Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy.

Abstract

We used an antibody to rat Clara cell 10 KD secretory protein (CC10) to compare the abundance of CC10 in non-ciliated epithelium of bronchioles and bronchi by immunohistochemistry and laser scanning confocal microscopy (LSCM) in the reflectance mode. Three zones of reflectance intensity (high, medium, low), directly related to CC10 content, were used to distinguish subcellular compartments of differing densities. Two major compartments contained CC10: granules and endoplasmic reticulum. Bronchiolar cell granules had relatively even reflectance, a high CC10 concentration (0.92 mg/ml), and occupied 7.5% of the cell volume. Bronchial cell granules were bi-zonal, lower in CC10 concentration (0.83 mg/ml), and occupied less cell volume (2.3%). Low-reflecting endoplasmic reticulum occupied a greater cell volume in bronchioles than in bronchi (32.8% and 22.1%, respectively). An intermediate-density zone consisted of endoplasmic reticulum close to granules. CC10 abundance, expressed as ng stored per unit surface area of epithelial basal lamina, was more than three times greater in bronchioles than in proximal bronchi (1.50 ng/mm2 vs 0.42 ng/mm2). These data demonstrate that the process of CC10 secretion differs markedly in non-ciliated epithelium of bronchi and bronchioles. Identifiable mucous-goblet cells did not contain CC10. The LSCM provides a method for quantifying intracellular CC10 pools in intact lung cells and has the potential for quantifying other proteins in subcellular compartments.