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     

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     

白醋加碘酸钾不同含量稳定性的四年观察研究

目的 摸清碘酸钾加入白醋中不同含量的稳定性。方法 配成含碘50、100、200mg／L的碘醋，各按500ml装瓶，密封室温阴凉处放置备用。第一年每隔两个月，第二、三、四年每隔半年测定一次其碘含量。结果 50、100、200mg／L的碘醋一年碘保存率分别为：96.06％、97．76％、95.20％；二年碘保存率分别为：86．06％、92．42％、92.85％；三年碘保存率分别为：76.98％、87.52％、90.95％；四年碘保存率分别为：71．78％、83．26％、89．35％，感观上均没有变化。结论 碘醋第一年的稳定性顺序为：100、50、200mg/L，第二、三、四年的稳定性顺序为：200、100、50mg／L。证明白醋加碘酸钾是稳定可行的。     

槲皮素磷酸酯钾对实验性心肌梗塞的影响

iv10～20mg/kg(木解)皮素磷酸酯钾使兔急性心肌梗塞范围明显缩小,ST段明显降低,并能对抗冠脉结扎后引起的血清CPK含量增加及心律失常,这些作用与普萘洛尔相似。但iv40mg/kg后作用不明显。     

Inward rectifying potassium channels in human malignant glioma cells

Human glioma cells obtained from established cell lines (Tp-276MG, Tp-301MG, Tp-378MG, Tp-483MG and U-251MG) were analyzed for the presence of ion channels with the tight-seal voltage clamp technique. The current-voltage relation revealed a marked inward rectification at hyperpolarizing voltages, due to the presence of inward rectifying K-channels in cells from all studied cell lines. These channels were conducting when the membrane potential was more negative than the K-equilibrium potential. The slope conductance for the inward K-currents (g_Ki) was affected both by [K⁺]_i and [K⁺]₀. g_Ki was proportional to [K⁺]₀ raised to 0.35 or 0.50, of which the larger value was measured in the presence of low [K⁺]_i (25mM). The rectification was not significantly different in cells perfused with Mg-free EDTA-buffered internal solution. Tl⁺ was 3.5 times more permaant than K⁺. g_ki was blocked by Cs⁺ (1 mM) in a voltage-dependent way (more effective in the hyperpolarized membrane), and by Na⁺ (154 mM) depending on voltage and time. From measurements of unitary current events in membrane patches (outside out or cell attached) the conductance of the single inward rectifying channel was estimated to be 27 ± 7 pS. This type of ion channel may be important for K-uptake by glial cells and hence for the K-homeostasis in the brain.     

氯胺酮对离体大鼠心室肌细胞内向整流钾电流的影响

目的观察氯胺酮对离体大鼠心室肌细胞内向整流钾电流(I_(k1))的影响。方法酶解法分离大鼠心室肌细胞,采用全细胞膜片钳技术记录 I_(k1),观察100μmol/L 氯胺酮在不同钳制电压下以及不同浓度(50～5000μmol/L)氯胺酮在-120mV 钳制电压下对大鼠心室肌细胞 I_(k1)的影响。结果 100 μmol/L 氯胺酮抑制 I_(k1),但不改变 I_(k1)翻转电压以及电流-电压曲线的形状；I_(k1)灌流液冲洗后,I_(k1)能够完全恢复。保持电压-40mV、钳制电压-120mV 下5～5000μmol/L 氯胺酮呈浓度依赖性抑制 I_(k1)其 IC_(50)为(162.3±8.4)μmol/L。结论氯胺酮呈浓度依赖性抑制大鼠心室肌细胞 I_(k1),可能延长动作电位时程导致心率变慢。