Macrophage alteration induced by inflammatory toxins isolated from Tityus discrepans scorpion venom. The role of Na+/Ca2+ exchangers

We study the effect of all Tityus discrepans venom components on macrophage alterations. Only seven toxins called “Inflammatory Toxin” (InfTx1-7) induced cell changes. Incubation with InfTx1 through InfTx5 rose macrophage NO level at 2 h toxin exposure. Cells rose NO release by 4 h exposure with InfTx2 and InfTx5, the NO levels reached concentrations similar or higher than the induced by lipopolysaccharides (LPS) incubation. InfTx2, -6 and -7 increased cell TNF-α release. InfTx2 as LPS roses cell TNF-α secretion gradually in time. Macrophages were loaded with fluorescent dyes, exposed to all toxins and observed with a 3D wide field deconvolution setup. Cells exposed to whole venom or InfTx4 through InfTx7 developed pseudopodia, cytoplasm prolongations, blebs, and loss their rounded form. The molecular masses and N-terminal sequences of InfTx4 through InfTx7 were analyzed by MALDI-TOF mass spectrometry and Edman degradation. InfTx4-7 induced a remarkable increase of intracellular Ca2+ levels ([Ca2+]i), measured as a rise of normalized cell green fluorescence intensity (FI) ×2.7, ×2.6, ×95 and ×2.9 the controls, respectively. InfTx6-7 action mechanisms were studied under different conditions. Results suggested that InfTx6 interact with a membrane sodium channel inducing cell depolarization with a consequent increase on intracellular [Na+], this would activate Na+/Ca2+ exchanger 3 (NCX) in the reverse mode and the phospholipase C inositol 1,4,5-trisphosphate (PLC-IP3) signaling pathway inducing [Ca2+]i overload. Inftx7 should activate the NCX in reverse mode and/or should activate the Na+/H+ exchanger, increasing intracellular [Na+] which indirectly induce the activation of NCX3rv and the PLC-IP3 signaling pathway. All these mechanisms would cooperate with the [Ca2+]i overload. A rise of [Ca2+]i activates the synthesis and secretion of inflammatory molecules like TNF-α, which in turn, increases the gene transcription for inducible nitric oxide synthase, resulting on NO production.