Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.
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.
Teng
B
B
Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298-0711, USA.
Murthy
K S
KS
Kuemmerle
J F
JF
Grider
J R
JR
Sase
K
K
Michel
T
T
Makhlouf
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003106:Colon / Q000166:cytology / Q000201:enzymology; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000201:enzymology; D006801:Humans; D007421:Intestine, Small / Q000166:cytology / Q000201:enzymology*; D007583:Jejunum / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D017978:Receptors, Growth Factor / Q000096:biosynthesis / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013270:Stomach / Q000166:cytology / Q000201:enzymology*; D014158:Transcription, Genetic
D017931:DNA Primers; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor