ENDOTHELIAL NITRIC-OXIDE SYNTHASE IS EXPRESSED IN CULTURED HUMAN BRONCHIOLAR EPITHELIUM

Citation
Pw. Shaul et al., ENDOTHELIAL NITRIC-OXIDE SYNTHASE IS EXPRESSED IN CULTURED HUMAN BRONCHIOLAR EPITHELIUM, The Journal of clinical investigation, 94(6), 1994, pp. 2231-2236
Citations number
44
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Medicine, Research & Experimental
ISSN journal
0021-9738
Volume
94
Issue
6
Year of publication
1994
Pages
2231 - 2236
Database
ISI
SICI code
0021-9738(1994)94:6<2231:ENSIEI>2.0.ZU;2-#
Abstract
Nitric oxide (NO) is an important mediator of physiologic and inflamma tory processes in the lung. To better understand the role of NO in the airway, we examined constitutive NO synthase (NOS) gene expression an d function in NCI-H441 human bronchiolar epithelial cells, which are b elieved to be of Clara cell lineage. NOS activity was detected by [H-3 ]arginine to [H-3]citrulline conversion(1,070+/-260 fmol/mg protein pe r minute); enzyme activity was inhibited 91% by EGTA, consistent with the expression of a calcium-dependent NOS isoform. Immunoblot analyses with antisera directed against neuronal, inducible, or endothelial NO S revealed expression solely of endothelial NOS protein. Immunocytoche mistry for endothelial NOS revealed staining predominantly in the cell periphery, consistent with the association of this isoform with the c ellular membrane. To definitively identify the NOS isoform expressed i n H441 cells, NOS cDNA was obtained by degenerate PCR. Sequencing of t he H441 NOS cDNA revealed 100% identity with human endothelial NOS at the amino acid level. Furthermore, the H441 NOS cDNA hybridized to a s ingle 4.7-kb mRNA species in poly(A)(+) RNA isolated from H441 cells, from rat, sheep, and pig lung, and from ovine endothelial cells, coinc iding with the predicted size of 4.7 kb for endothelial NOS mRNA. Guan ylyl cyclase activity in H441 cells, assessed by measuring cGMP accumu lation, rose 6.6- and 5.4-fold with calcium-mediated activation of NOS by thapsigargin and A23187, respectively. These findings indicate tha t endothelial NOS is expressed in select bronchiolar epithelial cells, where it may have autocrine effects through activation of guanylyl cy clase. Based on these observations and the previous identification of endothelial NOS in a kidney epithelial cell line, it is postulated tha t endothelial NOS may be expressed in unique subsets of epithelial cel ls in a variety of organs, serving to modulate ion flux and/or secreto ry function.