LIPOPOLYSACCHARIDE-STIMULATED TNF-ALPHA RELEASE FROM CULTURED RAT KUPFFER CELLS - SEQUENCE OF INTRACELLULAR SIGNALING PATHWAYS

We recently hypothesized that lipopolysaccharide (LPS) stimulation of rat Kupffer cells to induce tumor necrosis factor alpha (TNF-alpha) re lease requires internalization of LPS, acidification of endosomes, ele vation of intracellular calcium, protein kinase C (PKC) activation, an d protein tyrosine kinase (PTK) activation. This study uses inhibitors in pulse-chase experiments to determine the sequence of events of int racellular signals required for LPS-stimulated TNF-alpha release from Kupffer cells, Inhibitors of internalization (cytochalasin B, monodans ylcadaverine) prevented LPS-stimulated TNF-alpha release when added si multaneously with LPS but when added 10 min after LPS, no significant inhibition occurred. The inhibitor of PTK, tyrphostin AG, blocked TNF- alpha release by only 39 +/- 4 % (P < 0.001 compared with TNF-alpha re lease when added simultaneously with LPS) tr;when added 10 min after L PS, Inhibitors of endosomal acidification (bafilomycin A, monensin) in hibited LPS-stimulated TNF-alpha release by 92 +/- 11% (P < 0.001 when no inhibitor was used) when added 10 min after LPS and their effect w as totally abrogated when added 45 min after LPS, The PKC inhibitor, H -7, blocked TNF-alpha release by 94 +/- 9% (P < 0.001 when no inhibito r was used) when added 30 min after LPS. The calcium channel blocker, nisoldipine, still inhibited LPS-stimulated TNF-alpha release when add ed 45 min after LPS. These data support the hypothesis that for I,PS-s timulated TNF-alpha release in Kupffer cells, LPS must first be intern alized, which may stimulate PTK( activation. An intermediate step of s ignaling involves endosomal acidification. Elevation of intracellular calcium and PI(C activation occur as late intracellular signaling even ts.