The facilitatory actions of snake venom phospholipase A2 neurotoxins at the neuromuscular junction are not mediated through voltage-gated K channels

Electrophysiological investigations have previously suggested that phospholipase A₂ (PLA₂) neurotoxins from snake venoms increase the release of acetylcholine (Ach) at the neuromuscular junction by blocking voltage-gated K⁺ channels in motor nerve terminals.We have tested some of the most potent presynaptically-acting neurotoxins from snake venoms, namely β-bungarotoxin (BuTx), taipoxin, notexin, crotoxin, ammodytoxin C and A (Amotx C & A), for effects on several types of cloned voltage-gated K⁺ channels (mKv1.1, rKv1.2, mKv1.3, hKv1.5 and mKv3.1) stably expressed in mammalian cell lines. By use of the whole-cell configuration of the patch clamp recording technique and concentrations of toxins greater than those required to affect acetylcholine release, these neurotoxins have been shown not to block any of these voltage-gated K⁺ channels. In addition, internal perfusion of the neurotoxins (100 μg/ml) into mouse B82 fibroblast cells that expressed rKv1.2 channels also did not substantially depress K⁺ currents. The results of this study suggest that the mechanism by which these neurotoxins increase the release of acetylcholine at the neuromuscular junction is not related to the direct blockage of voltage-activated Kv1.1, Kv1.2, Kv1.3, Kv1.5 and Kv3.1 K⁺ channels.     

Corn oil enhancing hepatic lipid peroxidation induced by CCl4 does not aggravate liver fibrosis in rats

Lipid peroxidation (LPO) is known to be associated with liver fibrosis in chronic liver injury. However, direct effects of the products of LPO on liver fibrogenesis have not been demonstrated. In this study, we examined the LPO products of carbon tetrachloride (CCl4)+corn oil to evaluate the effect of LPO products on liver fibrosis. CCl4 was given twice a week for 8 weeks. Corn oil was given daily to rats at a dose of 2 or 10ml/kg via gastrogavage throughout the whole experiment period. CCl4 induced both cyclooxygenase (COX)-2 independent and COX-2 dependent LPO. COX-2 independent LPO was enhanced by corn oil treatment while no effect was reflected on COX-2 dependent LPO. CCl4-induced liver fibrosis in rats was not aggravated by corn oil treatment. In addition, the amount of fatty liver induced by CCl4 was increased by corn oil treatment. Though the inflammation-related UCP-2 mRNA expression was induced by CCl4, it was not aggravated by the enhancement of corn oil. Conclusion: corn oil enriches polyunsaturated fatty acids through COX-2 independent pathways to increase LPO products that do not enhance liver fibrosis induced by CCl4.     

Bioisosteric phentolamine analogs as selective human alpha(2)- versus alpha(1)-adrenoceptor ligands

Phentolamine is known to act as a competitive, non-subtype-selective alpha-adrenoceptor antagonist. In an attempt to improve alpha(2)- versus alpha(1)-adrenoceptor selectivity and alpha(2)-adrenoceptor subtype-selectivity, two new chemical series of bioisosteric phentolamine analogs were prepared and evaluated. These compounds were evaluated for binding affinities on alpha(1)- (alpha(1A)-, alpha(1B)-, alpha(1D)-) and alpha(2)- (alpha(2A)-, alpha(2B)-, alpha(2C)-) adrenoceptor subtypes that had been stably expressed in human embryonic kidney and Chinese hamster ovary cell lines, respectively. Methylation of the phenolic hydroxy group and replacement of the 4-methyl group of phentolamine with varying lipophilic substituents yielded bioisosteric analogs selective for the alpha(2)- versus alpha(1)-adrenoceptors. Within the alpha(2)-adrenoceptors, these analogs bound with higher affinity at the alpha(2A)- and alpha(2C)-subtypes as compared to the alpha(2B)-subtype. In particular, the t-butyl analog was found to be the most selective, its binding at the alpha(2C)-adrenoceptor (Ki=3.6 nM) being 37- to 173-fold higher than that at the alpha(1)-adrenoceptors, and around 2- and 19-fold higher than at the alpha(2A)- and alpha(2B)-adrenoceptors, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities of selected compounds from the bioisosteric series on human alpha(1A)- and alpha(2C)-adrenoceptors. Thus, the results with these bioisosteric analogs of phentolamine provide a lead to the rational design of potent and selective alpha(2)-adrenoceptor ligands that may be useful in improving the therapeutic profile of this drug class for human disorders.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

α-Methyl-5-HT, a 5-HT2 receptor agonist, stimulates β2-adrenoceptors in guinea pig airway smooth muscle

Alpha-methyl-5-HT is widely used as a high-affinity 5-HT(2) receptors agonist, though some studies have postulated that this drug also activates other serotonergic receptors. In the present work, we found that a wide range of concentrations of alpha-methyl-5-HT induced biphasic responses (contraction followed by relaxation) in guinea pig tracheal rings. The relaxing phase caused by 32microM alpha-methyl-5-HT was blocked by 0.1microM propranolol. Furthermore, during an ongoing histamine-induced contraction, alpha-methyl-5-HT (0.1-100microM) produced a concentration-dependent relaxation starting at 10microM. This relaxation was fully abolished by 0.1microM propranolol or 1microM ICI 118,551 (a selective beta(2)-adrenoceptor antagonist). Additionally, in electrophysiological recordings, 32microM alpha-methyl-5-HT also enhanced the membrane K(+) currents of single tracheal myocytes, an effect reverted by propranolol and ICI 118,551, and mimicked by 0.1microM salbutamol. Thus, we concluded that alpha-methyl-5-HT activates beta(2)-adrenoceptors in guinea pig tracheal smooth muscle at concentrations >or=10microM. This effect must be taken into account when this drug is used in airway smooth muscle and in other tissues expressing beta(2)-adrenoceptors.     

Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors

At present the cryo-electron microscopy structure at 4 Å resolution is known for the Torpedo marmorata nicotinic acetylcholine receptor (nAChR), and high-resolution X-ray structures have been recently determined for bacterial ligand-gated ion channels which have the same type of spatial organization. Together all these structures provide the basis for better understanding functioning of muscle-type and neuronal nAChRs, as well as of other Cys-loop receptors: 5HT3-, glycine-, GABA-A and some other. Detailed information about the ligand-binding sites in nAChRs, necessary both for understanding the receptor functioning and for rational drug design, became available when the X-ray structures were solved for the acetylcholine-binding proteins (AChBP), excellent models for the ligand-binding domains of all Cys-loop receptors. Of special value in this respect are the X-ray structures of AChBP complexes with agonists and antagonists. Among the latter are the complexes with polypeptide and peptide antagonists, that is with protein neurotoxins from snake venoms and peptide neurotoxins (α-conotoxins) from poisonous marine snails of Conus genus. The role of a bridge between the AChBP and nAChRs is played by the X-ray structure of the ligand-binding domain of α1 subunit of nAChR in the complex with α-bungarotoxin.The purpose of this review is to show the role of well-known and new polypeptide and peptide neurotoxins, from the earlier days of nAChRs research until present time, in identification of different nAChR subtypes and mapping their binding sites.     

Phospholipase A2 inhibitors (βPLIs) are encoded in the venom glands of Lachesis muta (Crotalinae, Viperidae) snakes

Phospholipase A₂ inhibitors (PLIs) are glycoproteins secreted by snake liver into the circulating blood aiming the self-protection against toxic venom phospholipases A₂. In the present study, we describe the first complete nucleotide sequence of a βPLI from venom glands of a New World snake, Lachesis muta. The deduced primary structure was compared to other known βPLIs and recent literature findings of other possible roles of PLIs in snakes are discussed.     

Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2.

An important group of toxins, whose action at the molecular level is still a matter of debate, is secreted phospholipases A(2) (sPLA(2)s) endowed with presynaptic or beta-neurotoxicity. The current belief is that these beta-neurotoxins (beta-ntxs) exert their toxicity primarily due to their extracellular enzymatic action on the plasma membrane of motoneurons at the neuromuscular junction. However, the discovery of several extra- and intracellular proteins, with high binding affinity for snake venom beta-ntxs, has raised the question as to whether this explanation is adequate to account for all the observed phenomena in the process of presynaptic toxicity. The purpose of this review is to critically examine the various published studies, including the most recent results on internalization of a beta-ntx into motor nerve terminals, in order to contribute to a better understanding of the molecular mechanism of beta-neurotoxicity. As a result, we propose that presynaptic neurotoxicity of sPLA(2)s is a result of both extra- and intracellular actions of beta-ntxs, involving enzymatic activity as well as interaction of the toxins with intracellular proteins affecting the cycling of synaptic vesicles in the axon terminals of vertebrate motoneurons.     

A study of the nicotinic agonist SIB-1553A on locomotion, and attention as measured by the five-choice serial reaction time task

SIB-1553A is a novel ligand with reputed agonist selectivity at nicotinic receptors containing the β₄ subunit. As such, it represents an interesting pharmacological tool with which to probe the function of nicotine receptor subtypes. In the present studies, we compared SIB-1553A with nicotine in its ability to stimulate locomotion and to enhance attention in rats as assessed using the five-choice serial reaction time task (5-CSRTT). In nicotine-naive rats, SIB-1553A (10–40 mg/kg) induced a comparable increase in locomotion to nicotine (0.4 mg/kg), whereas in nicotine-sensitised rats, an enhanced locomotor response was seen to nicotine (0.4 mg/kg) but not to SIB-1553A (10–80 mg/kg). Similarly, chronic treatment with either SIB-1553A or nicotine did not lead to a cross-sensitised locomotor response. Unlike nicotine, SIB-1553A-induced locomotion was insensitive to antagonism by either mecamylamine (1 mg/kg) or DHβE (3 mg/kg), suggesting a non-nicotinic mechanism. In young and aged rats, nicotine (0.4 mg/kg) enhanced attention as demonstrated by an increase in response accuracy and speed. SIB-1553A (3–10 mg/kg) did not mimic any of these changes and at the highest dose tended to disrupt performance. These results lend further support to the involvement of a high affinity site, possibly α₄β₂, in the locomotor and attentional-enhancing properties of nicotine.     

Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A(2)

Myotoxic phospholipases A(2) account for most of the muscle necrosis that results from envenenomation by crotaline snakes. In this study, we investigated the protective effect of fucoidan, a natural sulfated polysaccharide obtained from the brown seaweed Fucus vesiculosus, against the cytotoxic and myotoxic activities of a group of phospholipase A(2) myotoxins from crotaline snake venoms: Bothrops asper myotoxins I, II, III, and IV, Cerrophidion godmani myotoxins I and II, Atropoides nummifer myotoxins I and II, and Bothriechis schlegelii myotoxin I. All of the toxins tested were efficiently inhibited by fucoidan, in both their cytotoxic and myotoxic effects. The basis for this inhibition appears to be the rapid formation of complexes between fucoidan and myotoxins, as evidenced by turbidimetric analysis. The possible binding site of fucoidan on the myotoxins was investigated using short synthetic peptides that represent the membrane-damaging region (residues 115-129) for three of these toxins. Fucoidan clearly inhibited the cytolytic activity of the peptides, indicating its ability to interact with the C-terminal myotoxic region of these phospholipases A(2). Fucoidan significantly inhibited muscle damage in mice, when administered locally, immediately after experimental envenomation with crude venom from B. asper. These results encourage further studies of sulfated fucans as compounds of potential use to improve the treatment of envenomations by crotaline snakes.     

Inhibition of recombinant N-type and native high voltage-gated neuronal Ca channels by AdGABA: Mechanism of action studies

High-voltage activated Ca²⁺ (Ca_V) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca²⁺ concentration. These channels consist of a pore-forming Ca_Vα₁ protein and three auxiliary subunits (Ca_Vβ, Ca_Vα₂δ and Ca_Vγ). Ca_Vα₂δ is an important component of Ca_V channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca_Vα₂δ and reduces Ca²⁺ currents by modulating the expression and/or function of the Ca_Vα₁ subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca_V channels. However, the importance of the interaction of AdGABA with the Ca_Vα₂δ subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca²⁺ currents acting on Ca_Vα₂δ only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the l-amino acid transport system. Interestingly, a mutation in the Ca_Vα₂δ that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca_Vα₂δ ligand that regulates Ca_V channels.     

Role of α1- and β3-adrenoceptors in the modulation by SR59230A of the effects of MDMA on body temperature in the mouse

Background and purpose:

We have investigated the ability of the β₃-adrenoceptor antagonist 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A) to affect the hyperthermia produced by methylenedioxymethamphetamine (MDMA) in conscious mice and whether α₁-adrenoceptor antagonist actions are involved.

Experimental approach:

Mice were implanted with temperature probes under anaesthesia, and allowed 2 week recovery. MDMA (20 mg·kg⁻¹) was administered subcutaneously 30 min after vehicle or test antagonist and effects on body temperature monitored by telemetry.

Key results:

Following vehicle, MDMA produced a slowly developing hyperthermia, reaching a maximum increase of 1.8°C at 130 min post injection. A low concentration of SR59230A (0.5 mg·kg⁻¹) produced a small but significant attenuation of the slowly developing hyperthermia to MDMA. A high concentration of SR59230A (5 mg·kg⁻¹) revealed a significant and marked early hypothermic reaction to MDMA, an effect that was mimicked by the α₁-adrenoceptor antagonist prazosin. Functional and ligand binding studies revealed actions of SR59230A at α₁-adrenoceptors.

Conclusions and implications:

1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4,-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride in high concentrations modulates the hyperthermic actions of MDMA in mice in two ways: by blocking an early α₁-adrenoceptor-mediated component to reveal a hypothermia, and by a small attenuation of the later hyperthermic component which may possibly be β₃-adrenoceptor-mediated (this seen with the low concentration of SR59230A). Hence, the major actions of SR59230A in modulating the actions of MDMA on temperature involve α₁-adrenoceptor antagonism.     

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.
2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

     

Cloning, characterization and phylogenetic analyses of members of three major venom families from a single specimen of Walterinnesia aegyptia.

Walterinnesia aegyptia is a monotypic elapid snake inhabiting in Africa and Mideast. Although its envenoming is known to cause rapid deaths and paralysis, structural data of its venom proteins are rather limited. Using gel filtration and reverse-phase HPLC, phospholipases A(2) (PLAs), three-fingered toxins (3FTxs), and Kunitz-type protease inhibitors (KIns) were purified from the venom of a single specimen of this species caught in northern Egypt. In addition, specific primers were designed and PCR was carried out to amplify the cDNAs encoding members of the three venom families, respectively, using total cDNA prepared from its venom glands. Complete amino acid sequences of two acidic PLAs, three short chain 3FTxs, and four KIns of this venom species were thus deduced after their cDNAs were cloned and sequenced. They are all novel sequences and match the mass data of purified proteins. For members of each toxin family, protein sequences were aligned and subjected to molecular phylogenetic analyses. The results indicated that the PLAs and a Kunitz inhibitor of W. aegyptia are most similar to those of king cobra venom, and its 3FTxs belongs to either Type I alpha-neurotoxins or weak toxins of orphan-II subtype. It is remarkable that both king cobra and W. aegyptia cause rapid deaths of the victims, and a close evolutionary relationship between them is speculated.     

Effects of castration on α1-adrenoceptor subtypes in the rat aorta

The expression of α₁-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the α₁-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the α_1B/α_1D-adrenoceptor alkylating agent chloroethylclonidine resulted in ≈1600-fold rightward shift in the concentration–response curves to noradrenaline. The pA₂ values found for WB 4101, benoxathian (α_1A-selective) and BMY 7378 (α_1D-selective) indicate that α_1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pK_B estimated through the lowest effective concentrations of the α_1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pK_B estimated in aorta from control rats is consistent with the interaction with α_1D-adrenoceptors (7.58±0.06), while that calculated in organs from control rats is consistent with α_1A-adrenoceptors (8.76±0.09). These results suggest that castration induces plasticity in the α₁-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.     

Snake venom disintegrins and cell migration

Cell migration is a key process for the defense of pluricellular organisms against pathogens, and it involves a set of surface receptors acting in an ordered fashion to contribute directionality to the movement. Among these receptors are the integrins, which connect the cell cytoskeleton to the extracellular matrix components, thus playing a central role in cell migration. Integrin clustering at focal adhesions drives actin polymerization along the cell leading edge, resulting in polarity of cell movement. Therefore, small integrin-binding proteins such as the snake venom disintegrins that inhibit integrin-mediated cell adhesion are expected to inhibit cell migration. Here we review the current knowledge on disintegrin and disintegrin-like protein effects on cell migration and their potential use as pharmacological tools in anti-inflammatory therapy as well as in inhibition of metastatic invasion.     

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the ��2-adrenoceptors

BACKGROUND AND PURPOSE

Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

EXPERIMENTAL APPROACH

In binding experiments with ³H-rauwolscine, we studied the interactions of green mamba venom fractions with α₂-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α₂-adrenoceptors expressed in mammalian cells.

KEY RESULTS

ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of ³H-rauwolscine binding to the three α₂-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α_2A-adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA₂ values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively.

CONCLUSIONS AND IMPLICATIONS

ρ-Da1b is the first toxin identified to specifically interact with α₂-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α₂-adrenoceptor subtypes.     

Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E(2) and D(2) and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA(2)) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA(2) from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD(2) and PGE(2), with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. Inhibition of cPLA(2) by AACOCF(3), but not of iPLA(2) by PACOCF(3) or BEL, significantly reduced PGD(2), PGE(2) and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA(2) inhibition did not modify the effects of CB, whereas iPLA(2) inhibition reduced PGD(2) and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs' synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo.