Overview of all used inhibitors.

<p>In general, we expect complete inhibition of the target at concentrations about 10 times above the IC₅₀ value <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087685#pone.0087685-Ko1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087685#pone.0087685-Hourani1" target="_blank">[56]</a>.</p

Preparation of PCLS from GPs and humans and their histology.

<p>A) GP: Tracheotomy; B) GP: lung filled with agarose; C) tissue cores; D) GP: PCLS during videomicroscopy; E) human: lung lobe filled with agarose; F) human: human lung tissue with airway and pulmonary artery; G) human: PCLS during videomicroscopy; H) GP: pulmonary artery (PA) with thick media and thrinkeld inner lining; I) GP: pulmonary vein (PV) with thin media; J) human: pulmonary artery (PA) and airway (AW); K) human: pulmonary artery (PA) with internal and external elastic lamina (diameter: 354 µm); L) human: pulmonary vein (PV) (diameter: 362 µm in width and 782 in height).</p

Vascular effects of milrinone in the GP isolated perfused lung.

<p><b>A)</b> Effect of milrinone (mil) on the pulmonary arterial pressure (P_PA) after and without pre-constriction with 20 nM BP0104: (○) control (n = 6); () 10 µM mil (n = 3); (•) 20 nM BP0104 (n = 6); () 20 nM BP0104, 10 µM mil (n = 5) <b>B)</b> Effect of milrinone on the precapillary resistance (R_pre) after and without pre-constriction with 20 nM BP0104: (○) control (n = 6); () 10 µM mil (n = 3); (•) 20 nM BP0104 (n = 6); () 20 nM BP0104, 10 µM mil (n = 5) <b>C)</b> Effect of milrinone on the postcapillary resistance (R_post) after and without pre-constriction with 20 nM BP0104: (○) control (n = 6); () 10 µM mil (n = 3); (•) 20 nM BP0104 (n = 6); () 20 nM BP0104, 10 µM mil (n = 5). (<b>A–C</b>) Statistics was performed by a linear mixed model. P<0.05 are considered as significant: * p<0.05, ** p<0.01 and *** p<0.001.</p

Influence of milrinone on cAMP/PKA-signaling in naïve PVs.

<p><b>A)</b> (•) SQ 22536 (1 µM) (n = 4); () KT 5720 (1 µM) (n = 5); (□) KT 5823 (2 µM) (n = 5); (▪) L-NAME (100 µM) (n = 3); <b>B)</b> () milrinone (n = 7); () SQ 22536 (100 µM), milrinone (n = 7); (□) SQ 22536 (100 µM) (n = 4); <b>C)</b> () milrinone (n = 6); () KT 5720 (1 µM), milrinone (n = 6); (□) KT 5720 (1 µM) (n = 6). <b>A)</b> Statistics was performed by a linear mixed model. <b>B–C)</b> Asterics indicate different EC₅₀ values. P<0.05 are considered as significant: * p<0.05, ** p<0.01 and *** p<0.001.</p

Influence of milrinone on cGMP/NO-signaling in PVs with and without pre-constriction.

<p><b>A)</b> () milrinone (n = 8); () L-NAME (100 µM), milrinone (n = 8); (□) L-NAME (100 µM) (n = 3); <b>B)</b> (▪) BP0104 (1 nM), milrinone (n = 8); () BP0104 (1 nM), L-NAME (100 µM), milrinone (n = 8); (□) BP0104 (1 nM), L-NAME (100 µM) (n = 8); <b>C)</b> () milrinone (n = 6); () KT 5823 (2 µM), milrinone (n = 6); (□) KT 5823 (2 µM) (n = 6); <b>D)</b> (▪) BP0104 (1 nM), milrinone (n = 7); () BP0104 (1 nM), KT 5823 (2 µM), milrinone (n = 7); (□) BP0104 (1 nM), KT 5823 (2 µM) (n = 6). <b>A–C)</b> Asterics indicate different EC₅₀ values. <b>D)</b> Statistics was performed by the Mann-Whitney U test. P<0.05 are considered as significant: * p<0.05, ** p<0.01 and *** p<0.001.</p

Impact of K⁺channels on milrinone-induced relaxation in PVs.

<p><b>A)</b> () milrinone (n = 6); () glibenclamide (10 µM), milrinone (n = 6); (□) glibenclamide (10 µM) (n = 4); <b>B)</b> () milrinone (n = 6); () iberiotoxin (100 nM), milrinone (n = 6); (□) iberiotoxin (100 nM) (n = 3); <b>C)</b> () milrinone (n = 8); () 4-AP (5 mM), milrinone (n = 8); (□) 4-AP (5 mM) (n = 8). <b>A–C)</b> Asterics indicate different EC₅₀ values. <b>C)</b> The comparison of () and (□) was performed by the Mann Whitney U test. P<0.05 are considered as significant: * p<0.05, ** p<0.01 and *** p<0.001.</p

Relaxant potency of milrinone in human PAs and PVs.

<p><b>A)</b> () PA: milrinone (n = 4); () PV: milrinone (n = 3); <b>B)</b> (•) PA: BP0104 (100 nM) (n = 5); (▪) PV: BP0104 (50 nM) (n = 6); <b>C)</b> (•) PA: BP0104 (100 nM), milrinone (n = 5); (▪) PV: BP0104 (50 nM), milrinone (n = 5); <b>D)</b> Comparison of basal cAMP-levels of human PAs (n = 4) and human PVs (n = 8). (<b>B)</b> Statistics was performed by a linear mixed model. <b>C)</b> Asterics indicate different EC₅₀ values. <b>D)</b> The comparison of PAs and PVs was performed by the Mann Whitney U test. P<0.05 are considered as significant: * p<0.05, ** p<0.01 and *** p<0.001.</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

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

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