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 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 cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured 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 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 cytokine-exposed 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 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 molecular 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 islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.
Karlsen
A E
AE
Steno Diabetes Center, Gentofte, Denmark.
Andersen
H U
HU
Vissing
H
H
Larsen
P M
PM
Fey
S J
SJ
Cuartero
B G
BG
Madsen
O D
OD
Petersen
J S
JS
Mortensen
S B
SB
Mandrup-Poulsen
T
T
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016207:Cytokines / Q000494:pharmacology*; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology*; D007527:Isoenzymes / Q000096:biosynthesis; D008099:Liver / Q000201:enzymology; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases