Apoptosis induction in human leukemic cells by a novel protein Bengalin,isolated from Indian black scorpion venom: Through mitochondrial pathway and inhibition of heat shock proteins

Scorpion venom possesses protein toxins having numerous biological activities, some of which are potentially anticancerous. Previously we had reported antiproliferative activity of the venom of Indian black scorpion, Heterometrus bengalensis Koch. Here we have isolated and purified a novel protein named Bengalin (72 kDa) from the venom, responsible for antiproliferative and apoptogenic activities against human leukemic cells U937 (histiocytic lymphoma) and K562 (chronic myelogenous leukemia). N-terminal sequence of first 20 amino acids of Bengalin was G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin induced cell growth inhibition at IC₅₀ values of 3.7 and 4.1 μg/ml for U937 and K562 cells respectively did not significantly affect normal human lymphocytes. Inhibition of U937 and K562 cell proliferation occurred by apoptosis as evidenced from damaged nuclei, cell cycle arrest at sub G1 phase, increase of early apoptotic cells, augmentation of DNA fragmentation and also a reduction of telomerase activity. Further insights revealed that Bax:Bcl2 ratio was elevated after Bengalin treatment. Moreover Bengalin elicited loss of mitochondrial membrane potential (MMP) which commenced cytochrome c release in cytosol, decreased heat shock protein (HSP) 70 and 90 expression, activated caspase-9, caspase-3 and induced poly(ADP-ribose) polymerase (PARP) cleavage. We have also determined that HSP70 and 90 inhibitions correlated with Bengalin induced antiproliferation, caspase-3 upregulation, apoptogenesis and increased DNA fragmentation. These results hypothesize that Bengalin might provide a putative molecular mechanism for their anticancer effect on human leukemic cells which might be mediated by mitochondrial death cascade. Inhibition of HSPs might also play a crucial role in induction of apoptosis.     

An equilibrium ELISA for the dosage of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion venoms: set up and calibration

Scorpion stings are very frequent in Tunisia; yet a method for evaluating envenoming severity and consequently victim treatment, has never been adequately established nor has its efficiency been properly evaluated. Indeed, a management of envenomed patients requires the optimization of envenoming antivenom immunotherapy. This task requires either, an accurate evaluation of toxicokinetic parameters of scorpion venoms in absence and in presence of antivenom, using animals as models, and the establishment of a quantitative relationship between human blood scorption venom levels, envenoming severities and clinical symptoms. A performant sandwich ELISA was set up and calibrated for measuring scorpion venom levels in human and rabbit sera. This assays was performed with polyclonal F(ab')2 specific to the two North African scorpion (Androctonus australis garzonii; Aag and Buthus occitanus tunetanus: Bot) venoms. It is simple, rapid, very sensitive (detection limit = 0.9 ng/ml) and shows a good linearity for venom concentrations in human sera comprised between 0.5 and 15 ng/ml. The ELISA is also reproducible: the coefficient of variation, determined at different venom concentrations (low: 4 ng/ml; medium: 8 ng/ml and high: 12 ng/ml) prepared in a pool of sera collected from several healthy donors, were lower than 10%. Such an ELISA has been successfully used, either in experimental toxinokinetic and immunotherapeutic studies carried out in rabbits or for the quantification of Aag and Bot venom levels in the serum of human victims stung by these scorpion.     

Effects of 5-bromo-2′-deoxyuridine on arachidonic acid metabolism of neuroblastoma and leukemia cells in culture: A possible role of endogenous prostaglandins in tumor cell proliferation and differentiation

Effects of 5-bromo-2′-deoxyuridine (BrdU) were studied on two neuroblastoma and two leukemia cell lines, in terms of the relationship between prostaglandin (PG) synthesis and cell growth/differentiation. After treatment with BrdU (5 μg/ml), cell growth of the 4 cell lines was inhibited and one neuroblastoma cell line (GOTO) showed flattened morphology with positive S-100 protein, one of the differentiation markers for Schwann or glial cells. In the 4 cell lines, BrdU treatment reduced [1-¹⁴C]-arachidonic acid incorporation into phosphatidylinositol and phosphatidylethanolamine and was associated with an increase into phosphatidylcholine and triglyceride. BrdU treatment also increased fractions of 6-keto-PGF_1α and PGF_2α , with a decreased TXB₂ fraction. The decreased ratio of TXB₂ /6-keto-PGF_1α or increased 6-keto-PGF_1α fraction correlated significantly with cell growth inhibition, suggesting that the changes in the balance of endogenous PGs might be associated with BrdU-induced cell growth inhibition with or without differentiation of neuroblastoma and leukemia cells in culture.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.     

Structure-activity relationship of an alpha-toxin Bs-Tx28 from scorpion (Buthus sindicus) venom suggests a new alpha-toxin subfamily

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na(+), K(+), Ca(+) or Cl(-). An alpha-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3+/-2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain alpha-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJalpha-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na(+) channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known alpha- and alpha-like toxins suggests the presence of a new and separate subfamily of scorpion alpha-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD(50) 0.088 and 14.3microg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC(50)=0.01microg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na(+)-channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical alpha-mammal (AaH-II), alpha-insect (Lqh-alphaIT), and alpha-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a "necklace-like" structure differing significantly in all alpha-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned alpha-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion alpha-mammal toxins.     

蝎毒抗癌组分SVⅡ分离法的优化研究

目的比较三种柱径的分子筛G-50凝胶层析柱分离东亚钳蝎蝎毒的柱效;并对分离所得组分作MTT（酶反应比色法）抗肿瘤活性作用研究,为从中研制和开发出高效、低毒的新型抗癌特效药筛选出目标组分。方法（1）采用三种规格的分子筛层析柱分离蝎毒;（2）HPLC色谱分析比较各组分的指纹图谱;（3）MTT法观察不同浓度（1、10、100mg／L）的蝎毒及其组分对四种肿瘤细胞（HL-60、A549、K562／ADR、K562／S等）的毒性作用。结果经过分子筛柱层析,可从蝎毒（Scorpion venom,SV）获取三个组分SVⅠ、SVⅡ、SVⅢ;经HPLC色谱分析,各组分明显含有四种以上单体成分;MTT法研究表明,SVⅡ对四种肿瘤细胞的细胞毒性较原毒强,剂量-效应关系较好,而SVⅠ、SVⅢ对四种肿瘤细胞抑制作用不明显。结论（1）利用大柱径的层析柱分离蝎毒的柱效较高;（2）组分SVⅡ是蝎毒抗癌的目标组分,且其对耐药细胞株（K562／ADR）的抑制作用比阳性对照组强,有待进一步的分离纯化,筛选出色谱纯的抗癌活性成分（多肽单体）。     

OD1, the first toxin isolated from the venom of the scorpion Odonthobuthus doriae active on voltage-gated Na+ channels

In this study, we isolated and pharmacologically characterized the first alpha-like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage-gated Na+ channels expressed in Xenopus laevis oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80+/-14 nM) while Na(v)1.2/beta1 still was not affected at concentrations up to 5 microM. Na(v)1.5/beta1 was influenced at micromolar concentrations.     

Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

蝎毒对人肝癌细胞（SMMC7721）和宫颈癌（Hela）细胞抑制作用的研究

目的：探讨蝎毒粗粉及经初步纯化的蝎毒对人肝癌细胞（SMMC7721）和Hela细胞株的抑制作用。方法：以四甲基偶氮唑盐法（MTT）,Western Blotting,免疫细胞化学以及荧光标记的方法,对人肝癌细胞（SMMC7721）和Hela细胞株的凋亡相关蛋白进行检测。结果：蝎毒粗毒及经初步纯化的蝎毒具有诱导SMMC7721和Hela凋亡的作用。结论：蝎毒粗毒及经初步纯化的蝎毒对人肝癌细胞（SMMC7721）及Hela细胞的生长具有明显的抑制作用。     

Indian red scorpion venom modulates spontaneous activity of rat right atria through the involvement of cholinergic and adrenergic systems.

The effect of Indian red scorpion (Mesobuthus tamulus concanesis, Pocock; MBT) venom was investigated on isolated rat right atrial preparations. MBT venom (0.001-3.0 micrograms/ml) exhibited a peculiar concentration-response pattern with respect to rate. The venom concentrations between 0.001-0.01 microgram/ml increased the atrial rate (phase I), followed by a relative decrease with 0.03-0.3 microgram/ml (phase II), and then an abrupt increase with 0.6-3.0 micrograms/ml (phase III). On the other hand, the force was unaltered by venom at phases I and II, while an increase was seen at phase III (3.0 micrograms/ml). Propranolol (0.1 microM) completely blocked the cardiostimulant action of venom at phase III. Further, this stimulant action of venom was absent in atria obtained from reserpinized animals. Pretreatment with atropine (0.3 microM), produced tachycardia at concentrations 0.1-0.3 microgram/ml of venom. But, hexamethonium (30 microM) had no influence on the venom (0.1 microgram/ml)-induced alterations in rate. However, MBT venom increased the acetylcholinesterase (AChE) activity (2-3 fold) in a concentration-dependent manner. Tetrodotoxin (2 microM), did not block the increase in rate produced by 0.01 microgram/ml of venom. Results suggest that, MBT venom-induced alterations of cardiac rhythmicity are mediated through cholinergic as well as adrenergic mechanisms depending upon the concentrations. The modulation of atrial rate at very low concentrations may be due to the direct action of venom on the atrium.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.     

江浙蝮蛇蛇毒蛋白诱导K562细胞调亡的研究

目的：研究江浙蝮蛇蛇毒蛋白诱导K562细胞调亡。方法：通过电镜观察蛇毒蛋白作用后K562细胞的形态变化;MTT检测蛇毒蛋白对细胞增值的影响,同时应用流式细胞仪检测细胞凋亡数及其对细胞周期的影响;采用琼脂糖凝胶电泳观测凋亡片断。结果：蛇毒蛋白作用K562细胞后,能显著抑制细胞增值;LC50为4．96μg／mL,电镜可观察到凋亡形态学改变;电泳呈现典型的阶梯状条带,流式细胞仪检测到凋亡峰。结论：江浙蝮蛇蛇毒蛋白可诱导K562细胞调亡。     

盐酸罗哌卡因对骨肉瘤细胞增殖、侵袭、凋亡的影响及其机制*

目的:探讨盐酸罗哌卡因对骨肉瘤细胞增殖、侵袭、凋亡的影响及分子机制。方法:采用逐步增加药物剂量诱导法建立骨肉瘤多柔比星耐药细胞株(U2OS/DOX),用浓度分别为0、20、50、100 μg/ml的盐酸罗哌卡因处理U2OS/DOX细胞,作为不同浓度盐酸罗哌卡因处理组;将pcDNA3.1、pcDNA3.1-Livin转染至U2OS/DOX细胞中再用浓度为100 μg/ml的盐酸罗哌卡因处理,记为盐酸罗哌卡因100 μg/ml+pcDNA3.1组、盐酸罗哌卡因100 μg/ml+pcDNA3.1-Livin组。MTT检测细胞增殖抑制率及细胞半数抑制浓度(IC₅₀);蛋白质印迹(Western blot)法检测细胞周期蛋白依赖性激酶抑制剂1A(P21)、活化的半胱氨酸天冬氨酸蛋白酶-3(Cleaved Caspase-3)、上皮钙黏蛋白(E-cadherin)、基质金属蛋白酶2(MMP-2)、Livin蛋白表达;克隆形成实验检测细胞克隆形成数;流式细胞术检测细胞凋亡;Transwell检测细胞迁移和侵袭;实时荧光定量PCR(RT-qPCR)检测Livin mRNA表达水平。结果:多柔比星浓度大于1 μg/ml时,骨肉瘤细胞U2OS增殖抑制率显著升高,且具有剂量依赖性(P＜0.05);多柔比星浓度大于10 μg/ml时,骨肉瘤细胞骨肉瘤耐药细胞U2OS/DOX增殖抑制率显著升高,且具有剂量依赖性(P＜0.05)。盐酸罗哌卡因处理的U2OS/DOX细胞中P21、Caspase-3、E-cadherin表达水平显著升高,MMP-2表达水平显著降低,细胞增殖抑制率显著升高,克隆形成数显著降低,细胞凋亡率显著升高,细胞迁移、侵袭数显著降低,Livin表达水平显著降低,且呈浓度依赖性(P＜0.05)。过表达Livin部分逆转了盐酸罗哌卡因对细胞U2OS/DOX增殖、迁移、侵袭的抑制作用及凋亡的促进作用。结论:盐酸罗哌卡因能明显抑制对多柔比星具有耐药性的骨肉瘤细胞的增殖,迁移和侵袭,明显促进骨瘤细胞凋亡,其机制可能与Livin有关。     

Scorpion Venom Heat-Resistant Peptide (SVHRP) Enhances Neurogenesis and Neurite Outgrowth of Immature Neurons in Adult Mice by Up-Regulating Brain-Derived Neurotrophic Factor (BDNF)

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2’-dexoxyuridine (BrdU)-positive cells, BrdU- positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.     

Effects of Tityus serrulatus scorpion venom and one of its purified toxins (toxin gamma) on the isolated guinea-pig heart.

1. The effects of Tityus serrulatus scorpion venom and its most important toxin (toxin gamma) were investigated on isolated guinea-pig hearts, perfused with Locke solution, by the Langendorff's method. 2. The cardiac contraction, the coronary flow and the electrocardiogram (ECG) were simultaneously recorded. 3. Bolus injections of 25, 50 or 100 micrograms of scorpion venom and 2.5, 5 or 10 micrograms of toxin gamma in the heart evoked complex effects which were divided into 3 phases: an initial phase (tachycardia or bradycardia associated with an increase in contractile force), an intermediate phase (oscillations of cardiac rate, contractile force and coronary flow, due to wandering pacemakers) and a third phase (sinus tachycardia). 4. The bradycardia and the oscillations of rhythm were prevented by atropine, whereas the tachycardia and the increase in contractile force were prevented either by reserpine or propranolol. 5. Scorpion venom or toxin gamma induced a ST segment displacement in the ECG, explained by a transitory myocardial hypoxia, due to an increase in the contractile force and a simultaneous decrease of the coronary flow. 6. Perfusion of the heart with Locke solution containing 2% scorpion antivenom prevented almost totally the effects elicited by the venom. 7. It is concluded that the complex effects induced by scorpion venom and toxin gamma are due to the simultaneous release of acetylcholine and catecholamines from postganglionic nerve fibers in the heart.     

Indian red scorpion (Mesobuthus tamulus concanesis, Pocock) venom prolongs repolarization time and refractoriness of the compound action potential of frog sciatic nerve in vitro through calcium dependent mechanism

Effects of Indian red scorpion (M. tamulus concanesis; MBT) venom on the compound action potential (CAP) of sciatic nerve in vitro were examined. MBT venom (0.1-6.0 micrograms/ml) prolonged the repolarization time and refractory period of the CAP in a concentration-dependent manner with maximal potentiation occurring at 6 micrograms/ml (about 100-200 times of the initial). At 1 microgram/ml of venom the prolongations were 40-50 times the initial durations and this concentration was used for subsequent experiments. Rise time, threshold, and conduction velocity of CAP were not altered by MBT venom (1 microgram/ml). In Ca(2+)-free medium, the venom-induced prolongations were only 2-6 times the initial response but addition of Ca2+ in the same medium then prolonged than by 50-70 times. The Ca2+ channel antagonists (nifedipine, 10 microM or Mg2+ ions, 5 mM) attenuated the venom (1 microgram/ml)-induced prolongation of repolarization time and refractory period. However, venom-induced prolongation of CAP responses were still significantly greater than the control in presence of these antagonists. The results indicate that MBT venom-induced increases in repolarization time and refractory period of the action potential greatly depend upon the presence of Ca2+ ions in the medium. The Ca2+ influx was through the L-type of Ca2+ channels.     

Bradycardia induced by Mesobuthus tamulus scorpion venom involves muscarinic receptor-G-protein-coupled cell signaling pathways

Indian red scorpion (Mesobuthus tamulus; MBT) envenomation produces various cardio-respiratory abnormalities including cardiac dysrhythmias. The underlying cell signaling pathways for the cardiac dysrhythmias produced by MBT venom are not known. The present study was therefore conducted to delineate the second messenger signaling pathways involved in MBT venom-induced atrial rhythm changes. The effects of venom and various antagonists were examined on spontaneously beating rat right atrial preparations in vitro. The MBT-venom produced an increase (35%), a decrease (45%) and again an increase (50%) in rate at 0.03, 0.3 and 3.0 microg/ml of venom, respectively. On the other hand, force of contraction exhibited a concentration-dependent rise (up to 40%) at all concentrations of venom. Pretreatment with atropine (0.3 microM) blocked the decrease in atrial rate at 0.3 microg/ml concentration of venom while no such blockade was seen in force of contraction. Submaximal concentration of ACh (0.1 nM) decreased the atrial rate by 25%. In the presence of MBT venom (0.3 microg/ml), ACh-induced fall in atrial rate was enhanced. The venom-induced fall in atrial rate and augmentation of ACh response were blocked by pertussis toxin (PTx; a Gi-inhibitor) or methylene blue (a G-cyclase inhibitor). The results indicate that the decrease in atrial rate produced by venom is mediated muscarinic by receptors via Gi-guanylyl cyclase mediated cell signaling pathways.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.