Differential engagement of inhibitory and excitatory cardiopulmonary reflexes by capsaicin and phenylbiguanide in C57BL/6 mice

Abstract

The Bezold–Jarisch reflex is a powerful inhibitory reflex initiated by activation of cardiopulmonary vagal nerves during myocardial ischemia, hemorrhage, and orthostatic stress leading to bradycardia, vasodilation, hypotension, and vasovagal syncope. This clinically relevant reflex has been studied by measuring heart rate (HR) and mean arterial pressure (MAP) responses to injections of a variety of chemical compounds. We hypothesized that reflex responses to different compounds vary due to differential activation of vagal afferent subtypes and/or variable coactivation of excitatory afferents. HR and MAP responses to intravenous injections of the transient receptor potential vanilloid-1 (TRPV1) agonist capsaicin and the serotonin 5-HT₃ receptor agonist phenylbiguanide (PBG) were measured in anesthetized C57BL/6 mice before and after bilateral cervical vagotomy. Capsaicin and PBG evoked rapid dose-dependent decreases in HR and MAP followed by increases in HR and MAP above baseline. Bezold–Jarisch reflex responses were abolished after vagotomy, whereas the delayed tachycardic and pressor responses to capsaicin and PBG were differentially enhanced. The relative magnitude of bradycardic versus depressor responses (↓HR/↓MAP) in vagus-intact mice was greater with capsaicin. In contrast, after vagotomy, the magnitude of excitatory tachycardic versus pressor responses (↑HR/↑MAP) was greater with PBG. Although capsaicin-induced increases in MAP and HR postvagotomy were strongly attenuated or abolished after administration of the ganglionic blocker hexamethonium, PBG-induced increases in MAP and HR were mildly attenuated and unchanged, respectively. We conclude that responses to capsaicin and PBG differ in mice, with implications for delineating the role of endogenous agonists of TRPV1 and 5-HT₃ receptors in evoking cardiopulmonary reflexes in pathophysiological states.

INTRODUCTION

The Bezold–Jarisch reflex is a powerful inhibitory reflex characterized by bradycardia, sympathoinhibition, hypotension, and apnea triggered by activation of cardiopulmonary vagal afferent nerves (1–5). This reflex is of physiological and pathophysiological significance (for example during myocardial ischemia/reperfusion, severe hemorrhage, and orthostatic hypotension/syncope; 6–8). Interestingly, hypotension during orthostasis in humans may result from decreases in heart rate (HR) and/or stroke volume and/or total peripheral resistance (8–10). The factors determining which mechanisms come into play are not well understood.

The Bezold–Jarisch reflex can be evoked by numerous chemical compounds injected into the venous system, atria, or ventricles including veratrum alkaloids, nicotine, serotonin, phenylbiguanide (PBG), and capsaicin (1, 4, 5, 11–14). This approach is often used as an experimental tool to evoke the reflex under differing conditions or in different diseases. Although these compounds elicit reflexes by activating vagal afferents, they do so via different mechanisms and may activate different types of afferents with receptive fields located throughout the cardiopulmonary region, including spinal afferents that typically elicit sympathoexcitatory pressor reflexes (15–18). Moreover, some of these compounds may also influence arterial blood pressure (BP) and HR via direct vascular and/or cardiac actions. Engagement of sympathoexcitatory reflexes and/or direct vasoconstriction may oppose the Bezold–Jarisch reflex and/or generate delayed responses to drugs administered intravenously. For example, the transient receptor potential vanilloid-1 (TRPV1) agonist capsaicin activates excitatory as well as inhibitory reflexes and may contract vascular muscle, either directly or indirectly (18–23). The serotonin (5-hydroxytryptamine) 5-HT₃ receptor agonists PBG and phenyldiguanide are more selective in activating sympathoinhibitory reflexes (24, 25), although a reflex increase in cardiac sympathetic activity has been observed coincident with inhibition of lumbar sympathetic activity (14). Few studies have compared reflex responses to more than one agonist and evaluated responses postvagotomy, and no such studies to our knowledge have been performed on mice.

The major goals of this study were to compare the magnitude of decreases in HR and BP evoked by intravenous injections of capsaicin and PBG in anesthetized C57BL/6 mice with intact vagus nerves and determine the extent of engagement of sympathoexcitatory reflexes and their contributions to the changes in BP and HR by repeating the injections of capsaicin and PBG after bilateral cervical vagotomy and after administration of the ganglionic blocker hexamethonium. Distinct early (phase I) and late (phase II) responses were observed after injections of capsaicin and PBG, suggesting more than one site of action of each drug. The results demonstrate differential engagement of inhibitory and excitatory cardiopulmonary reflexes by capsaicin and PBG in C57BL/6 mice with implications for interpreting the results of studies using these drugs to evoke the Bezold–Jarisch reflex and investigating the role of endogenous agonists of TRPV1 and 5-HT₃ receptors in physiological and pathological states.

MATERIALS AND METHODS

Animals

All experimental procedures were carried out under the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Institutional Animal Care and Use Committee at The University of Iowa. Twenty-five male C57BL/6J mice were purchased from Jackson Laboratory and maintained in a 12:12 h light-dark cycle (6:00 AM–6:00 PM), with access to normal mouse chow and water ad libitum.

Surgical Procedures

Adult, male C57BL/6J mice (age 12–19 wk) were anesthetized with an intraperitoneal injection of ketamine (91 µg/g) and xylazine (9.1 µg/g; 26). Supplemental doses equivalent to 10% of the initial dose were given as needed. Appropriate depth of anesthesia was monitored by absence of the withdrawal reflex in response to noxious toe pinch and stable recordings of BP, HR, and respiration. The withdrawal reflex was tested at ∼15-min intervals before collecting baseline data and when BP, HR, or respiration were judged unstable. Body temperature was monitored via rectal probe and maintained at 37°C with a heating pad.

A midline incision was made in the ventral surface of the neck, and the trachea was cannulated with PE 90 tubing allowing animals to breath room air (21% O₂) spontaneously throughout the experimental protocol. The vagus nerves (bilateral) were isolated and looped with 5-0 silk suture in the cervical region. The right jugular vein was cannulated with stretched MRE040 tubing (Braintree Scientific) with the tip positioned adjacent to the right atrium. The left femoral artery was also cannulated with stretched MRE040 tubing (Braintree Scientific) to measure arterial BP and HR.

Drugs

A stock solution of capsaicin (2 mg/mL, Millipore) was prepared in 100% ethanol and stored at 4°C. A stock solution of PBG (0.25 mg/mL, Tocris) was prepared in isotonic saline and stored at −20°C. Working solutions were prepared from stock daily by diluting with isotonic saline. The ganglionic blocker hexamethonium bromide (30 mg/mL, Sigma) was dissolved in isotonic saline.

Experimental Protocols

Separate animals were used for capsaicin and PBG experiments. Animals were allowed to stabilize for 20 min after surgery followed by a 5-min baseline recording. Vehicle injections (50 µL over ∼2 s) were performed first followed by injections of either capsaicin (25 and 50 ng/g; 50 µL over ∼2 s) or PBG (50 and 100 ng/g; 50 µL over ∼2 s). The drug dosages were chosen based on previous studies on mice and rats (11, 20, 22, 27–34) and preliminary experiments performed in our laboratory. Higher absolute concentrations of PBG (than capsaicin) were injected to enable comparable reflex responses to the two drugs in vagus nerve-intact mice. The vagus nerves were then sectioned bilaterally in the cervical region. Animals were allowed to stabilize for 20 min and the previous protocol was repeated.

In separate groups of animals, bilateral cervical vagotomy was performed at the beginning of the protocol. After a 20-min stabilization period, either capsaicin (50 ng/g) or PBG (100 ng/g) was injected. After a 20-min recovery, hexamethonium bromide (30 µg/g iv) was administered and the previous protocol was repeated.

Data Analysis

The BP signal was amplified (Quad Bridge Amplifier, AD Instruments), digitized (Powerlab 16/35, AD Instruments), and recorded using LabChart 7 software (AD Instruments). Baseline values were obtained by averaging a 2-min segment of data just before injections of vehicle, capsaicin, or PBG. Both drugs induced initial decreases in mean arterial pressure (MAP) and HR within the first 10 s of injection (phase I), followed by increases in MAP and HR above baseline 10–20 s after injection (phase II). Peak changes in MAP and HR measured over 2–4 s were calculated during each phase. To estimate the contribution of changes in HR to the changes in MAP, we calculated the ratio of the change in HR (beats/min) to the change in MAP (mmHg; ΔHR/ΔMAP). Data were analyzed by paired t test, one-way ANOVA, or two-way ANOVA, the latter testing effects of drug dosage (vehicle vs. low dose vs. high dose), vagotomy, and their interaction. Post hoc analysis was performed with Tukey’s honestly significant difference (HSD) test to determine differences in responses to each drug dose and the effect of vagotomy at each dose. Summary data are expressed as means ± SE. Differences were considered statistically significant at a critical value of P < 0.05.

RESULTS

Baseline MAP and HR

Baseline values of MAP and HR measured in anesthetized mice are shown in Table 1. Vagotomy did not significantly affect baseline MAP in the vehicle, capsaicin, or PBG experimental groups. As expected, vagotomy significantly increased HR in all groups of mice (P < 0.01, two-way ANOVA).

Table 1.

Baseline values of MAP and HR with vagus nerves intact and after bilateral cervical vagotomy

	Vagus Nerves Intact			After Vagotomy
	Vehicle	Capsaicin (25 ng/g)	Capsaicin (50 ng/g)	Vehicle	Capsaicin (25 ng/g)	Capsaicin (50 ng/g)
n	6	4	5	4	4	4
MAP, mmHg	95.6 ± 2.9	100.2 ± 8.0	89.4 ± 2.1	103.3 ± 2.9	93.6 ± 4.3	89.2 ± 1.7
HR, beats/min	327 ± 19	283 ± 18	310 ± 19	416 ± 10†	334 ± 15†	396 ± 11†

	Vehicle	PBG (50 ng/g)	PBG (100 ng/g)	Vehicle	PBG (50 ng/g)	PBG (100 ng/g)
n	5	5	5	4	5	5
MAP, mmHg	94.2 ± 2.4	89.9 ± 3.0	89.6 ± 3.0	87.6 ± 2.0	87.8 ± 1.8	89.8 ± 1.7
HR, beats/min	279 ± 14	276 ± 11	274 ± 9	361 ± 13†	371 ± 11†	379 ± 11†

†P < 0.01 vs. corresponding vagus nerves intact. Statistical analysis was performed with by two-way ANOVA. n, number of animals. HR, heart rate; MAP, mean arterial pressure; PBG, phenylbiguanide.

Effects of Capsaicin on MAP and HR

In mice with vagus nerves intact, intravenous injection of capsaicin elicited an initial Bezold–Jarisch reflex with rapid reductions in MAP and HR (phase I), followed by delayed pressor and tachycardic responses (phase II; Fig. 1, left). After the bilateral cervical vagotomy, the phase I depressor and bradycardic responses were abolished, and the phase II pressor response was augmented (Fig. 1, right; Fig. 2). Inhibitory and excitatory responses to capsaicin were significant and dose dependent (Fig. 2). Injection of vehicle solution did not significantly change MAP or HR (Fig. 2).

Figure 1.

Representative traces showing ABP (top) and HR (bottom) responses to intravenous injection of the TRPV1 receptor agonist capsaicin (50 ng/g, 50 mL) with the vagus nerves intact (left) and after vagotomy (right) in the same mouse. Arrows indicate drug injection. ABP, arterial blood pressure; HR, heart rate.

Figure 2.

Summary data showing peak changes in MAP (A and B) and HR (C and D) during phase I (A and C) and phase II (B and D) of the response to intravenous injection of vehicle (n = 6 animals) or two doses of capsaicin (n = 4 animals). Data are presented as means ± SE with individual data points overlayed. Statistical analysis was performed by two-way ANOVA followed by post hoc Tukey’s HSD test. **P < 0.01. MAP, mean arterial pressure; HSD, honestly significant difference.

The depressor response to capsaicin (phase I) was reversed to a pressor response after bilateral vagotomy (Fig. 2A), whereas the bradycardic response was abolished but not reversed to tachycardia (Fig. 2C). The delayed pressor and tachycardic responses to capsaicin (phase II) were significant in mice with intact vagus nerves (Figs. 2, B and D). Interestingly, the pressor response during phase II was significantly enhanced after vagotomy (Fig. 2B), whereas the tachycardic response was similar to that observed in mice with intact vagus nerves (Fig. 2D).

Effects of PBG on MAP and HR

As was observed with capsaicin, intravenous injection of PBG in mice with intact vagus nerves elicited rapid decreases in MAP and HR (phase I) with the bradycardia being abolished after vagotomy (Fig. 3 and Fig. 4, A and C). The depressor response to PBG was markedly attenuated after vagotomy (Fig. 3 and Fig. 4A); it was not reversed to a pressor response as was observed with capsaicin. Injection of vehicle solution did not significantly change MAP or HR (Fig. 4).

Figure 3.

Representative traces showing ABP (top) and HR (bottom) responses to intravenous injection of the 5-HT₃ receptor agonist phenylbiguanide (100 ng/g, 50 µL) with the vagus nerves intact (left) and after vagotomy (right) in the same mouse. Arrows indicate drug injection. ABP, arterial blood pressure; HR, heart rate; PBG, phenylbiguanide.

Figure 4.

Summary data showing peak changes in MAP (A and B) and HR (C and D) during phase I (A and C) and phase II (B and D) of the response to intravenous injection of vehicle (n = 5 animals) or two doses of phenylbiguanide (PBG; n = 5 animals). Data are presented as means ± SE with individual data points overlayed. Statistical analysis was performed by two-way ANOVA followed by post hoc Tukey’s HSD test. *P < 0.05; **P < 0.01. HR, heart rate; HSD, honestly significant difference; MAP, mean arterial pressure.

The higher dose of PBG (100 ng/g iv) induced delayed, phase II pressor and tachycardic responses in mice with intact vagus nerves that were significantly greater than the responses to vehicle injection (Fig. 4, B and D). Phase II responses to the lower dose of PBG (50 ng/g) were not significantly different than responses to vehicle (Fig. 4, B and D). After vagotomy, the lower dose of PBG increased MAP and HR significantly, and the tachycardic responses to both doses were significantly greater than the responses measured in mice with vagus nerves intact (Fig. 4, B and D).

Similarities and Differences between Responses to Capsaicin and PBG

As shown in sections Effects of Capsaicin on MAP and HR and Effects of PBG on MAP and HR, both capsaicin and PBG injected intravenously caused biphasic changes in MAP and HR in intact mice, with rapid decreases in phase I (Bezold–Jarisch reflex) followed by increases in phase II. The bradycardia in phase I induced by both drugs was abolished after vagotomy. Despite these similarities, responses to capsaicin and PBG differed in many respects. The depressor response to capsaicin was reversed to a pressor response after vagotomy (Fig. 2A), whereas the depressor response to PBG was only attenuated (Fig. 4A). In addition, the delayed phase II pressor response to capsaicin was significantly enhanced after vagotomy (Fig. 2B), whereas the pressor response to PBG was not enhanced (Fig. 4B). Conversely, the phase II tachycardic response to PBG (but not capsaicin) was significantly enhanced after vagotomy (Fig. 2D and Fig. 4D).

To estimate the relative contribution of changes in HR to the changes in MAP after injection of capsaicin versus PBG, we calculated the ratio ΔHR/ΔMAP. For capsaicin- and PBG-induced decreases in HR and MAP in intact mice (phase I), ↓HR/↓MAP was significantly larger for capsaicin than PBG (Fig. 5A). Conversely, for capsaicin- and PBG-induced increases in HR and MAP in vagotomized mice (phase II), ↑HR/↑MAP was significantly larger for PBG than capsaicin (Fig. 5B).

Figure 5.

ΔHR/ΔMAP ratio for capsaicin (n = 4 animals) and PBG (n = 5 animals) during inhibitory phase I with vagus nerves intact (A), and excitatory phase II after vagotomy (B). Data are presented as means ± SE with individual data points overlayed. Statistical analysis was performed with one-way ANOVA followed by post hoc Tukey’s HSD test. *P < 0.05; **P < 0.01. HR, heart rate; HSD, honestly significant difference; MAP, mean arterial pressure; PBG, phenylbiguanide.

Contribution of Excitatory Reflexes to Pressor and Tachycardic Responses

In addition to activating autonomic reflexes, capsaicin and PBG may potentially affect MAP and HR via nonreflex mechanisms (19, 21, 22, 35, 36). To evaluate this possibility, we measured MAP and HR responses (phase II) to intravenous injections of capsaicin and PBG in vagotomized mice, both before and after injection of the ganglionic blocker hexamethonium. Pretreatment with hexamethonium markedly attenuated the capsaicin-induced increase in MAP and essentially abolished the capsaicin-induced increase in HR (Fig. 6, A and B). Although hexamethonium significantly attenuated the PBG-induced increase in MAP, the effect was modest resulting in a significant residual pressor response (Fig. 6C). Interestingly, hexamethonium did not affect the PBG-induced increase in HR (Fig. 6D).

Figure 6.

Summary data showing peak phase II excitatory MAP (A) and HR (B) responses to capsaicin (50 ng/g; n = 4 animals), and MAP (C) and HR (D) responses to phenylbiguanide (PBG 100 ng/g; n = 5 animals) before and after ganglionic blockade (GB) with hexamethonium bromide in vagotomized mice. Data are presented as means ± SE with individual data points overlayed. Statistical analysis was performed with paired t tests. *P < 0.05; **P < 0.01; N.S., not significant before vs. after GB. HR, heart rate; MAP, mean arterial pressure.

DISCUSSION

The major findings of this study are the following: 1) capsaicin and PBG each induce a dose-dependent Bezold–Jarisch reflex in C57BL/6J mice that is abolished after bilateral cervical vagotomy; 2) capsaicin and PBG also elicit delayed excitatory responses, with vagotomy significantly enhancing the capsaicin-induced increase in BP (but not the increase in HR) and the PBG-induced increase in HR (but not the increase in BP); 3) the ↓HR/↓MAP ratio during phase I inhibitory responses is greater with capsaicin versus PBG, whereas the ↑HR/↑MAP ratio during phase II excitatory responses is greater with PBG; and 4) although capsaicin-induced increases in MAP and HR postvagotomy are strongly attenuated or abolished after preinjection of the ganglionic blocker hexamethonium, the PBG-induced increase in MAP is only mildly blunted and the increase in HR is unchanged. We conclude that while capsaicin and PBG each activate the Bezold–Jarisch reflex in mice, the contribution of bradycardia to the hypotension and the excitatory responses measured in vagotomized mice differ markedly between these two drugs. The relationship of our results to those of previous studies, limitations in our study, and directions for future research are discussed.

Bezold–Jarisch Reflex

Capsaicin, a potent TRPV1 receptor agonist, and PBG, a selective 5-HT₃ receptor agonist, have been used to elicit the Bezold–Jarisch cardiopulmonary reflex in numerous studies and in a variety of species (11–14, 27, 29, 31, 33, 34, 37–40). Relatively few studies have measured HR and BP responses to intravenous injections of PBG or capsaicin in mice (11, 27, 30–32, 37), and to the best of our knowledge, no previous studies have compared responses to each agonist in mice. The reflex decreases in HR and BP to these and other agonists appear to be similar across species and abolished after bilateral vagotomy, consistent with the reflex being mediated by activation of vagal afferent nerves.

An important question relates to the relative contribution of decreases in HR and cardiac output versus the decrease in peripheral vascular resistance to the reflex fall in BP. Our finding that the ratio ↓HR/↓MAP was higher for responses to capsaicin than responses to PBG (Fig. 5A) suggests that these compounds may evoke different reflexes; with more cardiac parasympathetic activation and/or less peripheral sympathetic inhibition and vasodilation with capsaicin compared with PBG. In addition, PBG-induced increases in cardiac sympathetic activity as observed previously in rats (14) might have contributed to the lower ↓HR/↓MAP ratio with PBG. Previous studies have provided evidence that capsaicin and PBG (or 5-HT or phenyldiguanide) activate different types of vagal afferent fibers (25, 28, 29, 41).

The carotid sinus (and aortic depressor) nerves were intact in our study, so one might expect some baroreflex buffering of the capsaicin and PBG-induced changes in BP. Chemical activation of cardiopulmonary vagal afferents strongly decreases baroreflex sensitivity, thereby limiting baroreflex buffering of the BP changes (39). We chose not to denervate the arterial baroreceptors as a method to remove their influence on the Bezold–Jarisch reflex. The Bezold–Jarisch reflex is known to be enhanced after baroreceptor denervation, due to the high baseline sympathetic activity and HR after denervation and the dominating strength of the Bezold–Jarisch reflex (39).

Excitatory HR and BP Responses to Capsaicin and PBG

After vagotomy, the rapid (phase I) MAP response to intravenous (right atrial) injection of capsaicin was converted to a pressor response (Fig. 2A), suggesting that the Bezold–Jarisch reflex was masking a rapid excitatory MAP response to capsaicin in intact mice. Moreover, capsaicin increased MAP and HR during phase II in vagus-intact and vagotomized mice (Fig. 2, B and D). Phase II pressor and tachycardic responses to PBG were also observed (Fig. 4, B and D), but the increase in MAP was less than that observed with capsaicin, whereas the increase in HR was greater than that observed with capsaicin.

Capsaicin-induced increases in MAP and HR were markedly attenuated or abolished after injection of the ganglionic blocker hexamethonium (Fig. 6, A and B), whereas PBG-induced increases in MAP and HR were not (Fig. 6, C and D). These results clearly implicate a reflex increase in sympathetic nerve activity in mediating capsaicin-induced increases in MAP and HR but raise questions as to the mechanism of the PBG-induced reflex. The failure of hexamethonium to block PBG-induced increases in BP and HR postvagotomy does not rule out a reflex increase in sympathetic activity. Cholinergic muscarinic neurotransmission in sympathetic ganglia may mediate the hexamethonium-resistant increases in BP and HR (42–44).

What is the mechanism by which capsaicin and PBG increase sympathetic activity? TRPV1 and 5-HT₃ receptors are expressed in many types of sensory nerves including spinal sympathetic afferents in the cardiopulmonary region and visceral organs (15–18, 45). Application of capsaicin to the surface of the anterior left ventricle elicits the cardiac sympathetic afferent reflex, a powerful excitatory reflex known to drive increased efferent sympathetic outflow in heart failure (16, 46, 47). Epicardial application of capsaicin produces robust increases in MAP in anesthetized, ventilated mice demonstrating that TRPV1 containing afferents reside on the epicardial surface (23). Thus, activation of spinal sympathetic afferents likely contributes to the reflex increases in sympathetic activity, HR and BP induced by intravenous injections of capsaicin in vagotomized mice observed in the current study. Other studies provide evidence that direct vasoconstriction by capsaicin or endothelin mediates the increase in MAP following intravenous injection of capsaicin (19–22). Although we cannot rule out direct vascular actions of capsaicin or endothelin, the ability of hexamethonium to significantly attenuate MAP and abolish HR responses to capsaicin strongly suggests that a reflex mechanism predominates in mice.

The ability of PBG to elicit excitatory cardiac reflexes has received much less attention than capsaicin. Fu and Longhurst (45) reported that injection of PBG into the left ventricle of cats with the vagus nerves intact produced bradycardia and hypotension, whereas tachycardia and pressor responses were observed after vagotomy. These authors also reported that the ventricular injection of PBG increased the activity of a subpopulation of spinal afferents innervating the abdomen, suggesting that these afferents may contribute to sympathoexcitatory responses to PBG in vagotomized cats (45).

Serotonin and the 5-HT₃ receptor agonist 2CH3-5-HT injected intravenously each increased HR in conscious dogs; responses that were blocked by a 5-HT₃ receptor antagonist but not blocked by the β-adrenergic receptor blocker propranolol (35). Serotonin (5-HT) as well as several 5-HT₃ receptor agonists including PBG increased HR in acutely isolated guinea pig atria; an effect that was inhibited by a 5-HT₃ receptor antagonists, a calcitonin gene-related peptide (CGRP) receptor antagonist, and other interventions that disrupt CGRP stores and release (48–50). These results suggest that activation of 5-HT₃ receptors on cardiac sensory nerves triggers CGRP release that ultimately increases HR. These mechanisms that promote tachycardia may account for the higher ratio of ↑HR/↑MAP we observed during the excitatory phase II response to PBG compared with the response to capsaicin in vagotomized mice (Fig. 5).

Limitations of Study

There are several limitations to our study. Capsaicin, PBG, and other agonists are typically injected intravenously in studies investigating the Bezold–Jarisch reflex. Agonists injected intravenously activate pulmonary and cardiac afferents, with potential to activate additional afferents further downstream (e.g., spinal afferents innervating visceral organs and arterial chemoreceptors). We positioned the tip of the venous catheter at the level of the right atrium to provide consistent drug delivery to the cardiopulmonary circulation. We did not attempt to anatomically define the site of action of capsaicin and PBG (e.g., heart vs. lung) in this study. We measured responses to only two agonists, PBG and capsaicin, so comparisons to other agonists and interactions between multiple agonists were not investigated.

We also limited our measurements to HR and BP and did not measure sympathetic nerve activity or respiratory responses to drug injections. Although activation of the Bezold–Jarisch reflex consistently inhibits renal and lumbar sympathetic activity, responses vary in other end-organs. Right atrial injections of PBG in rats increase and/or decrease cardiac sympathetic nerve activity, with excitation predominating (14). Right atrial (or intravenous) injections of PBG (24) and capsaicin (51) in rats decrease and increase adrenal sympathetic nerve activity, respectively, with the opposite responses possibly reflecting the use of different agonists and/or the different types of adrenal nerves from which recordings were obtained (24, 51). In addition, we only studied male C57BL/6J mice, so sex differences remain to be investigated.

Chemical activation of cardiopulmonary receptors elicits not only reflex bradycardia and hypotension but also apnea and/or tachypnea (rapid shallow breathing; 11, 29, 31, 33, 34, 38, 40). These responses raise the possibility that subsequent hypoxemia might influence the HR and BP responses to injections of capsaicin and PBG. We did not measure breathing in the mice. Brief apneas (a few seconds in duration) were visibly evident in vagi-intact mice injected with capsaicin or PBG, consistent with the results of previous studies of rats and mice in which breathing was measured (11, 29, 31, 33, 34). We suspect that such brief periods of apnea would have negligible effects on the almost simultaneously occurring decreases in HR and BP and their rapid recovery postdrug injection. The overshoot of BP to levels above baseline after evoking the capsaicin-induced Bezold–Jarisch reflex (Phase II, Figs. 1 and 2) does not support a confounding vasodilator influence of hypoxemia in this study. More severe hypoxemia and/or hypercapnia during longer periods of apnea may very well influence BP and HR. A 1-min period of acute hypoxemia markedly prolongs the subsequent apnea induced by right atrial injections of capsaicin by ∼16-fold in rats (34).

Perspectives and Significance

Interest in the Bezold–Jarisch reflex has persisted for over 150 yr despite doubts raised by some as to its physiological and pathophysiological significance. The major experimental approach has relied on measuring the hallmark reflex responses (bradycardia and hypotension) to intravenous injections of a wide variety of chemical compounds/agonists (often studied in isolation). The realization that different agonists evoke the Bezold–Jarisch reflex through differential (as well as overlapping) mechanisms impacting vagal afferent, autonomic efferent, and efferent-end organ activity open new opportunities for investigation. For example, interactions between multiple agonists can be explored by measuring responses to two or more agonists injected simultaneously at physiologically or pathophysiologically relevant concentrations. Of course, measuring responses to injection of agonists (as done in this study) are limited in terms of understanding the contributions of endogenous agonists of TRPV1 and 5-HT3 receptors (and other receptors/ion channels) to cardiovascular reflexes in pathophysiological states. This will require selective pharmacological blockade and/or gene deletion of these receptors/ion channels. Zhang et al. (52) produced convincing evidence that hemorrhage-induced inhibition of renal sympathetic nerve activity is abolished in mice pretreated with the TRPV1 antagonist capsazepine and in TRPV1 globally deficient mice. Off-target effects of systemically administered receptor/ion channel antagonists and use of whole body knockout mice can be confounding. Further studies are needed to more selectively target TRPV1, 5-HT₃ receptors, and other receptors/ion channels expressed on sensory nerves innervating the cardiopulmonary region to define the functional roles of these reflexes in animal models of disease.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

Part of this work was performed during R. A. Larson’s tenure as the Lundbeck Fellow of the American Autonomic Society. This work was also supported by the National Institutes of Health (P01 HL14388 to M.W. Chapleau and F32 HL140880 to R.A Larson).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.A.L. and M.W.C. conceived and designed research; R.A.L. performed experiments; R.A.L. and M.W.C. analyzed data; R.A.L. and M.W.C. interpreted results of experiments; R.A.L. prepared figures; R.A.L. drafted manuscript; R.A.L. and M.W.C. edited and revised manuscript; R.A.L. and M.W.C. approved final version of manuscript.