Expression of nitric oxide synthase isoforms in normal human tracheobronchial epithelial cells in vitro: dependence on retinoic acid and the state of differentiation.

The retinoic acid (RA) and differentiation dependence of constitutive expression of the nitric oxide synthase (NOS) isoforms, iNOS, eNOS, and bNOS, was examined by reverse transcriptase polymerase chain recitation (RT-PCR) in cultured, normal, human, tracheobronchial epithelial (NHTBE) cells. In the presence of RA (RA+), early passage NHTBE cells grown in air-liquid interface (ALI) cultures undergo mucous differentiation; in the absence of RA (RA-), they undergo metaplastic squamous differentiation. Under both conditions the respective differentiated phenotype develops around day 10 of culture. We found that iNOS mRNA levels were much higher in RA+ cultures, expressing the mucous phenotype, than in RA- cultures, expressing the metaplastic squamous phenotype. In contrast, eNOS mRNA levels were much higher in RA- cultures than in RA+ cultures. Expression of bNOS was not significantly affected by the RA status. The pattern of expression of NOS isoforms was then studied during the course of development of the two cellular phenotypes. During the early stages of differentiation, expression of iNOS (RA+) and eNOS (RA-) was very low, indicating that the expression of these two isoforms was not only dependent on the presence or absence of RA, but also on the degree of differentiation. The differentiation dependence of bNOS mRNA was less obvious. Four days of RA treatment of RA- cultures, which reverses the squamous phenotype and restores mucous differentiation, induced iNOS expression in a concentration-dependent manner. eNOS expression was depressed by 10(-8) M RA, while bNOS mRNA levels were slightly reduced by 10(-6) M RA. No NOS proteins were detected in unstimulated RA+ and RA- cultures. iNOS protein was induced by cytokine treatment in RA+ cultures, in contrast to eNOS and bNOS protein levels, which were unaffected. Our studies show that constitutive expression of the NOS isoforms is differentially regulated and that iNOS and eNOS mRNA levels are dependent on the stage of mucous and squamous differentiation, respectively. bNOS expression was only marginally affected by the RA or differentiation status.