Stimulatory Effect of 5-Hydroxytryptamine (5-HT) on Rat Capsaicin-Sensitive Lung Vagal Sensory Neurons via Activation of 5-HT3 Receptors

Abstract

5-hydroxytryptamine (5-HT) is an inflammatory mediator known to be released in lung. Capsaicin-sensitive lung vagal (CSLV) afferents function as a primary sensor for detecting chemical stimuli and produce consequent reflexes during lung inflammation. To characterize the effect of 5-HT on CSLV afferents, responses of cardiorespiratory reflexes and single-unit C-fiber afferents to right-atrial injections of 5-HT were investigated in anesthetized Sprague-Dawley rats. Bolus injection of 5-HT (8 μg/kg) caused an immediate augmented breath and apnea, accompanied by hypotension and bradycardia. These initial responses were then followed by a brief pressor response and a more sustained depressor response. After a perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 5-HT still triggered the augmented breath, but no longer evoked the apnea, bradycardia and hypotension, indicating an involvement of C-fiber activation. The remaining augmented breath induced by 5-HT after perineural capsaicin treatment was totally eliminated by vagotomy. To further study the effect of 5-HT on CSLV afferents, activities arising from these afferents were determined using the single-fiber recording technique. Right-atrial injection of 5-HT evoked an intense discharge in CSLV afferents in a dose-dependent manner. The highest dose of 5-HT (16 μg/kg) activated 79% (19/24) of CSLV afferents which were also sensitive to capsaicin (0.8 μg/kg). The pretreatment of tropisetron, a selective antagonist of the 5-HT₃ receptor, completely blocked CSLV-afferents stimulation induced by 5-HT but did not affect that by capsaicin. Furthermore, a similar afferent response of CSLV afferents was mimicked by phenylbiguanide, a selective agonist of the 5-HT₃ receptor. In isolated rat lung vagal C neurons, 5-HT induced intense calcium transients in a dose-dependent manner. The highest concentration (3 μM) of 5-HT activated 67% (18/27) of the CSLV neurons. The 5-HT-induced response was totally abolished by pretreatment of tropisetron. In conclusion, 5-HT exerts an intense stimulatory effect on lung C-fiber terminals mediated through an activation of the 5-HT₃ receptor, which may contribute to the airway hypersensitivity under lung inflammation.

Keywords: C-fiber; afferent; airway; inflammation; reflex; serotonin.

FIGURE 1

Experimental records illustrating the effect of perineural capsaicin treatment (PCT) and vagotomy of both cervical vagi on cardiorespiratory responses to injections of 5-HT and capsaicin in an anesthetized rat (body weight: 320 g). Left panels: responses to right-atrial injections of 5-HT (8 μg/kg) before (as control) (A), 15 min after PCT (B), and 20 min after vagotomy (C). Right panels: responses to right-atrial injections of capsaicin (0.75 μg/kg) before (as control) (A), 15 min after PCT (B) and 20 min after vagotomy (C). Injection (0.1 ml) was first slowly injected into the catheter (dead space, 0.2 ml) and then flushed (at arrow) into the circulation by a bolus with saline (0.3 ml). V_T, tidal volume; ABP, arterial blood pressure.

FIGURE 2

Apneic responses to intravenous injections of 5-HT in anesthetized, spontaneously breathing rats. (A) Effect of increasing doses of 5-HT on apneic responses in a group of 6 rats. Symbols ^∗ and # depict significantly different (P < 0.05) from the responses to 4 and 8 μg/kg of 5-HT, respectively. (B) Apneic (upper panel) and augmented breath (lower panel) responses to 5-HT (8 μg/kg) before and after pretreatment of tropisetron (15 μg/kg) or its vehicle (saline) in two groups of rats (6 for each group). Data are mean ± SEM. One-way and two-way ANOVA tests followed by a post hoc Fisher’s least significant difference test for (A,B), respectively. ^∗, significantly different from the corresponding responses before tropisetron (P < 0.05); #, significant difference when corresponding data between vehicle and tropisetron were compared (P < 0.05).

FIGURE 3

(A,C,D) Effect of perineural capsaicin treatment (PCT) and vagotomy on apneic responses to right-atrial injections of 5-HT, capsaicin (0.8 μg/kg) and lung inflation, respectively. (B) Effect of PCT and vagotomy on augmented breath responses to right-atrial injections of 5-HT. Data are mean ± SEM. One-way ANOVA test followed by a post hoc Fisher’s least significant difference test. ^∗, significantly different from the control responses (P < 0.05).

FIGURE 4

Effect of increasing doses of 5-HT on activity of capsaicin-sensitive lung vagal (CSLV) afferents in anesthetized, open-chest and ventilated rats. (A–C) Experimental records illustrating responses of a CSLV afferent to right-atrial injections (arrows) of 4, 8, and 16 μg/kg 5-HT in an anesthetized and open-chest rat (body weight: 405 g), respectively. ΔFA, difference between peak FA (5-s average) occurring within 20 s after the injection and baseline FA (10-s average). AP, action potential; P_t, tracheal pressure; ABP, arterial blood pressure. (D) Effect of increasing doses of 5-HT on afferent responses of 10 CSLV fibers in 8 rats. Data are mean ± SEM. One-way ANOVA test followed by a post hoc Fisher’s least significant difference test. ^∗ and #, significantly different (P < 0.05) from the responses to 4 and 8 μg/kg of 5-HT, respectively.

FIGURE 5

Experimental records illustrating the responses of a capsaicin-sensitive lung vagal afferent arising from the right upper lobe to 5-HT (16 μg/kg), capsaicin (0.8 μg/kg), lactic acid (18 mg/kg), or phenylbiguanide (PBG; 6 μg/kg) before and after pretreatment with tropisetron in an anesthetized, open-chest and ventilated rat (body weight: 410 g). (A) Responses before tropisetron pretreatment (15 μg/kg). (B) Responses to 5-HT, capsaicin, lactic acid and PBG at 15 min after tropisetron pretreatment, respectively. Stimulant injections were marked by the arrows. AP, action potential; P_t, tracheal pressure; ABP, arterial blood pressure.

FIGURE 6

Effect of pretreatment with tropisetron (15 μg/kg) on capsaicin sensitive lung vagal afferent responses to various stimulants in anesthetized, open-chest rats. The afferent responses to right-atrial injections of 5-HT (16 μg/kg, n = 15; A), capsaicin (0.8 μg/kg, n = 15; B), lactic acid (9 or 18 mg/kg, n = 11; C) and phenylbiguanide (PBG, 4–8 μg/kg, n = 12; D). ΔFA, difference between peak FA (2-s average for capsaicin and lactic acid; 5-s average for 5-HT and PBG) occurring within 20 s after the injection and baseline FA (10-s average). Data are mean ± SEM. Paired t-test ^∗, significantly different (P < 0.05) from the corresponding responses before tropisetron.

FIGURE 7

5-HT-evoked calcium transients in isolated rat capsaicin-sensitive lung vagal (CSLV) neurons. (A) An experimental record illustrating that 5-HT concentration-dependently evoked calcium transients in a jugular ganglion neuron (diameter: 28 μm). 5-HT and capsaicin (Cap; 1 μM) was applied for 30 s each, and potassium chloride (KCl; 60 mM, 15 s) was applied to test cell viability at the end of experiments. Time between 2 consecutive 5-HT challenges was 15 min. [Ca²⁺]_i, intracellular concentration of Ca²⁺. (B) Group data of 5-HT evoked increase of [Ca²⁺]_i (Δ[Ca²⁺]_i) obtained from 21 CSLV neurons in 12 rats. Data are mean ± SEM. One-way ANOVA test followed by a post hoc Fisher’s least significant difference test. Symbols ^∗ and # depict significantly different (P < 0.05) from the responses to 0.3 and 1 μM of 5-HT, respectively.

FIGURE 8

Effect of tropisetron on 5-HT-induced calcium transients in isolated rat capsaicin-sensitive lung vagal (CSLV) neurons. (A) An experimental record illustrating that pretreatment with tropisetron (Trop; 10 nM, 5 min) almost completely prevented the Ca²⁺ transient evoked by 5-HT (3 μM, 30 s) in a nodose ganglion neuron (diameter: 24 μm). (B) Group data of 5-HT evoked increase of [Ca²⁺]_i (Δ[Ca²⁺]_i) before and after tropisetron pretreatment obtained from 8 CSLV neurons in 5 rats. Time between 2 consecutive 5-HT challenges was 15 min. Data are mean ± SEM. One-way ANOVA test followed by a post hoc Fisher’s least significant difference test. ^∗ and #, significantly different (P < 0.05) from the responses before and immediately after tropisetron, respectively.