| 查看: 162 | 回复: 0 | |||
| 当前主题已经存档。 | |||
| 【有奖交流】积极回复本帖子，参与交流，就有机会分得作者 niniweiwei 的 30 个金币 | |||
[交流]
【求助】求大虾帮忙翻译两篇专业论文，在线翻译的就别发了
|
|||
|
Inward and outward rectifying potassium currents set membrane potentials in activated rat microglia Activation of cultured rat microglial cells with lipopolysaccharide (LPS), induced outward rectifying K+ (KV) current in addition to already existing inward rectifying K+ current (KIR). By measuring zero-current membrane-potentials using whole-cell patch-clamp method, we showed that KV current plays a direct role in setting membrane potential to near -45 mV. Since the membrane potentials of microglia show two prominent peaks at -45 and -70 mV, we hypothesize that KIR current might set the membrane potential to near -70 mV. We observed that cells with larger KIR current had a zero-current membrane-potential at around -70 mV, and that blocking of KIR current with Ba2+ depolarized membrane potentials to near -45 mV. These results indicate that the amounts of KIR, and KV current determine the zero-current membrane-potentials in LPS-activated microglia. Ó 1999 Elsevier Science Ireland Ltd. All rights reserved. Microglia are known to have an important function as brain macrophages during immunological processes, oncogenesis and regeneration in the central nervous system [3,5,10]. Brain damage such as trauma, stroke, and Alzheimer’s disease lead to the conversion of resting microglia to macrophage-like cells, which are also called activated microglia [7,14,15]. Inward rectifying K(KIR) channels exists predominantly in the resting microglia, whilst both inward rectifying Kchannels and outward rectifying K (KV) channels exist in the microglia activated by lipopolysaccharide (LPS) [9,11,12]. We have already shown that a ‘shaker’ type delayed rectifier, Kv1.5 channel, is induced in microglia when they are activated by LPS in vivo [8], as well as, in vitro [13]. Of the two dominant Kcurrents, KV current was shown to play a direct role in maintaining the resting membrane potential of this non-excitable cell to near 45 mV [1,2]. However, the functional role of the other dominant current, KIR current, is not yet known. Since membrane potentials of microglia show two prominent peaks at 45 and 70 mV, and there exists a negative slope region in the I–V relation of Kcurrents [12], we hypothesize that KIR current set the membrane potential to near 70 mV Microglia and astrocytes were co-cultured from the cerebral cortices of 1–3-day-old Sprague–Dawley rats as previously described with some modification [2]. In brief, animals were killed by decapitation, meninges were removed, and cortices were minced and gently dissociated by trituration in Hank’s balanced salt solution. Cells were collected by centrifugation at 200 g for 10 min, resuspended in minimum essential medium, supplemented with 5% fetal bovine serum (Gibco, Gaithersburg, MD), plated onto 75cm2 culture flask (5 1010 cells/dish), and incubated at 37C in a humidified atmosphere of 95% air/5% CO2. After 14 days of culture under LPS-free conditions, floating microglia were harvested by shaking the flask vigorously and re-seeding into poly-D-lysine coated 35 mm petri dishes to give pure microglial cultures. When LPS (500 ng/ml; Sigma, St. Louis, MO) was added for 12–24 h, microglia became circular in shape with ruffled edges (diameter, 15–23 mm). Potasiumcurrent and zero-current membrane-potential were recorded using whole-cell mode of the patchclamp technique [6] from activated microglia. All the experiments were performed at room temperature (19–23C). The bath (extracellular) solution contained (in mM): NaCl, 140; KCl, 5; MgCl2, 3; N-2-hydroxyethylpiperazine- N-2-ethanesulphonic acid (HEPES), 10; adjusted to pH 7.4 with NaOH. The pipette (internal) solution contained (in mM): KCl, 140; NaCl, 5; MgCl2, 3; HEPES, 10; Mg- ATP, 1; Na-GTP, 0.5; adjusted to pH 7.4 with KOH. Calcium2 was not included in either of the bath or pipette solutions to minimize the Ca2-dependent currents. The resistance of the electrodes was 2–5 MQ when they were filled with internal solution. The patch-clamp studies were performed with the patch amplifier (Axopatch-1D; Axon Instruments, Foster City, CA). Voltage-clamp and currentclamp protocols were generated with a personal computer (IBM). All compounds were infused at a flow rate of 2 ml/ min. Data were acquired and analyzed using pCLAMP programs version 6.0 (Axon Instruments). Evoked currents were filtered at 1 kHz, and sampled at 10 kHz. As reported earlier LPS-activated microglia express both of KIR, and KV currents [12]. We have already shown that the specific blocker of KV current, 4-aminopyridine, depolarizes the membrane potential [2], and the reduction of KV current induced by external acidification results in similar membrane depolarization [1]. These results strongly suggest that KV currents participate in maintaining the resting membrane potential. We postulate that by expressing large amount of KV current, LPS-activated microglia are more hyperpolarized than control cells. To confirm this, zero-current membrane-potentials of control and LPS-activated cells were compared. The frequency histograms membrane potentials from 41 control (Fig. 1A) and 86 LPS-activated cells (Fig. 1B) show two distinct groups. In both cases, one group of cells show zero-current membrane-potentials at around 70 mV. In the other group of cells, zero-current membrane-potentials are clearly different, between control and LPS-activated cells. In control cells, membrane potentials show a broad distribution between 25 and 50 mV with a mean value of 32.2 mV. In LPS-activated microglia, a clear right shift in membrane potential was observed with a peak at 44.3 mV. These results confirm that KV current induced in LPS-activated microglia set zero current membrane potential to near 45 mV. In order to address the function of KIR current in LPSactivated microglia, Ba2was used to block the current. As shown in Fig. 2A the addition of 100 mM Ba2to bath solution blocked a large amount of KIR current. In seven cells tested 69.2% (7.8%; SD) of KIR current was blocked by 100 mM Ba2at 120 mV. The blockage of KIR current by Ba2was completely reversible (data not shown). In Fig. 2B the difference in current between control and Ba2trace in Fig. 2A is shown, representing the current blocked by Ba2. It is clear that Ba2blocked KIR current but not IK current. When we monitored zero-current membrane-potentials, while 100 mM Ba2was added, there was rapid depolarization upon the addition of Ba2. In six cells showing zerocurrent membrane-potential at around 70 mV, 100 mM Ba2elicited depolarization by 28.5 mV (3.7 mV; SD). However, in eight cells showing zero-current membranepotential at around 45 mV, Ba2did not change membrane potential at all. These results indicate that KIR current may function to bring zero-current membrane-potential to around 70 mV. Thus, it is possible that the relative amount of KIR and KV currents would decide zero-current membrane- potential to either near 45 or 70 mV. A large amount of outward KIR current might bring the membrane potential to near 70 mV, by overcoming the effect of KV currents. In contrast, when KIR current is small, KV current might set the membrane potential to 45 mV. In order to address these possibilities, we measured both of the amounts of outward currents carried by KIR channels at a holding potential of 60 mV, and zero-current membrane-potentials from 25 different LPS-activated microglia. At this voltage most of the current was blocked by 100 mM Ba2, implying that it was KIR current (Fig. 2A). It is apparent that zerocurrent membrane-potentials of cells with large outward currents are at around 70 mV (Fig. 3). With only few exceptions, zero-current membrane-potentials of cells, with greater than 50 pA of KIR current, are more negative than 70 mV, while the membrane potentials of cells with less than 50 pA of KIR current are less negative than 48 mV. There was no difference in cell membrane capacitance between two groups of cells. Three different cells identified as ‘A’, ‘B’ and ‘C’ within open circles on the left side of Fig. 3 represents cells whose zero-current membrane-potentials were less than 70 mV, initially. After 100 mM Ba2was added currents and membrane potentials were measured from the same cells, and plotted on the right side of Fig. 3. In these cells, the outward KIR currents measured at holding voltage of 60 mV were inhibited by 100 mM Ba2as expected, and zero-current membrane-potentials were depolarized to near 45 mV. Therefore, the inhibition of KIR current by Ba2made these cells similar to cells with membrane potential at near 45 mV. These results confirm that the large amount of KIR current would bring the membrane potential to around 70 mV. Outward rectifying KV current expressed in microglia when they are activated by inflammatory stimuli such as LPS and interferon-g [11]. The function of this current is suggested to counteract depolarization, which would occur during inflammation or cellular damage. In addition to this possibility, KV current plays a direct role in maintaining the resting membrane potential in LPS-activated microglia [1,2]. Thus, the large KV current induced by LPS in activated microglia might be participating in setting zero-current membrane-potential (Fig. 1). It was suggested that K and Clcurrents might be responsible for the two stable membrane potentials of microglia at 85 and 30 mV, respectively [16]. Thus, it is possible that with the large increase of KV current in LPS-activated microglia, it regulates the membrane potential along with already existing Cl conductance. In contrast to KV current, the density of inward KIR current was not changed much by LPS-treatment. KIR currents of control and LPS-activated microglia exhibited similar voltage dependence and similar kinetic characteristics, suggesting that the KIR channels in the two states of microglia are identical [12]. While KV current sets the membrane potential to near 45 mV, KIR current contributes to set zero current membrane potential to near 70 mV (Fig. 3). Similar to this observation, significantly more positive resting membrane potentials were reported in microglia that lacked KIR current [4]. Our results show that both of KV and KIR currents contribute to set the membrane potentials in LPS-activated microglia. Membrane potential might be set either at 45 mV or 70 mV depending on the relative amount of two currents. Thus, they constitute two stable alternative membrane potentials.This work was supported by KOSEF research grant (981- 0706-046-2) to S.C. Special thanks to Drs. I. Chung and J. Song for reading manuscript. |
回复此楼» 猜你喜欢
招贤纳博（已结束）
已经有28人回复
-
表征测试机构后台私聊我
已经有0人回复
-
分析化学论文润色/翻译怎么收费?
已经有128人回复
-
Coordination Chemistry Reviews
已经有1人回复
-
请问现在还有电池材料方向的博导招人吗
已经有1人回复
