ating that the EFS-induced responses depended on neural transmission in all species. Having proven the neural origin of EFS-induced airway contraction in PCLS, the types of nerves inducing bronchoconstriction were determined. The muscarinic antagonist atropine was used to address order GSK1278863 parasympathetic cholinergic innervation. Atropine significantly reduced airway contractions following repeated EFS of rat, sheep, marmoset and human PCLS. Of note, in the case of rat PCLS the acetylcholine esterase inhibitor neostigimine was used to augment the response, because initial responses were only moderate and insufficient to show significance. In guinea pigs, PCLS had to be pre-incubated with atropine before the first stimulation in order to demonstrate cholinergic airway innervation, since reversible stimulation was impossible due to an incomplete relaxation to the IAA. Neurally triggered airway contraction may not be restricted to cholinergic innervation, but can also be due to eNANC innervation. Therefore, we studied the effect of the TRPV1 agonist capsaicin, which leads to the tachykinin release from eNANC nerves. Capsaicin induced strong airway contractions in guinea pigs and modest contractions in human; of note, however, in three out of seven human lungs none of the studied PCLS responded to capsaicin. In mouse, rat, sheep and marmoset capsaicin did not contract airways. To further characterize the strong response to capsaicin in guinea pigs and to test whether TRP channels are pharmacologically accessible in PCLS, PCLS were incubated with the unspecific TRP channel blockers SKF96365 or with ruthenium red prior to stimulation by capsaicin. Both inhibitors significantly reduced the response to capsaicin. The observation that in guinea pigs airways did not relax back to the IAA after an EFS train, even if recovery periods were prolonged, suggested that a non-cholinergic mechanism may be involved. Magnesium competes with calcium at the terminal synapse and prevents the release of 
neurotransmitters resulting in a neuromuscular block. 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 mean6SEM; , p,0.05; , p,0.01 in Mann-Whitney test; {{, p,0.01 in t-test; 11, p,0.01 in paired t-test. doi:10.1371/journal.pone.0047344.t001 Discussion In the present study an electric field was applied to PCLS from different mammals allowing the comparison of neuronally triggered airway responses under identical conditions. Species were heterogeneous in their response: In rats, sheep, guinea pigs, marmosets and humans bronchoconstriction was caused by cholinergic activation. Only in PCLS from guinea pigs and humans did activation of eNANC nerves contribute to the airway contractions. General considerations on EFS of PCLS Recently, we introduced rat EFS as a means to study neural responses in PCLS and found the following conditions to be effective: F = 50 Hz, B = 1 ms, A = 200 mA and TW = 2.5 s. The same parameters were used to stimulate PCLS in the present study and evoked characteristic airway contractions in each species, except for the mouse. The neural mediation of these responses was demonstrated by the inhibitory effe