Role Of TRPC1in FMLP-stimulated Neutrophil Directional Migration

BackgroundPolymorphonuclear leukocytes (PMNs) share of60%to70%of the total number of leukocytes in the blood. They are the first-line defense against microorganism invasion in the innate immunity system, as is tightly related with their directed migration known as chemotaxis. Directional migration is basic biology character of eukaryotic cell, and it includes two parts of polarity and chemokines. Eukaryotic cell polarity is a fundamental characteristics. Cell polarity(cell polarity) on the cell migration, cytokinesis and the tissue, and many play a key role in biological processes. Cell polarity refers to that the cell in order to exercise specific function or produce directional differentiation, intracellular signaling molecules in the asymmetric distribution caused cells to generate asymmetry phenotype.Its Characteristic is specific expression in intracellular proteins and lipids in the asymmetric distribution of cell structure remodeling. Characteristic changes of cell polarity within the cell cytoskeleton required before the polar molecules in the cell to form a rich filamentous actin in lamellipodia, in the formation of the posterior pole of the end of foot. Cell chemotaxis is that the cells sense the source of chemotactic substances that inside and outside the cell and oppose the concentration of chemotactic substances to arrive the position where the chemoattractant substances exists and play a role in Biophysics. Therefore, directional movement is the basic functions of cells.Neutrophils have a strong directional migration. Upon exposure to chemoattractant, PMNs quickly characterized as a distinct asymmetric shape with actin-rich leading edge (pseudopod) and tail (uropod), and arrive the position where the chemoattractant substances exist to play a role in bactericidal and anti-inflammatory. Although the physiological function of neutrophils is so important,the premise of neutrophils playing a role in directed migration (chemotaxis), bactericidal and anti-inflammatory depends on the formation of polar. The basis of directional migration is cells,polarity and the cell chemotaxis depend on cells’ polarity. Upon exposure to chemoattractant, PMNs quickly characterized as a distinct asymmetric shape with actin-rich leading edge (pseudopod) and tail (uropod). Failure to govern any molecules of this event may lead to neutrophil inappropriate activation,which develop such several inflammatory pathologies as ARDS, COPD, asthma, ischemia-reperfusion, and so on. In this regard, inhibiting any signaling pathway may be candidate pharmaceuticals target. Therefore, unraveling of the mechanisms on regulating neutrophil polarization could potentially lead to novel therapeutic strategies for counteracting chronic activation of PMNs which leads to tissue damage.There has been recent progress in understanding how Ca2+signaling takes part in the activation of neutrophil reactions such as chemotaxis and migration, release of reactive oxygen species(ROC). The precise temporal and spatial regulation of [Ca2+]i is required for cell polarization. It is well known that an increase of cytosolic intracellular free calcium results from both a stores rapid depletion from endoplasmic reticulum (ER) and a external Ca2+influx. Because of lack of voltage-gated calcium entry, Ca2+influx is predominantly mediated by store-operated Ca2+entry (SOCE) in fMLP-stimulated human neutrophils. There are good evidence that Orail and STIM1are two essential molecullars comprising SOCE entity to be involved in neutrophil polarization and NADPH products, however, the role of transient receptor potential canonical1(TRPC1), as Ca2+-permeable channel in neutrophil polarization is not available. TRP is considered to be the most likely molecular basis of receptor-operated calcium channel and calcium channel of manipulation of calcium store. The reason may be closely related to intracellular calcium ion concentration and may be activated. TRPC is one of the main member of the TRP family and there is a very wide range of expression in mammals. The TRPC including seven subtypes of TRPC1-7, the distribution and function of each subtype are different. TRPCl is widely distributed, and it’s expression is high-level in skeletal muscle, kidney, brain, skin, lung, prostate, heart, testes and ovaries. TRPC1the first to be discovered in the mammalian TRP channels, and it is recognized that role of calcium-dependent secretion and contraction and participating in receptor-mediated. The TRPC1and the protein about PLC and IP3receptor compose signaling complexes and participate in cell signal transduction, the function of this complex can be achieved through assembly in the membrane lipid environment. As important candidate molecules about SOCE, TRPC1proteins are non-selective calcium hannel on the cell membrane, the role of polarity and chemotaxis about TRPCl proteins is unclear. Therefore, using the patch-clamp technique to study the role of TRPC1in the process of neutrophil polarity and chemotaxis from the point of view of the electrophysiology, to study TRPC1membrane current characteristic is particularly important in the process of neutrophil polarity and provide Electrophysiology theoretical basis for the study of neutrophil directional movement.Neutrophils can be activated by a variety of chemoattractants, including interleukin8(IL-8), Platelet activating factor (PAF), complement component factor5a(C5a) and formylated peptides such as formyl-Met-Leu-Phe(fMLP). On being stimulated with chemoattractants, adopts a polarized morphology with actin-rich leading edge(pseudopod) and tail (uropod). Reconstruction of cytoskeletal structures and promote the formation of polarization. In this process, it is not known the mechanisms that the cell quickly start a complex series of intracellular polarity signaling. It has not been reported on how the process TRPC1and/or lipid rafts regulation of the polarity of the membrane currents. We explored the effects of TRPC1channel in fMLP-induced neutrophil polarization by the membrane currents.ObjectiveIn this study, through the use of uniform, low concentrations exogenous chemotactic agent fMLP role of neutrophils in the establishment of cell polarity model. We use TRPC1channel antibody、blockers (GsMTx-4) to close and block TRPC1protein channel, record neutrophil membrane capacitance and the current diversification about TRPC1under various processing factors and compare the corresponding current density that is recorded by using the patch-clamp technique. Zigmond Chamber was used to observe the impact of these drugs on neutrophil polarity, Transwell chambers to observate TRPC1antibody and blocking agent (GsMTx-4) make an impact on neutrophil migration. To investigate whether TRPC1participates in the process of neutrophil polarity and the chemoattractant and the current characteristics about TRPC1.Methods1.The neutrophils were freshly isolated by standard isolation protocol. This protocol comprises three successive steps mainly:to sedimentate by Dextran, to centrifugate by standard density gradient and to crack the residual red blood cells by hypotonic. Firstly, using dextran T-500(Dextran-T500) to sedimentate Most of the red blood cells, secondly, centrifugating with density and gradien by lymphocyte separation medium, lastly, cracking remnants of red blood cells.Confirmed isolated Neutrophils purity by Wright-Giesma and Cell viability by trypan blue.2.Drawing microelectrode with filament by5step, Polishing and using patch clamp Instrument to detected electrode resistance.3.Using patch clamp instruments to seal and rupture the neutrophils, recording changes in membrane capacitance and processing factors membrane currents.4.In the case of exogenous chemotactic agent fMLP exist, Zigmond chamber were used to observe the diversification of cells morphological and ensure the optimum concentration of TRPC1Ab.5.Detecting the changes in the capacity of the migration of neutrophils through the Transwell chamber experiments.Result1.The microelectrode with filament resistance through the step5was drawn and polishing process is5-7MΩ.2.Neutrophil membrane capacitance(Cm) is14.58±3.09(pF)3.TRPC1Ab、GsMTx-4can cause neutrophil membrane current changes, compared with the control group and/or fMLP group, current density changes were statistically significant (P<0.05)4.TRPC1Ab can reduce the rate of fMLP induced neutrophil polarity.5.Both TRPC1Ab and GsMTx-4are able to reduced neutrophil chemoattractant capacity. Conclusion1.TRPC1Ab and GsMTx-4can reduce the membrane currents of the fMLP induced the neutrophil polarity of TRPC1related, play a role in blocking TRPC1current and restrain the function of cell directional movement.2.The TRPC1can mediate diversification of membrane current and participate directional movement of neutrophil, the orientation chemotactic ability will be reduce if the TRPC1is inhibited.