Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of G(Sα)), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th) 1.2- positive lymphoma cells deficient in G(Sα). The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that G(Sα) is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP- ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H] AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.
ASJC Scopus subject areas
- Infectious Diseases