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 and (RA) and differentiation dependence of constitutive e xpression of the nitric oxide synthase (NOS) isoforms, iNOS, eNOS, and bNOS, was examined by reverse transcriptase polymerase chain recitati on (RT-PCR) in cultured, normal, human, tracheobronchial epithelial (N HTBE) cells. In the presence of RA (RA(+)), early passage NHTBE cells grown in air-liquid interface (ALI) cultures undergo mucous differenti ation; 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 l evels were much higher in RA(+) cultures, expressing the mucous phenot ype, than in RA(-) cultures, expressing the metaplastic squamous pheno type. In contrast, eNOS mRNA tenets were much higher in RA(-) cultures than in RA(+) cultures. Expression of bNOS was not significantly affe cted by the RA status. The pattern of expression of NOS isoforms was t hen studied during the course of development of the two cellular pheno types. 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 an the presence or absenc e of RA, but also on the degree of differentiation. The differentiatio n 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-dep endent manner. eNOS expression was depressed by 10(-8) M RA, while bNO S mRNA levels were slightly reduced by 10(-6) M RA. No NOS proteins we re 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 regulate d and that iNOS and eNOS mRNA levels are dependent on the stage of muc ous and squamous differentiation, respectively. bNOS expression was on ly marginally affected by the RA or differentiation status.