DEVELOPMENTAL EXPRESSION OF NOS ISOFORMS IN FETAL-RAT LUNG - IMPLICATIONS FOR TRANSITIONAL CIRCULATION AND PULMONARY ANGIOGENESIS

To better understand the role of nitric oxide (NO) in fetal lung devel opment, specifically in the transition of the fetal circulation at bir th, we studied the timing of cell-specific expression of NO synthase ( NOS) isoforms from formation of lung buds (13th day of gestation) to 7 days postnatal. Expression of NOS was studied using immunohistochemic al labeling with antibodies against the three known NOS isoforms and t he NADPH diaphorase technique (NADPH-d). Endothelial NOS (eNOS) immuno reactivity was found in the cells of the 14-day fetal lung. As gestati on proceeded, the quantity of these immunopositive cells increased, an d they coalesced to form an inner (endothelial) layer of pulmonary ves sels. This process of angiogenesis marked by eNOS-positive cells was s een from 15 days of gestation to at least 7 days postnatal. A majority of the eNOS immunoreactivity appeared densely in one focal spot in th e cytoplasm, indicating that during development the eNOS may be primar ily located in a cytoplasmic organelle. Epithelial cells of the rat ai rway from the same developmental period were positively stained with b oth brain NOS antibody (bNOS) and NADPH-d at the beginning of 13 days of gestation. Then the intensity of stainings began to decrease and re ached the lowest level in the 16-day fetal lung. However, the NOS stai nings of the epithelium, especially in small canalicular structures of the airways, began to increase at 18 days of gestation and was dramat ically elevated at 20 days of gestation (term is 22 days). Postnatally , NOS in epithelium was decreased in distal airways in conjunction wit h the formation of alveolar structure. Inducible NOS (iNOS) immunoreac tivity was also found in the epithelium of rat lung airways after 16 d ays of gestation. Unlike the bNOS staining, iNOS immunoreactivity exhi bited a pattern of a small dotlike staining within epithelial cytoplas m during gestation and the first day postnatal, then changed to a patt ern of diffuse cytoplasmic staining by the 7th postnatal day. This stu dy concludes that 1) expression of three isoforms of NOS is present an d regulated during lung development; 2) markedly increased NOS in epit helium near term supports a role for NO in mediating the pulmonary tra nsition from fetal to neonatal life; and 3) eNOS immunohistochemistry serves as an effective marker to follow the process of pulmonary angio genesis and suggests the concept of in situ formation of endothelial v esicles in developing mesenchyme.