Quantitative immunocytochemistry shows calcitonin gene-related peptide-like immunoreactivity in lung neuroendocrine cells is increased by chronic hypoxia in the rat

We have previously shown that the vasodilator calcitonin gene-related peptide (CGRP) is increased in pulmonary neuroendocrine cells in response to hypoxia. To quantify the change, we have now examined lung of adult male Wistar rats exposed to hypoxia (FIO2 = 0.1) for 1 wk and littermate controls. Sections of lung were immunostained simultaneously using rabbit antiserum to rat alpha-CGRP with the peroxidase antiperoxidase technique. The area and integrated optical density of each group of endocrine cells were measured using an image analyzer. For each animal, the summed integrated optical density of endocrine cells divided by the sum of their areas was used as a measure of CGRP-like immunoreactivity. The intensity of immunostaining of endocrine cells in the respiratory portion of the lung was 43% greater than that of endocrine cells along the conducting airways (P less than 0.001). The intensity of staining was increased by approximately 12% (P less than 0.04) after 7 d of hypoxia with no apparent difference in the response of central and peripheral endocrine cells. Measurements of staining intensity of CGRP-coupled agarose beads indicated that a 12% change in staining intensity corresponded to a 15 to 20% change in the concentration of CGRP or CGRP-like immunoreactive material. The supra-optimal dilution technique (measurement of the increase in the number of immunoreactive cells upon sequential immunostaining with a supra-optimal and then an optimal dilution of primary antiserum) detected the increase in CGRP-like immunoreactivity after 7 d of hypoxia with a high degree of statistical significance (P less than 0.005) using the same number of sections.