Visentin S, Agresti C, Patrizio M, Levi G
Laboratory of Pathophysiology, Istituto Superiore di Sanitá, Rome, Italy.
J Neurosci Res. 1995 Nov 1;42(4):439-51. doi: 10.1002/jnr.490420402.
In order to study the voltage-dependent ion channels in microglia, and their possible modulation by pro-inflammatory substances like lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) we employed the patch-clamp technique on purified rat microglial cell subcultures grown for 1 - 5 days in control condition or after a 24 hour treatment with those agents. Regardless of the culture condition, almost 100% of the cells presented inward-rectifying (IR) K+ currents identified by the following features: (a) extracellular K(+)-dependence of Vrev and whole-cell conductance; (b) inward-rectifying property; (c) channel blocking mechanism by Cs+; and (d) single channel conductance of 27 pS. A 'n' type outward-rectifying (OR) K+ current was present in 30% of the cells during the first 2 days of subcultivation. Its occurrence was strongly dependent on the preparation, varying from 0% to almost 80%, and it decreased to 13% of the cells after three days in culture. It showed the following features: (i) threshold of activation close to -30 mV; (ii) sigmoid current onset; (iii) voltage-dependent kinetics; and (iv) sensitivity to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Furthermore, we detected two ion currents not previously described in microglia: (i) a slowly activating outward current which appeared at potentials more positive than +20 mV and with a reversal potential close to 0 mV, tentatively identified as a proton current; and (ii) a Cl- conductance identified in ion substitution experiments as the current sensitive to the Cl- channel blocker SITS. The two agents, LPS (20 - 2,000 ng/ml) and IFN-gamma (10 - 100 u/ml), shared the following effects: (a) enhancement of membrane capacitance, and (b) increase of OR current amplitude and frequency of occurrence. Moreover, IFN-gamma was also able to increase IR current density, especially in cells with ameboid morphology, while LPS was ineffective. We conclude that the voltage-dependent ion channel pattern of microglia is more complex than previously thought and that activating agents such as LPS and IFN-gamma share some electrophysiological effects, but differ in others.
为了研究小胶质细胞中的电压依赖性离子通道，以及它们如何被诸如脂多糖（LPS）和干扰素-γ（IFN-γ）等促炎物质调控，我们运用膜片钳技术，对在对照条件下生长1至5天，或用这些物质处理24小时后的纯化大鼠小胶质细胞亚培养物进行研究。无论培养条件如何，几乎100%的细胞都呈现内向整流（IR）钾离子电流，其具有以下特征：（a）Vrev和全细胞电导对细胞外钾离子的依赖性；（b）内向整流特性；（c）铯离子对通道的阻断机制；（d）单通道电导为27 pS。在亚培养的前两天，30%的细胞中存在一种“n”型外向整流（OR）钾离子电流。其出现强烈依赖于细胞制备，从0%到近80%不等，培养三天后，该电流在细胞中的占比降至13%。它具有以下特征：（i）激活阈值接近-30 mV；（ii）电流起始呈S形；（iii）电压依赖性动力学；（iv）对4-氨基吡啶（4-AP）和四乙铵（TEA）敏感。此外，我们检测到两种先前未在小胶质细胞中描述过的离子电流：（i）一种缓慢激活的外向电流出现在电位高于+20 mV时，反转电位接近0 mV，初步鉴定为质子电流；（ii）在离子置换实验中鉴定出的一种氯离子电导，该电流对氯离子通道阻滞剂4-乙酰苯磺酰氟（SITS）敏感。两种物质，LPS（20 - 2000 ng/ml）和IFN-γ（10 - 100 u/ml），具有以下共同作用：（a）增强膜电容，（b）增加OR电流幅度和出现频率。此外，IFN-γ还能够增加IR电流密度，特别是在具有阿米巴样形态的细胞中，而LPS则无效。我们得出结论，小胶质细胞的电压依赖性离子通道模式比之前认为的更为复杂，并且诸如LPS和IFN-γ等激活剂具有一些共同的电生理效应，但在其他方面存在差异。
