Regulation of immune complexes binding of macrophages by pectic polysaccharide from Bupleurum falcatum L.: pharmacological evidence for the requirement of intracellular calcium/calmodulin on Fc receptor up-regulation by bupleuran 2IIb.
The pectic polysaccharide, bupleuran 2IIb, up-regulates Fc-receptor (FcR) expression on peritoneal macrophages in a dose-dependent manner. The intracellular signal transduction by bupleuran 2IIb leading to the expression of FcR was studied. Neither the protein kinase C (PKC) inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride, nor the structurally distinct PKC antagonist, calphostin C, inhibited bupleuran 2IIb-induced up-regulation of FcR, whereas two direct activators of PKC, L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulphonamide were unable to up-regulate the expression of FcR. The protein kinase A (PKA) inhibitor, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide dihydrochloride also did not inhibit bupleuran 2IIb-induced up-regulation of FcR. Fluorescence image analysis using the calcium-sensitive dye, Fura-2, demonstrated that bupleuran 2IIb induced a rapid increase in intracellular levels of calcium (Ca2+). When macrophages were treated with calcium antagonist, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, bupleuran 2IIb-induced up-regulation of FcR was inhibited in a dose-dependent manner. The bupleuran 2IIb-induced up-regulation of FcR was also blocked by two structurally distinct calmodulin antagonists, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride. Furthermore, elevation of intracellular Ca2+ using the calcium ionophore, A23187, led to up-regulation of the FcR expression in a dose-dependent manner. These results suggest that bupleuran 2IIb induces the up-regulation of FcR on macrophages by a mechanism dependent on an increase in intracellular Ca2+ followed by activation of the calmodulin, but not by a PKC or PKA pathway.