Previous studies from this section have shown that human retina expresses both constitutive brain form of nitric oxide synthase (NOS) as well as inducible form. In this report studies regarding molecular cloning, sequencing and expression of human retinal nitric oxide synthase genes are being presented. Full length cDNAs were constructed using polymerase chain reaction using DNA templates obtained from mRNA's by reverse transcriptase reaching combined with 5' and 3'-RACE. One of the CDNAs consists of 4302 in the open reading frame encoding for a protein of 1434 amino acids. This had 99.2% homology to human brain NOS cDNA. The deduced amino acid sequence of retinal NOS was one amino acid longer than brain NOS. The retinal NOS cDNA was cloned into an expression vector and transiently transfected into CHO-K1 cells. The cells expressed a protein with high level of NOS activity. The size was found to be, 160 kDa. The enzyme was calcium-calmodulin dependent and required NADPH. The Km for argenine was found to be 4.4 pM. And the enzyme was inhibited by N-nitroso L-argenine in a competitive fashion. The human retinal cDNA after insertion into different vectors are being expressed in fruit flies (Drosophila melanogaster) for investigation into the role of nitric oxide in memory. Two inducible forms of NOS are also found to be expressed in human retina. The cDNA's have been cloned, sequenced and are being expressed in CHO-K1 cells. The size of mRNA were shown to be 4.2 and 4.5 kb. The deduced aminoacid sequence of 4.2kb inducible NOS of human retina was found to be similar to chondrocytes and human adeno carcinoma cells (DLD-1) except for one amino acid difference. The 4.5 kb appears to be due to differential splicing and a longer untranslated 5' and 3' region. The role of inducible NOS is not clear. However the finding that human retina expresses both constitutive brain form of NOS as well as inducible forms gives further credence to the possible role of nitric oxide in retinal function and disease.
