RESUMEN
Amyloid ß (Aß) protein is responsible for Alzheimer's disease, and one of its important fragments, Aß(25-35), is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aß(25-35)'s toxicity in cells even though they share similar primary structures with Aß(25-35). Here, we seek to understand the molecular mechanisms of how these peptides interact with Aß(25-35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aß(25-35), NK, Kassinin, Substance P, and mixtures of the latter three with Aß(25-35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of Aß(25-35), which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aß(25-35), NK, Kassinin, and Substance P do not form hexamers with a ß-sheet structure like Aß(25-35). The hexamer structure of Aß(25-35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aß(25-35) are discussed.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Taquicininas , Enfermedad de Alzheimer/metabolismo , Amiloide/química , Péptidos beta-Amiloides/química , Humanos , Kasinina/química , Fragmentos de Péptidos/química , Sustancia P/química , Taquicininas/químicaRESUMEN
Amyloids are associated with diseases, including Alzheimer's, as well as functional roles such as storage of peptide hormones. It is still unclear what differences exist between aberrant and functional amyloids. However, it is known that soluble oligomers formed during amyloid aggregation are more toxic than the final fibrils. Here, we perform molecular dynamics simulations to study the aggregation of the amyloid-ß peptide Aß25-35, associated with Alzheimer's disease, and two functional amyloid-forming tachykinin peptides: kassinin and neuromedin K. Although the three peptides have similar primary sequences, tachykinin peptides, in contrast to Aß25-35, form nontoxic amyloids. Our simulations reveal that the charge of the C-terminus is essential to controlling the aggregation process. In particular, when the kassinin C-terminus is not amidated, the aggregation kinetics decreases considerably. In addition, we observe that the monomeric peptides in extended conformations aggregate faster than those in collapsed hairpin-like conformations.
Asunto(s)
Péptidos beta-Amiloides/química , Kasinina/química , Simulación de Dinámica Molecular , Neuroquinina B/química , Multimerización de Proteína , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
Protein aggregation and amyloid formation are associated with both pathological conditions in humans such as Alzheimer's disease and native functions such as peptide hormone storage in the pituitary secretory granules in mammals. Here, we studied amyloid fibrils formation by three neuropeptides namely physalaemin, kassinin and substance P of tachykinin family using biophysical techniques including circular dichroism, thioflavin T, congo red binding and microscopy. All these neuropeptides under study have significant sequence similarity with Aß(25-35) that is known to form neurotoxic amyloids. We found that all these peptides formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells. However, the extent of amyloid formation, structural transition, and morphology were different depending on the primary sequences of peptide. When Aß(25-35) and Aß40 were incubated with each of these neuropeptides in 1:1 ratio, a drastic increase in amyloid growths were observed compared to that of individual peptides suggesting that co-aggregation of Aß and these neuropeptides. The electron micrographs of these co-aggregates were dissimilar when compared with individual peptide fibrils further supporting the possible incorporation of these neuropeptides in Aß amyloid fibrils. Further, the fibrils of these neuropeptides can seed the fibrils formation of Aß40 and reduced the toxicity of preformed Aß fibrils. The present study of amyloid formation by tachykinin neuropeptides is not only providing an understanding of the mechanism of amyloid fibril formation in general, but also offering plausible explanation that why these neuropeptide might reduce the cytotoxicity associated with Alzheimer's disease related amyloids.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Kasinina/química , Fragmentos de Péptidos/química , Fisalemina/química , Algoritmos , Secuencia de Aminoácidos , Amiloide/farmacología , Amiloide/ultraestructura , Péptidos beta-Amiloides/ultraestructura , Benzotiazoles , Línea Celular Tumoral , Dicroismo Circular , Rojo Congo/química , Heparina/química , Humanos , Kasinina/farmacología , Microscopía Electrónica , Neuronas/química , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/ultraestructura , Fisalemina/farmacología , Unión Proteica , Estructura Secundaria de Proteína , Sustancia P/química , Sustancia P/farmacología , Tiazoles/química , Pruebas de Toxicidad/métodosRESUMEN
Tachykinins are among the most widely-studied families of regulatory peptides characterized by a highly-conserved C-terminal -Phe-X-Gly-Leu-Met.amide motif, which also constitutes the essential bioactive core. The amphibian skin has proved to be a rich source of these peptides with physalaemin from the skin of Physalaemus fuscomaculatus representing the archetypal aromatic tachykinin (X=Tyr or Phe) and kassinin from the skin of Kassina senegalensis representing the archetypal aliphatic tachykinin in which X=Val or Ile. Despite the primary structures of both mature peptides having been known for at least 30 years, neither the structures nor organizations of their biosynthetic precursors have been reported. Here we report the structure and organization of the biosynthetic precursor of kassinin deduced from cDNA cloned from a skin secretion library. In addition, a second precursor cDNA encoding the novel kassinin analog (Thr(2), Ile(9))-kassinin was identified as was the predicted mature peptide in skin secretion. Both transcripts exhibited a high degree of nucleotide sequence similarity and of open-reading frame translated amino acid sequences of putative precursor proteins. The translated preprotachykinins each consisted of 80 amino acid residues encoding single copies of either kassinin or its site-substituted analog. Synthetic replicates of each kassinin were found to be active on rat urinary bladder smooth muscle at nanomolar concentrations. The structural organization of both preprotachykinins differs from that previously reported for those of Odorrana grahami skin indicating a spectrum of diversity akin to that established for amphibian skin preprobradykinins.
Asunto(s)
Anuros/metabolismo , Kasinina/química , Kasinina/farmacología , Músculo Liso/efectos de los fármacos , Piel/metabolismo , Taquicininas/farmacología , Vejiga Urinaria/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Técnicas In Vitro , Kasinina/síntesis química , Kasinina/genética , Masculino , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Taquicininas/síntesis química , Taquicininas/química , Taquicininas/genéticaRESUMEN
In frog skin, tachykinins stimulate the ion transport, estimated by measuring the short-circuit current (SCC) value, by interacting with NK1-like receptors. In this paper we show that Kassinin (NK2 preferring in mammals) increases the SCC, while Enterokassinin has no effect. Therefore, either 2 Pro residues or 1 Pro and 1 basic amino acid must be present in the part exceeding the C-terminal pentapeptide. Eledoisin (NK3 preferring in mammals) stimulation of SCC is reduced by CP99994 and SR48968 (NK1 and NK2 antagonists) and not affected by SB222200 (NK3 antagonist). None of the three antagonists affects Kassinin stimulation of SCC.
Asunto(s)
Eledoisina/farmacología , Transporte Iónico/fisiología , Kasinina/farmacología , Rana esculenta/fisiología , Piel/metabolismo , Taquicininas/farmacología , Secuencia de Aminoácidos , Animales , Benzamidas/farmacología , Eledoisina/metabolismo , Kasinina/metabolismo , Piperidinas/farmacología , Quinolinas/farmacología , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Estereoisomerismo , Taquicininas/metabolismoRESUMEN
Two tachykinin peptides, bufokinin and Xenopus neurokinin A (X-NKA) were recently isolated from Xenopus laevis. In this study we investigated the tachykinin receptors in the Xenopus gastrointestinal tract. In functional studies using stomach circular muscle strips, all peptides had similar potencies (EC50 values 1-7 nM). The rank order of potency to contract the intestine was physalaemin (EC50 1 nM)> or =bufokinin (EC50 3 nM)>substance P (SP)> or =cod SP>NKA>>X-NKA (EC50 1,900 nM). No maximum response could be obtained for [Sar9,Met(O2)11]SP, eledoisin and kassinin. In stomach strips, the mammalian tachykinin receptor antagonists RP 67580 (NK1) and MEN 10376 (NK2) had agonistic effects but did not antagonize bufokinin or X-NKA. In intestinal strips, RP 67580 (1 microM) reduced the maximal response to X-NKA but not bufokinin, while MEN 10376 was ineffective. [125I]BH-bufokinin bound with high affinity to a single class of sites, of KD 213+/-35 (stomach) and 172+/-9.3 pM (intestine). Specific binding of [125I]BH-bufokinin was displaced by bufokinin> or =SP>NKA> or =eledoisin approximately kassinin>X-NKA, indicating binding to a tachykinin NK1-like receptor. Selective tachykinin receptor antagonists were weak or ineffective. Other iodinated tachykinins ([125I]NKA and [125I]BH-eledoisin) displayed biphasic competition profiles, with the majority of sites preferring bufokinin rather than X-NKA. In conclusion, there is evidence for two different tachykinin receptors in Xenopus gastrointestinal tract. Both receptors may exist in stomach, whereas the bufokinin-preferring NK1-like receptor predominates in longitudinal muscle of the small intestine. Antagonists appear to interact differently with amphibian receptors, compared with mammalian receptors.
Asunto(s)
Neuroquinina A/análogos & derivados , Fisalemina/análogos & derivados , Receptores de Taquicininas/química , Receptores de Taquicininas/efectos de los fármacos , Especificidad de la Especie , Sustancia P/análogos & derivados , Xenopus/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Cardias/citología , Cardias/efectos de los fármacos , Cardias/metabolismo , Relación Dosis-Respuesta a Droga , Eledoisina/farmacología , Femenino , Indoles/farmacología , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Radioisótopos de Yodo , Isoindoles , Kasinina/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Neuroquinina A/antagonistas & inhibidores , Neuroquinina A/química , Neuroquinina A/farmacología , Fragmentos de Péptidos/farmacología , Fisalemina/farmacología , Receptores de Taquicininas/fisiología , Sustancia P/farmacologíaRESUMEN
1. Numerous studies have demonstrated that the urinary bladder is particularly sensitive to tachykinins; rat, rabbit and guinea pig bladders, besides human detrusor, have been the most extensively studied, whereas very little is known about most large animal detrusors. The aim of this work was to study natural tachykinin activity on the lower urinary tract of ovine to make a comparison with data obtained in laboratory animals. 2. As in other animal species, tachykinins are also able to contract ovine bladder smooth muscle. 3. The results reported in this study indicate that in ovine bladder, neurokinin 2 (NK2) receptors are expressed most. In fact, on lamb and sheep bladder neurokinin A (NKA), a NK2- almost selective peptide, was shown to be > 100% more active than the natural tachykinins kassinin (KASS) and eledoisin (ELED). Eledoisin was shown to be 50% less active than KASS, which is typical behaviour for an almost exclusively NK2 receptor population. Moreover, NK1- preferential peptides, namely substance P (SP) and physalaemin (PHYS), showed a lack of activity even when applied at high concentrations. 4. The results reported in this study show that lamb and sheep detrusor represent a good alternative model for the characterization of NK2-selective tachykinins.
Asunto(s)
Músculo Liso/efectos de los fármacos , Taquicininas/metabolismo , Sistema Urinario/efectos de los fármacos , Animales , Eledoisina/farmacología , Femenino , Cobayas , Técnicas In Vitro , Kasinina/farmacología , Masculino , Neuroquinina A/farmacología , Péptidos Cíclicos/farmacología , Receptores de Neuroquinina-2/efectos de los fármacos , Ovinos , Sustancia P/farmacologíaRESUMEN
OBJECTIVES: In order to determine the active sites for salivation of various tachykinins, the regulatory roles of the N-terminal portion of various newly-synthesized tachykinins were studied after i.p. injection of rats using the submandibular glands as model organs. METHODS: N-shortened oligopeptides from kassinin, eledoisin, neurokinins A (NKA) and NKB were synthesized by the multipin peptide synthesis method. Amino acids were eliminated one by one to form octa- to undeca-peptides adjoining the inactive or less active heptapeptides and various heptapeptides, in which an amino acid in position 8 (Xaa8), numbering as in an undecapeptide, was replaced with Tyr, Phe, Ile or Val. RESULTS: The N-terminal amino acids in positions 1 to 4 could be activators or inhibitors, depending on whether the C-terminal heptapeptide was inactive or less active. The Xaa8 residue, in combination with amino acids in positions 5 and 6, seemed to be very important in determining the sialogogic activity of a heptapeptide. The discrimination between NKA and NKB appeared due to the N-terminal amino acid sequence in positions I to 4 including Phe or Ser in position 6. CONCLUSIONS: It is concluded that the N-terminal amino acids in positions I to 4 serve as either activators or inhibitors depending upon the sialogogic activity of the C-terminal heptapeptide, in which particular amino acids in positions 5, 6 and 8 regulate its activity.
Asunto(s)
Salivación/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , Taquicininas/farmacología , Análisis de Varianza , Animales , Eledoisina/farmacología , Inyecciones Intraperitoneales , Isoleucina/farmacología , Kasinina/farmacología , Masculino , Modelos Animales , Neuroquinina A/farmacología , Neuroquinina B/farmacología , Fenilalanina/farmacología , Ratas , Ratas Sprague-Dawley , Serina/farmacología , Estadística como Asunto , Glándula Submandibular/metabolismo , Taquicininas/administración & dosificación , Tirosina/farmacología , Valina/farmacologíaRESUMEN
Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.
Asunto(s)
Kasinina/química , Modelos Moleculares , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Dicroismo Circular , Kasinina/metabolismo , Espectroscopía de Resonancia Magnética , Fosforilcolina/metabolismo , Conformación Proteica , AguaRESUMEN
The localisation of NK(3) tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3. 5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK(1) receptors was blocked by the NK(1) receptor antagonist, CP-99994. NK(3) receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK(3) receptors. In myenteric ganglia, NK(3) receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK(1) receptor-IR neurons. In submucosal ganglia, NK(3) receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK(3) receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways.
Asunto(s)
Íleon/metabolismo , Kasinina/metabolismo , Neuroquinina A/metabolismo , Neuronas/metabolismo , Receptores de Neuroquinina-3/metabolismo , Animales , Carbocianinas/química , Recuento de Células , Femenino , Colorantes Fluorescentes/química , Cobayas , Técnicas In Vitro , Kasinina/química , Masculino , Microscopía Confocal , Plexo Mientérico/citología , Plexo Mientérico/metabolismo , Neuroquinina A/química , Neuronas/citología , Piperidinas/farmacología , Receptores de Neuroquinina-3/antagonistas & inhibidores , Plexo Submucoso/citología , Plexo Submucoso/metabolismoRESUMEN
Neuromedins, smooth-muscle-stimulating peptides, are commonly divided into four groups: bombesin-like, kassinin-like, neurotensin-like and neuromedins U. In the present review, current data on the synthesis and mechanism of action of neuromedins on hypothalamo-pituitary-adrenal (HPA) axis will be presented. These neuropeptides and their receptors are localized to all components of the HPA axis, the only exemption seems to be neurokinin B, which is not detected in the adenohypophysis. Neuromedins exert a manifold effect on HPA axis, and their action on the adrenal suggests their involvement in the regulation of growth, structure and function of the adrenal cortex. Neuromedins may exert both direct and indirect effects on the adrenal cortex. Direct effect is proven by the stimulation of mineralo- and glucocorticoid output by isolated or cultured adrenocortical cells and by mobilisation of intracellular [Ca2+]i. Indirect effects, on the other hand, may be mediated by ACTH, arginine-vasopressin, angiotensin II, catecholamines or by other regulatory substances of medullary origin.
Asunto(s)
Corteza Suprarrenal/fisiología , Neuropéptidos/farmacología , Corteza Suprarrenal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Bombesina/genética , Bombesina/farmacología , Humanos , Kasinina/genética , Kasinina/farmacología , Datos de Secuencia Molecular , Neuropéptidos/genética , Neurotensina/genética , Neurotensina/farmacologíaRESUMEN
Administration of substance P and kassinin into the solitary tract nucleus of anesthetized rats induced a dose-dependent increase in ventilation, tidal volume, inspiratory muscle activity, and a decrease in the mean blood pressure and heart rate. Microinjections of peptides caused a decrease in ventilatory response to hypoxia and an inhibition of the Breuer-Hering reflex. The data obtained suggest involvement of tachykinins in the respiratory and circulatory control via the solitary tract nucleus.
Asunto(s)
Hemodinámica/efectos de los fármacos , Kasinina/farmacología , Respiración/efectos de los fármacos , Núcleo Solitario/efectos de los fármacos , Sustancia P/farmacología , Animales , Femenino , Hemodinámica/fisiología , Kasinina/fisiología , Masculino , Microinyecciones , Ratas , Receptores de Neuroquinina-1/agonistas , Receptores de Neuroquinina-2/agonistas , Núcleo Solitario/fisiología , Sustancia P/fisiologíaRESUMEN
Peptides present in a methanol extract prepared from skin of the Costa Rican frog Agalychnis callidryas of the Phyllomedusinae subfamily were studied by sequence analysis and pharmacological tests. Members of five different peptide families-tachykinins, bradykinins, caerulein, opioid peptides and sauvagine-were found. In particular, the extract contained a number of tachykinins with the following sequences: Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asp-Arg(Lys)-Phe-Tyr-Pro-Gly-Met-NH2, pGlu-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Pro-Gly-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Tyr-Pro-Val-Met. The latter three peptides have the unusual C-terminal sequence Pro-Gly(or Val)-Met-NH2 rather than Gly-Leu-Met-NH2 found in many other members of the tachykinin family. The observed amino acid substitutions may be the reason for the marked decrease in the biological activity observed in all in vitro and in vivo tests, even through the spectrum of tachykinin activities was retained. A kassinin-like peptide, with the sequence Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, was also found in the A. callidryas skin. While kassinin has a much higher affinity for NK-3 than for NK-1 receptors, the opposite is true for this A. callidryas peptide. The extract from A. callidryas skin also contained a new caerulein (pGlu-Asp-Tyr(HSO3)-Lys-Gly-Trp-Met-Asp-Phe-NH2) and a phyllokinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Tyr), as well as the opioid peptides dermorphin and [Hyp6]dermorphin, both previously isolated from different Phyllomedusa species.
Asunto(s)
Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Piel/química , Taquicininas/química , Taquicininas/aislamiento & purificación , Animales , Anuros , Bioensayo , Bradiquinina/análogos & derivados , Bradiquinina/química , Bradiquinina/aislamiento & purificación , Bradiquinina/metabolismo , Ceruletida/análogos & derivados , Ceruletida/química , Ceruletida/aislamiento & purificación , Ceruletida/metabolismo , Costa Rica , Kasinina/análogos & derivados , Kasinina/química , Kasinina/aislamiento & purificación , Kasinina/metabolismo , Oligopéptidos/metabolismo , Péptidos Opioides , Taquicininas/metabolismoRESUMEN
An antiserum raised against locustatachykinin I, one of four myotropic peptides that have been isolated from the locust brain and corpora cardiaca, was characterized by enzyme-linked immunosorbent assay (ELISA) and used for immunocytochemical detection of neurons and endocrine cells in the nervous system and intestine of the blowfly Calliphora vomitoria. The ELISA characterization indicated that the antiserum recognizes the common C-terminus sequence of the locustatachykinins I-III. Hence, the cross reaction with locustatachykinin IV is less, and in competitive ELISAs no cross reaction was detected with a series of vertebrate tachykinins tested. It was also shown that the antiserum recognized material in extracts of blowfly heads, as measured in ELISA. In high-performance liquid chromatography the extracted locustatachykinin-like immunoreactive (LomTK-LI) material eluted in two different ranges. A fairly large number of LomTK-LI neurons was detected in the blowfly brain and thoracicoabdominal ganglion. A total of about 160 LomTK-LI neurons was seen in the proto-, deuto-, and tritocerebrum and subesophageal ganglion. Immunoreactive processes from these neurons could be traced in many neuropil regions of the brain: superior and dorsomedian protocerebrum, optic tubercle, fan-shaped body and ventral bodies of the central complex, all the glomeruli of the antennal lobes, and tritocerebral and subesophageal neuropil. No immunoreactivity was seen in the mushroom bodies or the optic lobes. In the fused thoracicoabdominal ganglion, 46 LomTK-LI neurons could be resolved. The less evolved larval nervous system was also investigated to obtain additional information on the morphology and projections of immunoreactive neurons. In neither the larval nor the adult nervous systems could we identify any efferent or afferent immunoreactive axons or neurosecretory cells. The widespread distribution of LomTK-LI material in interneurons suggests an important role of the native peptide(s) as a neurotransmitter or neuromodulator within the central nervous system. Additionally a regulatory function in the intestine is indicated by the presence of immunoreactivity in endocrine cells of the midgut.
Asunto(s)
Sistema Nervioso Central/metabolismo , Dípteros/metabolismo , Hormonas de Insectos/metabolismo , Proteínas de Insectos , Mucosa Intestinal/metabolismo , Taquicininas/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Ganglios de Invertebrados/metabolismo , Inmunohistoquímica , Hormonas de Insectos/inmunología , Kasinina/inmunología , Larva , Datos de Secuencia Molecular , Vías Nerviosas/fisiología , Fragmentos de Péptidos/análisis , Péptidos/análisis , Taquicininas/inmunología , Extractos de Tejidos/químicaRESUMEN
Capsaicin at low concentrations increases the short circuit current (SCC) across frog skin. Simultaneous measurements of both transepithelial fluxes of 22Na or 36Cl demonstrate that the SCC increase is due to stimulation of sodium active absorption. Capsaicin acts through the liberation of several peptides; thus these peptides were tested on the SCC across frog skin. Those more active are, in order of potency: Cyclic Calcitonin Gene Related Peptide (CGRP), Kassinin and Eledoisin, Substance P (SP) and Neurokinin A. Neurokinin B and Vasoactive Intestinal Peptide (VIP) have no effect. Also the actions of SP and CGRP are due mainly to stimulation of Na+ active absorption. A strict parallelism regarding the sensitivity to inhibitors (Naproxen, SQ22536 and CP96345) between SP, CGRP and Capsaicin strengthens the hypothesis that SP and CGRP are liberated by Capsaicin in this tissue.
Asunto(s)
Capsaicina/farmacología , Fenómenos Fisiológicos de la Piel , Sodio/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/farmacología , Conductividad Eléctrica , Eledoisina/farmacología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Kasinina/farmacología , Neuroquinina A/farmacología , Rana esculenta , Piel/efectos de los fármacos , Sustancia P/farmacologíaRESUMEN
We are interested in the presence and function in insects of neuropeptides related to the vertebrate tachykinins. Hence, we have used antisera raised against the tachykinins substance P and kassinin, and against the insect neuropeptide leucokinin I, for localization studies and immunochemical analysis of related peptides in the nervous system of the blowfly Phormia terraenovae. In radioimmunoassays (with antisera against kassinin and leucokinin I) used in combination with reverse-phase HPLC, it was shown that the antisera recognize immunoreactive material with distinctly hydrophobic properties and each antiserum appear to detect several forms of immunochemically related peptides. With immunocytochemistry it was shown that the kassinin and leucokinin antisera each reacted with material in a distinct set of neurons. The leucokinin-immunoreactive material is present both in interneurons and in neurosecretory cells, suggesting roles of native leucokinin-like peptides as neuromodulators in the nervous system and as neurohormones acting on peripheral targets. The kassinin immunoreactivity was seen in interneurons, but could not be conclusively localized in neurosecretory cells, possibly indicating a role only within the nervous system.
Asunto(s)
Dípteros/química , Neuropéptidos/análisis , Secuencia de Aminoácidos , Animales , Química Encefálica/fisiología , Cromatografía Líquida de Alta Presión , Inmunohistoquímica , Kasinina/análisis , Datos de Secuencia Molecular , Neuronas/química , Oligopéptidos/análisis , Radioinmunoensayo , Homología de Secuencia de Aminoácido , Sustancia P/análisisRESUMEN
1. Circumoesophageal ganglia and foot muscle of the garden snail, Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE). 2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum. 3. SP and NKA radioimmunoassays were performed on extracts of circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum. 4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide. 5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-terminal structural characteristics similar to mammalian SP.
Asunto(s)
Caracoles Helix/química , Neuronas/química , Sustancia P/análisis , Taquicininas/análisis , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Eledoisina/inmunología , Técnica del Anticuerpo Fluorescente , Kasinina/inmunología , Datos de Secuencia Molecular , Sistema Nervioso/química , Neuroquinina A/inmunología , Radioinmunoensayo , Sustancia P/inmunología , Taquicininas/inmunologíaRESUMEN
Repeated sodium depletions produce a persistent, enhanced need-free salt intake in the rat, particularly in the female. The neurochemical mechanisms underlying the phenomenon are still unknown. The present studies evaluated the effect on the enhanced need-free salt intake of the female rat (1) of pharmacological interference with the natriorexigenic hormones angiotensin II and aldosterone and (2) of the central injection of the tachykinin peptides, which are endowed with antinatriorexic activity. The need-free salt intake of the female rat is not modified by treatment with the angiotensin-converting enzyme inhibitor captopril or by the aldosterone receptor antagonist RU-28318. On the other hand, the behavior is highly sensitive to the inhibitory effect of central tachykinins, suggesting the possibility that need-free salt intake might be linked to modification (down-regulation) of the inhibitory tachykininergic system.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Sodio en la Dieta , Taquicininas/farmacología , Aldosterona/farmacología , Angiotensina II/farmacología , Animales , Captopril/farmacología , Regulación hacia Abajo/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Eledoisina/administración & dosificación , Eledoisina/farmacología , Femenino , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Kasinina/administración & dosificación , Kasinina/farmacología , Antagonistas de Receptores de Mineralocorticoides/farmacología , Ratas , Ratas Endogámicas , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Receptores de Taquicininas , Espironolactona/análogos & derivados , Espironolactona/farmacología , Sacarosa , Taquicininas/administración & dosificaciónRESUMEN
We have measured the affinity of various analogs and fragments of the tachykinin substance P for the cloned rat NK1, NK2, and NK3 receptors heterologously expressed in Chinese hamster ovary cells. The hydrophobic carboxyl-terminal pentapeptide sequence substance P-(7-11) binds with similar affinity (2-20 microM) to all three receptors. Our data suggest that addition of one to three amino-terminal residues to this sequence results in the optimization of its interaction within the binding pocket of the NK1 receptor. The addition of Pro-Gln-Gln to the carboxyl-terminal pentapeptide sequence increases affinity for the NK1 receptor, either by providing additional binding interactions or by modifying the conformation of the carboxyl-terminal sequence. This latter hypothesis is supported by the observation that physalaemin and phyllomedusin, which also contain a proline residue in the position analogous to the proline residue 4 of substance P, are also selective for NK1 receptors. Tachykinins that lack this proline have no higher affinity for NK1 than [pGlu] substance P-(6-11). Conversely, addition of Pro-Gln-Gln to the carboxyl-terminal pentapeptide sequence is unfavorable for NK2 and NK3 receptor binding. Preliminary data suggest that tachykinins with high affinity (Kd less than 500 nM) for NK2 receptors contain an aspartate residue in the position analogous to residue 5 of substance P, suggesting that an ionic interaction with the receptor may contribute binding energy. Further experiments will be required to determine the structural determinants of the NK1, NK2, and NK3 receptors responsible for these binding properties.