Effect of capsaicin on airway tone in precision-cut lung slices (PCLS).

<p>Capsaicin was applied to PCLS from the indicated species at a concentration of 10 µM and airway contraction was monitored. Data are shown as mean – SEM (species/number of PCLS/number of subjects: mouse/6/6, rat/10/4, guinea pig/9/9, sheep/4/4, marmoset/3/3, human responder/6/4 and human non-responder/5/3.</p

Electrically activated airway contractions in mouse PCLS require higher electric fields and are neurally mediated.

<p>A. Frequency response curve: The frequency (F) was increased from 1 Hz to 200 Hz, while the other EFS settings were kept constant at B = 1 ms/2 ms, A = 200 mA≙40 V and TW = 2.5 s; data are shown as mean±SEM (1 ms: n = 5 PCLS from 5 mice; 2 ms: n = 6 PCLS from 6 mice). Frequency response curves were calculated by four parameter logistic regression and compared by the F-test; ***, p<0.001. B. Pulse duration was increased in EFS of murine PCLS from 0.5 ms to 4 ms, while the other parameters were kept constant at F = 50 Hz, A = 200 mA≙40 V and TW = 2.5 s; data are shown as mean±SEM (n = 6 PCLS from 6 mice). C. PCLS from mice were stimulated repeatedly at F = 50 Hz, B = 2 ms A = 200 mA and TW = 2.5 s each minute for 3.3 min. Upper panel: Control stimulations, each EFS train was conducted without pharmacological interference. Lower panel: 10 mM MgSO₄ was added 30 min prior to the second EFS train. Magnesium was able to block airway responses indicating specific neurally-induced bronchoconstriction. Data are shown as mean±SEM (n = 5 PCLS from 5 mice); ***, p<0.001 in the t-test on the minimal airway area before and after the application of magnesium.</p

Cholinergic responses in EFS-triggered airway contraction.

<p>The PCLS from either rats (n = 5) (<b>A</b>), sheep (n = 4) (<b>B</b>), marmosets (n = 5) (<b>C</b>) or human (n = 4) (<b>D</b>) were stimulated repeatedly at F = 50 Hz, B = 1 ms, A = 200 mA, TW = 2.5 s, TR = 60 s for 3.3 min, i.e. 4×EFS. As control, the first stimulation (1^st EFS) was always carried out without any cholinergic interference. In subsequent EFS separated by at least 15 min to the preceding stimulation, neostigmine and/or atropine were added to augment or inhibit cholinergic airway contraction. The response in each single PCLS before and after treatment is plotted and matched to each other by a line *, p<0.05 and **, p<0.01 for atropine <i>vs.</i> no atropine (paired t-test). <b>E.</b> To prove cholinergic responses in guinea pig (n = 6), PCLS were electrically stimulated once (F = 50 Hz, B = 1 ms, A = 200 mA, TW = 2.5 s) either after pre-incubation with atropine or without interference. The mean with SEM is indicated by the horizontal line and the error bars, respectively. ***, p<0.001 in unpaired t-test.</p

EFS-induced airway contractions are neurally mediated.

<p>Magnesium competes with calcium at the terminal synapse and prevents the release of neurotransmitters resulting in a neuromuscular block <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047344#pone.0047344-delCastillo1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047344#pone.0047344-Feldman1" target="_blank">[28]</a>. The muscarinic receptor agonist methacholine was used to confirm that Mg²⁺ did not affect airway smooth muscle directly. Con., contraction in marmoset PCLS, i.e. deviation below baseline airway area before EFS; EFS; electric field stimulation; IAA, initial airway area; Mg²⁺, magnesium; Rel., relaxation in marmoset, i.e. deviation above baseline airway area before EFS; w/o, without; data are shown as mean±SEM (species/number of PCLS/number of subjects: rat/5/5, guinea pig/4/4, sheep/4/4, marmoset/6/5, human/6 EFS, 5 methacholine/6 EFS, 5 methacholine); *, p<0.05; **, p<0.01 in Mann-Whitney test; ^††, p<0.01 in t-test; ^§§, p<0.01 in paired t-test.</p

Characterization of peripheral eNANC airway responses in guinea pigs.

<p><b>A.</b> Capsaicin was added to precision-cut lung slices (PCLS) at a concentration of 10 µM, either after 15 min pre-incubation with an unspecific transient receptor potential (TRP) channel antagonist (ruthenium red, SKF96365) or without pre-incubation (control). Data are shown as mean – SEM; n = 5 PCLS from 5 guinea pigs. 100% of the initial airway area (IAA) was considered as baseline and the area above the curve (AAC), i.e., the area enclosed by the baseline and the curve, is depicted as shading. Statistical analysis was performed on the AAC; *, p<0.05 in ANOVA followed by the Tukey test. <b>B.</b> Electric field stimulation (EFS; F = 50 Hz, B = 1 ms, A = 200 mA, SD = 2.5 s) was carried out on PCLS either after pre-incubation with TRP channel blockers or without pre-treatment (control). The minimal airway area obtained in the EFS of PCLS was used in statistical analysis showing an effect for both TRP channel antagonists. Data are shown as mean - SEM; n = 6 PCLS from 6 guinea pigs; *, p<0.05 and ^##, p<0.01 for the comparison of the control group to the ruthenium red and SKF96365 group, respectively, in the Tukey multiple comparison test.</p

Frequency dependence across species.

<p>Frequency response curves were performed on PCLS from the species indicated. Except for the frequency (F), which was increased from 0.4 Hz to 100 Hz after a single train at the distinct frequency each minute (TR = 60 s), EFS settings were kept constant at B = 1 ms, A = 200 mA and TW = 2.5 s. Sigmoidal curves, in which the top was constrained to 100% of the initial airway area [IAA], were plotted and the half-maximal response frequencies (EF₅₀±SEM) were determined: rat 16.5±0.3 Hz, guinea pig 7.0±0.5 Hz, sheep 0.4±0.1 Hz, marmoset 22.8±3.6 Hz, human 10.2±0.4 Hz. The four parameters in logistic regression were compared by the F-test and except for the guinea pig (p = 0.23), EF₅₀ values from all species (rats: p<0.001; sheep: p<0.01; marmoset (p<0.05) were significantly different from the human EF₅₀. Data are shown as mean±SEM (species/number of PCLS/number of subjects: rat/7/6, guinea pig/5/5, sheep/9/9, marmoset/4/3 and human/5/5).</p