Amyloid-like fibril formation by tachykinin neuropeptides and its relevance to amyloid ß-protein aggregation and toxicity.

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.

Kassina senegalensis skin tachykinins: molecular cloning of kassinin and (Thr2, Ile9)-kassinin biosynthetic precursor cDNAs and comparative bioactivity of mature tachykinins on the smooth muscle of rat urinary bladder.

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.

Eledoisin and Kassinin, but not Enterokassinin, stimulate ion transport in frog skin.

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.

Characterization of receptors for two Xenopus gastrointestinal tachykinin peptides in their species of origin.

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.

Effects of natural tachykinins on ovine lower urinary tract smooth muscle.

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.

The roles of the N-terminal portions of various tachykinins in promoting salivation.

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.

Role of neuromedins in the regulation of adrenocortical function.

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.

[The effect of tachykinins microinjections into the solitary tract nucleus on respiration and blood circulation in rats]. / Vliianie mikroin''ektsií takhikininov v oblast' iadra solitarnogo trakta na dykhanie i krovoobrashchenie u krys.

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.

Action of capsaicin and related peptides on the ionic transport across the skin of Rana esculenta.

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.

Central tachykinin injection potently suppresses the need-free salt intake of the female rat.

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.

Determination of the amino acid residues in substance P conferring selectivity and specificity for the rat neurokinin receptors.

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.

Effects of tachykinins and selective tachykinin receptor agonists on vascular permeability in the rat lower urinary tract: evidence for the involvement of NK-1 receptors.

1. Intravenous administration of three mammalian tachykinins (substance P, neurokinin A and neurokinin B) and three non-mammalian tachykinins (physalaemin, eledoisin and kassinin) induced dose-dependent increases in vascular permeability, as measured by Evans blue leakage technique, in various segments of the lower urinary tract (bladder dome and neck, proximal urethra, ureters) in urethane-anaesthetized rats. 2. Plasma extravasation induced by substance P (3.71 nmol kg-1 i.v.) was unaffected by pretreatment with antihistaminic drugs or methysergide. 3. A comparison of the relative potencies of various tachykinins did not allow characterization of a distinct type of receptor involved in the increase in vascular permeability. 4. The effects of tachykinin-related peptides which are selective agonists at the NK-1 (substance P-methylester, [Pro9]-SP-sulphone), NK-2 receptor [( Nle10]-NKA(4-10] or NK-3 receptor [( MePhe7]-NKB(4-10) and Senktide) indicated that NK-1 agonists are effective in the whole lower urinary tract, while NK-2 or NK-3 agonists are inactive or weakly active. 5. [beta-Ala4, Sar9]-SP(4-11)-sulphone, a selective NK-1 receptor agonist devoid of histamine-releasing properties, was highly potent and effective in producing plasma extravasation in the rat lower urinary tract. 6. These findings indicate that NK-1 receptors mediate the effect of intravenous tachykinins on vascular permeability in the rat lower urinary tract, through a histamine-independent mechanism.

NK-1 receptor mediation of neurogenic plasma extravasation in rat skin.

1. Plasma extravasation was induced by electrical nerve stimulation and by perfusion of tachykinins over a vacuum-induced blister base on rat footpad. 2. Stimulation of the sciatic nerve (18 V, 15 Hz, 0.5 ms) for 20 min produced a significant increase in the protein content of the perfusate. The response in capsaicin pretreated rats was only 4% of the control response. This indicates that the electrically-induced plasma extravasation response was mediated by capsaicin-sensitive sensory fibres. 3. Exogenous perfusion of the mammalian tachykinins substance P, neurokinin A and neurokinin B and the non-mammalian tachykinins physalaemin, kassinin and eledoisin was used to determine the tachykinin receptor type mediating the plasma extravasation response. Dose-response curves of the tachykinins (10(-9) M-10(-4) M) gave a rank order of potency of substance P = physalaemin greater than eledoisin greater than or equal to kassinin greater than neurokinin B = neurokinin A. 4. In addition, specific agonists of neurokinin receptors were perfused. Perfusion of [Glp6, D-Pro9] SP6-11 and [Glp6, L-Pro9]SP6-11 demonstrated that the L-Pro isomer was much more potent than the D-Pro isomer. 5. The rank order of potency and the greater potency of [Glp6, L-Pro9]SP6-11 over its D-isomer indicate an NK-1 neurokinin receptor mediates plasma extravasation in rat footpad skin.

Contractile effects of substance P and other tachykinins on the mouse isolated distal colon.

1. Substance P (SP), physalaemin, eledoisin and kassinin induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The responses were not antagonized by atropine (1.5 x 10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), timolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). They were enhanced by tetrodotoxin (TTX, 1.5 x 10(-7) M). These observations indicate that the contractile responses to the tachykinins result from a direct activation of smooth muscle cells. 2. The contractile activity provoked by SP and physalaemin was inhibited by nifedipine (a Ca2+-entry blocker) and was abolished in Ca2+-free EGTA solution. Such data suggest that the myogenic effects of SP and physalaemin are mainly dependent on their ability to promote Ca2+ influx. 3. Eledoisin and kassinin evoked a contractile response in the absence of external Ca2+ and their myogenic activity was, to some extent, resistant to the inhibitory effect of nifedipine. This may indicate that an additional process, probably the release of an intracellularly bound Ca2+ store, participates in the mechanism by which eledoisin and kassinin contract the mouse distal colon. 4. After desensitization of the mouse distal colon to SP, the contractile activity provoked by SP or physalaemin was totally abolished whilst the responses evoked by eledoisin and kassinin were barely affected. These observations and other experimental findings indirectly support the assumption that the mouse distal colon could possess different tachykinin-binding sites.

Suppression of salt intake in the rat by neurokinin A: comparison with the effect of kassinin.

The present study investigates the effect of the mammalian tachykinin neurokinin A on salt intake in the rat. Intracerebroventricular injection of neurokinin A inhibited salt intake elicited by sodium depletion, by subchronic deoxycorticosterone treatment and by adrenalectomy. It also inhibited the need-free salt intake of female rats that had been previously depleted of sodium. Since different brain mechanisms elicit salt intake in these experimental models, it is concluded that neurokinin A exerts a general antinatriorexic effect. Apparently, its inhibitory effect on salt intake is not due to malaise or competing behaviors, as shown by the fact that the doses of neurokinin A which suppress salt intake do not suppress milk intake. In comparison to the amphibian tachykinin kassinin, neurokinin A possesses a similar spectrum of antinatriorexic activities, but is markedly less potent and less effective in all the experimental models investigated. These findings suggest that activation of neurokinin A receptors cannot solely account for the potent antinatriorexic effect of kassinin and of other nonmammalian tachykinins.

The development in infant rats of kassinin's potent and selective control of cell-dehydration thirst.

In infant rats, kassinin exerts its antidipsogenic effect in the very early stages of neonatal life (2nd-3rd day). The inhibition of cell-dehydration drinking appears in rats of 2 days, and attains adult levels in pups of 9 days. Instead, the thirsts induced by suckling deprivation or by intracerebroventricular angiotensin II are inhibited by kassinin precociously (3rd day), but are unaffected by it in rats of 12-15 days. Kassinin also inhibits milk intake very early (3rd day) and this effect also disappears at 12 days of age. The pattern of ontogenetic results described here may be that of a brain kassinin-like tachykinin that, in the course of the development of the neural structures on which it acts, gains potent and selective control of cell-dehydration thirst.

Effects of kassinin, a tachykinin of the skin of the African frog Kassina senegalensis, on body fluid homeostasis in rats.

In Wistar rats loaded with SC hypertonic NaCl solution kassinin produces an inhibition of cell-dehydration drinking which lasts several hours. The long duration of the effect does not depend on a renal excretion of sodium but, hypothetically, on a redistribution of electrolytes between the extra- and the intra-cellular compartments of body fluids.

Differential effects of tachykinins injected intranigrally on striatal dopamine metabolism.

The effects of intranigral injection of kassinin, eledoisin, and substance P on striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents as well as circling behavior were studied in rats. Kassinin and eledoisin produced a marked dose-dependent increase of DOPAC concentrations in the ipsilateral striatum, as well as in the number of contralateral circlings. Substance P produced a similar but weaker effect. At the larger dose (5 nmol), the three tachykinins also induced an increase of DA concentrations in the ipsilateral striatum. The rank order of activity was kassinin greater than eledoisin greater than substance P. These results suggest that tachykinins stimulated the nigro-striatal dopaminergic system by accelerating the dopamine metabolism in striatum.

Inhibitory effect of intracranial injections of tachykinins on angiotensin-induced drinking in the cat.

The tachykinins eledoisin, substance P and kassinin were administered by pulse intracerebroventricular (ICV) injections to cats made thirsty by ICV angiotensin II, 100 ng per cat. Eledoisin, 100 ng per cat, produced an inhibition of drinking which was larger (56.0 vs. 45.2%) and lasted longer than that evoked by 400 ng per cat of substance P. Kassinin, 100 ng per cat, did not evoke any effect at all. The treatment with these peptides neither produced signs of discomfort nor induced any other behavioural alteration. The results of present experiments suggest that the antidipsogenic effect of tachykinins is a phenomenon of general interest among mammals.

Effects of synthetic kassinin on splanchnic circulation and exocrine pancreas in dogs.

Effects of intravenously administered synthetic kassinin on splanchnic circulation and exocrine pancreatic secretion were examined in six anesthetized dogs. Kassinin caused dose-related increases in the blood flow in superior mesenteric artery and portal vein, and produced an initial increase followed by a decrease in pancreatic blood flow, but did not affect the exocrine pancreatic secretion. This study demonstrates that kassinin affects splanchnic blood flow in dogs, and suggests that kassinin or a kassinin-like substance functions as a neuropeptide controlling the splanchnic circulation in mammalian species.