RESUMEN
The platypus (Ornithorhynchus anatinus), a uniquely Australian species, is one of the few living venomous mammals. Although envenomation of humans by many vertebrate and invertebrate species results in pain, this is often not the principal symptom of envenomation. However, platypus envenomation results in an immediate excruciating pain that develops into a very long-lasting hyperalgesia. We have previously shown that the venom contains a C-type natriuretic peptide that causes mast cell degranulation, and this probably contributes to the development of the painful response. Now we demonstrate that platypus venom has a potent action on putative nociceptors. Application of the venom to small to medium diameter dorsal root ganglion cells for 10 s resulted in an inward current lasting several minutes when the venom was diluted in buffer at pH 6.1 but not at pH 7.4. The venom itself has a pH of 6.3. The venom activated a current with a linear current-voltage relationship between -100 and -25 mV and with a reversal potential of -11 mV. Ion substitution experiments indicate that the current is a nonspecific cationic current. The response to the venom was blocked by the membrane-permeant Ca(2+)-ATPase inhibitor, thapsigargin, and by the tyrosine- and serine-kinase inhibitor, k252a. Thus the response appears to be dependent on calcium release from intracellular stores. The identity of the venom component(s) that is responsible for the responses we have described is yet to be determined but is probably not the C-type natriuretic peptide or the defensin-like peptides that are present in the venom.
Asunto(s)
Calcio/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ornitorrinco , Ponzoñas/toxicidad , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Carbazoles/farmacología , Cationes/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Concentración de Iones de Hidrógeno , Alcaloides Indólicos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Nociceptores/citología , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Wistar , Tapsigargina/farmacologíaRESUMEN
The venom of the male Australian duck-billed platypus contains a family of four polypeptides of appox. 5 kDa, which are referred to as defensin-like peptides (DLPs). They are unique in that their amino acid sequences have no significant similarities to those of any known peptides; however, the tertiary structure of one of them, DLP-1, has recently been shown to be similar to beta-defensin-12 and to the sodium neurotoxin peptide ShI (Stichodactyla helianthus neurotoxin I). Although DLPs are the major peptides in the platypus venom, little is known about their biological roles. In this study, we determined the three-dimensional structure of DLP-2 by NMR spectroscopy, with the aim of gaining insights into the natural function of the DLPs in platypus venom. The DLP-2 structure was found to incorporate a short helix that spans residues 9-12, and an antiparallel beta-sheet defined by residues 15-18 and 37-40. The overall fold and cysteine-pairing pattern of DLP-2 were found to be similar to those of DLP-1, and hence beta-defensin-12; however, the sequence similarities between the three molecules are relatively small. The distinct structural fold of the DLP-1, DLP-2, and beta-defensin-12 is based upon several key residues that include six cysteines. DLP-3 and DLP-4 are also likely to be folded similarly since they have high sequence similarity with DLP-2. The DLPs, and beta-defensin-12 may thus be grouped together into a class of polypeptide molecules which have a common or very similar global fold. The fact that the DLPs did not display antimicrobial, myotoxic, or cell-growth-promoting activities implies that the nature of the side chains in this group of peptides is likely to play an important role in defining the biological function(s).
Asunto(s)
Proteínas/química , Ponzoñas/química , Secuencia de Aminoácidos , Animales , Defensinas , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Ornitorrinco , Conformación Proteica , Pliegue de Proteína , Homología de Secuencia de AminoácidoRESUMEN
A peptide which relaxes rat uterine smooth muscle and exhibits homology with the mammalian C-type natriuretic peptide (CNP) has previously been identified in platypus (Ornithorhynchus anatinus) venom from its partial N-terminal amino acid sequence. In this study we describe the purification, detailed structure, synthesis and pharmacological characteristics of this peptide, which has been designated ovCNP-39 (Ornithorhynchus venom C-type natriuretic peptide). Elucidation of the 39-residue amino acid sequence confirms the homology with mammalian CNPs. These peptides produce hypotension in vivo and relax smooth muscle in vitro, but are poorly characterised in terms of physiological function. ovCNP-39 is equipotent with human/rat/porcine CNP-22 in eliciting cyclic guanosine 5'-monophosphate (cGMP) elevation in cultured vascular smooth muscle cells, suggesting that, like CNP, it acts through the ANPB natriuretic peptide receptor subtype. The direct elevation of cGMP in vascular smooth muscle by ovCNP-39 may underlie the vasodilatory effects of platypus envenomation.
Asunto(s)
Péptido Natriurético Tipo-C/química , Péptido Natriurético Tipo-C/farmacología , Ponzoñas/química , Secuencia de Aminoácidos , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Femenino , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Péptido Natriurético Tipo-C/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Ornitorrinco , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Porcinos , Contracción Uterina/efectos de los fármacosRESUMEN
In this study we characterise the ability of a C-type natriuretic peptide from platypus (Ornithorhynchus anatinus) venom (ovCNP-39) to relax the rat uterus in vitro and we investigate the possibility that ovCNP-39 contributes to the acute effects of envenomation, which include oedema, pain and erythema. We have found that both ovCNP-39 and the endogenous C-type natriuretic peptide, CNP-22, produce oedema in the rat paw and release histamine from rat peritoneal mast cells. Two synthetic peptides, ovCNP-39(1-17) and ovCNP-39(18-39), corresponding to the N- and C-termini, respectively, are equipotent histamine releasers, suggesting that ovCNP-39 and other natriuretic peptides do not act through conventional natriuretic peptide receptors on mast cells.