蜘蛛毒素研究进展

其中剧毒蜘蛛有10多种［1-2］。蜘蛛毒素含有多种化学成分，根据其相对分子质量大小分为小分子物质、蛋白质和多肽类毒素三类。其中最为丰富也是目前研究最多的是相对分子质量在（1～10）×103的多肽类毒素，包括多肽神经毒素、激肽类似物、凝集活性肽和抗菌肽等。在这些多肽类毒素中，神经毒素又是多肽毒素中含量最多的成分，其主要生物学功能是作用于靶细胞膜上的各种离子通道，如钾、钙、钠、酸敏感通道等［3-4］。此外蜘蛛毒液中还含有无机盐、三磷酸腺苷、氨基酸、核苷酸、单胺类以及多胺类等，统称为蜘蛛毒素中的小分子物质［5-6］。由于蜘蛛毒素成分具有多样性和多功能性，蜘蛛毒素不仅在分子毒理学、分子药理学和神经生物学等领域有着广泛应用，此外在临床新药研究和农药生产开发等方面也有着广泛的前景［7］。本文主要对近年蜘蛛毒素几类成分的研究进展进行一个综述，为更好地对蜘蛛毒素进行研究奠定基础。     

生物毒素的研究与应用:选择性蜘蛛毒揭示Nav1.1通道在机械痛中的作用

电压门控性钠(Nav)通道参与启动大多数神经元动作电位,其中也包括痛觉初级传入神经纤维。局部麻醉剂可通过非特异性阻断所有Nav通道亚型来抑制疼痛,但探求选择性亚型调节因子将更有助于分析Nav通道亚型对化学、机械或热痛的作用及机制。美国加州大学旧金山分校的Jeremiah D.Osteen,Volker Herzig和David Julius等人通过运用电生理、组织化学及行为学,筛选和鉴定出一种蜘蛛毒素可选择性激活Nav1.1亚基,并阐明其在伤害感受和疼痛中的作用。在非神经炎症的情况下,Nav1.1的激活诱导了疼痛反应,产生了机械性痛觉过敏,而对热痛并无影响。在肠道中,高阈值机械敏感性神经纤维表达Nav1.1,且肠易激综合征模型小鼠也表现为毒素敏感性增强,证实了Nav1.1通道在调节机械痛的感觉神经纤维兴奋性中的作用。     

心肌细胞钠、氯离子通道与室性心律失常的关系研究进展

正心肌细胞内外带电离子通过离子通道进出心肌细胞产生电位差,使心肌有自主电活动,并兴奋心肌细胞、传导生物电活动,引起心肌收缩来完成心脏的泵血功能[1]。心肌细胞上存在着多种通道,主要包括钠、钾、钙和氯离子通道。它们参与了心肌的正常代谢,同时也是多种心脏疾病发生发展的基础[2-4]。因此,离子通道及与其相关的基因、分子结构、功能等为众多科     

钠通道在肺泡液体清除中的作用

肺水增多是急性肺损伤的主要特征.肺泡上皮细胞具有主动转运肺泡液体的作用.最近研究认为,该机制主要和肺泡Ⅱ型上皮细胞顶膜的钠通道主动转运钠离子并随之带动肺泡液体转运有关.探讨钠通道功能调节影响因素及信号转导机制在因急性肺损伤导致的肺水的清除中具有重要的临床意义.     

不同浓度一氧化氮供体对培养海马神经元存活率影响的相关研究

目的:观察不同浓度一氧化氮供体硝普钠对培养海马神经元存活率的影响以及不同类型钾通道阻断剂对硝普钠诱导的神经元死亡的保护作用。方法:实验于2004-11/2005-04在南方医科大学基础医学院神经生物学教研室实验室进行。①取新生两天以内SD大鼠海马组织做神经元分散细胞原代培养,培养第8天时,分别以不同浓度硝普钠(0.01mmol/L,0.1mmol/L,0.5mmol/L,1mmol/L)处理18h,采用MTT法观察硝普钠对神经元存活率的影响。②硝普钠(0.1mmol/L)处理前10min培养液内预先分别给予钾通道阻断剂四乙铵1mmol/L、4-氨基砒碇1mmol/L、Iberiotoxin(IBTX)100nmol/L或paxilline100μmol/L,18h后测定神经元存活率。结果:①低浓度硝普钠(0.01mmol/L)对培养海马神经元存活无影响(P>0.05),增大硝普钠浓度至0.1mmol/L,0.5mmol/L或1mmol/L可引起神经元存活率浓度依赖性地降低(P<0.001)。②硝普钠(0.1mmol/L)处理前10min给予不同类型钾通道阻断剂,广谱钾通道阻断剂1mmol/L四乙铵或A型钾通道阻断剂1mmol/L4-氨基砒碇均不能降低硝普钠诱导的神经元死亡(P>0.05);两种不同的特异性大电导钙激活钾通道阻断剂100nmol/LIBTX或10μmol/Lpaxilline均可完全保护硝普钠诱导的神经元死亡(P<0.001)。结论:一氧化氮供体硝普钠剂量依赖性地诱导培养新生大鼠海马神经元死亡,大电导钙激活钾通道阻断剂对一氧化氮诱导的神经元死亡具有完全保护作用,提示阻断大电导钙激活钾通道可能作为临床治疗缺血性脑损伤的新靶点。     

蛇床子素对大鼠心室肌细胞钠通道的影响

目的探讨蛇床子素对大鼠心室肌细胞钠离子通道电流(INa)的影响。方法采用Langendorff装置,恒压恒温灌流及酶解消化等方法对大鼠心肌细胞进行分离和处理。应用膜片钳技术,观察给予不同浓度蛇床子素(Ost)后钠离子通道电流特征的变化。结果 Ost(100μmol/L)能明显抑制钠电流,其作用呈浓度依赖性(500μmol/L几乎完全阻断)和时间依赖性(10 min左右抑制力达到最强)。100μmol/L和300μmol/L Ost使钠电流I-V曲线明显上移,峰值钠电流(-29.8±4.21)p A/p F分别降为(-20.1±3.7)p A/p F和(-17.7±5.7)p A/p F(P0.05),但激活电位和峰电位没有改变。对于钠通道失活曲线,不同浓度的Ost能使其向超级化方向移动,V1/2(空白,100μmol/L,300μmol/L)分别为(-81.10±0.35)、(-91.62±1.06)、(-100.60±0.21)m V(P0.01)。Ost还能显著延长钠通道失活后恢复时间,τ(空白,100μmol/L,300μmol/L)分别为(15.09±0.78)、(23.41±1.23)、(31.62±0.97)ms(P0.01)。结论 Ost对大鼠心室肌钠通道电流有明显的抑制作用。     

背根神经节钠通道与疼痛

慢性疼痛是周围神经、组织损伤或炎性刺激所诱导的周围神经性病变的一种主要症状。最近一些研究表明背根神经节(dorsalrootganglion,DRG)钠通道表达及位置的改变与某些病理性疼痛有关,钠通道基因表达的可塑性及电生理的改变导致DRG细胞呈高兴奋性、产生自发动作电位及异常高频电活性,DRG钠通道在疼痛的病理生理中起做重要的作用。通过选择性地影响伤害性神经元产生动作电位及阻滞特异性钠通道可缓解神经性及炎性疼痛,有希望成为疼痛治疗的又一新领域。     

蛇毒研究进展——蛇毒中的心脏毒素

蛇毒的主要致死毒性成份大致可以分为神经毒素及心脏毒素两类。目前已从多种蛇毒中分离出50多种神经毒,并搞清了它们的一级结构,合成了两种神经毒,即Cobrotoxin和α-Bungarotoxin。心脏毒素是广泛存在于眼镜蛇科多种蛇毒中的一种强碱性多肽,对机体有广泛的毒理作用,根据其某一作用     

背根神经节钠通道与疼痛

慢性疼痛是周围神经、组织损伤或炎性刺激所诱导的周围神经性病变的一种主要症状。最近一些研究表明背根神经节（dorsal root ganglion,DRG）钠通道表达及位置的改变与某些病理性疼痛有关，钠通道基因表达的可塑性及电生理的改变导致DRG细胞呈高兴奋性、产生自发动作电位及异常高频电活性，DRG钠通道在疼痛的病理生理中起做重要的作用。通过选择性地影响伤害性神经元产生动作电位及阻滞特异性钠通道可缓解神经性及炎性疼痛，有希望成为疼痛治疗的又一新领域。     

The sea anemone toxins BgII and BgIII prolong the inactivation time course of the tetrodotoxin-sensitive sodium current in rat dorsal root ganglion neurons

We have characterized the effects of BgII and BgIII, two sea anemone peptides with almost identical sequences (they only differ by a single amino acid), on neuronal sodium currents using the whole-cell patch-clamp technique. Neurons of dorsal root ganglia of Wistar rats (P5-9) in primary culture (Leibovitz's L15 medium; 37 degrees C, 95% air/5% CO2) were used for this study (n = 154). These cells express two sodium current subtypes: tetrodotoxin-sensitive (TTX-S; K(i) = 0.3 nM) and tetrodotoxin-resistant (TTX-R; K(i) = 100 microM). Neither BgII nor BgIII had significant effects on TTX-R sodium current. Both BgII and BgIII produced a concentration-dependent slowing of the TTX-S sodium current inactivation (IC50 = 4.1 +/- 1.2 and 11.9 +/- 1.4 microM, respectively), with no significant effects on activation time course or current peak amplitude. For comparison, the concentration-dependent action of Anemonia sulcata toxin II (ATX-II), a well characterized anemone toxin, on the TTX-S current was also studied. ATX-II also produced a slowing of the TTX-S sodium current inactivation, with an IC50 value of 9.6 +/- 1.2 microM indicating that BgII was 2.3 times more potent than ATX-II and 2.9 times more potent than BgIII in decreasing the inactivation time constant (tau(h)) of the sodium current in dorsal root ganglion neurons. The action of BgIII was voltage-dependent, with significant effects at voltages below -10 mV. Our results suggest that BgII and BgIII affect voltage-gated sodium channels in a similar fashion to other sea anemone toxins and alpha-scorpion toxins.     

Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug.

Class I antiarrhythmic drugs inhibit the sodium channel by binding to a drug receptor associated with the channel. In this report we show that in vivo administration of the class I antiarrhythmic drug mexiletine to rats induces sodium channel upregulation in isolated cardiac myocytes. The number of sodium channels was assessed with a radioligand assay using the sodium channel-specific toxin [3H]batrachotoxinin benzoate ([3H]BTXB). The administration of mexiletine to rats induced a dose-dependent increase in [3H]BTXB total specific binding (Bmax) on isolated cardiac myocytes. Sodium channel numbers were 15 +/- 5, 29 +/- 9, and 54 +/- 4 fmol/10(5) cells after 3 d treatment with 0, 50 mg/kg per d, and 150 mg/kg per d mexiletine (P less than 0.001, analysis of variance). Sodium channel number increased monoexponentially to a steady-state value within 3 d with a half-time of increase of 1.0 d. After cessation of treatment with mexiletine the number of sodium channels returned to normal within 12 d. Finally, treatment with mexiletine altered only sodium channel number; the Kd for [3H]BTXB and the IC50 for mexiletine were not different for myocytes prepared from control and mexiletine-treated rats.     

1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a potent and highly selective small molecule blocker of the large-conductance voltage-gated and calcium-dependent K+ channel

The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.     

Molecular cloning and functional characterization of a unique mammalian cardiac Na(v) channel isoform with low sensitivity to the synthetic inactivation inhibitor (-)-(S)-6-amino-alpha-[(4-diphenylmethyl-1-piperazinyl)-methyl]-9H-purine-9-ethanol (SDZ 211-939)

Cardiac voltage-dependent sodium channels (Na(v)) are drug targets for synthetic inactivation inhibitors typified by (+/-)-4- [3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxy propoxy]-1H-indole-2-carbonitrile (DPI 201-106), of which the molecular mode of action is not yet defined. The previous observation by Mevissen and coworkers in 2001 of the electrophysiological ineffectiveness of DPI 201-106 in the bovine heart, in contrast to other species, offers the opportunity for investigating these open questions. We now report about the molecular cloning, expression in Xenopus laevis oocytes, and electrophysiological characterization of a unique bovine heart sodium channel. Although the predicted 2022-amino acid bovine heart sodium channel (bH1) shares 92% identity with the rat and human isoforms and normal gating properties, it displays drastically reduced sensitivity to (-)-(S)-6-amino-alpha-[(4-diphenylmethyl-1-piperazinyl)-methyl]-9H-purine-9-ethanol (SDZ 211-939). Experimental results with Anemonia sulcata toxin II (0.1-2.5 microM) exclude the possibility of an overall insensitivity of this isoform to various sodium channel modulators. The binding of SDZ 211-939 seems to be largely unaffected (EC(50) of 10.3 and 10.6 microM for bovine and rat isoforms, respectively) but the corresponding efficacy in bovine (V(m) of 0.15) is approximately 5 times smaller compared with the rat heart isoform (V(m) of 0.69). The comparison of the primary structure of bH1 to other sodium channels and the gating properties obtained in presence or absence of SDZ 211-939 revealed a high degree of similarity. Whether the mechanism of channel modulation depends on the interaction of synthetic modulators with some possibly voltage-independent part of the inactivation machinery needs to be determined.     

The Effect of Oral Deferoxamine on Iron Absorption in Humans

Introduction.Tricyclic antidepressant (TCA) poisoning is a relatively common occurrence and remains a significant cause of mortality and morbidity. Deaths from TCA toxicity are typically due to cardiovascular events such as arrhythmias and hypotension. Cardiovascular toxicity may be multifactorial. However, the primary mechanism is a TCA-induced membrane-depressant or “quinidine-like” effect on the myocardium resulting in slowing down of phase 0 depolarization of the cardiac action potential and subsequent impairment of conduction through the His-Purkinje system and myocardium. This effect is manifest as QRS prolongation on the EKG, atrioventricular (AV) block, and impairment in automaticity leading to hypotension and ventricular dysrhythmia. Primary treatment strategies include sodium bicarbonate, hypertonic saline, and correction of any conditions that may aggravate this toxicity such as acidosis, hyperthermia, and hypotension. In cases of severe TCA toxicity, administration of sodium bicarbonate may be insufficient to correct the cardiac conduction defects. Use of lidocaine or phenytoin, both Vaughan Williams Class IB antiarrhythmic agents, has been reported as an effective adjunctive therapy in cases of severe cardiotoxicity.?Methods.?Thirty articles of interest were identified by searching PubMed, abstracts from meetings, and the reference sections of related primary and review articles and toxicological texts. Role of lidocaine and phenytoin. Lidocaine and phenytoin also cause sodium channel blockade, but unlike Class IA or IC agents do not depress phase 0 depolarization in healthy cardiac tissue. Lidocaine and phenytoin dissociate relatively quickly from cardiac sodium channels. Sodium channels have faster recovery times after exposure to lidocaine (1–2 s) and phenytoin (0.71 s), than with some TCAs such as amitriptyline (13.6 s), but not others (e.g., imipramine at 1.6 s). In experimental models of amitriptyline poisoning, lidocaine co-administration resulted in decreased sodium channel blockade compared to amitriptyline alone. This correlated with clinical improvement, including normalization of QRS interval, improved hypotension, and decreased mortality. It is postulated that lidocaine's rapid binding to the sodium channel may directly displace slower acting agents from the channel, leaving more channels unbound, and therefore be able to facilitate cardiac conduction. Phenytoin may act through a similar mechanism as lidocaine, although experimental studies suggest that it does not compete directly for the same sodium channel binding site as TCAs. Allosteric modulation of the TCA binding site may occur in the setting of phenytoin use. The evidence for using phenytoin in treating TCA-induced sodium channel blockade is less convincing than that for lidocaine. Human trials are limited to case series and, in most human exposures in which there appeared to be efficacy, the toxicity was not severe.?Conclusions.?Although there appears to be more evidence for the use of lidocaine than phenytoin as adjunctive treatment for TCA-associated cardiotoxicity, specific clinical indications and dosing recommendations remain to be defined. We recommend the use of lidocaine in cases in which cardiotoxicity (arrhythmias, hypotension) is refractory to treatment with sodium bicarbonate or hypertonic saline, or in which physiological derangement (e.g., severe alkalosis or hypernatremia) limits effective use of these primary strategies.     

Models of drug interaction with the sodium channel.

The local anesthetic-class of anti-arrhythmic drugs block the inward sodium current in nerve and cardiac muscle. A number of models for the interaction of these drugs with the neuronal sodium channel have been extended to cardiac muscle. The models assume a single binding site for the entire class of agents. The kinetics of drug interaction with this site depend on the Na channel conformation, open and inactivated channels having greater affinity than resting channels. An alternative formulation considers drug-receptor affinity as fixed, but access to the binding site is controlled by channel gating. Several clinically relevant predictions, such as competitive displacement of multiple agents, can be made from these models.     

Potassium channel toxins

Many venom toxins interfere with ion channel function. Toxins, as specific, high affinity ligands, have played an important part in purifying and characterizing many ion channel proteins. Our knowledge of potassium ion channel structure is meager because until recently, no specific potassium channel toxins were known, or identified as such. This review summarizes the sudden explosion of research on potassium channel toxins that has occurred in recent years. Toxins are discussed in terms of their structure, physiological and pharmacological properties, and the characterization of toxin binding sites on different subtypes of potassium ion channels.     

Involvement of voltage-gated sodium channels blockade in the analgesic effects of orphenadrine

Orphenadrine is a drug acting on multiple targets, including muscarinic, histaminic, and NMDA receptors. It is used in the treatment of Parkinson’s disease and in musculoskeletal disorders. It is also used as an analgesic, although its mechanism of action is still unknown. Both physiological and pharmacological results have demonstrated a critical role for voltage-gated sodium channels in many types of chronic pain syndromes. We tested the hypothesis that orphenadrine may block voltage-gated sodium channels. By using patch-clamp experiments, we evaluated the effects of the drug on whole-cell sodium currents in HEK293 cells expressing the skeletal muscle (Nav1.4), cardiac (Nav1.5) and neuronal (Nav1.1 and Nav1.7) subtypes of human sodium channels, as well as on whole-cell tetrodotoxin (TTX)-resistant sodium currents likely conducted by Nav1.8 and Nav1.9 channel subtypes in primary culture of rat DRG sensory neurons. The results indicate that orphenadrine inhibits sodium channels in a concentration-, voltage- and frequency-dependent manner. By using site-directed mutagenesis, we further show that orphenadrine binds to the same receptor as the local anesthetics. Orphenadrine affinities for resting and inactivated sodium channels were higher compared to those of known sodium channels blockers, such as mexiletine and flecainide. Low, clinically relevant orphenadrine concentration produces a significant block of Nav1.7, Nav1.8, and Nav1.9 channels, which are critical for experiencing pain sensations, indicating a role for sodium channel blockade in the clinical efficacy of orphenadrine as analgesic compound. On the other hand, block of Nav1.1 and Nav1.5 may contribute to the proconvulsive and proarrhythmic adverse reactions, especially observed during overdose.     

The genetic basis for cardiac dysrhythmias and the long QT syndrome.

Cardiac muscle excitation is the result of ion fluxes through cellular membrane channels. Any alterations in channel proteins that produce abnormal ionic fluxes will change the cardiac action potential and the pattern of electrical firing within the heart. The idiopathic long QT syndrome (LQTS) is an inherited cardiac pathology localized to mutated genes encoding for myocardial, voltage-activated sodium and potassium ion channels. The expression of abnormal sodium and potassium channels results in aberrant ionic fluxes that produce a prolonged ventricular repolarization. This prolonged time to repolarization is the electrophysiologic basis for prolongation of the QT interval. Individuals with LQTS are at significant risk for developing lethal ventricular dysrhythmias due to an abnormal pattern of cardiac excitation. Identification of a genetic basis for LQTS has had significant implications for genetic counseling, the development of effective antidysrhythmic drug therapies, and nursing interventions.     

Depolarizing action of a red-tide dinoflagellate brevetoxin on axonal membranes

The neurotoxic actions of T17 , a toxin isolated from the red-tide dinoflagellate Ptychodiscus brevis, on membrane excitability were investigated by the intracellular microelectrode technique on the crayfish giant axons and by the voltage clamp experiments on the squid giant axons. External application of T17 toxin caused a concentration-dependent depolarization, transient repetitive discharges, followed by depression of the action potential leading to a complete block of excitability. The reversibility of the depolarizing action upon washing decreased as the time of toxin treatment was increased. The T17 -induced depolarization was effectively reversed by 0.3 microM tetrodotoxin or 1 mM Na external solution. Pretreatment with tetrodotoxin completely antagonized the T17 depolarizing action. However, upon washing the axon with the normal external solution, depolarization occurred. Pretreatment with either procaine or dibucaine at high doses also offered protection against the depolarization. The toxin action was greatly potentiated by the sea anemone toxin, anthopleurin-A. The voltage clamp experiments showed that T17 toxin affected sodium current only. The activation voltage for sodium current was shifted in the hyperpolarizing direction by more than 35 mV. T17 toxin also greatly depressed the fast inactivation of sodium current. However, there was no significant change in the kinetics of the sodium tail current. These results indicate that T17 toxin depolarizes the membrane by selectively opening sodium channels at fairly negative potentials and by inhibiting the fast sodium inactivation. We also infer that the binding site for T17 toxin is different from those for tetrodotoxin and sea anemone toxin.