Reeve H L, Vaughan P F, Peers C
Department of Pharmacology, Leeds University, UK.
Pflugers Arch. 1995 Mar;429(5):729-37. doi: 10.1007/BF00373996.
The effects of phorbol esters on Ca2+ channel currents in human neuroblastoma SH-SY5Y cells were studied using whole-cell patch-clamp recordings. Bath application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu; 100 nM to 1 microM), known activators of protein kinase C (PKC), enhanced Ca2+ channel currents in a voltage-dependent manner similar to that of Bay K 8644. TPA also enhanced Ca2+ channel currents during cell dialysis with the PKC pseudosubstrate, PKC(19-36), and in cells which had been pre-incubated with 500 nM staurosporine, and which were exposed to staurosporine during recordings. Application of 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD; 100 nM), which does not activate PKC, caused current enhancement similar to the effects of TPA. However, intracellular dialysis of TPA was without effect on Ca2+ channel currents. Residual Ca2+ channel currents recorded after exposure to 1 microM omega-conotoxin GVIA were still enhanced by TPA, but in the presence of either Bay K 8644 (5 microM) or nifedipine (5 microM), TPA was without effect. When cells were pre-incubated for 10 min at 37 degrees C with 100 nM TPA, currents subsequently recorded in its absence were enhanced as compared to untreated cells; 5 microM nifedipine still inhibited currents to the same degree. This enhancement was not mimicked by 4 alpha-PDD, and was inhibited by staurosporine. Our results indicate that acute applications of phorbol esters (at concentrations commonly used to activate PKC) enhance L-type Ca2+ channel currents in SH-SY5Y cells via a PKC-independent mechanism which appears similar to that induced by Bay K 8644. By contrast, pre-incubation with TPA enhances both L- and N-type currents via activation of PKC.
利用全细胞膜片钳记录技术，研究了佛波酯对人神经母细胞瘤SH-SY5Y细胞中钙离子通道电流的影响。浴槽中加入已知的蛋白激酶C（PKC）激活剂12-O-十四酰佛波醇-13-乙酸酯（TPA）或佛波醇12,13-二丁酸酯（PDBu；100 nM至1 μM），以电压依赖性方式增强钙离子通道电流，类似于Bay K 8644的作用。TPA在用PKC假底物PKC(19-36)进行细胞透析期间，以及在预先用500 nM星形孢菌素预孵育并在记录期间暴露于星形孢菌素的细胞中，也增强了钙离子通道电流。应用不激活PKC的4α-佛波醇-12,13-二癸酸酯（4α-PDD；100 nM），引起的电流增强类似于TPA的作用。然而，TPA的细胞内透析对钙离子通道电流没有影响。暴露于1 μM ω-芋螺毒素GVIA后记录的残余钙离子通道电流仍被TPA增强，但在存在5 μM Bay K 8644或5 μM硝苯地平的情况下，TPA没有作用。当细胞在37℃用100 nM TPA预孵育10分钟后，随后在其不存在的情况下记录的电流与未处理的细胞相比增强；5 μM硝苯地平仍以相同程度抑制电流。这种增强未被4α-PDD模拟，且被星形孢菌素抑制。我们的结果表明，急性应用佛波酯（以通常用于激活PKC的浓度）通过一种不依赖PKC的机制增强SH-SY5Y细胞中的L型钙离子通道电流，该机制似乎类似于Bay K 8644诱导的机制。相比之下，用TPA预孵育通过激活PKC增强L型和N型电流。
