Partial cloning of constitutive and inducible nitric oxide synthases and detailed neuronal expression of NOS mRNA in the cerebellum and optic tectum of adult Atlantic salmon (Salmo salar)

Studies of different species have implicated nitric oxide (NO) synthase (NO S) in various physiological and pathological events. Three major NOS isofor ms are present in the brain of mammals; endothelial NOS (eNOS), neuronal NO S (nNOS) and inducible NOS (iNOS). Little is known about the significance o f the presence of these proteins in the brain. We report the first investig ation into the presence of nNOS and iNOS isoforms in a teleost, adult Atlan tic salmon (Salmo salar). Complementary DNA was synthesized from cerebellum and thymus mRNA using RT-PCR techniques with primers against conserved reg ions of NOS. Cloning and sequencing revealed a partial gene sequence of 560 bp corresponding to mammalian nNOS from cerebellum cDNA. The predicted pro tein sequence of identified salmon nNOS possessed 85% identity to that of m ammalian nNOS. Northern blot analysis of different tissues revealed express ion in brain and heart, and indicated expression of three different nNOS mR NAs in the brain. In addition, a 389 bp sequence corresponding to iNOS was identified in thymus cDNA. Salmon iNOS is almost identical to rainbow trout iNOS (95%), but shows much less amino acid identity to goldfish (65%) and mammalian (52%) iNOS. Phylogenetically, all vertebrate nNOS and iNOS homolo gues are clustered separately. Expression studies by means of in situ hybri dization revealed abundant nNOS mRNA transcripts in distinct neuronal popul ations throughout the Purkinje cell layer of the corpus cerebellum and the periventricular layer of the optic tectum. Our data show that adult Atlanti c salmon possess a gene encoding an nNOS isoform and putative alternatively spliced forms, which are expressed in distinct neuronal populations in the cerebellum and optic tectum, and in yet unidentified cell types in the hea rt. The data suggest that the arising of different vertebrate NOS isoforms is an evolutionary old event. The well conserved sequences present in salmo n and mammalian nNOS may reflect their importance in protein function, wher eas interspecies distributional differences in cellular expression of nNOS and sequence differences of iNOS may reflect variations and specializations in routes of NO action in the vertebrate phylogeny. (C) 2000 Elsevier Scie nce B.V. All rights reserved.