Novel adaptation of a method to assess responsiveness of bronchial segments in vitro

In vivo agonist delivery to the lungs is characterized by absorption through the mucosal layer followed by access to the smooth muscle. When agonists are applied to perfused bronchial segments, a difference in potency to agonists applied to the serosal (outside) or mucosal (inside) surface can be demonstrated. In order to elucidate this effect in canine bronchial segments, we adapted a method to assess responsiveness of agonists applied to the inside or outside surface of canine bronchial segments. A 2 cm "fluid-tight" length of bronchus was cannulated and mounted in a perfusion chamber. Auxotonic contraction of the bronchus displaced fluid inside the segment up a column and the change in height of fluid within the column (afterload) was measured as a change in hydrostatic pressure (volumetric). We assessed the optimal conditions for measuring bronchial responsiveness to acetylcholine. Neither stretching segments lengthways from 100 to 140% of resting length, nor altering the transmural pressure from 3 to 21 cmH2O had a significant effect on the potency of acetylcholine applied to the outside surface. Both acetylcholine (n=7) and methacholine (n=4) were approximately 10-fold more potent when applied to the outside surface than the inside surface of bronchial segments (p<0.0001). Furthermore, mechanical removal of the epithelium (n=6) led to a 67-fold increase in potency of acetylcholine applied to the inside surface compared with segments with epithelium. The advantages that this system has over previously reported methods include: 1) agonists can be injected at a low flow rate so as to minimize epithelial stress; 2) changes in luminal volume are measured under conditions of no flow; 3) after-load can be varied such that contraction is either practically isotonic or auxotonic; and 4) a "barrier effect" of the epithelium of canine bronchial segments can be demonstrated. This new adapted method will provide us with the means to assess the relationship between in vitro and in vivo responsiveness in human bronchi.