Stimulation of pulmonary vagal C-fibres by anandamide in anaesthetized rats: role of vanilloid type 1 receptors

Abstract

This study was carried out to determine the effect of intravenous injection of anandamide on pulmonary C-fibre afferents and the cardiorespiratory reflexes. In anaesthetized, spontaneously breathing rats, intravenous bolus injection of anandamide near the right atrium immediately elicited the pulmonary chemoreflex responses, characterized by apnoea, bradycardia and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C-fibres, anandamide no longer evoked these reflex responses. In open-chest, and artificially ventilated rats, anandamide injection evoked an abrupt and intense discharge in vagal pulmonary C-fibres in a dose-dependent manner. After injection of the high dose, the fibre discharge generally started within 1 s, reached a peak in approximately 2 s, and returned to baseline within 7 s. The stimulation of C-fibres by anandamide was completely and reversibly blocked by pretreatment with capsazepine, a competitive antagonist of the vanilloid type 1 receptor. Anandamide (0.4 mg kg(-1)) stimulated approximately 93 % of pulmonary C-fibres that were activated by capsaicin at a much lower dose (0.6 microg kg(-1)); the response to anandamide showed similar intensity, but had slightly longer latency and duration than that to capsaicin. In conclusion, intravenous bolus injection of anandamide evokes a consistent and distinct stimulatory effect on pulmonary C-fibre terminals, and this effect appears to be mediated through an activation of the vanilloid type 1 receptor.

Figure 1. Experimental records illustrating the effect of perineural capsaicin treatment of both cervical vagi on cardiorespiratory responses to injections of capsaicin and anandamide in an anaesthetized rat

A, C and E, responses to intravenous injections of capsaicin (0.8 μg kg⁻¹) near the right atrium before, and 5 and 60 min after perineural capsaicin treatment, respectively. B, D and F, responses to intravenous injections of anandamide (0.8 mg kg⁻¹) before, and 15 and 75 min after perineural capsaicin treatment, respectively. 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, 405 g. V_T, tidal volume; ABP, arterial blood pressure.

Figure 2. Effect of perineural capsaicin treatment of both cervical vagi on cardiorespiratory responses to intravenous injections of anandamide

Ventilatory and cardiovascular responses to anandamide (0.8–1.2 mg kg⁻¹) before (○) and 15 min after (•) perineural capsaicin treatment. f, respiratory frequency; V_T, tidal volume; HR, heart rate; ABP, arterial blood pressure. The vertical dashed line depicts time of injection. Data are means ±s.e.m. of 6 rats.

Figure 3. Apnoeic responses to intravenous injections of anandamide

A, effect of increasing doses of anandamide on apnoeic responses. ^a,b,c Significantly different (P < 0.05) from the responses to vehicle, and 0.4 and 0.8 mg kg⁻¹ anandamide, respectively. B, effect of perineural treatment (PNT) of both cervical vagi with capsaicin or vehicle on anandamide (0.8–1.2 mg kg⁻¹)-induced apnoea. □, before PNT; ▪, after PNT. * Significantly different (P < 0.05) from the corresponding responses before PNT. Apnoeic T_E, longest expiratory duration (T_E) within 3 s after the injection. Baseline T_E, average T_E over 10 control breaths. Dashed lines, 100 % level. Data of each group are means ±s.e.m. of 6 rats.

Figure 4. Experimental records illustrating responses of a pulmonary C-fibre arising from an ending in the right accessory lobe to intravenous injections of anandamide in an anaesthetized and open-chest rat

A, B, C and D, responses to injections (arrows) of vehicle, and 0.2, 0.4 and 0.6 mg kg⁻¹ anandamide, respectively. An interval of at least 15 min elapsed between two injections. Rat body weight, 385 g. AP, action potential; P_t, tracheal pressure; ABP, arterial blood pressure.

Figure 5. Dose dependence of anandamide-induced stimulatory effect on 8 pulmonary C-fibres in 6 anaesthetized and open-chest rats

A, average responses to intravenous injections (arrow) of vehicle (○), and 0.2 (•), 0.4 (▵) and 0.6 mg kg⁻¹ (▴) of anandamide. Fibre activity (FA) was measured in 0.5 s intervals. B, ΔFA, difference between peak FA (5 s average) after the injection and baseline FA (10 s average). ^{a, b, c} Significantly different (P < 0.05) from the responses to vehicle, and 0.2 and 0.4 mg kg⁻¹ anandamide, respectively. Data are means ±s.e.m.

Figure 6. Experimental records illustrating the responses of a pulmonary C-fibre arising from the right middle lobe to capsaicin or anandamide before and after pretreatment with capsazepine in an anaesthetized and open-chest rat

A, response to intravenous injection of capsaicin (Cap; 0.6 μg kg⁻¹). B, C and D, responses to intravenous injections of anandamide (Ana; 0.4 mg kg⁻¹) before, and 2 and ∼20 min after pretreatment with capsazepine (3 mg kg⁻¹), respectively. See legend to Fig. 4 for further explanation. Rat body weight, 398 g.

Figure 7. Effect of pretreatment with capsazepine or AM281 on pulmonary C-fibre responses to anandamide

A, afferent responses to injections of anandamide (0.4–0.6 mg kg⁻¹) before (□), and 2 (▪) and ∼20 min (recovery; ) after pretreatment with capsazepine (3 mg kg⁻¹; n = 11) or vehicle (n = 7). B, afferent responses to injections of anandamide (0.4–0.6 mg kg⁻¹) before, and 15 and 40 min after pretreatment with AM281 (0.3 mg kg⁻¹; n = 7). *P < 0.05, compared with the corresponding control. Data of each group are means ±s.e.m. See legend to Fig. 5 for further explanation.

Figure 8. Comparison between responses to anandamide and capsaicin in 32 pulmonary C-fibres from 19 anaesthetized and open-chest rats

Average responses to anandamide (•; 0.4 mg kg⁻¹) and capsaicin (▵; 0.6 μg kg⁻¹) injected (arrow) near the right atrium. Data are means ±s.e.m.