血吸虫电压门控性钙通道(VGCCs)亚单位与吡喹酮药物靶点的关系

吡喹酮作为当前治疗血吸虫病的首选药物,临床应用已达30余年,但其作用机制至今尚未完全阐明.电压门控性钙通道(voltage-gated calcium channels,VGCCs)为Ca2+的内流提供通道,同时也是多种药物与毒素的作用靶点.该文围绕"血吸虫钙通道亚单位-吡喹酮药物靶点"假说,讨论血吸虫VGCCs亚单位的结构与功能,尤其是变异的β亚单位(Cavβvar),及β亚单位作为吡喹酮抗血吸虫药物靶点的研究进展.     

吡喹酮抗血吸虫作用的研究进展

吡喹酮是目前惟一对人体5种血吸虫病均有效的治疗药物，其突出优点是口服方便、安全有效和疗程短。了解其抗血吸虫的作用机制，可能有益于发展新的广谱抗蠕虫药物。本文综述了近30年来国、内外实验室对吡喹酮抗血吸虫作用的研究进展。     

血吸虫对吡喹酮的抗药性研究进展

吡喹酮是高效、低毒的口服抗血吸虫首选药物,自1970年代研制出至今,已被大规模反复用于现场30余年。血吸虫是否会在药物选择压力下对吡喹酮产生抗药性已引起了高度关注。该文对血吸虫抗药性的定义、吡喹酮抗药性的实验和现场研究近况、抗药性产生机制、血吸虫对吡喹酮敏感性的检测、抗药性产生的相关因素及控制措施等作一综述。     

吡喹酮对日本血吸虫毛蚴攻击力的影响

吡喹酮对血吸虫虫卵无直接杀伤作用，但可促进血吸虫虫卵毛蚴的孵出，对毛蚴有明显的杀伤作用［１］。经吡喹酮作用后的毛蚴对钉螺的攻击力如何，国内外尚未见报道。本文观察了吡喹酮在体内条件下对日本血吸虫毛蚴攻击力的影响。材料与方法昆明种小鼠１８只，感染日本血吸...     

血吸虫对吡喹酮抗药性评价方法的研究进展

吡喹酮自问世以来，以其高效、低毒、使用方便、价格低廉等优点在血吸虫病临床治疗和防治工作中得到广泛应用，并成为目前治疗各种血吸虫病的首选药物。然而，近年来在非洲北塞内加尔采用标准剂量吡喹酮治疗曼氏血吸虫病的治愈率仅为18％～36％，增加治疗剂量并不能明显提高治愈率。继而又在埃及发现部分患者经反复治疗难以治愈。鉴于人群化疗仍是当前血吸虫病防治的一个重要措施，是否出现了吡喹酮抗性虫株的问题引起了科学界的重视。     

左旋吡喹酮与右旋吡喹酮治疗对体内日本血吸虫皮层…

感染日本血吸虫３５ｄ的小鼠用左旋吡喹酮（Ｌ－Ｐｒａ）１５０ｍｇ．ｋｇ＾－１，或右旋吡喹酮（Ｄ－Ｐｒａ）１５０．６００ｍｇ．ｋｇ＾－１次灌胃治疗，在给药后的１５ｍｉｎ，１、２、４、８、２４ｈ分别解剖取虫，以扫描电镜观察血吸虫的皮层变化。结果，发现，Ｌ－Ｐｒａ１５０ｍｇ．ｋｇ＾－１对血吸虫可引起明显和广泛的损害，包括皮层的严重肿胀、融合、糜烂和剥落，并伴有宿主白细胞的粘附；盘状感觉器官亦示有明显的肿胀     

吡喹酮抗血吸虫作用机理的奥秘

本文简要综述吡喹酮抗血吸虫作用的机理,指出吡喹酮对血吸虫发育不同阶段有不同的作用,且呈现间隔变化的模式,并对这一奇特现象提出开展研究的建议。     

吡喹酮影响日本血吸虫病兔血清Ca2+变化的实验研究

目的：探讨吡喹酮用药前后血清Ca^2 浓度的变化并观察疗效。方法：使用吡喹酮100mg/kg和50mg/kg剂量于用药前后不同时间取血清检测Ca^2 浓度，用灌流法检获虫体，计算减虫率及减雌率。结果：不同剂量用药后2，4，8，24h的血清Ca^2 浓度均明显低于正常水平，30d后血清Ca^2 浓度基本恢复正常；100mg/kg剂量减虫率为89．7％，50mg/kg为72．2％，减雌率均为100％，结论：日本血吸虫病兔经吡喹酮治疗期间的血清Ca^2 浓度低于正常。     

左旋吡喹酮、右旋吡喹酮和消旋吡喹酮的体外与体内抗不同发育期日本血吸虫的作用

目的：测定消旋吡喹酮（Ｐｒａ）、左旋吡喹酮（Ｌ－Ｐｒａ）和右旋吡喹酮（Ｄ－Ｐｒａ）对不同发育期日本血吸虫的作用。方法：用含２０％小牛血清的ＰＲＭＩ１６４０培养不同发育期的血吸虫，测定上述三种药物的体外抗血吸虫作用。体内试验系于小鼠感染血吸虫尾蚴后不同时间灌服（ｉｇ）Ｐｒａ、Ｌ－Ｐｒａ或Ｄ－Ｐｒａ，根据残留平均虫数评估药效。结果：依据药物引起虫的皮层损害程度，虫龄为２８ｄ（ｄ２８）和３５ｄ（ｄ３５）的成虫对Ｌ－Ｐｒａ最敏感，而１４ｄ（ｄ１４）童虫则最不敏感。在药物浓度为０．１－１μｇ／ｍｌ时，Ｌ－Ｐｒａ的抗血吸虫作用较Ｐｒａ的为强，即使Ｌ－Ｐｒａ的浓度减至Ｐｒａ最低有效药浓度的１／２亦有效。在体外，上述浓度的Ｄ－Ｐｒａ对不同发育期血吸虫无明显作用。感染小鼠ｉｇ单剂量的Ｌ－Ｐｒａ，Ｐｒａ或Ｄ－Ｐｒａ３００ｍｇ／ｋｇ或５００ｍｇ／ｋｇ，仅前２种药物对３ｈ（ｄｏ）、２１ｄ（ｄ２１）童虫和ｄ２８及ｄ３５成虫有较明显的疗效，而对３ｄ（ｄ３）、７ｄ（ｄ７）和ｄ１４童虫的疗效甚差或无效。与Ｌ－Ｐｒａ和Ｐｒａ相比，Ｄ－Ｐｒａ仅对ｄ３５成虫有较差的疗效。感染ｄ３５血吸虫成虫的小鼠用Ｌ－Ｐｒａ１５０ｍｇ／ｋｇｉｇ治疗，其疗效与用Pra 300mg/kg 治疗的相仿。D-Pra 的总剂量增至L -Pra 的2—6 倍时亦仅示很差的疗效。结论: 在消旋Pra 中,L-Pra 是抗血吸虫的活性成分。     

抗环子孢子蛋白保守II+区单克隆抗体的制备及鉴定

目的　获得针对恶性疟原虫环子孢子蛋白 (CSP)保守II⁺ 区 (RegionII⁺ )的单克隆抗体。　方法　采用恶性疟原虫保守II⁺ 区十二肽 (EWSPCSVTCGNG)免疫BALB/c小鼠 ,经融合 ,ELISA 3次筛选 ,获得 3株分泌针对保守II⁺ 区单克隆抗体的杂交瘤细胞株。　结果　ELISA检测结果显示单抗能与重组表达的恶性疟原虫CSP片段及天然CSP特异性反应。间接荧光抗体检测显示 ,单抗不仅能识别恶性疟原虫子孢子 ,也能识别约氏疟原虫子孢子。　结论　成功地获得了针对恶性疟原虫CSP保守II⁺ 区的单克隆抗体。     

近年来发展抗血吸虫新药的进展

全球有2亿人感染血吸虫,其治疗仅依赖吡喹酮一种药物是很不相适应的。吡喹酮虽有很好的治疗效果,但无预防作用,故发展抗血吸虫新药倍受关注。本文综述近年来报道的恶二唑-2-氧化物和甲氟喹等抗血吸虫新药的实验研究,阐述这些药物的发展过程,及其抗血吸虫特点。     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Effects of the Ca2+ sensitizers EMD 57033 and CGP 48506 on myocardial contractility and Ca2+ transients in human ventricular and atrial myocardium

Zusammenfassung Ca²⁺-Sensitizer, wie z.B. EMD 57033 (EMD) und CGP 48506 (CGP) erhöhen die Kontraktionskraft ohne dabei den intrazellulären Ca²⁺-Transienten zu erhöhen und sind somit möglicherweise für die Behandlung der menschlichen Herzinsuffizienz von Bedeutung. Es ist jedoch unklar, ob sich Ca²⁺-Sensitizer in ihrem pharmakokinetischen Wirkprinzip am menschlichen Myokard unterscheiden. Daher wurde der Einfluss von EMD und CGP auf die Kontraktionskraft und den intrazellulären Ca²⁺-Transienten (Fura 2) an linksventrikulären Papillarmuskelstreifen (PAP) von menschlichem insuffizientem Myokard sowie in rechtsatrialen Trabekeln (RA) von Patienten untersucht, die sich einer aortokoronaren Bypass-Operation unterziehen mussten. In PAP wurde die Kraft effizienter und stärker nach Applikation von EMD (EC₅₀ EMD: 4,7ǃ,0 7mol/l, max. PIE EMD: +12,0DŽ,0 mN/mm²) erhöht als nach CGP (EC₅₀: 16,9lj,6 7mol/l, max. PIE: +6,4DŽ,8 mN/mm²). Ähnliche Ergebnisse wurden an RA erhalten. Carbachol (100 7mol/l) hatte keinen Einfluss auf die positiv inotrope Wirkung von EMD und CGP. Beide Ca²⁺-Sensitizer erhöhten signifikant die Relaxationszeit und die diastolischen Spannung. EMD und CGP veränderten den intrazellulären Ca²⁺-Transienten nicht. Schlussfolgerung Die Ca²⁺-Sensitizer EMD und CGP erhöhen cAMP- und Ca²⁺-unabhängig die Kontraktionskraft am menschlichen Myokard. Da sie die Relaxation beeinträchtigen, ist ihr therapeutischer Nutzen für die Herzinsuffizienz begrenzt. Summary Ca²⁺ sensitizers like EMD 57033 (EMD) and CGP 48506 (CGP) may be advantageous for the treatment of human heart failure, as they increase force of contraction without increasing the intracellular Ca²⁺ transients or energy consumption. However, whether or not Ca²⁺ sensitizers differ in their mode of action in human myocardium is not fully understood. The present study investigates the influence of EMD and CGP on force of contraction (FOC) and the intracellular Ca²⁺ transient (fura-2 ratio method) in left ventricular papillary muscle strips from left ventricular failing human myocardium (DCM, n=28) as well as in right atrial trabeculae (RA, n=21) obtained from patients undergoing cardiac bypass surgery. In isolated trabeculae of DCM, FOC was more efficacious and potently increased after application of EMD (EC₅₀ EMD: 4.7ǃ.0 7mol/l, max. PIE EMD: +12.0DŽ.0 mN/mm²) than CGP (EC₅₀: 16.9lj.6 7mol/l, max. PIE: +6.4DŽ.8 mN/mm²). Similar results were obtained in RA. Application of carbachol (100 7mol/l) had no effect on the positive inotropic effect of EMD or CGP. Both Ca²⁺ sensitizers significantly increased time to half peak relaxation as well as diastolic tension in DCM. EMD (10 7mol/l) and CGP (30 7mol/l) did not affect the Ca²⁺ transients in RA. The Ca²⁺ sensitizers EMD and CGP increase cAMP and Ca²⁺ independently from the force of contraction in the human myocardium. However, their therapeutic use in human heart failure may be limited as they impair relaxation.     

NIP-141, a multiple ion channel blocker, terminates aconitine-induced atrial fibrillation and prevents the rapid pacing-induced atrial effective refractory period shortening in dogs.

AIMS: NIP-141 is a novel multiple ion channel blocker with atrial selective effects. In this study, we examined the effects of NIP-141 on aconitine-induced atrial fibrillation (AF) and rapid atrial pacing-induced atrial effective refractory period (ERP) shortening in dogs. METHODS AND RESULTS: Aconitine AF was induced by the application of aconitine on the right appendage. NIP-141 (10 mg/kg) converted AF to sinus rhythm in 5 of 6 dogs. The Na(+) channel blockers disopyramide (1 mg/kg) and phenytoin (10 mg/kg) also terminated AF, but the I(Kr) blocker (d-sotalol; 4 mg/kg) and a Ca(2+) channel blocker (verapamil; 0.3 mg/kg) did not terminate AF in this model. To clarify the mechanism of AF termination, we examined the effects on ERP and conduction time, but NIP-141 (10 mg/kg) had no significant effects. In a short-term rapid atrial pacing model, NIP-141 (2.5 mg/kg/10 min, followed by 0.033 mg/kg/min) prevented atrial ERP shortening. We also found NIP-141 bound to Na(+) channel site 2 receptor and L-type Ca(2+) channel, but not to Na(+) channel site 1 receptor using radioligands binding assay. CONCLUSION: NIP-141 terminated AF in aconitine-induced AF and prevented the atrial remodelling by short-term rapid pacing in dogs, possibly via the blocking of Na(+) and Ca(2+) channels.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Importance of sympathetic activation for the expression of Na+-Ca2+ exchanger in end-stage failing human myocardium.

AIMS: In end-stage heart failure, an alteration in the expression of the Na+-Ca2+ exchanger has been reported. Regulation of its expression is largely unknown. We sought to find out whether Na+-Ca2+ exchanger in human heart failure is regulated by sympathetic activation. In addition, since Na+-Ca2+-exchange is electrogenic, we conjectured whether increased expression of Na+-Ca2+ exchanger is associated with an increased incidence of cardiac arrhythmias. METHODS AND RESULTS: Twenty-three patients suffering from end-stage cardiac failure were examined in the hours preceding cardiac transplantation. Plasma levels of norepinephrine, epinephrine, atrial natriuretic peptide, renin activity, aldosterone, tumour necrosis factor (TNF)-alpha, and TNF-receptors were measured. All parameters were elevated relative to 21 healthy control subjects. As determined by immunoblots, protein levels of the Na+-Ca2+ exchanger were increased by 56% and protein levels of sarcoplasmic reticulum (SR) Ca2+-ATPase were decreased by 20% in left ventricles of the explanted failing hearts. A significant correlation between protein and neurohumoral levels was exclusively found for the Na+-Ca2+ exchanger with norepinephrine (r=0.64; P=0.01). Recent Holter ECGs revealed that patients with sustained or non-sustained ventricular tachycardia (more than three consecutive beats) had significantly higher Na+-Ca2+ exchanger protein and plasma norepinephrine levels than patients with a maximum of two consecutive beats (Na+-Ca2+ exchanger: 109+/-10 vs 83+/-7, n=11 each, P<0.05; norepinephrine: 1359+/-159 vs 656+/-88 pg. ml(-1), n=9 each, P<0.001). CONCLUSIONS: Sympathetic activation may enhance the expression of Na+-Ca2+ exchanger in end-stage heart failure. The data support the hypothesis that increased Na+-Ca2+-exchange could favour malignant ventricular arrhythmias.