RESPONSIVENESS OF INDIVIDUAL AIRWAYS TO METHACHOLINE IN ADULT-RAT LUNG EXPLANTS

We used a modified adult lung explant technique to directly measure the area of individual airways before and after methacholine (MCh) administration. Lungs were removed from 12-wk-old male Lewis rats under sterile conditions, filled with an agarose-containing solution at 37-degrees-C, and cooled to 4-degrees-C. Transverse slices (0.5-1.0 mm thick) were cut and cultured overnight. Concentration-response curves to MCh were determined for explant airways from lungs inflated to 25, 50, 75, and 100% total lung capacity (TLC) with a 1.0% agarose solution and to 75% TLC with 0.5 and 2.0% agarose solutions. MCh was added to the medium to achieve final concentrations ranging from 10(-9) to 10(-2) M. Airways were imaged before and 10 min after each increase in MCh concentration with an inverted microscope and video camera, and airway area was determined by computerized image processing. The maximal response (MR) {[1 - (minimal area/baseline area)] x 100) and concentration of MCh resulting in 50% MR (EC50) were determined. A total of 217 airways from 3-12 explants per rat constricted in a concentration-dependent manner. Baseline area was larger with both higher lung volumes and agarose concentrations. MR was greatest in the airways from the 25% TLC and 0.5% agarose explants. Although there was considerable heterogeneity toward MCh within rats (EC50 varied up to 5.46 X 10(5)-fold), the median EC50 was similar among all rats (range 1.96 X 10(-6)-5.87 X 10(-4) M). Lung inflation volume and agarose concentration affected baseline area and MR, suggesting that airway-parenchymal interdependence mechanisms are operative in this preparation. Adult lung explants provide a direct and efficient means of studying constriction of individual airways.