Regulation of single potassium channels by serotonin in the cell bodies of the tail mechanosensory neurons ofAplysia californica

Sensory neurons in the pleural ganglion ofAplysia mediate the afferent portion of the tail withdrawal reflex. Previous work has shown that in these neurons and in the siphon sensory neurons ofAplysia, serotonin modulates a steady-state non-inactivating potassium current called the S current. Using the technique of patch clamping, we have examined the kinetics of single potassium channels and found that they share the properties of the S potassium channel of the siphon sensory neurons. This channel has an elementary slope conductance of73 ± 9.98pS(x±S.E.M.) and shows Goldman rectification. It is active at the resting potential and does not inactivate with maintained depolarization. Bath application of serotonin in a majority of experiments decreased the functional number of channels in the patch.     

3种钾通道在哮喘豚鼠气道高反应中的作用

目的：探讨钙激活钾通道（KCa）、延迟整流型钾通道（Kdr）和ATP敏感型钾通道（KATP）在哮喘豚鼠气道高反应中的作用。 方法： 采用离体气管环张力实验，观察加入特异性钾通道阻断剂后，与不加阻断剂相比气管环对组胺反应的量效曲线的差异。 结果： （１）加入KCa阻断剂TEA后，对照组气管环对组胺的反应变化不显著，而哮喘组气管环对10-4mol/L、10-3mol/L组胺的收缩反应均显著低于不加TEA的气管环(P<0.01)，量效曲线明显下移；（2）加入Kdr阻断剂4-AP后，对照组气管环对10-3mol/L组胺的最大收缩反应明显下降(P<0.05)，组胺的量效曲线下移，哮喘组气管环对10-4mol/L、10-3mol/L组胺的收缩反应均低于不加4-AP的气管环(P<0.01)，且下降程度较对照组大（P<0.05），量效曲线明显下移；（3）加入KATP阻断剂glibenclamide后，对照组和哮喘组气管环对组胺的量效曲线与不加glibenclamide的气管环相比变化均无明显差异。 结论： 在豚鼠哮喘模型中，KCa和Kdr活性的降低在气道高反应的发生中起介导作用，KATP作用不明显。     

苯骈吡喃肟类化合物的合成及生物活性

苯骈吡喃类钾通道启开剂作为抗高血压药物正日益引人注目。根据现有构效关系，运用经典的药物设计方法，设计合成了２８个苯骈吡喃肟类化合物。初步药理研究表明，某些化合物有一定程度的扩血管活性。     

Interaction between thiol-modifying agents and P1075, a KATP channel opener, in rat isolated aorta

In vascular smooth muscle, openers of ATP-dependent potassium channels (K _ATP channels), such as P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the ⁸⁶Rb⁺ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in ⁸⁶Rb⁺ efflux induced by P1075 was taken as a qualitative measure of K⁺ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100μM), abolished specific binding of [³H]-P1075 with an IC₅₀ value of 7.6±1.2μM; at 30μM, the half time for inhibition was 38min. Two other thiol-oxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2’-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1mM), reduced [³H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In ⁸⁶Rb⁺ efflux experiments, thimerosal (3 to 100μM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC₅₀ value of 7±1μM. The inhibitory effect occurred with a half-time of approximately 8min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation.The data show that oxidation of thiol groups interferes with the binding of the K _ATP channel opener, P1075; concomitantly, the ⁸⁶Rb⁺ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K_ATP channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated ⁸⁶Rb⁺ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity. Received: 7 April / Accepted: 9 July 1997     

中国仓鼠肺成纤维细胞和HeLa细胞DNA氧化损伤的自身修复能力与比较

目的:研究不同氧化相关因素对中国仓鼠肺成纤维细胞(CHL)和HeLa细胞DNA损伤的自身修复情况.方法:将CHL细胞和HeLa细胞用不同氧化相关因素处理一定时间[CHL细胞:过氧化氢(H2O2)25 min,重铬酸钾(K2Cr2O7)105 min,阿霉素(Dox)75 min;HeLa细胞:H2O2 25 min,K2Cr2O7 105 min],随后立即去毒培养0、0.5、1、2、3 h,以碱性单细胞凝胶电泳技术检测DNA链断裂情况.结果:①CHL细胞经H2O2、K2Cr2O7、Dox作用后引起DNA链断裂,去毒培养1 h链断裂修复明显(P<0.01);去毒培养2～3 h,前两毒剂的损伤组完全修复,而Dox组链断裂仍高于未损伤组;②HeLa细胞经H2O2、K2Cr2O7作用后引起DNA链断裂,去毒培养0.5 h链断裂明显修复(P<0.01),去毒培养1 h则完全修复;③CHL细胞和HeLa细胞损伤后修复的拖尾率与修复时间的回归系数显著不同(P<0.05).结论:两种细胞在氧化性DNA损伤后均迅速启动自身修复,但HeLa细胞比CHL细胞有更快的修复能力;同时这两种细胞由Dox所致损伤修复能力均较H2O2、K2Cr2O7所致的差.     

An overview of new pharmacological treatments for cerebrovascular dysfunction after experimental subarachnoid hemorrhage

Cerebral vasospasm and the resulting cerebral ischemia occurring after subarachnoid hemorrhage (SAH) are still responsible for the considerable morbidity and mortality in patients affected by cerebral aneurysms. Mechanisms contributing to the development of vasospasm, abnormal reactivity of cerebral arteries and cerebral ischemia after SAH have been intensively investigated in recent years. It has been suggested that the pathogenesis of vasospasm is related to a number of pathological processes, including endothelial damage, smooth muscle cell contraction resulting from spasmogenic substances generated during lyses of subarachnoid blood clots, changes in vascular responsiveness and inflammatory or immunological reactions of the vascular wall.A great deal of experimental and clinical research has been conducted in an effort to find ways to prevent these complications. However, to date, the main therapeutic interventions remain elusive and are limited to the manipulation of systemic blood pressure, alteration of blood volume or viscosity, and control of arterial dioxide tension.Even though no single pharmacological agent or treatment protocol has been identified which could prevent or reverse these deadly complications, a number of promising drugs have been investigated. Among these is the hormone erythropoietin (EPO), the main regulator of erythropoiesis. It has recently been found that EPO produces a neuroprotective action during experimental SAH when its recombinant form (rHuEPO) is systemically administered.This topic review collects the relevant literature on the main investigative therapies for cerebrovascular dysfunction after aneurysmal SAH. In addition, it points out rHuEPO, which may hold promise in future clinical trials to prevent the occurrence of vasospasm and cerebral ischemia after SAH.     

Gating modifier peptides as probes of pancreatic beta-cell physiology.

Pancreatic beta-cells depolarize in response to glucose and fire calcium-dependent actions potentials that trigger insulin secretion. The major current responsible for action potential repolarization in these cells is a delayed rectifier and Kv2.1 subunits are thought be a major contributor of the delayed rectifier channels. Hence, blockers of Kv2.1 channels might prolong action potentials and enhance calcium influx and insulin secretion. However, the lack of specific small molecule Kv2.1 inhibitors has hindered the testing of this mechanism. Importantly, several gating modifier peptides inhibit Kv2.1 channels in a relatively specific fashion. Hanatoxin (HaTX) and guangxitoxin-1 (GxTX-1) are examples that have been used to probe the role of Kv2.1 channels in beta-cell physiology. Both HaTX and GxTX-1 strongly inhibit the Kv current of beta-cells from various species, arguing that Kv2.1 subunits contribute significantly to the beta-cell delayed rectifier. GxTX-1 prolongs glucose-triggered action potentials, enhances glucose-dependent intracellular calcium elevations and augments glucose-dependent insulin secretion. Taken together, these data suggest that blockers of Kv2.1 channels may be a useful approach to the design of novel therapeutic agents for the treatment of type 2 diabetes. These studies highlight the utility of gating modifier peptides in the study of physiological systems.     

The Protective Role of Glutathione, Cysteine and Vitamin C against Oxidative DNA Damage Induced in Rat Kidney by Potassium Bromate

The roles of glutathione (GSH), cysteine, vitamin C., liposome-encapsulated superoxide dismutase (L-SOD) and vitamin E in preventing oxidative DNA damage and cytotoxicity in the rat kidney after administration of potassium bromate (KBrO₃) to male F344 rats were investigated by measuring 8-hydroxydeoxyguanosine (8-OH-dG), an oxidative DNA product, lipid peroxidation (LPO) levels and relative kidney weight (RKW). Combined pre- and posttreatment of animals with 2 × 800 mg/kg GSH i.p. inhibited the increase of 8-OH-dG, LPO levels and RKW caused by 80 mg/kg KBrO₃ i.p. administration. In contrast, pretreatment with 0.3 ml/kg diethylmaleate (DEM) i.p., a depletor of tissue GSH, was associated with elevation of 8-OH-dG, LPO levels and RKW after a 20 mg/kg KBrO₃ i.p. treatment, which itself caused no change. Administration of KBrO₃ itself reduced renal non-protein thiol levels, but this was inhibited by the two doses of exogenous GSH. Combined treatment with DEM and KBrO₃ lowered the non-protein thiol level in the kidney more than did DEM treatment alone. Protective effects against the oxidative damage caused by KBrO₃ were also observed for pre- and posttreatment with 400 mg/kg cysteine i.p., another sulfhydryl compound, and daily i.g. application of 200 mg/kg vitamin C for 5 days. However, no influence was evident after pre- and posttreatment with 18,000 U/kg L-SOD i.p. or daily i.g. 100 mg/kg of vitamin E for 5 days. The results suggest that intracellular GSH plays an essential protective role against renal oxidative DNA damage and nephrotoxicity caused by KBrO₃.     

Blockade of the human cardiac K+ channel Kv1.5 by the antibiotic erythromycin

Erythromycin administration has been associated with a prolongation of cardiac repolarization in certain clinical settings. This could be due to blockade of voltage-dependent K⁺ channels in the human heart. For this reason we examined the effects of erythromycin on a rapidly activating delayed rectifier K⁺ channel (Kv1.5) cloned from human heart and stably expressed in human embryonic kidney cells. When examined using the whole-cell patch clamp technique, erythromycin (100 μM) blocked Kv1.5 current in a time-dependent manner but required prolonged exposure to do so. However, when we examined Kv1.5 current using inside-out macropatches, erythromycin applied to the cytoplasmic surface rapidly (within 1-2 min) inhibited Kv1.5 current with an IC₅₀ value of 2.6 x 10^-5M (1.7 - 3.9 x 10^-5M, 95% C.L.). The main effect of erythromycin was to accelerate the rate of Kv1.5 current decay thereby reducing the current at the end of a prolonged voltage-clamp pulse. Erythromycin also blocked Kv1.5 current in both a voltage- and frequency-dependent manner but had little effect on the activation kinetics, deactivation kinetics, or the steady-state inactivation properties of Kv1.5. These data suggest that erythromycin acts as a blocker of an activated state of the Kv1.5 channel and that it may access its binding site from the intracellular face of the channel. This study is the first to examine the effects of erythromycin on a cloned human cardiac K⁺ channel. It is concluded that erythromycin blocks Kv1.5 at clinically relevant concentrations. Blockade of voltage-dependent K⁺ channels in the heart could contribute to the alterations in cardiac repolarization that have been observed with erythromycin. Received: 22 November 1996 / Accepted: 26 February 1997