Developmental expression of NOS isoforms in fetal rat lung: implications for transitional circulation and pulmonary angiogenesis

Abstract

To better understand the role of nitric oxide (NO) in fetal lung development, specifically in the transition of the fetal circulation at birth, 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 immunohistochemical labeling with antibodies against the three known NOS isoforms and the NADPH diaphorase technique (NADPH-d). Endothelial NOS (eNOS) immunoreactivity was found in the cells of the 14-day fetal lung. As gestation proceeded, the quantity of these immunopositive cells increased, and they coalesced to form an inner (endothelial) layer of pulmonary vessels. This process of angiogenesis marked by eNOS-positive cells was seen from 15 days of gestation to at least 7 days postnatal. A majority of the eNOS immunoreactivity appeared densely in one focal spot in the cytoplasm, indicating that during development the eNOS may be primarily located in a cytoplasmic organelle. Epithelial cells of the rat airway from the same developmental period were positively stained with both brain NOS antibody (bNOS) and NADPH-d at the beginning of 13 days of gestation. Then the intensity of stainings began to decrease and reached the lowest level in the 16-day fetal lung. However, the NOS stainings of the epithelium, especially in small canalicular structures of the airways, began to increase at 18 days of gestation and was dramatically elevated at 20 days of gestation (term is 22 days). Postnatally, NOS in epithelium was decreased in distal airways in conjunction with the formation of alveolar structure. Inducible NOS (iNOS) immunoreactivity was also found in the epithelium of rat lung airways after 16 days of gestation. Unlike the bNOS staining, iNOS immunoreactivity exhibited a pattern of a small dot-like staining within epithelial cytoplasm during gestation and the first day postnatal, then changed to a pattern of diffuse cytoplasmic staining by the 7th postnatal day. This study concludes that 1) expression of three isoforms of NOS is present and regulated during lung development; 2) markedly increased NOS in epithelium near term supports a role for NO in mediating the pulmonary transition from fetal to neonatal life; and 3) eNOS immunohistochemistry serves as an effective marker to follow the process of pulmonary angiogenesis and suggests the concept of in situ formation of endothelial vesicles in developing mesenchyme.

Localization of endothelial NOS at the basal microtubule membrane in ciliated epithelium of rat lung.

“…There is evidence that exhaled NO may be derived, at least in part, from the respiratory tract (Gerlach et al, 1994), suggesting that NO originates from the airway epithelial cells (Xue et al, 1996;Rengasamy et al, 1994;Xue et al, 1994a,b;Kobzik et al 1993;Schmidt et al, 1992), consistent with the hypothesis that lung epithelial cells can produce NO. Furthermore, the NO receptor, soluble guanylate cyclase, was demonstrated in bronchial smooth muscle cells, indicating a paracrine role of NO in bronchial function (Rengasamy et al, 1994).…”

“…Their products are bNOS, iNOS, and eNOS (Asano et al, 1994;Shad et al, 1994). In vivo, immunohistochemical evidence shows that bNOS and iNOS are expressed and localized in the cytoplasm of the bronchial epithelium (Xue et al, 1996;Rengasamy et al, 1994;Kobzik et al, 1993;Xue et al, 1974a,b;Schmidt et al, 1792). However, expression and localization of eNOS remain unclear in these cells.…”

Cell-Specific Nitric Oxide Synthase-Isoenzyme Expression and Regulation in Response to Endotoxin in Intact Rat Lungs

“…Cellular expression of iNOS in the lung parenchyma has so far only been observed after experimental endotoxin stimulation or in sections from patients suffering from inflammatory lung disease (Kobzik et al, 1993;Robbins et al, 1994;Tracey et al, 1994). However, recently all three NOS isoforms were shown by immunohistochemistry to be expressed in the developing lung, and all were assumed to contribute to NO generation functioning in normal lung physiology (Sherman et al, 1999;Xue et al, 1996). A recent study performed in human airway tissue originating from patients with bronchial carcinoma undergoing lung resection showed iNOS expression in bronchial epithelial cells (Watkins et al, 1997).…”

Developmental Changes in Endothelial Vasoactive and Angiogenic Growth Factors in the Human Perinatal Lung

“…Previous data were obtained in animal studies and exhibited some apparent discrepancies. For instance, eNOS and ET-1 expression were found to increase before birth in lambs and after birth in rats and to be similar before and after birth in the piglets (1,11,12). Differences in the timing of lung development may account for the differences observed between human and animal studies (1,20,25).…”

“…In experimental animals, the basal pulmonary vessel tone appears to be tightly regulated by a balance between endothelium-derived mediators, some of which display major vasodilating effects, such as NO, EDHF, and prostacyclin, and some potent vasoconstrictive effects, such as and ET-1 and leukotrienes (4 -10). NO, for instance, produced from L-arginine by the action of a constitutive eNOS, is clearly involved in the postnatal fall in PVR in rats, lambs, and piglets (1,11,12). The effects of ET-1, mediated through two different sets of receptors, ET-A and ET-B, are more ambiguous (13).…”