Site-specific gene expression of nNOS variants in distinct functional regions of rat gastrointestinal tract.
5' mRNA variants of neuronal nitric oxide synthase (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOSalpha) or alternative splicing encoding NH(2)-terminally truncated proteins (nNOSbeta/gamma) that lack the PDZ/GLGF domain for protein-protein interaction of nNOSalpha. We studied the expression of 5' nNOS mRNA forms and nNOS-interacting proteins (postsynaptic density protein-95; PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with COOH- and NH(2)-terminal nNOS antibodies. 5' nNOS mRNA variants showed a site-specific expression along the gastrointestinal tract with presence of all forms (nNOSalpha-a, -b, -c; nNOSbeta) in the muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of nNOSalpha-a and nNOSbeta mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOSalpha and nNOSbeta cDNAs revealed proteins of ~155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and NH(2)-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell-specific expression and distinct compartmentalization of nNOS proteins. Observed distribution of 5' nNOS mRNA variants and proteins argue for a complex control of nNOS expression by usage of separate promoters, cell- and site-specific splicing mechanisms, and translational initiation. These mechanisms could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOSalpha knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.
Saur
Dieter
D
Department of Internal Medicine II, Technical University of Munich, Ismaningerstrasse 22, 81675 Munich, Germany. Dieter.Saur@lrz.tu-muenchen.de
Neuhuber
Winfried L
WL
Gengenbach
Bernd
B
Huber
Andrea
A
Schusdziarra
Volker
V
Allescher
Hans-Dieter
HD
D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D003106:Colon / Q000294:innervation; D018076:DNA, Complementary; D004064:Digestive System / Q000294:innervation*; D000076122:Disks Large Homolog 4 Protein; D004386:Duodenum / Q000294:innervation; D017615:Enteric Nervous System / Q000201:enzymology*; D004947:Esophagus / Q000294:innervation; D015971:Gene Expression Regulation, Enzymologic; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000235:genetics*; D052248:Nitric Oxide Synthase Type I; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000502:physiology; D011708:Pylorus / Q000201:enzymology / Q000294:innervation; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013270:Stomach / Q000294:innervation; D013569:Synapses / Q000201:enzymology; D014158:Transcription, Genetic / Q000502:physiology
D018076:DNA, Complementary; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C033652:postsynaptic density proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat