Department of Pharmacy-Drug Science, University of Bari "Aldo Moro" Bari, Italy.
Front Physiol. 2014 Dec 9;5:476. doi: 10.3389/fphys.2014.00476. eCollection 2014.
Here we investigated on the role of the calcium activated K(+)-channels(BKCa) on the regulation of the neuronal viability. Recordings of the K(+)-channel current were performed using patch-clamp technique in human neuroblastoma cells (SH-SY5Y) in parallel with measurements of the cell viability in the absence or presence of the BKCa channel blockers iberiotoxin(IbTX) and tetraethylammonium (TEA) and the BKCa channel opener NS1619. Protein kinase C/A (PKC, PKA) activities in the cell lysate were investigated in the presence/absence of drugs. The whole-cell K(+)-current showed a slope conductance calculated at negative membrane potentials of 126.3 pS and 1.717 nS(n = 46) following depolarization. The intercept of the I/V curve was -33 mV. IbTX(10(-8) - 4 × 10(-7) M) reduced the K(+)-current at +30 mV with an IC50 of 1.85 × 10(-7) M and an Imax of -46% (slope = 2.198) (n = 21). NS1619(10-100 × 10(-6) M) enhanced the K(+)-current of +141% (n = 6), at -10 mV(Vm). TEA(10(-5)-10(-3) M) reduced the K(+)-current with an IC50 of 3.54 × 10(-5) M and an Imax of -90% (slope = 0.95) (n = 5). A concentration-dependent increase of cell proliferation was observed with TEA showing a maximal proliferative effect(MPE) of +38% (10(-4) M). IbTX showed an MPE of +42% at 10(-8) M concentration, reducing it at higher concentrations. The MPE of the NS1619(100 × 10(-6) M) was +42%. The PKC inhibitor staurosporine (0.2-2 × 10(-6) M) antagonized the proliferative actions of IbTX and TEA. IbTX (10 × 10(-9) M), TEA (100 × 10(-6) M), and the NS1619 significantly enhanced the PKC and PKA activities in the cell lysate with respect to the controls. These results suggest that BKCa channel regulates proliferation of the SH-SY5Y cells through PKC and PKA protein kinases.
在这里，我们研究了钙激活的钾通道（BKCa）在神经元存活调节中的作用。我们使用膜片钳技术在人神经母细胞瘤细胞（SH-SY5Y）中同时记录钾通道电流，并在不存在或存在 BKCa 通道阻滞剂 Iberiotoxin（IbTX）和四乙铵（TEA）以及 BKCa 通道开放剂 NS1619 的情况下测量细胞活力。在有/无药物的情况下研究了细胞裂解物中的蛋白激酶 C/A（PKC、PKA）活性。全细胞钾电流在去极化后于负膜电位下表现出 126.3 pS 和 1.717 nS 的斜率电导（n = 46）。I/V 曲线的截距为-33 mV。IbTX（10-8-4×10-7 M）在+30 mV 时降低 K+电流，IC50 为 1.85×10-7 M，Imax 为-46%（斜率=2.198）（n = 21）。NS1619（10-100×10-6 M）在-10 mV（Vm）时将 K+电流增强 141%（n = 6）。TEA（10-5-10-3 M）在 3.54×10-5 M 时降低 K+电流，IC50 为 90%（斜率=0.95）（n = 5）。用 TEA 观察到细胞增殖呈浓度依赖性增加，其最大增殖效应（MPE）为+38%（10-4 M）。IbTX 在 10-8 M 浓度下表现出 MPE 为+42%，而在较高浓度下则降低。NS1619（100×10-6 M）的 MPE 为+42%。蛋白激酶 C 抑制剂 Staurosporine（0.2-2×10-6 M）拮抗 IbTX 和 TEA 的增殖作用。IbTX（10×10-9 M）、TEA（100×10-6 M）和 NS1619 与对照组相比显著增强了细胞裂解物中的蛋白激酶 C 和蛋白激酶 A 活性。这些结果表明，BKCa 通道通过蛋白激酶 C 和蛋白激酶 A 蛋白激酶调节 SH-SY5Y 细胞的增殖。
