Involvement of protein kinases in the potentiation of lipopolysaccharide-induced inflammatory mediator formation by thapsigargin in peritoneal macrophages

We have explored the regulatory roles played by Ca2+-dependent signaling on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E-2 (PGE (2)), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) rel ease in mouse peritoneal macrophages. To elevate intracellular Ca2+, we use d thapsigargin (TG) and UTP. Although LPS alone cannot stimulate NO synthes is, co-addition with TG, which sustainably increased [Ca2+](i), resulted in NO release. UTP, via acting on P2Y(6) receptors, can stimulate phosphoinos itide (PI) turnover and transient [Ca2+](i) increase, however, it did not p ossess the NO priming effect. LPS alone triggered the release of PGE(2), TN F-alpha, and IL-6; all of which were potentiated by the presence of TG, but not of UTP. The stimulatory effect of LPS plus TG on NO release Tvas inhib ited by the presence of Ro 31-8220, Go6916, KN-93, PD 098059, or SB 203580, and abolished by BAPTA/AM and nuclear factor kappaB (NF-kappaB) inhibitor, PDTC. PGE(2), TNF-alpha, and IL-6 release by LPS alone were attenuated by Ro 31-8220, Go6916, PD 098059, SE 203580, and PDTC. Using L-NAME, soluble T NF-a receptor, IL-6 antibody, NS-398, and indomethacin, we performed experi ments to understand the cross-regulation by the four mediators. The results revealed that TNF-alpha up-regulated NO, PGE(2), and IL-6 synthesis; PGE(2 ) up-regulated NO, but down-regulated TNF-alpha synthesis; and PGE(2) and I L-6 mutually up-regulated reciprocally. Taken together, murine peritoneal m acrophages required a sustained [Ca2+](i) increase, which proceeds after TG , but not UTP, stimulation, to enhance LPS-mediated release of inflammatory mediators, particularly for NO induction. Activation of PKC-, ERK-, and p3 8 MAPK-dependent signaling also are essential for LPS action. The positive regulatory interactions among these mediators might amplify the inflammator y response caused by endotoxin.