afferent axons.

First, we examined the sensitivity of cardiac afferents to epicardial application of AA in the postischemic heart without inhibition of AA metabolism. A control response of the cardiac afferents to graded doses of AA (0.3, 3, and 30 nmol) was determined before the initiation of myocardial ischemia-reperfusion. The heart was then subjected to 30 min of ischemia by ligating the left anterior descending coronary artery (LAD) and a 15-min reperfusion by releasing the snare around the LAD. The ischemia and reflow were confirmed by sight: the ischemic area blanched pale and turned cyanotic with occlusion of LAD and regained normal color with release of the snare. At the end of 15-min reperfusion, we reexamined the afferent response to AA.

We then examined the afferent response to AA in the postischemic heart in separate groups after blockade of individual metabolic pathways with either INDO (5 mg/kg), NDGA (5 mg/kg), or 17-ODYA (1 mg/kg). AA metabolic inhibitors were administered intravenously 10 min before ischemia commenced, and immediately after the control, AA dose response was taken.

We conducted additional experiments to examine the effect of combined blockade of COX and CP450 by coadministration of INDO (5 mg/kg) and 17-ODYA (1 mg/kg) and the combined blockade of all three metabolic pathways by coadministration of INDO (5 mg/kg), 17-ODYA (1 mg/kg) and NDGA (5 mg/kg) in two groups of rats (n = 6).

Effects of AA metabolites on the activity of cardiac afferent axons. We examined the effect of several AA metabolites from each of the three metabolic pathways on the activity of the cardiac afferents to screen for potential active metabolites that stimulate the cardiac afferents. Among the COX metabolites, PGI2, PGE2, and PGF2alpha were tested in eight fibers obtained from five rats; TxA2 and TxB2 were tested in seven fibers obtained from four rats. CP450 metabolites (14,15-EET and 20-HETE) were tested in eight fibers from four rats. The 5-LOX metabolites (LTB4, LTC4, LTD4, and LTE4) were tested in six fibers from four rats. The 12-LOX metabolites (12-HETE and 12-HpETE) and 15-LOX metabolites (15-HETE and 15-HpETE) were tested in five fibers from four rats. The 15-LOX related derivatives, 8(S),15(S)-DiHETE and 8(R),15(S)-DiHETE, were also tested in eight fibers from four rats. All metabolites were applied to the epicardial loci at two doses, 0.3 and 3 nmol, in random order.

Effects of nonsubstrate fatty acids on the activity of cardiac afferent axons. To assess any nonspecific effect of AA on the activity of cardiac afferents, we examined the effect of three nonsubstrate fatty acids, palmitic acid, linoelaidic acid, and oleic acid, on the activity of cardiac vagal afferents in eight fibers from five rats. The fatty acids were applied to the epicardial loci at the same doses as AA (0.3, 3, and 30 nmol), in random order.

Data Analysis

The activity of cardiac afferent axons was expressed as the number of impulses per second. The baseline activity was calculated over 5 s of maximal activity during a 1-min control recording, and the activity in response to fatty acids or AA metabolites was measured over 5 s of the maximal activity during the 1-min test stimulus. AA-induced responses and the effects of AA metabolic inhibition on the responses were analyzed by two-way analysis of variance (ANOVA) for repeated measures and the differences in responses between doses and between control responses and treatments were isolated by Newman-Keuls tests. AA metabolite-induced responses were analyzed with one-way ANOVA for repeated measures, and Dunnet's multiple range test was used for comparisons between treatments and control. All values are expressed as means ± SE. Statistical significance was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reproducibility of AA-Induced Activation of Cardiac Afferent Axons

The response characteristics of the sensory fields associated with 10 afferent fibers were examined in 6 rats. Seven fibers originated from the left ventricle and three from the right ventricle. Because there were no obvious differences found in the discharging characteristics and in the responses to AA between the fibers from the left ventricle and right ventricle, the data were pooled for statistical analysis. Epicardial application of AA activated cardiac vagal afferents in a dose-dependent manner (Fig. 1A and Table 1) with a latency of 20-40 s (Fig. 1B). The afferent activity increased and reached its plateau rapidly and remained at the plateau level during the 1-min period of AA application. After the stimulus was removed and heart surface was washed with saline, afferent activity returned to the prestimulation level gradually (Fig. 1B). The response of cardiac afferents to AA was reproducible following intravenous infusion of vehicle for the metabolic inhibitors (see Table 1).


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  		Fig. 1.   Original recordings of action potentials (AP) and rate meter counts from a cardiac vagal afferent axon from the left ventricle. A: epicardial application of arachidonic acid (AA) increased discharge frequency (DF) in a dose-dependent manner. Numbers at the lower right of each recording indicate the dose of AA. B: rate meter counts of DF (top) from a cardiac vagal afferent axon showing the time course and pattern of neural activity as well as the accompanying arterial blood pressure (ABP; bottom) in response to epicardial application of 3.0 nmol of AA.


                              
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  		Table 1.   Reproducibility of AA-induced activation of cardiac vagal chemosensitive afferent axons in two consecutive tests before and after intravenous vehicle in nonischemic hearts

Epicardial AA at 0.3 and 3.0 nmol did not cause significant changes in arterial pressure (Fig. 1B) and heart rate. Thirty nanomoles of AA induced hypotension (from 76 ± 2 to 67 ± 2 mmHg of mean pressure, P = 0.0115) without altering heart rate (414 ± 6 to 414 ± 5 beats/min, P = 0.9595) before and after vehicle infusion.

Effects of COX, LOX, and CP450 Inhibition on AA Induced Activation of Cardiac Afferent Axons in Nonischemic Hearts

The afferent response to epicardial application of AA was attenuated but not abolished after COX inhibition by INDO (5 mg/kg iv) (Fig. 2A). Nine fibers (9 rats) were studied: seven from the left ventricle and two from the right ventricle. The hypotensive response induced by 30 nmol of AA was abolished by INDO (66 ± 5.0 vs. 70 ± 7.8 mmHg, P = 0.4241). INDO itself decreased resting heart rate (from 367 ± 13 to 334 ± 11 beats/min, P = 0.0328) without altering mean blood pressure (63 ± 6.3 to 67 ± 6.4 mmHg, P = 0.0717) or afferent activity (Table 2).


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  		Fig. 2.   DF of cardiac vagal afferent axons in response to epicardial AA before and after blockade of cyclooxygenase (COX) (A; n = 9), lipoxygenase (LOX) (B; n = 8), cytochrome P-450 (CP450) (C; n = 9), and COX + CP450 (D; n = 7) in nonischemic rat hearts. Afferent endings were not affected by vehicle (0 nmol of AA). Thus DF at 0 nmol represents resting (baseline) discharge. A-C: afferent responses to AA were significantly attenuated by blockade of either COX [A, indomethacin (INDO) 5 mg/kg] or CP450 [C, 17-octadecynoic acid (17-ODYA), 1 mg/kg], but not LOX [B, nordihydroguaiaretic acid (NDGA), 5 mg/kg]. D: combined blockade of COX and CP450 (INDO+17-ODYA) abolished the AA-induced activation of cardiac afferents. *P < 0.05; **P < 0.01, AA dose vs. baseline (0 nmol); dagger P < 0.05, treatment vs. control.


                              
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  		Table 2.   Effects of AA metabolic inhibitors on resting activity of cardiac vagal afferent axons in nonischemic hearts

The afferent response to AA also was attenuated after CP450 inhibition by 17-ODYA (1 mg/kg iv) (Fig. 2C). Nine fibers were studied: eight from the left ventricle and one from the right ventricle. 17-ODYA abolished the hypotensive response to 30 nmol of AA (68 ± 5.2 vs. 64 ± 4.6 mmHg, P = 0.5218). 17-ODYA did not affect resting afferent activity (see Table 2), heart rate (361 ± 9 vs. 353 ± 11 beats/min, P = 0.2797), or mean blood pressure (71 ± 4.0 vs. 75 ± 5.7 mmHg, P = 0.2242).

The AA-induced activation of the cardiac afferents was not affected by LOX inhibition by NDGA (5 mg/kg iv) (Fig. 2B). Eight fibers were studied: five from the left ventricle and three from the right ventricle. NDGA did not affect the hypotension induced by 30 nmol of AA (from 80 ± 4.5 to 72 ± 4.3 mmHg, P = 0.0448). NDGA itself decreased resting heart rate (from 371 ± 29 to 337 ± 17 beats/min, P = 0.0392) without altering mean blood pressure (73 ± 5.9 vs. 72 ± 5.4 mmHg, P = 0.3910) or afferent activity (Table 2).

Combined blockade of COX and CP450 by coadministration of INDO (5 mg/kg) and 17-ODYA (1 mg/kg) abolished AA-induced activation of the cardiac afferents (see Fig. 2D) and the accompanying hypotension at 30 nmol of AA (70 ± 8.9 vs. 77 ± 11.6 mmHg, P = 0.7202). Seven fibers were studied, all from the left ventricle. Combined blockade of COX and CP450 itself decreased resting heart rate (from 360 ± 19 to 323 ± 8 beats/min, P = 0.0078) without altering mean blood pressure (72 ± 7.5 vs. 80 ± 12.2, P = 0.3671) or afferent activity (Table 2).

Effects of COX, LOX, and CP450 Inhibition on AA-Induced Activation of Cardiac Afferent Axons in Postischemic Hearts

The resting activity of the cardiac afferents increased at the onset of ischemia (Table 3) and remained elevated through the end of the reperfusion period (Fig. 3). In addition, the afferent response to AA was enhanced after ischemia-reperfusion (see Fig. 3). Six fibers were examined in this group: four from the left ventricle (within the ischemic zone) and two from the right ventricle (outside the ischemic zone). Because the responses to AA after ischemia were enhanced similarly in fibers within and outside the ischemic zone, the data were pooled for statistical analysis. Similar to the effect in nonischemic hearts, 30 nmol of AA induced hypotension (from 83 ± 4 to 72 ± 3 mmHg mean pressure, P = 0.0118) in postischemic hearts without altering heart rate. Smaller doses of AA did not affect hemodynamics in this condition.

                              
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  		Table 3.   Effects of AA metabolic inhibitors on activity of cardiac vagal afferent axons during coronary occlusion



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  		Fig. 3.   DF of cardiac vagal afferent axons in response to epicardial application of AA in ischemic-reperfused (IR) rat hearts. The resting activity (0 nmol) and the afferent responses to AA were significantly increased after IR. Blockade of either COX (INDO 5 mg/kg), CP450 (17-ODYA, 1 mg/kg) and LOX (NDGA, 5 mg/kg) individually, or combined blockade of COX and CP450 (INDO+17-ODYA) attenuated the resting afferent activity (0 nmol) and the activity-induced by AA after IR. Increased resting activity and the AA-induced activation were abolished after simultaneous blockade of all three metabolic pathways (INDO + 17-ODYA + NDGA). *P < 0.05, **P < 0.01, AA dose vs. baseline (0 nmol) within treatments; #P < 0.05, IR vs. control; dagger P < 0.05, treatment vs. IR.

Ischemia-reperfusion caused premature beats and transient ventricular fibrillation in all rats. This occurred irregularly throughout the period of ischemia and the early stage of reperfusion, particularly during the first 1-2 min of reperfusion. Arrhythmias disappeared by the end of the 15-min reperfusion period, and neither resting mean arterial pressure (control 83 ± 6 vs. postischemic 84 ± 7 mmHg, P = 0.9240) nor the heart rate (402 ± 20 vs. 388 ± 24 beats/min, P = 0.6793) differed from control.

We examined the effect of COX inhibition (INDO, 5 mg/kg iv), CP450 inhibition (17-ODYA, 1 mg/kg iv), and LOX inhibition (NDGA, 5 mg/kg iv) on the afferent response to AA after myocardial ischemia-reperfusion. Similar to the nonischemic state, blockade of either COX (4 fibers within and 2 outside the ischemic zone) or CP450 pathway (5 fibers within and 1 outside the ischemic zone) attenuated the AA-induced activation of the cardiac afferent axons in postischemic hearts (Fig. 3). In contrast to the nonischemic heart, LOX inhibition (4 fibers within and 2 outside the ischemic zone) attenuated AA-induced activation in the postischemic condition.

Also, unlike the nonischemic state, combined COX and CP450 blockade attenuated but did not abolish AA-induced activation of the cardiac afferent endings (5 within and 1 outside the ischemic zone) in the postischemic heart (see Fig. 3). Simultaneous blockade of all three metabolic pathways was required to abolish activation of the cardiac afferent endings (5 within and 1 outside the ischemic zone) induced by AA (see Fig. 3). Moreover, blockade of AA metabolic pathways, either individually or in combination, attenuated the resting activity generated by cardiac afferent axons during the ischemic (see Table 3) and reperfusion (see Fig. 3) periods.

Effects of AA Metabolites and Nonsubstrate Fatty Acids on the Activity of Cardiac Afferent Axons

Among the COX metabolites, PGI2, PGE2, and TxA2 activated cardiac chemosensitive afferents. PGF2alpha and TxB2 were not effective (Figs. 4 and 5). Both CP450 epoxygenase and hydroxylase metabolites, 14,15-EET and 20-HETE, respectively, activated the cardiac afferents (Fig. 6). All of the tested LTs (LTB4, LTC4, LTD4, and LTE4) activated the cardiac afferents (Fig. 7). Neither 12-LOX metabolites (12-HETE and 12-HpETE) nor 15-LOX metabolites (15-HETE and 15-HpETE) affected cardiac afferent activity (Fig. 8). Similarly, 8(S),15(S)-DiHETE and 8(R),15(S)-DiHETE had no effect on the activity of the cardiac afferents (Fig. 9). Except for the high dose of PGI2 (3 nmol), which caused a hypotension (from 75 ± 7 to 49 ± 3 mmHg of mean pressure, P = 0.0107), no other tested AA metabolites affected either arterial pressure or heart rate. In contrast to AA, palmitic acid, linoelaidic acid, and oleic acid did not activate cardiac chemosensitive afferents (Table 4).


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  		Fig. 4.   DF of cardiac vagal afferent axons (n = 5) in response to prostaglandin (PG) I2 (PGI2), PGE2, and PGF2alpha . PGI2 (A) and PGE2 (B) activated the cardiac afferents, but PGF2alpha (C) did not. *P < 0.05, **P < 0.01, prostaglandin dose vs. baseline (0 nmol).



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  		Fig. 5.   DF of cardiac vagal afferent axons (n = 4) in response to thromboxane (Tx) A2 (TxA2) and TxB2. TxA2 (A) activated the cardiac afferents but TxB2 (B) did not. *P < 0.05, **P < 0.01, Tx dose vs. baseline (0 nmol).



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  		Fig. 6.   DF of cardiac vagal afferent axons (n = 4) in response to AA metabolites derived from CP450 pathway. 14,15-epoxyeicosatrienoic acid (EET) (A) and 20-hydroxyeicosatrienoic acid (HETE) (B) activated the cardiac afferents. *P < 0.05, **P < 0.01, CP450 metabolite dose vs. baseline (0 nmol).



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  		Fig. 7.   DF of cardiac vagal afferent axons (n = 4) in response to AA metabolites derived from 5-LOX pathway. A-D: leukotrienes (LTs) activated the cardiac afferents. *P < 0.05, **P < 0.01, LT dose vs. baseline (0 nmol).



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  		Fig. 8.   DF of cardiac vagal afferent axons (n = 4) in response to AA metabolites derived from the 12- and 15-LOX pathways. 12-HETE (A), 12-hydroperoxyeicosatetranoic acid (HpETE) (B), 15-HETE (C), and 15-HpETE (D) were not effective in activating the cardiac afferents.



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  		Fig. 9.   DF of cardiac vagal afferent axons (n = 4) in response to AA metabolites derived from 15-LOX pathway. 8(S), 15(S)-dihydroperoxyeicosatetranoic acid (DiHETE) (A) and 8(R), 15(S)-DiHETE (B) were not effective in activating the cardiac afferents.


                              
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  		Table 4.   Effects of nonsubstrate fatty acids on the activity of cardiac vagal afferent axons


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There are four major conclusions of this study. First, epicardial application of exogenous AA, but not related fatty acids, activates cardiac vagal chemosensitive afferent endings via COX, CP450 and LOX pathways in the rat heart. Second, in nonischemic hearts, AA induced activation of cardiac afferents is mediated by COX and CP450 pathways but not the LOX pathway. However, endogenous AA metabolism does not appear to contribute to the resting activity of cardiac vagal afferents in the nonischemic condition. Third, in postischemic hearts, AA-induced activation of the cardiac afferents is enhanced and is mediated by LOX as well as COX and CP450 pathways. Moreover, enhanced endogenous AA metabolism through each of the three major AA pathways contributes to the elevated tonic activity of the cardiac afferents observed during ischemia-reperfusion. Fourth, AA-induced activation of cardiac afferents is mediated by many, but not all, of its metabolites. Active metabolites include: COX metabolites, PGI2, PGE2, and TxA2; CP450 metabolites, 14,15-EET and 20-HETE; and 5-LOX metabolites, LTB4, LTC4, LTD4, and LTE4. Inactive metabolites include: COX metabolites PGF2alpha and TxB2; 12-LOX metabolites 12-HETE and 12-HpETE; and 15-LOX metabolites 15-HETE, 15-HpETE, 8(S),15(S)-DiHETE and 8(R),15(S)-DiHETE. These findings are illustrated in Fig. 10.


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  		Fig. 10.   Schematic summary of the effects of AA metabolic pathways and their metabolites on activation of cardiac vagal chemosensitive endings during nonischemic and ischemic conditions. Bold font, metabolites effective in stimulation of cardiac vagal afferents; italic font, metabolites not effective in stimulation of cardiac vagal afferents; regular font, metabolites not tested.

We chose INDO, NDGA, and 17-ODYA to inhibit COX, LOX, and CP450 enzymes, respectively, because they are the most commonly used AA metabolic inhibitors. On the basis of previous studies, we are confident that the doses of INDO, NDGA, and 17-ODYA adopted by the present study were adequate to effectively and selectively inhibit the corresponding enzymes. It has been shown (1, 30) that INDO at a dose of 5 mg/kg is able to effectively inhibit the formation of PGs from AA in rats, without inhibiting the production of LTs (19). NDGA, at a range of 0.3 to 6 mg/kg, has been shown to effectively inhibit the production of LTs, 12- and 15-HETES in rats (2, 36). 17-ODYA, at a dose used in the present study (1 mg/kg), has been shown to inhibit the activity of CP450 and does not affect COX activity even at a 250 times greater dose (37).

The possibility that afferent responses were influenced by hemodynamic changes in the present study are not likely. INDO and NDGA produced bradycardia in both normal and postischemic conditions, but we found no correlation between the magnitude of afferent responses to AA and the resting heart rate. High doses of AA (30 nmol) caused hypotension in addition to activating the cardiac afferents. This change in arterial pressure is unlikely to contribute to the activation of the cardiac afferents because none of the fibers examined in this study responded to manipulation of intracardiac pressure when we characterized the mechanical sensitivity of those fibers. Moreover, these endings responded to lower doses of AA without changes in arterial pressure. Concerns for nonspecific effects, such as the changes in pH, temperature, or mechanical touch over the receptive field of the fibers were negated by the fact that nonsubstrate fatty acids and vehicle (1% ethanol) were not effective in activating the cardiac afferents.

Two lines of evidence support the notion that AA-induced activation of the cardiac vagal afferent axons in rats is mediated via its metabolites but not by AA itself. First, the AA-induced activation was abolished by combined blockade of COX and CP450 in the nonischemic condition and by simultaneous blockade of COX, LOX, and CP450 pathways in the postischemic condition. Second, AA metabolites from each of the three major metabolic pathways were capable of activating the vagal cardiac afferents. Evidence from previous studies also supports this notion. Hintze et al. (14) and Panzenbeck et al. (26) reported that intracoronary infusion of AA produced reflex hypotension and bradycardia. The effect was prevented by blockade of COX with INDO.

Among COX metabolites, PGI2, PGE2, and TxA2 were found to activate cardiac afferent axons, but PGF2alpha and TxB2 did not. This is consistent with previous studies, in which intracoronary or epicardial administration of PGI2 (15), PGE2 (12, 26), and TxA2 (34) produced vagal-medicated hypotension and changes in heart rate. TxB2 was reported to increase pulmonary and systemic vascular resistance when given intravenously or intraventricularly (7, 18). To the best of our knowledge, its effect on cardiac reflex and/or the activity of cardiac afferents has never been tested. Our result is the first evidence showing that TxB2 does not affect the activity of the cardiac vagal afferent axons. In contrast to the previous studies (13), in which PGF2alpha produced vagal-mediated hypotension and bradycardia in cats, we did not see any effect of PGF2alpha on the activity of the cardiac afferents in the present study. The discrepancy may be due to the difference in the route by which PGF2alpha was administered (intracoronary vs. epicardial) and/or due to the difference in species (cat vs. rat).

This study is the first to document a significant role of CP450 metabolites in activation of cardiac vagal afferent axons in both the ischemic and nonischemic heart. CP450 enzymes are a family of enzymes, including epoxygenases and hydroxylases that metabolize AA to EETs and 19- and 20-HETE, respectively (9). 17-ODYA is known to be a nonselective CP450 inhibitor (37) and, thus, does not allow us to ascertain the relative contributions of these two CP450 pathways to AA-induced activation of the cardiac afferents in normal and postischemic conditions. However, 14,15-EET and 20-HETE were found to be nearly equipotent in activation of the cardiac vagal afferent endings. The relative potencies of other AA metabolites from CP450 pathways remain to be determined.

This study also documented a significant role of LOX products in the activation of cardiac vagal afferent axons but only during ischemia-reperfusion. LOXs are a family of enzymes, designated as 5-, 12-, and 15-LOX, that convert AA to HpETEs that ultimately are converted to LTs, 12-HETE, and 15-HETE, respectively. Studies have shown that both cardiac 5-LOX (27) and 12-LOX (21) activity is enhanced during ischemia-reperfusion. The function of 15-LOX during myocardial ischemia is not known. NDGA is known to be a nonselective LOX inhibitor and, thus, does not allow us to ascertain the relative contributions of these LOX pathways to AA-induced activation of the cardiac afferents. However, we found that all of the 5-LOX metabolites, i.e., LTs including LTB4, were nearly equipotent in activation of the cardiac vagal afferent endings. By contrast, neither 12-LOX nor 15-LOX metabolites, 12-HpETE, 15-HpETE, and their reduction products (12- and 15-HETE), were capable of stimulating cardiac vagal afferents in the present study. Similarly, two derivatives of 15-LOX, 8(S), 15(S)-DiHETE and 8(R), 15(S)-DiHETE, formed when 15-HETE is subjected to further oxidation by 15-LOX (22), also did not affect the activity of the cardiac vagal afferents. This is consistent with the study by Pan et al. (25), who found that topical application of 8(S), 15(S)-DiHETE, and 8(R), 15(S)-DiHETE onto the receptive fields of abdominal visceral C fibers did not affect afferent activity. Taken together, these results suggest that the 5-LOX pathway is the predominating LOX pathway participating in AA-induced activation of the cardiac afferents during ischemia-reperfusion.

We did not measure infarct size produced by left anterior coronary ligation, but it has been well documented in the literature. An ischemic period of 30 min in rats typically produces infarcts <30% of the ischemic zone. The effect of myocardial infarction on nerves that lie in and/or course through the infarct area should be considered in evaluating our findings. An important consideration is that cardiac nerves possess their own rich blood supply, much of which arises from extracardiac arteries (3, 17). Thus the blood supply of nerves coursing through a ventricular infarction is not interrupted when surrounding myocardial tissue becomes ischemic. A previous study (17) showed that nerves coursing through a ventricular infarction retain their normal function. The results of our study corroborate this fact. We found that afferent responses were consistent regardless of the location of the afferent endings with respect to the ischemic zone.

It has been shown (5) that altered AA metabolism in the myocardium as a consequence of ischemia-reperfusion is not limited to the ischemic area. Our results are consistent with these findings with respect to AA-induced activation of the afferent endings. Ustinova and Schultz (32) showed that myocardial ischemia-reperfusion increases the activity of the cardiac vagal afferents originating both in and outside the ischemic zone. In the present study, the enhancement of AA-induced activation of the cardiac afferents in the postischemic heart was observed in all chemosensitive fibers tested, regardless of their location.

We administered exogenous AA to the heart as a means to test the reactivity of the cardiac afferent endings to the various potential enzymatic pathways for AA, but the physiological significance of these effects ultimately depends on the level of endogenous AA production and metabolism in the myocardial tissue. Although we did not directly measure levels of AA or its metabolites in cardiac tissue, we obtained evidence of their functional role by observing the effects of the various AA metabolic inhibitors on the resting (tonic) activity of the cardiac afferent axons. None of the inhibitors (INDO, 17-ODYA, and NDGA), alone or in combination, affected the resting activity of cardiac vagal chemosensitive afferent axons in the nonischemic heart. These results imply that endogenous AA metabolism plays little role in tonic activation of cardiac afferents at rest and are consistent with observations that AA metabolism is normally low in cardiac tissue (21). By contrast, myocardial AA metabolism is enhanced during ischemia (21). In like manner, we found that the elevated tonic cardiac afferent activity during ischemia-reperfusion was attenuated by blockade of each AA metabolic pathway (COX, CP450, and LOX) alone, and was abolished by simultaneous blockade of all three metabolic pathways. These results indicate that a variety of endogenous metabolites from the three major pathways for AA metabolism contribute to the elevated afferent activity during ischemia-reperfusion. What is not clear from these observations is whether the enhanced afferent activity may be due to a sensitization of the afferent endings (25, 35) and/or due to more AA metabolites being produced because of an augmented AA metabolism (24, 28, 33). This issue will require further study.

We studied a population of cardiac vagal afferent axons that mediate what is known as the Bezold-Jarisch reflex (also known as the coronary chemoreflex) characterized by bradycardia, hypotension, and active cholinergic coronary vasodilatation (8) evoked by chemical stimulation of their afferent endings. The physiological significance of this reflex pathway remains unresolved but theoretically many believe it serves a protective function to reduce energy demands on myocardial tissue during stress (29). Several substances produced by myocardial tissue under stress, particularly PGs, have been implicated as the chemical mediators of this reflex (29, 36). The present study demonstrates that a variety of AA metabolites contribute to activation of the afferent arm of this reflex during myocardial ischemia-reperfusion. Further studies are needed to ascertain the extent to which AA metabolism via CP450 and LOX pathways influences coronary chemoreflex function in other physiological and pathological circumstances such as exercise and heart failure.

In summary, the results of the present study indicate that a wide spectrum of AA metabolites derived from COX, CP450, and 5-LOX pathways are capable of stimulating cardiac vagal chemosensitive afferent axons. AA metabolites derived from COX and CP450 pathways, but not the LOX pathways, can contribute to activation of cardiac vagal afferents in the nonischemic heart. However, endogenous AA metabolism does not appear to play a major role in maintaining the resting activity of cardiac vagal afferents in the nonischemic condition. During myocardial ischemia-reperfusion, metabolites from all three major AA metabolic pathways contribute to activation of these cardiac afferent endings. Induction of the LOX pathways during ischemia-reperfusion appears to contribute to the enhanced responsiveness of these cardiac afferent endings to AA in the postischemic heart. Our results reaffirm an important role of COX metabolites and unveil an important new role for CP450 and 5-LOX products as chemical mediators of coronary chemoreflex function during myocardial ischemia-reperfusion.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-52190 and a grant from the University of Nebraska College of Medicine. S.-Y. Sun was supported in part by a fellowship from the American Heart Association.


    FOOTNOTES

Address for reprint requests and other correspondence: H. D. Schultz, Dept. of Physiology and Biophysics, Univ. of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: hschultz{at}unmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 April 2000; accepted in final form 23 February 2001.


    REFERENCES
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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