Effects Of Ln³⁺ On Cell Proliferation And K Channels Of NIH3T3 And MC3T3 Cells

With the widely used of rare earth elements, especially in agriculture and stock breeding in china, they are more and more entered into entironment and food cycle. So the researches of their physiology, toxicology and pharmacology are extensively interested. Researches on their biological effects have been a hot spot. Lanthanide compounds display a variety of biochemical and physiological effects, but the exact mechanism is not clear. it is limit to reasonably and safely use them. Ion channels play an important role in the processes of cell differentiation and upgrowth in non-excitable cells. So it is significant to study the relationship and it's mechanism between ion channel and cell proliferation. Effects of two lanthanide ions of La3+and Yb3+ on potassium currents and it's kinetics in non-excitable NIH3T3 cells and MC3T3 cells were investigated using the whole-cell voltage-clamp technique, and influences of La3+and Yb3+ on cell proliferation, cell apoptosis and cell cycles were studied using cell biological methods. We aim at posting some evidence to reveal the function and it's molecular mechanism of the effect on potassium channel by rare earth elements. The results show as below:Cell proliferation and cell apoptosis in NIH3T3 cells and MC3T3 cells were mensurated by MTT assay and by flow cytometer. (1) La3+observably increased cell proliferation of NIH3T3 cells in a dose-and time-dependent manner, with a 46.7% increase in 100μM La3+treated cells for 72 h. (2) At the same time, Ca2+can also promote proliferation of NIH3T3 cells. However, it is lower than that of La3+'s. Furthermore, there have synergistic effects between La3+and Ca2+on increasing proliferation. All these results showed that exposure to La3+was able to increase NIH3T3 cell growth which was correlated with Ca2+and Ca2+is absolutely necessarily in cell proliferation. (3) La3+also increased cell proliferation of MC3T3 cells in a dose-and time-dependent manner, with a 38.7% increase in 50μM La3+ treated cells for 72 h. (4) Oppositely, Yb3+ inhibited observably cell proliferation of NIH3T3 cells in a dose-and time-dependent manner, with a 57.2% increase in 1mM Yb3+treated cells for 72h. (5) Yb3+had no obvious effect on cell proliferation of NIH3T3 cells. (6) In addition, under research condition of time and dose, La3+and Yb3+had no obvious effects on apoptosis of NIH3T3 cells.Cell cycles in cultured NIH3T3 cells and MC3T3 cells were assessed by flow cytometer. (1) La3+observably decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase of NIH3T3 cells in a dose-and time-dependent manner, with 6.6% increase to 26.1% in 100μM La3+ treated cells. These showed that La3+ promoted cell proliferation of NIH3T3 cells through promoted G1/S cell cycle progression. (2) At the same time, Ca2+also can decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase of NIH3T3 cells. However, it is not observably. Furthermore, 100μM LaCl3 and 5mM CaCl2 increased the percentage of cells in S phase of NIH3T3 cells from 26.1% to 30.6%. These indicated that La3+and Ca2+have synergistic effect on increase in the percentage of cells in S phase. (3) La3+had no visible effect on cell cycle of MC3T3 cells with different concentration of La3+treated cells for 72h. These results showed that there was different mechanism of La3+promoted cell proliferation between NIH3T3 cells and MC3T3 cells induced by different cell style. (4) The effect of Yb3+on cell cycle of NIH3T3 cells is slender, with little increase in the percentage of cells in G2/M phase. These showed that Yb3+inhibited cell proliferation through promoted G2/M cell cycle progression. (5) Yb3+had no obvious effect on cell cycle of MC3T3 cells.Outward potassium currents (Ik) in cultured NIH3T3 cells were recorded by whole cell patch-clamp technique. (1) The outward potassium currents of NIH3T3 cells were concentration-dependent of Ca2+ and the EC50 value of Ca2+was calculated to be 252 nmol/L. Contrast to excitable cells, the currents density are very small. These may be the reason of non-excitable cell few channel or little open rate. (2) La3+and Yb3+all inhibited the outward potassium current in a concentration-dependent manner and EC50 was 1.58 and 10.63μmol/L respectively. The inhibition effect of La3+was stronger than that of Yb3+ due to his small inhibition EC50. (3) The kinetics of blockade of La3+and Yb3+on IK indicated that the activation curves was shifted by La3+to positive potential for 16mV and the inactivation curves was shifted to negative potential for 9.48mV; contrarily, the activation curves was shifted by Yb3+to negative potential for 11.41mV and the inactivation curves no changed. The suspendment of activation and the advance of inactivation all decreased current intensity. This was the main reason of La3+inhibition stronger than that of Yb3+.Outward potassium currents (IK) in cultured MC3T3 cells were recorded by whole cell patch-clamp technique. (1) The outward potassium currents of MC3T3 cells were concentration-dependent of Ca2+and the EC50 value of Ca2+was calculated to be 39.8 nmol/L. (2) La3+inhibited the outward potassium current in a concentration-dependent manner and EC50 was 8.23μmol/L. (3) The kinetics of blockade of La3+indicated that the activation curves was shifted by La3+to positive potential for 21mV and the inactivation curves was shifted to negative potential for 9.66mV. (4) The activation curves and the inactivation curves by Yb3+had no changed. These showed that it is a different mechanism between La3+and Yb3+on K+currents and its kinetics.In summary, in this dissertation, it has been researched the effects of La3+ and Yb3+on cell proliferation, cell apoptosis and cell cycles of NIH3T3 cells and MC3T3 cells; it has been studied, for the first time, the effects of La3+ and Yb3+on potassium currents and its kinetics of activation and inactivation in NIH3T3 cells and MC3T3 cells by whole cell patch-clamp technique. It is demonstrated that potassium channel plays a crucial roles in cell growth from channel level. In conclusion, our experiments have revealed the rare earth element biology effect and their molecular mechanism further. These results are significant for the further understand biology effect of rare earth element and its potential used in medicine.