Protein kinase C independent activation of inducible nitric oxide synthaseby tumor necrosis factor-alpha in TM4 Sertoli cells

To investigate the nitric oxide (NO) production and its signalling mechanis m in TM4 Sertoli cells, the cells were treated with recombinant tumor necro sis factor-alpha (rTNF-alpha), recombinant interleukin-1 alpha (rIL-1 alpha ), or lipopolysaccharide (LPS), either alone or in combination with recombi nant interferon-gamma (rIFN-gamma), and NO production was measured by using the Griess method. TM4 Sertoli cells produced a small amount of NO upon tr eatment with rIFN-gamma. The effect of rIFN-gamma was drastically increased by cotreatment with rTNF-alpha in a dose-dependent manner. However, combin ation of rIL-1 alpha or LPS with rIFN-gamma did not synergize to activate c ells. RIFN-gamma in combination with rTNF-alpha showed marked increase of t he expression of iNOS protein. Protein kinase C inhibitors did not inhibit the production of NO induced by rIFN-gamma plus rTNF-alpha. These results s uggest that the role of TNF-alpha is to provide TM4 Sertoli cells with the active cofactor for NO production and TNF-alpha-induced signaling for induc tion of NO synthesis is not dependent on protein kinase C activation.