Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells

Pasteurella multocida toxin, both native and recombinant, is an extremely potent mitogen for Swiss 3T3 cells and acts to enhance the formation of total inositol phosphates (Rozengurt, E., Higgins, T., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). P. multocida toxin also stimulates diacylglycerol production and activates protein kinase C (Staddon, J. M., Chanter, N., Lax, A. J., Higgins, T. E., and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848). Here we analyze, by [³H]inositol labeling and high performance liquid chromatography, the inositol phosphates in recombinant P. multocida toxin-treated cells. Recombinant P. multocida toxin stimulated increases in [³H]inositol 1,4,5-trisphosphate ([³H]InS(1,4,S)P₃) and its metabolic products, including Ins(1,3,4,5)P₄, Ins(1,3,4)P₃, Ins(1,4)P₂, Ins(4/5)P, and Ins(1/3)P. The profile of the increase in the cellular content of these distinct inositol phosphates was very similar to that elicited by bombesin. Furthermore, recombinant P. multocida toxin, like bombesin, mobilizes an intracellular pool of Ca²⁺. Recombinant P. multocida toxin pretreatment greatly reduces the Ca²⁺-mobilizing action of bombesin, consistent with Ca²⁺ mobilization from a common pool by the two agents. The enhancement of inositol phosphates and mobilization of Ca²⁺ by recombinant P. multocida toxin were blocked by the lysosomotrophic agents methylamine, ammonium chloride, and chloroquine and occurred after a dose-dependent lag period. The stimulation of inositol phosphate production by recombinant P. multocida toxin persisted after removal of extracellular toxin, in contrast to the reversibility of the action of bombesin. Recombinant P. multocida toxin, unlike bombesin and guanosine 5′-O-(γ-thiotriphosphate), did not cause the release of inositol phosphates in permeabilized cells. These data demonstrate that recombinant P. multocida toxin, acting intracellularly, stimulates the phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate.