NO对新生大鼠下丘脑钙激活钾通道的直接激活作用

目的：研究NO对下丘脑神经元钙激活钾通道（K_Ca）的作用及其机制。方法：采用膜片钳技术内面向外式。结果：NO可显著提高通道的开放概率，这种增强作用是通过延长通道开放时间及增加开放频率实现的。结论：下丘脑神经元中NO可直接激活K_Ca，它的病理生理作用还有待于进一步研究。     

毛细胞PMCA2在小鼠内耳Ca2+调节和听觉平衡中的作用

目的： 研究小鼠内耳毛细胞细胞膜Ca²⁺-ATP酶2型蛋白（PMCA2）在听觉平衡生理中的作用及意义。 方法：利用不同基因型小鼠PMCA2^-/-(突变纯合子)、PMCA2^+/-(杂合子)和PMCA2^+/+(野生型)为实验对象，采用听觉脑干反应(ABR)、畸变产物耳声发射(DPOAE)和耳蜗内电位(EP)检测等方法，分别检测不同基因型小鼠的内耳生理功能。结果：PMCA2^+/+野生型鼠的听力正常，ABR的短声(click)阈值为(13.75±11.08)dB SPL；EP均值为(91.3±11.0)mV。PMCA2^+/-杂合子小鼠听力低于同窝PMCA2^+/+野生型鼠，ABR的短声阈值为(63.89±12.90)dB SPL，与PMCA2^+/+小鼠比较差异显著(P<0.01)；PMCA2^+/-小鼠没有检测出DPOAE的高频区辐值，其EP均值为(80.7±9.0)mV。PMCA2^-/-小鼠的ABR在100 dB SPL无反应，表现出全聋和平衡功能失调，其EP均值为(56.6±13.0)mV；PMCA2^-/-小鼠没有检测出DPOAEs。结论：PMCA2是内耳毛细胞纤毛丛上的重要Ca²⁺转运通道，对维持内耳的Ca²⁺代谢和听觉平衡功能有重要作用。     

Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium‐dependent vasodilation in the femoral artery of developing piglets

Mechanisms mediating endothelium‐dependent vasodilation were investigated in femoral artery rings from <2‐day‐old (newborn) and 2‐week‐old piglets. Based on previous results we hypothesized an age difference in the relative contribution of nitric oxide(NO)‐cyclic 3′,5′‐guanosine monophosphate (cGMP) and K⁺ channel‐activation to acetylcholine (ACh)‐induced vasodilation. Changes in vascular tone were studied in organ baths in the absence or presence of NO synthase(NOS) inhibition or K⁺ channel blockade and the intra‐arterial accumulation of cGMP in response to ACh was measured with radioimmunoassay (RIA). In control experiments, relaxant responses to ACh were equal in the two age groups. In the presence of the NOS‐inhibitors N ^G‐monomethyl‐L ‐arginine acetate (L ‐NMMA; 100 μM ) or N^G‐nitro‐L ‐arginine (L ‐NOARG; 1–100 μM ), however, relaxation was significantly more reduced in femoral artery rings from 2‐week‐old than from newborn, with lower pD₂ values in the older age group. Inhibition of large (BK_Ca) conductance calcium‐sensitive K⁺ channels with tetraethylammonium chloride (TEA; 1 mM ), gave a significant rightward shift in the concentration‐response curves to ACh which was of the same magnitude in both age groups. The ACh‐induced vasodilation was abolished in both age groups by high K⁺ (20 mM ) in combination with L ‐NOARG (100 μM ). The relative increase in cGMP levels after addition of ACh (10 nM ) was significantly larger in rings from newborn compared with 2‐week‐old piglets (12‐ vs. four‐fold). In summary, sensitivity to NOS inhibition increased with age while the effect of K⁺ channel blockade with TEA was the same in femoral artery rings from newborn to 2‐week‐old piglets. Lower sensitivity to NOS inhibition and a larger increase in cGMP in response to ACh could indicate a higher efficacy of the NO/cGMP pathway in this vessel in the newborn piglet.     

Physiological mechanisms of TRPC activation

TRPC (canonical transient receptor potential) channels are vertebrate homologs of the Drosophila photoreceptor channel, TRP. Considerable research has been brought to bear on the seven members of this family, especially with regard to their possible role in calcium entry. Unfortunately, the current literature presents a confusing picture, with different laboratories producing widely differing results and interpretations. It appears that ectopically expressed TRPC channels can be activated by phospholipase C products (generally, diacylglycerols), by stimulation of trafficking to the plasma membrane, or by depletion of intracellular Ca²⁺ stores. Here, I discuss the possibility that these diverse experimental findings arise because TRPC channels can, under both experimental as well as physiological conditions, be activated in three distinct ways, possibly depending on their subunit composition and/or signaling complex environment. The TRPCs may be unique among ion-channel subunit families in being able to participate in the assembly and function of multiple types of physiologically important ion channels.     

失血性休克对大鼠血管平滑肌BKCa通道酪氨酸磷酸化水平的影响

目的:探讨失血性休克对大鼠肠系膜上动脉血管平滑肌BK_Ca通道酪氨酸磷酸化的影响以及NO对BK_Ca通道酪氨酸磷酸化的调控。方法:建立大鼠失血性休克模型[(35±5)mmHg]并提取肠系膜上动脉总裂解蛋白,采用免疫沉淀及免疫印迹技术,观察休克血管平滑肌BK_Ca通道酪氨酸磷酸化的变化情况;采用原代培养的大鼠肠系膜上动脉血管平滑肌细胞,观察NO对BK_Ca通道酪氨酸磷酸化的调控及其时-效、量-效关系。结果:失血性休克2 h及4 h后大鼠肠系膜上动脉血管平滑肌BK_Ca通道α亚基酪氨酸磷酸化明显增强 (P＜0.01 );L-精氨酸(5×10^-5-5×10^-4 mol/L)孵育30 min即可诱导培养血管平滑肌细胞BK_Ca通道α亚基酪氨酸磷酸化增强,2 h内无明显下降;且L-精氨酸诱导BK_Ca通道α亚基酪氨酸磷酸化具有剂量依赖性。结论:重症失血性休克可增强血管平滑肌BK_Ca 通道酪氨酸磷酸化,且NO参与了该调控过程。     

COPD合并慢性缺氧患者肺血管对急性缺氧反应变化中肺动脉平滑肌细胞钾通道的作用

目的：以人离体肺动脉环缺氧张力变化为对象研究钾通道在缺氧性肺血管收缩（HPV）发生中的作用。 方法： 从手术室切取人肺动脉环，进行人肺动脉环张力试验，分正常对照组，单纯慢性阻塞性肺病（chronic obstructive pulmonary disease ，COPD）组和COPD合并慢性缺氧组，分别利用相应的特异性阻断剂观察电压门控性钾通道（KV），钙离子激活的钾通道（KCa）， ATP敏感的钾通道（KATP），在缺氧性肺血管收缩（HPV）的作用。 结果： （1）3组肺动脉环在急性缺氧时血管收缩张力均增加，同时COPD＋慢性缺氧组增加百分比显著低于对照组；COPD组与对照组无显著差异。（2）4-AP阻断Kv后较阻断前，3组肺血管环缺氧张力增加幅度均显著降低（P<0.05），同时正常对照组和COPD组降低幅度明显大于慢性缺氧组。TEA阻断KCa后以及格列苯脲（glybenclamide）阻断KATP后二者较阻断前，3组肺血管环缺氧张力均显著增加（P<0.05），同时COPD＋慢性缺氧组增加的幅度明显大于正常组（分别是P<0.01和P<0.05）。 结论： （1）慢性缺氧可降低肺血管的收缩性及肺血管对急性缺氧的收缩反应；（2）Kv在3组人离体肺动脉环HPV反应中均起介导作用，慢性缺氧可使此介导作用加强；KCa和KATP在3组人离体肺动脉环HPV反应中均起调节作用，慢性缺氧可使此调节作用加强。     

Sodium conductance in calcium channels of guinea-pig ventricular cells induced by removal of external calcium ions

An inward current characterized by a slow inactivation, was induced when the extracellular Ca^2– concentration was reduced by EGTA. It was suppressed by replacing external Na^– with Tris⁺ or by D-600, increased by epinephrine, and was not affected by TTX. These findings suggest that this current is carried by Na⁺ ions through the Ca channels. The Na current decreased in amplitude as the concentration of external divalent cations was elevated. Blocking the Na current by divalent cations could be approximated by a bimolecular interaction between divalent cation and channel, with a dissociation constant of 1.2 M for Ca²⁺ and 60 M for Mg²⁺. Single channel currents were recorded in the cell-attached configuration. With a pipette solution of pCa=7.5 or pCa>8, the single channel I-V relationship was linear and the slope conductance was 70–75 pS. For 40 mV depolarizations from the resting potential, unitary currents were smaller at pCa=6 than at pCa=7.5. However, single channel events, which were observed after the repolarizing step to the resting potential, were much the same amplitude. The open time histogram was fitted with a single exponential having a time constant of 1.9 ms at around –40 mV (pCa>8, with 5 M Bay K 8644 in the bath solution), which was decreased with increasing the Ca²⁺ concentration in the pipette solution. Noise power spectra of patch currents at pCa=6 revealed a high-frequency component at around 1500 Hz. These results suggest that Ca binding to the sites with a high affinity for Ca²⁺ blocks the Na conductance in Ca channels. Reduction of the unitary current at higher concentrations of Ca²⁺ might be attributed to a rapid block by Ca²⁺.     

Intracellular calcium changes in rat aortic smooth muscle cells in response to fluid flow

Vascular smooth muscle cells (VSM) are normally exposed to transmural fluid flow shear stresses, and after vascular injury, blood flow shear stresses are imposed upon them. Since Ca²⁺ is a ubiquitous intracellular signaling molecule, we examined the effects of fluid flow on intracellular Ca²⁺ concentration in rat aortic smooth muscle cells to assess VSM responsiveness to shear stress. Cells loaded with fura 2 were exposed to steady flow shear stress levels of 0.5–10.0 dyn/cm² in a parallel-plate flow chamber. The percentage of cells displaying a rise in cytosolic Ca²⁺ ion concentration ([Ca²⁺]_i) increased in response to increasing flow, but there was no effect of flow on the ([Ca²⁺]_i) amplitude of responding cells. Addition of Gd³⁺ (10 M) or thapsigargin (50 nM) significantly reduced the percentage of cells responding and the response amplitude, suggesting that influx of Ca²⁺ through ion channels and release from intracellular stores contribute to the rise in ([Ca²⁺]_i) in response to flow. The addition of nifedipine (1 or 10 M) or ryanodine (10 M) also significantly reduced the response amplitude, further defining the role of ion channels and intracellular stores in the Ca²⁺ response. © 2002 Biomedical Engineering Society. PAC2002: 8716Uv, 8719Uv, 8716Dg, 8719Ff     

Expression of voltage-dependent sodium and transient potassium currents in an identified sub-population of dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos

The electrophysiological properties of a subset of dorsal root ganglion (DRG) neurons microdissected from 12-day-old (E12) mouse embryos and acutely isolated were analyzed as soon as 3 after their isolation. Two classes of neurons were defined according to their mean diameter. The larger diameter class was examined in this study. They display uniform cytoskeletal properties with co-expression of vimentin and neurofilament triplet proteins. Patch-clamp methods also revealed a homogeneous and limited repertoire of ionic channels that included (1) a TTX-sensitive Na⁺ current whose properties are similar to that reported in mature mammalian neurons, and (2) two types of K⁺ currents that can be compared with the delayed rectifier (I _k ) and the transient (I _a) potassium currents found in other mammalian preparations. It may be possible to use this in vitro model to examine the development of new types of currents, such as Ca²⁺ currents during neuronal growth and differentiation.     

Mechanisms of the inhibition of Shaker potassium channels by protons

Potassium channels are regulated by protons in various ways and, in most cases, acidification results in potassium current reduction. To elucidate the mechanisms of proton-channel interactions we investigated N-terminally truncated Shaker potassium channels (K_v1 channels) expressed in Xenopus oocytes, varying pH at the intracellular and the extracellular face of the membrane. Intracellular acidification resulted in rapid and reversible channel block. The block was half-maximal at pH 6.48, thus even physiological excursions of intracellular pH will have an impact on K⁺ current. The block displayed only very weak voltage dependence and C-type inactivation and activation were not affected. Extracellular acidification (up to pH 4) did not block the channel, indicating that protons are effectively excluded from the selectivity filter. Channel current, however, was reduced greatly due to marked acceleration of C-type inactivation at low pH. In contrast, inactivation was not affected in the T449V mutant channel, in which C-type inactivation is impaired. The pH effect on inactivation of the wild-type channel had an apparent pK of 4.7, suggesting that protonation of extracellular acidic residues in Kv channels makes them subject to pH regulation.