Fitzgerald E M, Dolphin A C
Department of Pharmacology, Royal Free Hospital School of Medicine, London, UK.
Eur J Neurosci. 1997 Jun;9(6):1252-61. doi: 10.1111/j.1460-9568.1997.tb01480.x.
Influx of calcium through voltage-dependent calcium channels (VDCCs) has been implicated in the processes of cell growth and differentiation. Various signalling proteins, including nerve growth factor (NGF), p21-ras and src tyrosine kinases, have been suggested to have a role in the regulation of neuronal VDCCs. Using the whole-cell patch-clamp technique we have investigated the role of endogenous p21-ras in the regulation of VDCCs in primary cultured dorsal root ganglion (DRG) neurons obtained from neonatal rats. Neutralization of endogenous p21-ras by microinjection of p21-ras antibody (Y13-259) reduced the maximum peak barium current, I(max), whereas microinjection of oncogenic p21-K-ras increased the current. Thus, endogenous p21-ras is involved in the tonic regulation of calcium currents in these cells. Intracellular application of a phosphopeptide, Trk 490, which prevents the binding of the adaptor protein shc to the activated NGF receptor, so blocking p21-ras activation, reduced I(max). Similarly, deprivation of NGF by overnight incubation in NGF-free medium also reduced I(max). Together, these results suggest that NGF receptor tyrosine kinase activation of p21-ras is likely to be involved in the tonic regulation of VDCCs in DRG neurons. Deprivation of NGF combined with microinjection of p21-ras antibody (Y13-259), however, caused an even greater reduction of I(max). Thus, NGF activation can only partially explain the regulation of these currents by endogenous p21-ras. Src tyrosine kinases have been suggested to activate p21-ras. In DRG neurons, microinjection of purified src tyrosine kinase, pp60c-src, increased I(max) in these cells. However, co-microinjection of pp60c-src with Y13-259 antibody prevented the increase in I(max), implying that pp60c-src can also regulate calcium currents via the activation of endogenous p21-ras. Further support for the involvement of tyrosine kinases in VDCC regulation was provided by the application of the general tyrosine kinase inhibitor, genistein, which also reduced I(max). Thus, VDCCs in rat DRG neurons appear to be tonically up-regulated by endogenous p21-ras. This effect appears largely to involve NGF receptor tyrosine kinase activation of p21-ras. In addition, src tyrosine kinase may also regulate VDCCs, possibly via p21-ras.
通过电压依赖性钙通道（VDCCs）的钙内流与细胞生长和分化过程有关。包括神经生长因子（NGF）、p21 - ras和src酪氨酸激酶在内的各种信号蛋白，被认为在神经元VDCCs的调节中起作用。我们使用全细胞膜片钳技术，研究了内源性p21 - ras在从新生大鼠获得的原代培养背根神经节（DRG）神经元中对VDCCs的调节作用。通过显微注射p21 - ras抗体（Y13 - 259）中和内源性p21 - ras，降低了最大峰值钡电流I(max)，而显微注射致癌性p21 - K - ras则增加了该电流。因此，内源性p21 - ras参与了这些细胞中钙电流的紧张性调节。细胞内应用一种磷酸肽Trk 490，它可阻止衔接蛋白shc与活化的NGF受体结合，从而阻断p21 - ras的活化，降低了I(max)。同样，在无NGF培养基中过夜孵育剥夺NGF也降低了I(max)。这些结果共同表明，NGF受体酪氨酸激酶对p21 - ras的激活可能参与了DRG神经元中VDCCs的紧张性调节。然而，剥夺NGF并结合显微注射p21 - ras抗体（Y13 - 259），导致I(max)进一步大幅降低。因此，NGF激活只能部分解释内源性p21 - ras对这些电流的调节作用。有人提出src酪氨酸激酶可激活p21 - ras。在DRG神经元中，显微注射纯化的src酪氨酸激酶pp60c - src增加了这些细胞中的I(max)。然而，将pp60c - src与Y13 - 259抗体共同显微注射可阻止I(max)的增加，这意味着pp60c - src也可通过激活内源性p21 - ras来调节钙电流。通用酪氨酸激酶抑制剂染料木黄酮的应用也降低了I(max)，这进一步支持了酪氨酸激酶参与VDCC调节的观点。因此，大鼠DRG神经元中的VDCCs似乎受到内源性p21 - ras的紧张性上调。这种作用似乎很大程度上涉及NGF受体酪氨酸激酶对p21 - ras的激活。此外，src酪氨酸激酶也可能调节VDCCs，可能是通过p21 - ras。
