Cloning and Expression of Cytokine-Inducible Nitric Oxide Synthase cDNA From Rat Islets of Langerhans

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the β-cells of interleuldn (IL)-1β–exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokineexposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH β-cells cultured in the absence of cytokines. Addition of IL-1α alone or in combination with tumor necrosis factor-α induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokineexposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the Larginine analogs, Nω-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pi values in the range of 6.8 to 7.0. In conclusion, the IL-1β-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.