豚鼠耳蜗单离Hensen细胞钾电流特性及三磷酸腺苷对其影响

目的 研究豚鼠耳蜗单离Hensen细胞的钾离子电流及其特性以及三磷酸腺苷(adenosine triphosphate，ATP)对Hensen细胞电生理特性的影响。方法 采用传统全细胞膜片钳技术，对单离Hensen细胞进行记录。ATP通过压力注射仪对单离Hensen细胞给药。结果 Hensen细胞的钾电流呈明显的外向整流性，只有延迟整流性钾电流(delayed rectification potassiu mcurrent，IK)，没有瞬间外向性钾电流(transient outward potassium current，IA)。低浓度(0．1μmol/L，1μmol／L，10μmol／L)ATP可以使Hensen细胞的钾电流的幅度明显降低，且呈浓度依赖性，浓度越高，降幅越大。高浓度ATP(100μmol／L，1mmoL／L，10mmol／L)可以引起Hensen细胞的内向离子流，呈浓度依赖性，浓度越高，升幅越大。ATP的这两种作用均可被ATP受体拮抗剂(100μmol／L舒拉明)所逆转。结论 对Hensen细胞不同电压的刺激可以诱发出延迟整流性钾电流，低浓度ATP对Hensen细胞的钾电流有明显抑制作用，且呈浓度依赖性。高浓度ATP可以引起Hensen细胞的非选择性内向离子流，为钾离子依赖性，而且是通过ATP受体起作用。     

听力学

20 0 4 0 82 0豚鼠耳蜗单离Hensen细胞钾电流特性及三磷酸腺苷对其影响 /李建雄… / /中华耳鼻咽喉科杂志 2 0 0 3;38(5 ) 343～ 346目的 :研究豚鼠耳蜗单离Hensen细胞的钾离子电流及其特性以及三磷酸腺苷 (ATP)对Hensen细胞电生理特性的影响。方法 :采用传统全细胞膜片钳技术 ,对单离Hensen细胞进行记录。ATP通过压力注射仪对单离Hensen细胞给药。结果 :Hensen细胞的钾电流呈明显的外向整流性 ,只有延迟整流性钾电流 (delayedrectificationpotassiumcurrent ,IK) ,没有瞬间外向性钾电流 (transientoutwordpotassiumcur rent ,IA)…     

硝苯地平对三磷酸腺苷引起的Hensen细胞内向性离子流的抑制作用

目的探讨硝苯地平对高浓度三磷酸腺苷（ATP）引起的豚鼠耳蜗单离Hensen细胞非选择性内向性离子流的影响。方法采用酶消化和机械分离的方法单离豚鼠耳蜗Hensen细胞，选择胞膜清晰、胞质透明的单离细胞通过压力注射仪分别给予0．1mmol／LATP、1mmol／LATP、10mmol／LATP、0．1mmol／LATP＋0．1mmol／L舒拉明、单独细胞外液、140mmol／LCsCl＋1mmol／LATP、40mmol／L四乙基铵（tetraethylammonium，TEA）＋1mmol／LATP、1mmol／LATP＋10μmol／L硝苯地平，并行全细胞膜片钳记录。结果当分别给予Hensen细胞0．1mmol／L（n=10），1mmol／L（n=10），10mmol／L（n=6）的ATP刺激时，均可记录到内向电流，并随ATP浓度的增加而增强，呈现浓度依赖性。该内向电流可被0．1mmol／L舒拉明（n=5），140mmol／LCsCl（n=5）和40mmol／LTEA（n=5）所抑制。单独给予细胞外液刺激后未见内向及外向离子流。当同时给予1mmol／LATP和10μmol／L硝苯地平刺激时，内向性电流消失，转而出现外向性电流。结论高浓度ATP可引起Hensen细胞的内向性电流，此电流与钾通道密切相关，并无机械转化通道参与，硝苯地平可抑制这种内向性电流并出现与正常情况相似的外向性电流。提示硝苯地平可通过Hensen细胞改善钾离子循环，起到部分保护耳蜗功能的作用。     

利诺吡啶对豚鼠耳蜗外毛细胞和支持细胞钾电流的影响

目的　观察KCNQ家族钾通道特异性阻滞剂利诺吡啶 (linopirdine)对豚鼠耳蜗单离外毛细胞和Deiters细胞 (支持细胞 )总钾电流的影响 ,初步探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的分布。方法　运用膜片钳技术 ,在全细胞模式下记录正常细胞外液中 8个外毛细胞和5个Deiters细胞的总钾电流 ,并观察 10 0 μmol/L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果　在正常细胞外液中 ,单离外毛细胞可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流 (theK+ currentactivatedatnegativepotential,IKn)两种钾电流 ,而单离Deiters细胞中只记录到外向整流性钾电流。加入 10 0 μmol/L利诺吡啶后 ,外毛细胞中的四乙基二乙胺敏感的钾电流减小 ,IKn被完全抑制 ;而Deiters细胞中的外向整流性钾电流大小无变化。结论KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞 ,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分 ,并构成全部的IKn;但KCNQ家族钾通道不存在于豚鼠耳蜗Deiters细胞。     

豚鼠耳蜗单离外毛细胞的外向整流钾电流

目的 :观察豚鼠耳蜗单离外毛细胞 (OHC)的电生理特性 ,记录不同长度OHC的外向整流钾电流 ,分析区分外向整流钾电流所包含的通道电流成分 ,研究外向整流钾电流的动力学特征。方法 :采用酶消化法及机械分离OHC。运用全细胞膜片钳技术 ,在电压钳下记录K+ 通道电流。结果 :OHC的全细胞膜电容为 (30 .96±2 .79) pF(n =2 9) ,零电流电位 (30± 2 .1)mV(n =16 ) ,反转电位为 (- 5 1.6 7± 1.84 )mV(n =9)。不同长度OHC的外向整流钾电流存在系统差异 ,短OHC表现出大的钾电导 ,长OHC则相反。 10 0 μmol/L的氯化镉 (Cd Cl2 )抑制了OHC外向整流钾电流的最大电流幅度的 6 0 % ,且改变了电流的动力学特征 ,对峰电流的影响明显大于稳态电流 (P<0 .0 1,n =5 ) ;1mmol/L的四氨基吡啶 (4 AP)抑制了最大电流幅度的 4 3% ,没有改变电流的动力学特征。外向整流钾电流的激活符合Boltzmann方程 ,V1/ 2 =(- 11.0 7± 0 .2 6 )mV ,S =(6 .6 2± 1.74 )mV(n=13)。结论 :外向整流钾电流包含有钙离子激活的钾离子电流、外向延迟整流钾电流和A型电流     

豚鼠耳蜗单离Hensen细胞的分离技术及其钾通道

目的　探讨适合膜片钳技术的单离Hensen细胞的分离及对其钾通道的研究。方法　取豚鼠耳蜗 ,获得Corti器 ,采用酶消化和机械分离方法对Hensen细胞进行分离 ,并采用传统全细胞膜片钳技术 ,记录单离Hensen细胞的钾电流。结果　每只耳蜗可以获得活性良好的单离Hensen细胞 12± 3个。Hensen细胞的钾电流呈明显的外向整流性 ,只有迟电流 (IK) ,没有峰电流 (IA)。结论　酶消化结合机械分离的方法可获得适用于膜片钳技术研究的单离Hensen细胞 ,并用于讨论了所记录到的Hensen细胞钾电流     

豚鼠耳蜗单离Deiters细胞的钾电流

目的　研究豚鼠耳蜗单离Deiters细胞的钾电流及其特性。方法　运用膜片钳技术 ,在全细胞模式下记录正常细胞外液中钾电流 ,不同K 浓度的细胞外液对细胞反转电位和外向钾电流的影响 ,四氨基吡啶 (4 aminopyridine ,4 AP)和四乙基胺 (tetraethylammonium ,TEA)对钾电流成分的阻滞作用 ,探讨外向钾电流通道的激活和失活动力学。结果　单离Deiters细胞具有电压依赖的外向整流离子选择性通道 ;钾通道阻滞剂 4 AP和TEA可使峰电流和迟电流幅度下降 ,表明存在两种类型的钾通道 ,或者这种通道有两种不同的状态 ;通道的激活和失活符合Boltzmann方程。未记录到Deiters细胞的内向钙电流。结论　Deiters细胞外向整流钾电流的作用可能是缓冲细胞周围间隙钾离子浓度     

豚鼠耳蜗外毛细胞乙酰胆碱敏感性钾电流特性

目的 研究豚鼠耳蜗外毛细胞乙酰胆碱(acetylcholine,ACh)敏感性钾电流离子特性及其受体的药理学特性.方法 健康豚鼠38只,断头后取出基底膜,经胶原酶Ⅳ消化后获取外毛细胞.采用全细胞记录膜片钳技术检测新鲜单离外毛细胞ACh-敏感性钾电流对细胞外钙依赖性钾电流阻断剂和N型胆碱能受体抑制剂的敏感性.结果 ①细胞外ACh激活一快速去敏感化的外向性钾电流,其平均(-x±s,以下同)反转电位为(-67.3±8.2)mV(n=10);-50 mV钳制电压下,100 μmol/LACh激活电流的幅值为(506.6±186.3)pA(n=9).②ACh-敏感性钾电流对细胞外四乙铵(tetraethylammonium,TEA,10 mmol/L)、蜂毒明肽(apamin,1 μmol/L)敏感,而细胞外的IBTX(iberiotoxin,200 nmol/L)对ACh-敏感性钾电流幅值无抑制作用.③ACh-敏感性钾电流的半数激活浓度(EC50)为(33.5±5.7)μmol/L(n=7).④ACh-敏感性钾电流对细胞外γ-氨基丁酸(gammaaminobutyric acid,GABA)-A受体阻断剂荷包牡丹碱(bicuculline)和α9-N型胆碱能受体(α9受体)特异性抑制剂士的宁(strychnine)敏感.士的宁和荷包牡丹碱对ACh-敏感性钾电流的抑制作用具有浓度依赖性,其半数抑制浓度(IC50)分别为(22.3±2.6)nmol/L(n=7)和(1.2±0.4)μmol/L(n=6).结论 细胞外ACh激活豚鼠耳蜗外毛细胞产生小电导钙依赖性钾电流(SK),此电流可能由α9受体介导.     

豚鼠耳蜗血管纹边缘细胞钾离子通道特性的研究

目的 研究单个豚鼠耳蜗血管纹边缘细胞的基本电生理特性。方法 应用耳蜗血管纹组织块培养技术和全细胞膜片钳技术，观察单个培养的豚鼠血管纹边缘细胞在电压钳模式下的电流特性。结果 边缘细胞上记录到钾离子电流，该钾通道有以下特性：①具有电压依赖性；②通道的活动可被4-氨基吡啶（4-aminopyridine，4-AP）和四乙铵（tetraethylammonium，TEA）在相对高浓度下阻滞；③细胞外钙离子浓度的减少可导致该钾通道电流的降低。结论 豚鼠耳蜗血管纹边缘细胞钾离子通道电流包括钙离子依赖性钾电流、外向延迟整流钾电流和A型钾电流。     

顺铂对大鼠耳蜗螺旋神经节细胞外向延迟整流钾电流的影响

目的:观察记录顺铂作用下急性分离新生大鼠耳蜗螺旋神经节细胞(SGNs)延迟整流钾通道的电流曲线,分析顺铂对SGNs钾电流激活动力学的影响,并初步探讨其耳毒性机制。方法:采用全细胞膜片钳技术记录SGNs外向延迟整流钾电流及顺铂对此电流的影响。结果:钳制电压为-60mV,刺激电压从-60mV到 80mV逐渐去极化,阶跃电压为10mV,持续时间为500ms,可在SGNs上记录到外向钾电流,该电流对TEA-Cl(4-乙基胺)敏感,具有延迟整流特性;在细胞外液中加入10μmol/L顺铂,能明显抑制SGNs延迟整流钾电流;顺铂对此电流的抑制作用与细胞外液中顺铂的浓度呈剂量依赖性;外液洗脱后SGNs电流可基本恢复正常。结论:钾通道与SGNs动作电位的产生密切相关,顺铂可抑制SGNs钾通道电流,导致听觉功能障碍。     

Saturation of outer hair cell receptor currents causes two-tone suppression

Zwicker [Biol. Cybern. 35, 243-250, (1979); J. Acoust. Soc. Am. 80, 163-176 (1986)] has previously proposed that many nonlinear phenomena in the mammalian cochlea can be explained by saturation of a positive feedback process which enhances mechanical sensitivity, although the site of the nonlinearity producing this saturation has so far remained obscure. In this paper we present evidence suggesting that the nonlinearity of mechano-electrical transduction in the outer hair cells is the dominant nonlinearity producing two-tone suppression in the mammalian cochlea. In particular, we show that: (i) suppression of the extracellular summating potential (SP), recorded from a particular place within the organ of Corti, has characteristics similar to the suppression of activity in the auditory-nerve; (ii) that SP suppression occurs at approximately constant basilar membrane displacement, inferred from the SP iso-response contours; and that (iii) the onset of SP suppression with suppressor tones on the tail of the frequency tuning curve closely parallels the onset of nonlinearity in the local cochlear microphonic. Since previous studies (Patuzzi et al., 1989) have demonstrated that the vibration of the basilar membrane at its characteristic frequency is very sensitive to changes in outer hair cell receptor current, we consider that interference in outer hair cell currents caused by nonlinearity in mechano-electrical transduction is an adequate explanation of two-tone suppression. This requires that outer hair cell receptor currents deviate from linearity at a suppressor tone level below that required to produce a significant DC receptor potential within the inner hair cells, and that the active process within the cochlea is distributed along a local region of the cochlea, basal of the vibration peak.     

Recording of mechano-electrical transduction currents by a nystatin perforated-patch method

Mechano-electrical transduction (MET) currents in isolated cochlear hair cells of chicks were recorded by use of a nystatin perforated-patch method. The membrane of a cell-attached patch was permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without loss of cytoplasmic compounds. The current-voltage relationship was linear for the inward-going MET current at negative membrane potentials, but outward currents were reduced at positive membrane potentials, evidence of inward-going rectification. Elevation of the intracellular concentration of calcium at positive membrane potential, mediated via a voltage-dependent Ca2+ channel, may suppress the outward-going MET current by acting from within the cell.     

The effect of air currents on the naso-respiratory mucosa.

The direction of air currents through the nose have been studied by many investigators. However, in injury of the nose followed by deformities, a marked change in the nasal configuration occurs. Many patients will present localized areas of crusting and attention is not drawn to this condition until the patient is seen with an epistaxis. In this study, we are interested in the character of the mucosa and their changes as affected by long-term injury from the trauma of the inspiratory and expiratory air currents, which, on sniffling or snorting, may reach hurricane speeds. In the study of the naso-respiratory mucosa from these areas by electron-microspy, it was found metaplastic respiratory epithelium where the cells have undergone mucinous transformation and areas exposed to air jets how metaplastic changes followed by erosion. We wish to thank Nikolajs Cauna, M.D. for his assistance and making available the micrographs used in this paper.     

Whole cell currents and mechanical responses of isolated outer hair cells

Outer hair cells (OHCs) exhibit electrically induced cell movements which are considered to enhance the frequency selectivity and sensitivity of basilar membrane vibration. Using simultaneous whole cell voltage clamp and video analysis, we demonstrate that the mechanical response of OHCs is not altered by agents which alter membrane currents under voltage clamp. Thus the underlying mechanism of OHC movements appears to be dependent upon membrane potential, rather than transmembrane currents.     

Frog saccular hair cells dissociated with protease VIII exhibit inactivating BK currents,K(V) currents,and low-frequency electrical resonance

Outward K currents and electrical resonance of frog (Rana esculenta) saccular hair cells isolated enzymatically with bacterial protease VIII were investigated using the perforated patch-clamp method. Under voltage-clamp conditions we identified two K currents, a voltage-dependent K (K(V)) current, and a partially inactivating iberiotoxin-sensitive K (BK) current. The K(V) current activated at a membrane potential of approximately -50 mV (from a holding potential of -70 mV). Its activation rate was rather slow, having a time constant in the range 5-8 ms at 0 mV. The K(V) current was resistant to tetraethylammonium (10 mM), but was inhibited by 4-aminopyridine (1 mM). A striking feature of the BK current was its inactivation; this was monoexponential and had fast kinetics (tau(inact)=2.7 ms +/-1.2, at -10 mV; n=8). Inactivation of the current was incomplete, a residual sustained component remaining. This varied considerably among hair cells (mean ratio between peak transient and sustained component was 1.22+/-0.18, range 0.53-1.8; n=8). In current-clamp mode steady depolarizing current pulses evoked membrane potential oscillatory responses, with mean frequencies varying between 30 and 100 Hz for membrane potentials from -60 to -40 mV (n=18). Most hair cells (14/18) exhibited damped oscillations, and in the remainder a few initial damped oscillations were succeeded by smaller, undamped voltage oscillations. The peak quality factor and the characteristic frequency assessed on 14 cells displaying only damped oscillatory responses were 2.4+/-1.3 and 59+/-39 Hz, respectively. In contrast, papain-dissociated frog saccular hair cells possess solely a sustained BK current, and exhibited significantly higher resonant frequencies and quality factors. In conclusion, the K currents and the electrical resonance of hair cells dissociated in protease VIII differ markedly from those dissociated with papain, but are similar to those reported for in situ preparations, suggesting that our dissociation procedure preserves the electrophysiological profile of in situ frog saccular hair cells.     

Regional distribution of calcium currents in frog semicircular canal hair cells

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.     

Chlorogenic acid alters the voltage-gated potassium channel currents of trigeminal ganglion neurons

Chlorogenic acid（5-caffeoylquinic acid, CGA） is a phenolic compound that is found ubiquitously in plants, fruits and vegetables and is formed via the esterification of caffeic acid and quinic acid. In addition to its notable biological functions against cardiovascular diseases, type-2 diabetes and inflammatory conditions, CGA was recently hypothesized to be an alternative for the treatment of neurological diseases such as Alzheimer＇s disease and neuropathic pain disorders. However, its mechanism of action is unclear.Voltage-gated potassium channel（Kv） is a crucial factor in the electro-physiological processes of sensory neurons. Kv has also been identified as a potential therapeutic target for inflammation and neuropathic pain disorders. In this study, we analysed the effects of CGA on the two main subtypes of Kv in trigeminal ganglion neurons, namely, the IK,Aand IK,Vchannels. Trigeminal ganglion（TRG）neurons were acutely disassociated from the rat TRG, and two different doses of CGA（0.2 and 1 mmol·L21） were applied to the cells.Whole-cell patch-clamp recordings were performed to observe alterations in the activation and inactivation properties of the IK,Aand IK,Vchannels. The results demonstrated that 0.2 mmol·L21CGA decreased the peak current density of IK,A. Both 0.2 mmol·L21and1 mmol·L21CGA also caused a significant reduction in the activation and inactivation thresholds of IK,Aand IK,V. CGA exhibited a strong effect on the activation and inactivation velocities of IK,Aand IK,V. These findings provide novel evidence explaining the biological effects of CGA, especially regarding its neurological effects.