AbstractDistinct activities of toxin B up-regulate PLCβ3 or down-regulate PLCβ4-dependent IP3-Ca2+ coupling.Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca2+ responses to Gαi-linked receptors, including the C5aR, but reduced responses to Gαq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca2+ responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCβ isoform-deficient BMDM, we found that ToxB inhibited Ca2+ signaling through PLCβ4 but enhanced signaling through PLCβ3. Effects of ToxB on GPCR Ca2+ responses correlated with GPCR use of PLCβ3 versus PLCβ4. ToxB inhibited UDP Ca2+ signaling without reducing InsP3 production or the sensitivity of cellular Ca2+ stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca2+coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca2+ signaling by C5a was prevented by inhibition of PLA2 or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca2+ signaling by different GPCR-linked PLCβ isoforms in macrophages.