TRPV1 antagonists attenuate antigen-provoked cough in ovalbumin sensitized guinea pigs

Abstract

We examined the molecular pharmacology and in vivo effects of a TRPV1 receptor antagonist, N-(4-Tertiarybutylphenyl)-4(3-cholorphyridin-2-yl)-tetrahydro-pyrazine1(2H) - carboxamide (BCTC) on the guinea pig TRPV1 cation channel. BCTC antagonized capsaicin-induced activation and PMA-mediated activation of guinea pig TRPV1 with IC50 values of 12.2 +/- 5.2 nM, and 0.85 +/- 0.10 nM, respectively. In addition, BCTC (100 nM) completely blocked the ability of heterologously expressed gpTRPV1 to respond to decreases in pH. Thus, BCTC is able to block polymodal activation of gpTRPV1. Furthermore, in nodose ganglia cells, capsaicin induced Ca2+ influx through TRPV1 channel was inhibited via BCTC in a concentration dependent manner. In in vivo studies capsaicin (10 - 300 muM) delivered by aerosol to the pulmonary system of non-sensitized guinea pigs produced an increase in cough frequency. In these studies, the tussigenic effects of capsaicin (300 muM) were blocked in a dose dependent fashion when BCTC (0.01-3.0 mg/kg, i.p.) was administered 30 minutes before challenge. The high dose of BCTC (3.0 mg/kg, i.p) produced a maximum inhibition of capsaicin-induced cough of 65%. We also studied the effects of BCTC (0.03 and 3.0) when administered 60 minutes before capsaicin. Under these conditions, BCTC (3.0 mg/kg, i.p) produced a maximum decrease in capsaicin-induced cough of 31%. In ovalbumin passively sensitized guinea pigs, we found that BCTC (1 and 3 mg/kg, i.p.) attenuated antigen ovalbumin (0.3%) cough responses by 27% and 60%, respectively. We conclude that TRPV1 channel activation may play role in cough mediated by antigen in sensitized guinea pigs. Our results supports increasing evidence that TRPV1 may play a role in the generation of the cough response.

Figure 1

Inhibition of TRPV1 polymodal activation by BCTC in HEK293^OFF cells. Panel A shows that BCTC antagonizes capsaicin (10 nM) and PMA-mediated (50 nM) activation of gpTRPV1. Panel B shows that inclusion of 100 nM BCTC completely inhibits gpTRPV1 respond to decreases in pH. Data are presented as percent maximal response in the absence of inhibitor (A). Data shown are representative of at least three separate experiments.

Figure 2

Intracellular Ca²⁺ in response to capsaicin (0.1 μM) was measured in isolated guinea pig nodose ganglia neurons and expressed as 334/380 ratio change. When cells were preincubated with BCTC, capsaicin-induced Ca2+ response was inhibited in a concentration dependent manner. * p < 0.05 compared with control (n = 5–12).

Figure 3

Tussigenic effects of capsaicin in non-sensitized conscious guinea pigs. Figures shows that aerosolized capsaicin (10 – 300 μM, 4 min exposure; n = 6–8 per treatment group) produces a dose-dependent increase in cough frequency in guinea pigs. The tussigenic response to a saline (which produced no coughing; n = 5) is not shown in the figure.

Figure 4

Effect of BCTC on capsaicin-induced cough in non-sensitized guinea pigs. Figure demonstrates the cough suppressant activity of BCTC (0.01 – 3.0 mg/kg, i.p.) administered at 30 and 60 minutes before capsaicin (300 μM) provocation. Each bar represents the Mean ± SEM of the number of coughs produced by a 4 min exposure to capsaicin. Control animals were guinea pigs that received oral vehicle instead of BCTC and were exposed to capsaicin provocation. (*p < 0.05 compared to control animals using a Kruskal-Wallis in conjunction with a Mann-Whitney-U; n = 8–9 per treatment group).

Figure 5

Effect of BCTC on cough responses elicited by antigen challenge in sensitized guinea pigs. BCTC (1 and 3 mg/kg, i.p.) blocked the increase in cough produced by antigen ovalbumin (0.3%) challenge. Also shown are the activities of a second TRPV1 antagonist (given by aerosol 4 min before antigen provocation*), capsazepine (300 μM) on allergic cough. Each bar represents the Mean ± SEM of the number of coughs produced by a 4 min exposure to capsaicin. (*p < 0.05 compared to controls (sensitized and administered vehicle) animals using a Kruskal-Wallis in conjunction with a Mann-Whitney-U; n = 9–16).