Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium.
Nitric oxide (NO) is an important mediator of physiologic and inflammatory processes in the lung. To better understand the role of NO in the airway, we examined constitutive NO synthase (NOS) gene expression and function in NCI-H441 human bronchiolar epithelial cells, which are believed to be of Clara cell lineage. NOS activity was detected by [3H]arginine to [3H]citrulline conversion (1,070 +/- 260 fmol/mg protein per 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 NOS revealed expression solely of endothelial NOS protein. Immunocytochemistry for endothelial NOS revealed staining predominantly in the cell periphery, consistent with the association of this isoform with the cellular membrane. To definitively identify the NOS isoform expressed in H441 cells, NOS cDNA was obtained by degenerate PCR. Sequencing of the H441 NOS cDNA revealed 100% identity with human endothelial NOS at the amino acid level. Furthermore, the H441 NOS cDNA hybridized to a single 4.7-kb mRNA species in poly(A)+ RNA isolated from H441 cells, from rat, sheep, and pig lung, and from ovine endothelial cells, coinciding with the predicted size of 4.7 kb for endothelial NOS mRNA. Guanylyl cyclase activity in H441 cells, assessed by measuring cGMP accumulation, rose 6.6- and 5.4-fold with calcium-mediated activation of NOS by thapsigargin and A23187, respectively. These findings indicate that endothelial NOS is expressed in select bronchiolar epithelial cells, where it may have autocrine effects through activation of guanylyl cyclase. Based on these observations and the previous identification of endothelial NOS in a kidney epithelial cell line, it is postulated that endothelial NOS may be expressed in unique subsets of epithelial cells in a variety of organs, serving to modulate ion flux and/or secretory function.
Shaul
P W
PW
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75235.
North
A J
AJ
Wu
L C
LC
Wells
L B
LB
Brannon
T S
TS
Lau
K S
KS
Michel
T
T
Margraf
L R
LR
Star
R A
RA
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001980:Bronchi / Q000201:enzymology* / Q000473:pathology; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000201:enzymology; D004848:Epithelium / Q000201:enzymology / Q000473:pathology; D006162:Guanylate Cyclase / Q000032:analysis; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D051381:Rats; D012756:Sheep; D013045:Species Specificity; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006162:Guanylate Cyclase