tram-34 and 2-aminoethoxydiphenyl-borate

Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions. Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation. Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation.

Topics: Boron Compounds; Calcium Channel Blockers; Calcium Release Activated Calcium Channels; Cell Proliferation; Cells, Cultured; Drug Synergism; Humans; Immunosuppressive Agents; Interferon-gamma; Interleukin-4; Intermediate-Conductance Calcium-Activated Potassium Channels; Kv1.3 Potassium Channel; Lymphocyte Activation; Mitogens; Pyrazoles; Receptors, Antigen, T-Cell; Sirolimus; T-Lymphocytes

Lysophosphatidylcholine (LPC) is a major atherogenic lipid which stimulates the recruitment of monocytes to atherosclerotic lesions. The physiological mechanisms underlying LPC-induced monocyte migration are poorly understood. Here we demonstrate that LPC activates non-selective cation channels, which are significantly involved in LPC-induced chemotaxis of monocytes. External LPC elicited the activation of non-selective cation currents in THP-1 monocytes, which occurred in a G protein and phospholipase C-independent manner. LPC-activated currents were almost completely inhibited by Gd(3+), La(3+), and TRAM-34. Furthermore, currents were partially reduced by either 2-aminoethoxydiphenyl borate (2-APB) or ruthenium red, while combined application of 2-APB and ruthenium red abolished LPC-activated currents. The 2-APB-sensitive current component was potentiated by flufenamic acid and Ca(2+)-free extracellular solution, while the ruthenium red-sensitive current component was abolished by capsazepine. This pharmacological profile suggests that LPC simultaneously activates TRPC6 and TRPV1 channels in monocytes. Furthermore, in the presence of Gd(3+), La(3+), TRAM-34, 2-APB, ruthenium red or capsazepine, LPC-induced chemotaxis of monocytes was substantially inhibited, indicating that activation of both channel types is required for optimal migration of LPC-stimulated monocytes. Thus, ion channel inhibition may represent a powerful strategy to attenuate the progression of atherosclerosis by reducing monocyte infiltration.

Topics: Boron Compounds; Cations; Cell Line; Cell Movement; Estrenes; Gadolinium; GTP-Binding Proteins; Humans; Ion Channel Gating; Ion Channels; Lanthanum; Lysophosphatidylcholines; Monocytes; Pyrazoles; Pyrrolidinones; Ruthenium Red; Type C Phospholipases