RESUMEN
BACKGROUND: CD26 is a multifunctional cell surface glycoprotein expressed by T and B cells. It exhibits a dipeptidyl-peptidase activity (DPP-IV) that cleaves the penultimate proline from the N-terminus of polypeptides, thereby regulating their activity and concentration. METHODS: Using CD26-/- mice resulting from targeted inactivation of the gene, we examined the consequences of a DPP-IV defect on behavioural response to nociceptive stimuli and concentration of the pain modulator peptides substance P (SP) and endomorphin 2, two DPP-IV substrates. RESULTS: CD26 inactivation induced a three-fold decrease in circulating endopeptidase activity while that found in brain extracts was normal, albeit very weak. CD26-/- mice had high SP concentrations in plasma (3.4+/-1 pg/ml versus 1.5+/-0.3 pg/ml, P<10(-3)) but not in brain extracts (35+/-12 pg/ml versus 32+/-9 pg/ml, P>0.05). Endomorphin-2 levels in the two groups were in the same range for plasma and brain extracts. CD26-/- mice displayed short latencies to nociceptive stimuli (hot plate test: 6.6+/-1.2 s versus 8.6+/-1.5 s, P<10(-4); tail pinch test: 3.1+/-0.6 s versus 4.2+/-0.8 s, P<10(-3)). Administration of an SP (NK1) receptor antagonist or DPP-IV to CD26-/- mice normalised latencies. DPP-IV inhibitors decreased latencies only in CD26+/+ mice. CONCLUSIONS: Our observations represent the first fundamental evidence showing that DPP-IV influences pain perception via modulation of the peripheral SP concentration. Our work also highlights the role of peripheral NK1 receptors in nociception.
Asunto(s)
Dipeptidil Peptidasa 4/fisiología , Dolor/metabolismo , Análisis de Varianza , Androstanos/farmacología , Animales , Bencimidazoles/farmacología , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligopéptidos/sangre , Dolor/dietoterapia , Dolor/genética , Dimensión del Dolor/métodos , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Sustancia P/sangreRESUMEN
The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.
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Músculo Esquelético/metabolismo , Neuronas/metabolismo , Venenos de Escorpión/genética , Canales de Sodio/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Femenino , Inyecciones Intraventriculares , Inyecciones Subcutáneas , Dosificación Letal Mediana , Potenciales de la Membrana/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Xenopus laevisRESUMEN
CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.
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Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Venenos de Escorpión , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Dicroismo Circular , Simulación por Computador , Disulfuros/química , Humanos , Canal de Potasio Kv.1.2 , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/química , Canales de Potasio Calcio-Activados/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Ratas , Venenos de Escorpión/síntesis química , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Venenos de Escorpión/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Scorpion neurotoxins acting on ion channels share some structural features but differ in antigenic and immunogenic properties. They are highly structured peptides, 60-70 amino acids long. Monoclonal antibodies have been obtained for Androctonus australis hector scorpion venom neurotoxin II (AahII) and a nontoxic synthetic analog ((Abu)(8) AahII). In this study, no antibody response was elicited in mice of various strains injected with AahI, the other important toxin of the venom, in a native or an inactive ((Abu)(8) AahI) form. We found that AahI was only immunogenic in BALB/c or C57BL/6 mice if it was coupled to a carrier protein. The helper protein molecule could be BSA, KLH, or the nontoxic analog of AahII. We obtained a panel of high-affinity mAbs with these immunogens. Two of these mAbs, including the very high-affinity antibody 9C2 (K(D)=0.11x10(-11) M), were used to set up a two-site ELISA, sensitive enough for the quantification of AahI in the biological fluids of envenomed animals. The detection limit of the assay was 75 pg/ml.
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Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Neurotoxinas/inmunología , Radioinmunoensayo/métodos , Venenos de Escorpión/inmunología , Escorpiones/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Antivenenos/inmunología , Proteínas Portadoras/inmunología , Inmunoglobulina G/análisis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Conejos , Resonancia por Plasmón de SuperficieRESUMEN
Scorpion stings represent a medical problem in numerous countries. The scorpion Androctonus australis hector produces three alpha toxins (Aah I to III), which are responsible for most of the lethality in mammals. These toxins act on sodium channel and do not cross-react immunologically. We used RIA and ELISA to measure the concentrations of these three toxins in plasma, urine and different organs after i.v. and s.c. injections of water extracts of venoms in rabbits or mice. In both animals, the toxins rapidly appeared in plasma after s.c. injection as it was previously described for the whole venom. However, the toxins disappeared from the blood more quickly than did other main components of the venom. Thus, serotherapy must be initiated immediately to prevent the toxin from reaching its target. We also detected the toxins in urine, kidneys, heart and lungs, but not in the brain. However, the concentration of Aah II was always lower than that of Aah I. Analysis of five samples of venom collected in different areas of southern Tunisia showed that a large polymorphism exists for the three toxins. This is yet another difficulty for serotherapy as there is no cross-antigenicity between them.
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Neurotoxinas/farmacocinética , Venenos de Escorpión/farmacocinética , Animales , Infusiones Intravenosas , Inyecciones Subcutáneas , Riñón/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Miocardio/metabolismo , Neurotoxinas/administración & dosificación , Conejos , Venenos de Escorpión/administración & dosificación , Distribución TisularRESUMEN
Antibody-based therapy is the only specific treatment for scorpion envenomation. However, there are still major drawbacks associated with its use; mainly because antivenoms are still prepared from immune equine serum raised against crude venoms, whereas only a limited number of neurotoxins are responsible for the lethality of the venom. Using a murine hybridoma that secretes a well-characterized neutralizing IgG directed to neurotoxins AahI and AahIII from the venom of the scorpion Androctonus australis, we constructed a recombinant Fab (rFab) fragment, which was produced and purified from transformed bacteria. It recognized toxin AahI with a high affinity (KD = 8.2 x 10(-11)) equivalent to the homologous pFab prepared by papain digestion of whole IgG. Although the AahI-neutralizing capacity of protein L-purified rFab was low compared to other recombinant antibody formats (scFv and diabody) investigated in parallel, the antibody engineering approach presented here provides an innovative way to synthesize novel toxin-neutralizing molecules. It may serve as a strategy for designing a new generation of antivenoms.
Asunto(s)
Fragmentos de Inmunoglobulinas/química , Inmunoglobulina G/química , Neurotoxinas/química , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Cartilla de ADN , Escherichia coli/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neurotoxinas/antagonistas & inhibidores , Neurotoxinas/inmunología , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venenos de Escorpión/antagonistas & inhibidores , Venenos de Escorpión/inmunologíaRESUMEN
We studied the effects of scorpion (Androctonus australis hector) venom on hemodynamics and on the release of catecholamines, neuropeptide Y (NPY), endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) in dog model of severe scorpion envenomation. Nine mongrel anesthetized dogs were submitted to mechanical ventilation through intubation and were administered intravenously purified dried scorpion venom (Androctonus autstralis) 0.05 mg/kg. Measurements including pulmonary artery catheter derived parameters, serum toxin levels and humoral variables were performed at baseline (before venom injection) and 5, 15, 30 and 60 min after venom injection. Humoral variables included: serum lactate, epinephrine (EP), norepinephrine (NE), NPY, ET-1 and ANP plasma concentrations. Scorpion venom caused rapid and transient increase of mean arterial pressure (MAP) and PAOP associated with a marked and sustained decline in cardiac output (-55% at 60 min; P < 0.001). Hemodynamic changes were associated with a rapid and significant increase of all measured hormones. The highest increase was for NE (28-fold) and EP (25-fold). MAP was closely correlated with NE and less significantly correlated with toxin levels. Similarly, significant correlation was observed between PAPO and ANP plasma levels. These findings support the implication of excessive catecholamines release in hemodynamic disturbances of severe SE and suggest that NPY and ET-1 could be involved in this process. Serum toxin does not appear to consistently contribute to these effects. Through its correlation with PAOP, ANP could be a reliable and useful marker of cardiac dysfunction in SE.
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Hemodinámica/efectos de los fármacos , Neurotransmisores/metabolismo , Venenos de Escorpión/toxicidad , Picaduras de Arañas/sangre , Picaduras de Arañas/fisiopatología , Animales , Factor Natriurético Atrial/metabolismo , Catecolaminas/metabolismo , Perros , Endotelina-1/metabolismo , Inyecciones Intravenosas , Neuropéptido Y/metabolismo , Neurotoxinas/sangre , Neurotoxinas/toxicidad , Venenos de Escorpión/sangreRESUMEN
BACKGROUND: The modulation of extracellular adenosine concentration by opioids provides evidence that the antinociceptive effects of these compounds involve endogenous adenosine. The aim of this study was to determine whether there is a relation between the inhibition of brain synaptosome adenosine uptake by opioid agonists and the analgesic effects of these compounds. METHODS: The authors used the hot plate and tail-pinch tests to evaluate in mice (C57BL/6 females; weight, 25-30 g) the effects of caffeine, a nonspecific adenosine receptor antagonist, on the antinociceptive effect induced by the intracerebroventricular administration of oxymorphone as a mu agonist, SNC80 as a delta agonist, or U69593 as a kappa agonist. They also investigated the effect of these opioid receptor agonists on the uptake of adenosine by whole brain synaptosomes. RESULTS: Caffeine decreased the analgesic effects induced by oxymorphone or SNC80 but not those induced by U69593. Oxymorphone and SNC80 inhibited adenosine uptake by brain cells, but U69593 did not. CONCLUSION: The antinociceptive effects obtained with mu or delta (but not kappa) agonists administered supraspinally were indicative of the involvement of modulation of adenosine uptake.
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Adenosina/metabolismo , Analgésicos Opioides/farmacología , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Analgésicos Opioides/administración & dosificación , Animales , Química Encefálica/efectos de los fármacos , Cafeína/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Inyecciones , Inyecciones Intraventriculares , Ratones , Ratones Endogámicos C57BL , Dimensión del Dolor/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Médula Espinal , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.