Airway responses to aerosolized brevetoxins in an animal model of asthma

Abstract

Florida red tide brevetoxins are sodium channel neurotoxins produced by the dinoflagellate Karenia brevis. When aerosolized, the toxin causes airway symptoms in normal individuals and patients with airway disease, but systematic exposures to define the pulmonary consequences and putative mechanisms are lacking. Here we report the effects of airway challenges with lysed cultures of Karenia brevis (crude brevetoxin), pure brevetoxin-2, brevetoxin-3, and brevetoxin-tbm (brevetoxin-2 minus the side chain) on pulmonary resistance and tracheal mucus velocity, a marker of mucociliary clearance, in allergic and nonallergic sheep. Picogram concentrations of toxin caused bronchoconstriction in both groups of sheep. Brevetoxin-tbm was the least potent, indicating the importance of the side chain for maximum effect. Both histamine H(1)- and cholinergic-mediated pathways contributed to the bronchoconstriction. A synthetic antagonist, beta-naphthoyl-brevetoxin-3, and brevenal, a natural antagonist, inhibited the bronchoconstriction. Only crude brevetoxin and brevetoxin-3 decreased tracheal mucus velocity; both antagonists prevented this. More importantly, picomolar concentrations of the antagonists alone improved tracheal mucus velocity to the degree seen with mM concentrations of the sodium channel blocker amiloride. Thus, Karenia brevis, in addition to producing toxins that adversely affect the airways, may be a source of agents for treating mucociliary dysfunction.

Figure 1

Structures of PbTx-2, PbTx-3, PbTx-tbm, β-naphthoyl-PbTx-3, and brevenal.

Figure 2

Effect of inhaled toxins on pulmonary resistance (Rl) in allergic sheep. All toxins caused significant (p < 0.05, analysis of variance) concentration-dependent increase in Rl. Values are mean ± SEM for 6–12 sheep. *p < 0.05 versus all other toxins.

Figure 3

Time course of Rl response to inhaled PbTx-3 in allergic sheep. Values are mean ± SEM for 4 sheep. *p < 0.05 versus baseline (BSL). P-PbTx-3 = post-PbTx-3.

Figure 4

Effects of inhaled PbTx-3 on Rl in allergic (n = 7) and nonallergic (n = 4) sheep. Values are mean ± SEM. There were no differences between the groups.

Figure 5

Effect of β-naphthoyl-PbTx-3 on toxin-induced bronchoconstriction in allergic sheep. Data are presented as mean ± SEM % inhibition of the maximum response to 10 pg/ml PbTx-2 and PbTx-3. The IC₅₀ of β-naphthoyl-PbTx-3 for PbTx-2 and PbTx-3 was 6.9 and 7.9 pg/ml, respectively (calculated by interpolation from the mean curve). Values are for 4–7 sheep at each concentration. *p < 0.05 versus 1 and 3 pg/ml; ^p < 0.05 versus 10 pg/ml; + p < 0.05 versus 1 pg/ml.

Figure 6

Effect of brevenal on toxin-induced bronchoconstriction in allergic sheep. Data are presented as mean ± SEM % inhibition of the maximum response to 10 pg/ml PbTx-2 and PbTx-3. The IC₅₀ of brevenal for PbTx-2 and PbTx-3 was 10 and 44 pg/ml, respectively (by interpolation from the mean curve). Values are for 4–7 sheep at each concentration. *p < 0.05 versus 3 pg/ml; + p < 0.05 versus 3 and 10 pg/ml.

Figure 7

Effect of toxins on tracheal mucus velocity (TMV) in allergic sheep. Only PbTx-3 and crude PbTx reduced TMV. Values are mean ± SEM for 4–5 sheep. *p < 0.05 versus vehicle.

Figure 8

Effect of toxin antagonists and amiloride on TMV in allergic sheep. All agents increased TMV over the first 30 minutes after challenge. Amiloride was given at a dose of 3 mM; β-naphthoyl-PbTx-3 and brevenal were given at a concentration of 100 pg/ml. Values are mean ± SEM for 4–6 sheep. *p < 0.05 versus baseline (pretreatment value).