PRIMARY CULTURES OF RAT ISLET CAPILLARY ENDOTHELIAL-CELLS - CONSTITUTIVE AND CYTOKINE-INDUCIBLE MACROPHAGE-LIKE NITRIC-OXIDE SYNTHASES ARE EXPRESSED AND ACTIVITIES REGULATED BY GLUCOSE-CONCENTRATION

We have succeeded in obtaining cultures of pure rat islet capillary en dothelial cells. These multiply in vitro and exhibit the same antigeni c phenotype as expressed in situ: von willebrand factor(high), Ox43 (r at endothelial marker)(weak), and Ox2 (thymocyte and brain endothelium marker)(high). This phenotype differs from both exocrine endothelium stained in situ and rat aorta endothelial cells cultured in vitro unde r identical conditions. Islet and aorta endothelial cells were culture d in the presence of various glucose concentrations. Nitrite and citru lline concentrations in culture supernatants were measured as an indir ect quantification of nitric oxide formation. In islet endothelia, bot h nitrite and citrulline levels were found to be strongly glucose-depe ndent, with high levels at high glucose concentrations and vice versa, in contrast to aorta endothelial cells, where no glucose effect was f ound. Shifting islet endothelial cultures from high to low glucose lev els or the reverse led to a slow decrease or increase in nitrite and c itrulline formation with several cell generations needed to reach stea dy levels. Adding a combination of the cytokines interleukin-1 beta, t umor necrosis factor-alpha, and interferon-gamma to both endothelial c ell cultures led to a dramatic increase of nitric oxide formation. Aga in with islet but not with aorta endothelial cells a modulating effect by glucose concentrations was found. Reverse-transcription-polymerase chain reaction with specific primers demonstrated the presence of con stitutively expressed nitric oxide synthase-RNA in the islet capillary endothelial cells and confirmed the glucose effect. In addition, we f ound that cytokines indeed induce the expression of inducible cells, w hich was not found in the absence of cytokines. Electron paramagnetic resonance spectroscopy of islet endothelial cells confirmed intracellu lar synthesis of nitric oxide in the presence of cytokines. In conclus ion, we here for the first time provide evidence that constitutive nit ric oxide synthase is also expressed in capillary endothelium and that cytokine challenge leads to the expression of the inducible isoform i n these cells.