Ca2+ and calmodulin selectively regulate lipopolysaccharide-inducible cytokine mRNA expression in murine peritoneal...

The role of Ca2+ and Calmodulin in regulating LPS-induced cytokine gene expression in murine peritoneal macrophages has been investigated. Treatment of macrophages with three structurally distinct antagonists of Calmodulin (Trifluoperazine, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide, and 1,3-dihydro-1-(-((4-mentyl-4H,6H-pyrrolo(1,2-a) (4,1)-benzoxazepin-4-yl)methyl)-4-peperidinyl)-2H-benzimi dazol-2-one) resulted in a characteristic modulation of the expression of three LPS-inducible cytokine genes: IL-1 alpha mRNA levels were only modestly reduced, IL-1 beta mRNA levels were markedly suppressed and IP-10 mRNA levels were increased. The same pattern of modulation was seen when LPS-stimulated cells were also treated with two different Ca2+ antagonists (8-(diethylamono)-octyl-3,4,5-trimethoxybenzoate hydrochloride and bis-(o-amonophenoxy)-ethane-N,N,N'N'-tetraacetic acid). Although the suppression of IL-1 beta mRNA accumulation by N-(6-amonohexyl)-5-chloro-1-napthalene-sulfonamide or bis-(o-amonophenoxy)ethane-N,N,N'N'-tetraacetic acid occurred even if the antagonist was added after LPS, the potentiation of IP-10 mRNA levels required the use of the agent before or along with the LPS stimulus. Elevation of intracellular Ca2+ using ionomycin did not initaite cytokine gene expression and thus changes in Ca2+ cannot replace the LPS-initiated signal. Furthermore, removal of extracellular Ca2+ did not block the response to LPS. Calmodulin antagonists selectively increased the transcriptional activity of the IP-10 gene but decreased the stability of all three mRNA measured. Thus the mechanisms involved in Ca2+/Calmodulin control of macrophage gene expression are multifactorial and contribute to the diversity of macrophage inflammatory behavior. In concert with previous reports, the present results indicate that Ca2+, acting through Calmodulin may be a necessary but insufficient component of the signalling process that mediates intracellular response to LPS. Agents that alter intracellular Ca2+ levels without inducing cytokine gene expression may thereby indirectly regulate inflammation.