scholarly journals NOSIP and Its Interacting Protein, eNOS, in the Rat Trachea and Lung

2005 ◽  
Vol 53 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Peter König ◽  
Jürgen Dedio ◽  
Stefanie Oess ◽  
Tamara Papadakis ◽  
Axel Fischer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cellular Localization ◽  
Muscle Cells ◽  
Clara Cells ◽  
Rt Pcr ◽  
Interacting Protein ◽  
Endothelial Nitric Oxide ◽  
Rat Trachea

Endothelial nitric oxide synthase (eNOS), the major nitric oxide (NO)-generating enzyme of the vasculature, is regulated through multiple interactions with proteins, including caveolin-1, Hsp90, Ca2+-calmodulin, and the recently discovered eNOS-interacting protein, NOSIP. Previous studies indicate that NOSIP may contribute to the intricate regulation of eNOS activity and availability. Because eNOS has been shown to be abundantly expressed in the airways, we determined the expression and cellular localization of NOSIP in rat trachea and lung by RT-PCR and immunohistochemistry and examined the interaction of NOSIP with eNOS in lung by coimmunoprecipitation. In tracheal epithelium and lung, NOSIP mRNA expression was prevalent, as shown by RT-PCR, and the corresponding protein interacted with eNOS, as demonstrated by coimmunoprecipitation. Using immunohistochemistry, we found both NOSIP and eNOS immunoreactivity in ciliated epithelial cells of trachea and bronchi, while Clara cells showed immunoreactivity for NOSIP only. NOSIP and eNOS were present in vascular and bronchial smooth muscle cells of large arteries and airways, whereas endothelial cells, as well as bronchiolar and arteriolar smooth muscle cells, exclusively stained for NOSIP. Our results point to functional role(s) of NOSIP in the control of airway and vascular diameter, mucosal secretion, NO synthesis in ciliated epithelium, and, therefore, of mucociliary and bronchial function.

Induction and cDNA sequence of inducible nitric oxide synthase from canine aortic smooth muscle cells

1998 ◽  
Vol 275 (4) ◽  
pp. H1122-H1129 ◽  
Author(s):  
Xiaofang Wang ◽  
Christopher G. A. McGregor ◽  
Virginia M. Miller
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Smooth Muscle Cells ◽  
Allograft Rejection ◽  
Muscle Cells ◽  
Type Ii ◽  
Rt Pcr ◽  
Aortic Smooth Muscle ◽  
Oxide Synthase

An inducible isoform of nitric oxide synthase (type II, iNOS) is expressed in cardiac and vascular smooth muscle in response to inflammatory cytokines. The dog is an important large animal used for cardiovascular research including effects of exercise, heart failure, and allograft rejection. However, molecular probes for iNOS developed in other mammals have not been reliable for the study of iNOS induction in canine vascular smooth muscle. Experiments were designed to develop a molecular probe for canine iNOS. Smooth muscle cells were isolated from canine aortas. The cells ( passages 3–10) were incubated for 1, 3, 6, 12, 24, 48, or 72 h in the absence and presence of Escherichia coli lipopolysaccharide (LPS) to induce iNOS. Total RNA was isolated from the cells using standard techniques. RT-PCR with primers against conserved regions of all known iNOS enzyme was used to clone the iNOS cDNA. RT-PCR showed a single band only from cells treated with LPS. Cloned cDNA from cultured canine aortic smooth muscle cells has 84% homology to human, 81% to rat, and 81% to mouse iNOS gene. Identification of the cDNA for canine iNOS will be useful in the study of differential, transcriptional regulation of inducible (type II) compared with constitutive endothelial (type III) NOS in canine studies of allograft rejection and cardiovascular disease.

Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells

1998 ◽  
Vol 275 (2) ◽  
pp. G342-G351 ◽  
Author(s):  
B.-Q. Teng ◽  
K. S. Murthy ◽  
J. F. Kuemmerle ◽  
J. R. Grider ◽  
K. Sase ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Taenia Coli ◽  
Rt Pcr ◽  
Specific Primers ◽  
Gastric Smooth Muscle ◽  
Oxide Synthase

The aim of this study was to identify the nitric oxide synthase (NOS) isoform expressed in freshly dispersed rabbit gastric smooth muscle cells and in cultured rabbit gastric, human intestinal, and guinea pig taenia coli smooth muscle cells. RT-PCR products of the predicted size (354 bp) were obtained with endothelial NOS (eNOS)-specific primers, but not neuronal NOS (nNOS)- or inducible NOS (iNOS)-specific primers, in all smooth muscle preparations except guinea pig taenia coli. Control RT-PCR studies showed absence of the endothelial markers, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor receptor (VEGFR), and the interstitial cell marker, c- kit, from cultures of smooth muscle cells. Cloning and sequence analysis showed that the predicted amino acid sequence (117 residues) in rabbit and human smooth muscle cells differed by only one residue from that of human eNOS. Northern blot analysis, using the PCR-generated and cloned eNOS cDNA from rabbits and humans as probes, demonstrated the expression of eNOS mRNA (4.4 kb) in both species. eNOS, but not nNOS or iNOS, transcripts were localized by in situ RT-PCR in single, freshly dispersed rabbit gastric smooth muscle cells; expression was evident in the majority of cells in each preparation. We conclude that eNOS is selectively expressed in rabbit gastric and human intestinal smooth muscle cells. The results confirm functional evidence for the existence of a constitutive NOS in smooth muscle cells of the gut in different species, except for guinea pig taenia coli.

Thrombin Prevents the Expression of Inducible Nitric Oxide Synthase in Vascular Smooth Muscle Cells by a Proteolytically-Activated Thrombin Receptor

1995 ◽  
Vol 74 (03) ◽  
pp. 980-986 ◽  
Author(s):  
Valérie B Schini-Kerth ◽  
Beate Fißithaler ◽  
Thomas T Andersen ◽  
John W Fenton ◽  
Paul M Vanhoutte ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Inducible Nitric Oxide Synthase ◽  
Muscle Cells ◽  
Thrombin Receptor ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

SummaryProteolytically active forms of thrombin (α- and γ-thrombin) and thrombin receptor peptides inhibited the release of nitrite, a stable endproduct of nitric oxide, evoked by interleukin-1 β(IL-1 β) in cultured vascular smooth muscle cells while proteolytically inactive forms [D-Phe-Pro-Arg chloromethyl ketone-α-thrombin (PPACK-α- thrombin) and diisopropylphosphoryl-α-thrombin (DIP-α-thrombin)] had either no or only minimal inhibitory effects. Under bioassay conditions, perfusates from columns containing IL-1 β-activated vascular smooth muscle cells or cells treated with IL-1βplus PPACK-α-thrombin relaxed detector blood vessels. These relaxations were abolished by the inhibitor of nitric oxide synthesis, NG-nitro-L arginine. No relaxations were obtained with untreated cells or IL-1 β-treated cells in the presence of α-thrombin. The expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells by IL-1 β was impaired by α-thrombin. These results demonstrate that thrombin regulates the expression of the inducible nitric oxide synthase at a transcriptional level via the proteolytic activation of the thrombin receptor in vascular smooth muscle cells

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