2-aminoethoxydiphenyl borate stimulates pulmonary C neurons via the activation of TRPV channels

Abstract

This study was carried out to determine the effect of 2-aminoethoxydiphenyl borate (2-APB), a common activator of transient receptor potential vanilloid (TRPV) type 1, 2, and 3 channels, on cardiorespiratory reflexes, pulmonary C fiber afferents, and isolated pulmonary capsaicin-sensitive neurons. In anesthetized, spontaneously breathing rats, intravenous bolus injection of 2-APB elicited the pulmonary chemoreflex responses, characterized by apnea, bradycardia, and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 2-APB no longer evoked any of these reflex responses. In open-chest and artificially ventilated rats, 2-APB evoked an abrupt and intense discharge in vagal pulmonary C fibers in a dose-dependent manner. The stimulation of C fibers by 2-APB was attenuated but not abolished by capsazepine, a selective antagonist of the TRPV1, which completely blocked the response to capsaicin in these C fiber afferents. In isolated pulmonary capsaicin-sensitive neurons, 2-APB concentration dependently evoked an inward current that was partially inhibited by capsazepine but almost completely abolished by ruthenium red, an effective blocker of all TRPV channels. In conclusion, 2-APB evokes a consistent and distinct stimulatory effect on pulmonary C fibers in vivo and on isolated pulmonary capsaicin-sensitive neurons in vitro. These results establish the functional evidence demonstrating that TRPV1, V2, and V3 channels are expressed on these sensory neurons and their terminals.

Fig. 1

Pulmonary chemoreflex responses to intravenously injection of 2APB in anesthetized rats. A, B, C and D: experimental records illustrating the responses to intravenous injections of vehicle, and 1, 2 and 3 mg/kg 2APB, respectively, in an anesthetized rat breathing spontaneously. Injectate (0.1 ml) was first slowly injected into the catheter (dead space, 0.2 ml) and then flushed (at arrow), as a bolus with saline (0.3 ml). Rat body weight, 390 g. V_T, tidal volume; ABP, arterial blood pressure. E: effect of increasing doses of 2APB on apneic ratio (apneic duration/baseline T_E). Apneic duration, longest expiratory duration within 3 s after the injection. Baseline T_E, average expiratory duration over 10 control breaths. Dashed line, apneic ratio of 100% level (no apnea). * Significantly different (P < 0.05) from the response to vehicle. Data of each group are means ± SE of 12 rats.

Fig. 2

Experimental records illustrating the effect of perineural capsaicin treatment (PNCT) of both cervical vagi on cardiorespiratory responses to injections of capsaicin and 2APB in an anesthetized rat. A, C and E: responses to intravenous injections of capsaicin (1 μg/kg) before, 5 min and 60 min after PNCT of both vagi, respectively. B, D and F: responses to injections of 2APB (3 mg/kg) before, 15 min and 75 min after PNCT, respectively. Rat body weight, 365 g.

Fig. 3

Effect of perineural capsaicin treatment (PNCT) of both cervical vagi on ventilatory responses to intravenous injections of capsaicin and 2APB in anesthetized rats. A and B: ventilatory responses to capsaicin (1 μg/kg) and 2APB (3 mg/kg), repectively, before (open circle), and 5 (capsaicin) or 15 (2APB) min after (filled circle) PNCT of both vagi. f, respiratory frequency; V̇_E , minute ventilation. The vertical dashed line depicts time of injection. C and D: apneic responses to capsaicin and 2APB, respectively. Control (open bars), before PNCT; PNCT (solid bars), 5 (capsaicin) or 15 (2APB) min after PNCT; recovery (hatched bars), 60 (capsaicin) or 75 (2APB) min after PNCT. For calculation of apneic ratio, see legend of Fig. 1. Dashed line, apneic ratio of 100% level (no apnea). * Significantly different (P < 0.05) from the corresponding control. Data of each group are means ± SE of 6 rats.

Fig. 4

Effect of intravenous injection of 2APB on single pulmonary C-fiber afferents in anesthetized and open-chest rats. A, B and C: experimental records illustrating the responses to injections (arrows) of 1, 2 and 3 mg/kg 2APB, respectively. Receptor location, right accessory lobe; rat body weight, 430 g. AP, action potential; P_tr, tracheal pressure; ABP, arterial blood pressure. D: effect of increasing dose of 2APB on the response of pulmonary C fibers (n = 26). ΔFA, difference between peak FA (2-s average) after the injection and baseline FA (10-s average); imp, impulses. * Significantly different (P < 0.05) from the response to vehicle. †Significantly different (P < 0.05) from the response to 2 mg/kg 2APB. Data are means ± SE.

Fig. 5

Experimental records illustrating the responses of a pulmonary C fiber arising from the right middle lobe to capsaicin or 2APB before and after pretreatment with capsazepine in an anesthetized and open-chest rat. A and C: responses to intravenous injections of capsaicin (1 μg/kg) before and 2 min after intravenously infusion of capsazepine (0.3 mg/kg/min; 5 min), respectively. B and D: responses to intravenous injections of 2APB (3 mg/kg) before and 2 min after pretreatment with the same dose of capsazepine, respectively. Rat body weight, 405 g.

Fig. 6

Effect of pretreatment with capsazepine on averaged pulmonary C-fiber and cardiovascular responses to capsaicin and 2APB in anesthetized and open-chest rats. A and B: response to intravenous injections of capsaicin (1 μg/kg) and 2APB (3 mg/kg), respectively, before and 2 min after intravenously infusion of capsazepine (0.3 mg/kg/min; 5 min). Open circle, before capsazepine; filled circle, after capsazepine; FA, fiber activity; HR, heart rate; imp, impulses. Data are means ± SE of 11 and 17 C fibers in A and B, respectively. Note that capsazepine only slightly shortened but did not prevent the bradycardia induced by 2APB (panel B).

Fig. 7

Effect of pretreatment with capsazepine on pulmonary C-fiber responses to capsaicin and 2APB in anesthetized and open-chest rats. A: responses to intravenously injections of 2APB (3 mg/kg) before and 2 min after pretreatment with the vehicle of capsazepine. B and C: responses to intravenously injections of 2APB (3 mg/kg) and capsaicin (1 μg/kg), respectively, before and 2 min after pretreatment with capsazepine (0.3 mg/kg/min; 5 min). BL, baseline fiber activity averaged over 20-s; P, peak fiber activity averaged over 2-s; Veh, vehicle; CPZ, capsazepine; imp, impulses. * Significantly different (P < 0.05) from the corresponding baseline response. † Significantly different (P < 0.05) from the corresponding response before treatment with CPZ or vehicle. Data are means ± SE of 16, 17 and 11 in A, B and C, respectively.

Fig. 8

2APB-induced whole-cell inward current and effect of capsazepine on the currents induced by capsaicin and 2APB in isolated rat pulmonary capsaicin-sensitive neurons. A: experimental records illustrating that a small pulmonary nodose ganglia neuron (18.5 pF) was activated by application of three concentrations of 2APB (30, 100 and 300 μM; 4 s, added horizontal bars). B: group data showing the concentration-dependence of the whole-cell inward currents induced by 2APB (4-8 s). * Significantly different (P < 0.05) from the response to 2APB vehicle. Data are means ± SE of 14 neurons tested for each group, except the group of 300 μM (n = 24). C and E: experimental records illustrating the effect of pretreatment with capsazepine (10 μM; 5 min), a selective TRPV1 antagonist, on the inward currents induced by capsaicin (1 μM; 5 s) and 2APB (300 μM; 4 s), respectively. D and F: group data for responses to capsaicin (0.3-1 μM; 4-8 s) and 2APB (300 μM; 4-8 s), respectively, before (open bars), during (closed bars) and ∼60 min after (hatched bars) capsezepine (10 μM; 5 min). Cap, capsaicin; CPZ, capsazepine. * Significantly different (P < 0.05) from the response before capsazepine treatment. Data are means ± SE of 8 and 9 pulmonary capsaicin-sensitive neurons in D and F, respectively.

Fig. 9

Effect of ruthenium red on the whole-cell inward currents induced by capsaicin and 2APB in isolated rat pulmonary capsaicin-sensitive neurons. A and C: experimental records illustrating the effect of pretreatment with ruthenium red (5 μM; 5 min), a non-selective TRPV channels blocker, on the inward currents induced by capsaicin (1 μM; 6 s) and 2APB (300 μM; 8 s), respectively. B and D: group data for responses to capsaicin (0.3-1 μM; 4-8 s) and 2APB (300 μM; 4-8 s), respectively, before (open bars), during (closed bars) and ∼60 min after (hatched bars) ruthenium red (5 μM; 5 min). Cap, capsaicin; RR, ruthenium red. * Significantly different (P < 0.05) from the response before ruthenium red treatment. Data are means ± SE of 6 and 7 pulmonary capsaicin-sensitive neurons in B and D, respectively.