Neuronal isoform of nitric oxide synthase is expressed at low levels in human retina

Abstract

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 sequence showed a possibility of genetic polymorphism in comparison to human brain form. 2. Restriction fragment length polymorphism (RFLP) patterns of a partial cDNA fragment suggest that there is genetic polymorphism in the neuronal form of NOS. Important differences were observed in a certain region between human retinal and brain froms. This region is a result of frame shift by the addition of three cytidines. In this study, regions from human brain (cerebellum) and skeletal muscle as well as retina were sequenced to confirm the difference in this region. The sequences from these tissues were completely identical. This indicated that genetic polymorphism of nNOS gene was due to single base substitution and not frame shift phenomenon by addition or deletion of bases. 3. The nNOS mRNA of approximately 12 kb was detected by northern blot analysis. The lower level of the expression was distinguished in comparison to those of human brain and skeletal muscle. The cDNA transiently 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 approximately 160 kDa by both in vitro and in vivo translation systems. This NOS was calcium dependent and the K(m) for arginine was 4.4 microM. 4. The Ca+2, L-arginine and NADPH dependency along with the inhibitory effect of N-nitro-L-arginine on NOS activity were evaluated. The finding of a constitutive from of NOS in human retina, which is calcium-NADPH dependent, gives further credence to the possible role of nitric oxide in retinal function and neuronal diseases.