三七总皂苷对大鼠海马CA1区突触传递的作用及机制

目的研究三七总皂苷(Panax notoginseng saponins,PNS)对大鼠海马脑片CA1区锥体神经元兴奋性和抑制性突触传递的作用。方法断头法分离3~4周雄性Wistar大鼠海马半脑,用切片机切出400μm厚度的海马脑片,对CA1区锥体细胞采用"盲法"全细胞膜片钳技术记录,分别检测和分析PNS(0.05~0.4g/L)对刺激CA1传入纤维引出的兴奋性突触后电流(EPSCs)和抑制性突触后电流(IPSCs)的影响,继而以脉冲间隔为50ms的配对刺激代替单刺激,通过EPSC2/EPSC1(P2/P1)值的变化观察PNS对双脉冲易化(paired-pulse facilitation,PPF)的影响。结果0.1~0.4g/L PNS显著抑制EPSCs(P<0.05),且PNS在抑制P1、P2的同时明显升高P2/P1值(P<0.05),加强了双脉冲易化,但PNS对IPSCs无显著影响(P>0.05)。结论PNS显著减小大鼠海马CA1区锥体神经元的EPSCs而不影响IPSCs,说明PNS不是通过强化抑制性中间神经元的功能间接地抑制兴奋性神经元,而是对兴奋性突触传递直接产生抑制;PNS明显升高P2/P1值,说明PNS是通过突触前机制抑制CA1区兴奋性突触传递。     

Negatively charged residues located near the external entrance are required for the Kir2.1 channel to function

To understand the significance of negative charges in the extracellular loops of the inwardly rectifying K⁺ (Kir2.1) channel, single-point mutants (D112N, D114N, E125Q, D152E, D152K, D152N, E153D, E153K, and E153Q) and double-point mutants (D112N/D114N and D152N/E153Q) were constructed and transfected into COS-1 and HEK293 cells. All single-point mutants, except D152K, and D112N/D114N expressed functional channels. Cells transfected with the D152K and D152N/E153Q constructs did not show any inwardly rectifying K⁺ currents, although fluorescence images confirmed that the channel proteins produced by D152K and D152N/E153Q were transported to the cell surface. While a tandem tetramer with one D152N subunit and three D152N/E153Q subunits, D152N–(D152N/E153Q)₃, did not express functional channels, a tandem tetramer with one E153Q subunit and three D152N/E153Q subunits, E153Q–(D152N/E153Q)₃, and that with two D152N subunits and two D152N/E153Q subunits, (D152N)₂–(D152N/E153Q)₂, expressed channels having similar conductance and kinetics of single-channel currents to the wild-type channels. These results suggest that one negative charge of D152 or two negative charges of E153 are required for Kir2.1 channels to function. It is suggested that the contribution by D152 and E153 to the electronegative extracellular pore entrance is critical for the channel to function properly.     

Endothelin-1 modulates the arrhythmogenic activity of pulmonary veins

Objective: Endothelin-1 has important cardiovascular effects and is activated during atrial fibrillation. Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation. The aim of this study was to evaluate whether endothelin-1 affects PV arrhythmogenic activity.
Methods: Conventional microelectrodes were used to record the action potentials (APs) and contractility in isolated rabbit PV tissue specimens before and after the administration of endothelin-1 (0.1, 1, 10 nM). The ionic currents of isolated PV cardiomyocytes were investigated before and after the administration of endothelin-1 (10 nM) through whole-cell patch clamps.
Results: In the tissue preparation, endothelin-1 (1, 10 nM) concentration dependently shortened the AP duration and decreased the PV firing rates. Endothelin-1 (10 nM) decreased the resting membrane potential. Endothelin-1 (0.1, 1, 10 nM) decreased the contractility and increased the resting diastolic tension. In single PV cardiomyocytes, endothelin-1 (10 nM) decreased the PV firing rates from 2.7 ± 1.0 Hz to 0.8 ± 0.5 Hz (n = 16). BQ-485 (100 μM, endothelin-1 type A receptor blocker) reversed and prevented the chrono-inhibitory effects of endothelin-1 (10 nM). Endothelin-1 (10 nM) reduced the L-type calcium currents, transient outward currents, delayed rectifier currents, transient inward currents, and sodium–calcium exchanger currents in the PV cardiomyocytes with and without pacemaker activity. Endothelin-1 (10 nM) increased the inward rectifier potassium current, hyperpolarization-induced pacemaker current, and the sustained outward potassium current in PV cardiomyocytes with and without pacemaker activity.
Conclusion: Endothelin-1 may have an antiarrhythmic potential through its direct electrophysiological effects on the PV cardiomyocytes and its action on multiple ionic currents.     

Anoikis: a necessary death program for anchorage-dependent cells

Cell to matrix adhesion is a key factor for cellular homeostasis and disruption of such interaction has adverse effects on cell survival. It leads to a specific type of apoptosis known as “anoikis” in most non-transformed cell types. This kind of apoptosis following loss of cell anchorage is important for development, tissue homeostasis and several diseases. Integrins sense mechanical forces arising from the matrix, thereby converting these stimuli to downstream signals modulating cell viability. Anchorage-independent growth is a crucial step during tumorigenesis and in particular during the metastatic spreading of cancer cells. The disruption of the tight control leading an “homeless” cell to death is therefore able to violate the cell defences against transformation. This review analyses the recent investigations into the molecular mechanisms governing anoikis, discussing the different ways in which adhesion can influence this process and addressing the relevance of this unique apoptosis mode in the development of metastatic cancers, as well as in other diseases.     

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca_V) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca_V2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca6^84Q) developed progressive motor impairment and aggregation of mutant Ca_V2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca²⁺ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6^84Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant Ca_V2.1 channels.     

Chiral separation of racemate CPU86017, an anti-arrhythmic agent, produces stereoisomers possessing favourable ion channel blockade and less alpha-adrenoceptor antagonism

1. CPU86017 is an effective anti-arrhythmic agent of the Class III complex that has two chiral centres, 7N and 13aC. As a promising anti-arrhythmic agent, the blockade on I(Kr), I(Ks) and calcium influx may be modulated to be mild, moderate and potent, with less a-adrenoceptor blockade. In order to improve activity at ion channels, four stereoisomers, namely SS ((+)-7S,13aS-CPU86017), SR ((-)-7S,13aR-CPU86017), RR ((-)-7R,13aR-CPU86017) and RS ((+)-7R,13aS-CPU86017), have been separated. In the present study, the effects of these four isomers on I(Kr) and I(Ks), calcium channels and a-adrenoceptors were compared with the effects of the racemate CPU86017. 2. In the present study, I(Kr) and I(Ks) were measured as tail currents (I(Kr.tail) and I(Ks.tail), respectively) using the whole-cell patch-clamp technique. Antagonism of receptor-operated calcium channels and voltage-dependent calcium channels (VDC) in vascular smooth muscle by CPU86017 and the four isomers were tested as suppression of phenylephrine- or KCl-induced contractions of aortic rings, respectively. 3. For I(Kr.tail) inhibition, the IC(50) of SS, SR, RR, RS and CPU86017 was 2.86 +/- 1.20, 39.4 +/- 8.5, 3.48 +/- 0.80, 7.65 +/- 1.50 and 12.5 +/- 7.8 x 10(-9) mol/L, respectively; for I(Ks.tail) inhibition IC(50) values were 16.9 +/- 4.0, 20.0 +/- 2.1, 99.1 +/- 5.9, 160 +/- 81 and 65.0 +/- 4.7 x 10(-9) mol/L, respectively. The SR isomer showed balanced blockade of I(Kr) and I(Ks) that was associated with a loss of a-adrenoceptor antagonism but enhanced VDC blockade. 4. Configuration of 13aC critically determines I(Kr) blockade and the Ca(2+) antagonism of the isomers of CPU86017. The SR isomer exhibits mild blockade of I(Kr), moderately enhanced blockade of I(Ks) and Ca(2+) influx and less a-adrenoceptor antagonism compared with the racemate and may be promising as an anti-arrhythmic.     

Ionic currents expressed in a cell line derived from the organ of Corti of the Immortomouse

 We have investigated the maturation of adult hair cell electrophysiology in a population of precursor cells in a conditionally immortal cell line. The cell line, UB/OC-2, from the embryonic organ of Corti of the H-2Kb-tsA58 transgenic mouse, permits cells to grow proliferatively at 33°C and to differentiate at 39°C. Whole-cell patch-clamp recordings showed that proliferating cells had a different electrophysiology to differentiating cells. Differentiating cells had a conditionally expressed slowly activating inward current activated by hyperpolarization. The current was not blocked by extracellular application of 0.5 mM Ba²⁺, but was blocked reversibly by 2 mM Cs⁺. The current was found to be carried by both K⁺ and Na⁺ ions (P _K/P _Na=2.2) and activated by 10 μM forskolin. These properties identify the slowly activating current as I _h. A proportion of proliferating and differentiating cells exhibited a voltage-gated Na⁺ current, I _Na. I _Na was abolished in Na-free external medium and was inhibited reversibly by tetrodotoxin (TTX) with K _i=64 nM. Together these results suggest that proliferating and differentiating hair cell precursors in the immortal cochlear cell line UB/OC-2 express currents which are also found in developing hair cells. Received: 6 January 1999 / Received after revision: 1 February 1999 / Accepted: 2 February 1999     

缬沙坦对豚鼠心房肌细胞电生理的影响

 目的 从心肌细胞电生理的角度探讨血管紧张素Ⅱ受体阻断药(ARB)类药物抗房颤的可能机制.方法 采用全细胞膜片钳技术的电流钳方法记录单细胞动作电位,采用全细胞膜片钳技术的电压钳方法记录心房肌细胞的延迟整流钾电流(Ik).观察ARB类药物缬沙坦对豚鼠心房肌细胞动作电位及离子通道电流的影响.结果 缬沙坦100 μM可延长心房肌细胞动作电位时程,尤其是APD50从(52.2±6.5) ms延长至(58.6±7.8 ms,P<0.01),APD90从(94.0±11.7) ms延长至(105.3±14.0 ms,P<0.01),而对RMP、APA无显著影响.缬沙坦100 μM明显抑制心房肌细胞IK的峰值电流,从5.33±0.13(pA/pF)减小至4.49±0.48(pA/pF)(P<0.05),并呈电压依赖性.结论 高浓度缬沙坦延长心房肌细胞动作电位时程、APD50及APD90.缬沙坦抑制心房肌细胞延迟外向钾电流,可能是引起其动作电位时程延长的一个重要因素.缬沙坦延长心房肌动作电位时程,可能在房颤的转复及房颤转复后窦性心律的维持中有价值.     

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses

It has been known that Ca²⁺ plays an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. Synaptotagmin 1, 2, and 9 were found to act as the Ca²⁺ sensors for synchronous release and synaptotagmin 7 and Doc‐2 were proposed as the Ca²⁺ sensors for asynchronous release. Comparatively, the Ca²⁺ sensor for spontaneous release remains a mystery. At the Calyx of Held synapse, the Ca²⁺sensor for spontaneous release was found not identical to the sensor for synchronous release, synaptotagmin 2. As Ca²⁺ sensors have different sensitivity to Sr²⁺ and Ca²⁺ and induce significantly different rate of vesicle release, Sr²⁺ is traditionally used as a tool to examine the intrinsic properties of different Ca²⁺ sensors. Here, we employed cell‐attached patch recording and presynaptic/postsynaptic whole‐cell recording at the Calyx of Held synapses of synaptotagmin 2 knock‐out mice to assay the Sr²⁺ and Ca²⁺ influx into the nerve terminal at resting potential and observed the effects of Ca²⁺ and Sr²⁺ on spontaneous neurotransmitter release. We found that the dwell time of single voltage gated Ca²⁺ channel opening increased around threefold for Sr²⁺ than Ca²⁺ with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to Sr²⁺ than Ca²⁺. Thus, our study reveals some of the intrinsic properties of Ca²⁺ sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.