NEURONAL ISOFORM OF NITRIC-OXIDE SYNTHASE IS EXPRESSED AT LOW-LEVELS IN HUMAN RETINA

1. The expression of neuronal isoform of nitric oxide synthase (nNOS) was studied in human retinal tissues. The cDNA sequence was cloned in human retinal poly (A)(+) RNA by the RT-PCR method and encompassed an open-reading frame of 4,302 bp encoding 1,434 amino acids. This sequen ce showed a possibility of genetic polymorphism in comparison to human brain form. 2. Restriction fragment length polymorphism (RFLP) patter ns of a partial cDNA fragment suggest that there is genetic polymorphi sm in the neuronal form of NOS, Important differences were observed in a certain region between human retinal and brain forms, This region i s a result of frame shift by the addition of three cytidines. In this study, regions from human brain (cerebellum) and skeletal muscle as we ll as retina were sequenced to confirm the difference in this region, The sequences from these tissues were completely identical, This indic ated that genetic polymorphism of nNOS gene was due to single base sub stitution and not frame shift phenomenon by addition or deletion of ba ses. 3. The nNOS mRNA of similar to 12 kb was detected by northern blo t analysis. The lower level of the expression was distinguished in com parison to those of human brain and skeletal muscle. The cDNA transien tly transfected into CHO-K1 cells expressed a protein which contained a significant level of NOS activity. The size of the nNOS was found to be similar to 160 kDa by both in vitro and in vivo translation system s, This NOS was calcium dependent and the K-m for arginine was 4.4 mu M. 4. The Ca++, L-arginine and NADPH dependency along with the inhibit ory effect of N-nitro-L-arginine on NOS activity were evaluated, The f inding of a constitutive form of NOS in human retina, which is calcium -NADPH dependent, gives further credence to the possible role of nitri c oxide in retinal function and neuronal diseases.