SYNERGISTIC COOPERATION BETWEEN THAPSIGARGIN AND PHORBOL ESTER FOR INDUCTION OF NITRIC-OXIDE SYNTHESIS IN MURINE PERITONEAL-MACROPHAGES

The biochemical transductional events involved in NO synthesis are not fully understood. These studies, therefore, were undertaken to elucid ate the role of intracellular calcium and protein kinase C (PKC) in th e induction of nitric oxide (NO) synthesis in murine peritoneal macrop hages. Thapsigargin (TG), Ca2+-ATPase inhibitor of endoplasmic reticul um, had modest activity on NO synthesis by itself, whereas phorbol est er, PKC activator, alone had no effect. When TG was used in combinatio n with phorbol ester, there was a marked cooperative induction of NO s ynthesis in a dose-dependent manner. The optimal effect of phorbol est er was shown in the first 6 h after TG treatment. In addition, the abi lity of TG with phorbol ester on NO synthesis could be mimicked by ano ther chemically unrelated inhibitor of Ca2+-ATPase, 2,5-di-(t-butyl)-1 ,4-benzohydroquinone, and Ca2+ ionophore, A23187. This increase of NO synthesis was reflected as increased amount of NO synthase (NOS) mRNA, as determined by Northern blotting. Intracellular Ca2+ transient by T G was not affected in the presence or absence of extracellular Ca2+, i ndicating that TG must be effective on cytosolic Ca2+ pool. In additio n, chelation of intracellular Ca2+ by acetoxymethyl ester of -bis-(2-a minophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracel lular Ca2+ chelating agent, blocked TG- or TG + PMA-induced NO product ion. PKC inhibitors such as staurosporine or polymyxin B reduced only the synergistic cooperative effect of TG with phorbol ester without af fecting TO-induced NO production. In addition, when the cells were pre treated with phorbol ester before TG treatment, there was no synergy b etween TG and phorbol ester, indicating that PKC is not directly invol ved in the expression of NOS but involved in ''triggering'' signal. Se cretion of NO corresponded with tumor cell killing, but TG plus phorbo l ester-activated macrophages failed to kill tumor cell targets in the presence of N-G-monomethyl-L-arginine. Collectively, these data illus trate that mobilization of intracellular Ca2+ provides a ''priming'' s ignal for induction of NOS gene expression by itself and it also requi res PKC as a ''triggering'' signal for macrophage tumoricidal activity .