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.

[1]  P. Shaul,et al.  Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells. , 1994, American journal of respiratory cell and molecular biology.

[2]  R. Star,et al.  Localization and regulation of endothelial NO synthase mRNA expression in rat kidney. , 1994, The American journal of physiology.

[3]  S. Snyder,et al.  Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung. , 1994, The American journal of physiology.

[4]  S. Muallem,et al.  Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx. , 1994, The Journal of biological chemistry.

[5]  M. Lathrop,et al.  Gene structure, polymorphism and mapping of the human endothelial nitric oxide synthase gene. , 1994, Biochemical and biophysical research communications.

[6]  F. Murad,et al.  Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells. , 1994, The American journal of physiology.

[7]  J. Stamler,et al.  Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Stamler,et al.  Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. , 1993, American journal of respiratory cell and molecular biology.

[9]  A. Mukherjee,et al.  Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin. , 1993, The Journal of biological chemistry.

[10]  T. Michel,et al.  Endothelial nitric oxide synthase. N-terminal myristoylation determines subcellular localization. , 1993, The Journal of biological chemistry.

[11]  F. Murad,et al.  Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle , 1993, FEBS letters.

[12]  J. Drazen,et al.  Bronchodilator action of inhaled nitric oxide in guinea pigs. , 1992, The Journal of clinical investigation.

[13]  F. Murad,et al.  Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction. , 1992, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[14]  H. Geuze,et al.  Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung. , 1992, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[15]  S. Snyder,et al.  Nitric oxide, a novel biologic messenger , 1992, Cell.

[16]  K. Schappert,et al.  Molecular cloning and characterization of human endothelial nitric oxide synthase , 1992, FEBS letters.

[17]  S. Snyder,et al.  Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[18]  A. M. Paradiso,et al.  Intracellular Ca2+ and regulation of ion transport across rabbit Clara cells. , 1992, The American journal of physiology.

[19]  F. Salazar,et al.  Renal effects of prolonged synthesis inhibition of endothelium-derived nitric oxide. , 1992, Hypertension.

[20]  R. Johns,et al.  Nitric Oxide Alters Renal Function and Guanosine 3′,5′‐Cyclic Monophosphate , 1992, Hypertension.

[21]  O. Carretero,et al.  Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. , 1992, The Journal of clinical investigation.

[22]  F. Murad,et al.  Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides. , 1992, Science.

[23]  P. Shaul,et al.  Oxygen modulates endothelium-derived relaxing factor production in fetal pulmonary arteries. , 1992, The American journal of physiology.

[24]  S Moncada,et al.  Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. , 1991, Biochemical and biophysical research communications.

[25]  S. Snyder,et al.  Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase , 1991, Nature.

[26]  D. Hyde,et al.  Epithelial cells of the conducting airways : a species comparison , 1991 .

[27]  P. Cullen,et al.  Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[28]  S. Snyder,et al.  Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Hassid,et al.  Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. , 1989, The Journal of clinical investigation.

[30]  L. Ignarro,et al.  Endothelium-derived nitric oxide relaxes nonvascular smooth muscle. , 1989, European journal of pharmacology.

[31]  A. Gazdar,et al.  Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line. , 1988, Biochimica et biophysica acta.

[32]  P. Vanhoutte,et al.  Heterogeneity in the effects of epithelium removal in the canine bronchial tree. , 1987, Journal of applied physiology.

[33]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[34]  J. Minna,et al.  Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium. , 1986, Cancer research.

[35]  L. Ignarro,et al.  The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. , 1985, Annual review of pharmacology and toxicology.

[36]  Ordóñez Ng,et al.  Comparison of Ulex europaeus I lectin and factor VIII-related antigen in vascular lesions. , 1984 .

[37]  J. Batsakis,et al.  Comparison of Ulex europaeus I lectin and factor VIII-related antigen in vascular lesions. , 1984, Archives of pathology & laboratory medicine.

[38]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[39]  P. Leder,et al.  Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[40]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.