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 beta-cells of interleukin (IL)-1 beta-exposed rat isle ts, suggesting a role for NO in the pathogenesis of type I diabetes. T he aim of this study was to clone and characterize iNOS cDNA from cyto kine-exposed islets. Neither NO production nor iNOS transcription coul d be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cu ltured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO produ ction (measured as nitrite) from both islets and RIN cells. iNOS trans cripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence a nalysis revealed a near 100% identity to the recently published iNOS c DNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cel ls. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inh ibited by addition of the L-arginine analogs, N-omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected m olecular mass of 131 kDA and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat i slets and RIN cells is encoded by the same transcript as the iNOS indu ced in other cell types.