Conformational study of neurotensin

The conformation of the biologically-active tridecapeptide, neurotensin, has been predicted by a procedure including a continuum method for solvent effects on conformation. The neurotensin conformation is described as a β-type chain with breaks or “corners” most obvious at Tyr³, Asn⁵, Lys⁶, Arg⁸ and Tyr¹¹. The general problem of predicting conformation of larger peptides is emphasized by comparison of the structures of neurotensin and its fragments obtained by other workers with the calculation carried out here on the entire molecule including the effects of the solvent-dependent reaction field. This inclusion of the reaction field leads to a further, equally-plausible conformation, which is more justifiable physically as well as in terms of the pharmacological data available on neurotensin. Factors leading to different residue conformations and the relative merits of the different results obtained are analyzed in some detail to cast further light on the aspects that must be considered in the analysis and design of biologically-active analogues of neurotensin.     

Conformational energy calculations on Substance P

The conformational and spacial configurations of the biologically active undecapeptide, Substance P, were studied using conformational energy calculations. Low energy conformers of residues 1–5 and 6–11 were found by energy minimization and the two fragments were then combined to find low-energy structures for Substance P. Several configurational classes were found with different backbone conformations. A comparison of the final low-energy structures with data from biological tests on analogs of Substance P gives some insight into the conformation required for interaction at the biological receptor.     

Conformation study of neurotensin C-terminal heptapeptide Pro-Arg-Arg-Pro-Tyr-Ile-LeuOH

The conformational behavior of the active C-terminal heptapeptide of neurotensin (NT), Pro-Arg-Arg-Pro-Tyr-Ile-LeuOH, or NT^{7–13}, was investigated using empirical energy calculations. A sequential approach was used to display the specific contribution of each residue to induce stable conformations of the whole heptapeptide. The most stable conformations include numerous conformations of various types in a rather limited energy range. There is no preferential conformation contrary to the active C-terminal pentapeptide. These structures may coexist in the biological environment.     

Conformational study of two substance P hexapeptides by two-dimensional NMR

The conformation of two substance P (SP) related hexapeptides. Glp-Phe-Phe-(L-Pro)-Leu-Met.NH₂(I) and Glp-Phe-Phe-(D-Pro)-Leu-Met.NH₂ (II), in two solvents, chloroform-d and trifluoroethanol(TFE)-d₃/H₂O, was studied by two-dimensional NMR methods, including COSY, TOCSY, ROESY and HMQC. The study shows that these two peptides exist predominantly in the extended form in TFE/H₂O, but in general exhibit a reverse-turn structure in chloroform. I is clearly less ordered than II in both solvents. Furthermore, extensive Phe³-Pro⁴cis?trans isomerization was found in I but not in II. The differences in the conformational behavior of these two peptides, which are selective agonists for neurokinin NK1 and NK2 receptors, respectively, are discussed.     

Substance P antagonists and mucociliary activity in rabbit

Summary Substance P (SP) is known to accelerate mucociliary (m.c.) activity in the rabbit maxillary sinus in vivo. The physiological significance of this finding was investigated by testing three putative SP antagonists. [Arg⁵, d-Trp^{7, 9}, Nle¹¹]SP_5–11 could not be used as an antagonist because it stimulated m.c. activity. [d-Arg¹, d-Trp^{7, 9}, Leu¹¹]SP had no effect on the m.c. activity changes induced by SP. [d-Pro², d-Trp^{7, 9}]SP was found to be an effective antagonist, 1 mg/kg of this drug reversibly inhibiting both the effects of 0.1 g/kg SP and the stimulating effect of 1.0 g/kg bradykinin and 30.0g/kg capsaicin; the stimulating effect of 0.5 g/kg methacholine was not inhibited. It is suggested that bradykinin and capsaicin stimulate m.c. activity at least partly by releasing SP.The results of this investigation also support the view that the accelerating effect of SP on m.c. activity reflects physiological SP-mediated protective mechanisms in the airways. It is concluded that [d-Pro², d-Trp^{7, 9}]SP is a useful pharmacological tool for studying the role of SP in the control of m.c. actvity in rabbits.     

Substance P enhancement of passive and active avoidance conditioning in mice

In a series of seven experiments we explored the effects of peripherally administered substance P on passive and active avoidance conditioning in mice of two genotypes. The peripheral post-trial administration of substance P significantly enhanced the retention of a single-trial passive avoidance task. This effect was dose dependent; 1 ng/g of substance P enhanced the retention of this habit, whereas higher and lower doses were either less effective or ineffective. In heterogeneous strain (HS) mice, substance P administered before training on an active avoidance task did not alter the rate at which these animals learned this habit. However, animals that had been trained with substance P were significantly more resistant to extinction than were animals that had been injected with vehicle. Similarly, C57Bl/6J mice that had been treated with substance P immediately after active avoidance training were more resistant to extinction than were mice that had been given control injections. The enhancement of retention of the passive avoidance habit with substance P was reversed in animals that had been pretreated with naltrexone. Substance P enhancement of the retention of the passive avoidance habit, and its reversal with naltrexone, was observed in both sham operated and adrenalectomized mice.     

Substance P mediates atropine-sensitive response of guinea-pig ileum to serotonin

The action of serotonin (5HT) was investigated on guinea-pig isolated ileum. Low concentrations of 5HT (10(-8) - 2.5 x 10(-7) mol x 1(-1)) produced contractile responses which were abolished by atropine and by methionine enkephalin but not by methysergide. Higher concentrations of 5HT (5 x 10(-7) - 5 x 10(-6) mol x 1(-1)) produced contractions which were inhibited by methysergide. The atropine-sensitive response was also abolished following desensitization of preparations to substance P and by the substance P antagonist [D-Pro2,D-Phe7,D-Trp9]SP. It was concluded that the atropine-sensitive response to 5HT is mediated by substance P.     

Substance P and antistrychnine activity

Summary The administration to mice of crude substance P prepared from bovine brain inhibited strychnine convulsions. After alumina column chromatography, this antistrychnine activity was observed mainly in the fraction which was not adsorbed, while those fractions containing substance P or its satellite polypeptides (Zetler, 1963) were found ineffective. The regional distribution of such an antistrychnine activity in bovine brain was also found to be different from that of substance P. Experiments in which inactivation with proteases and dialysis were investigated suggested that the component or components bearing the antistrychine activity were of a polypeptide nature.     

Synthesis of (pGlu-5, MePhe-8, Sar-9) Substance P (5–11) (DiMe-C7) using a polyacrylamide resin and biological activity on guinea pig ileum and tracheal smooth muscle

DiMe-C7 (pGlu-Gln-Phe-MePhe-MeGly-Leu-MetNH₂), a metabolically stable analogue of Substance P, was prepared by solid-phase peptide synthesis using a polyacrylamide resin and a labile anchorage derived from glycolic acid. Myotropic activities in guinea pig ileum (ED₅₀ = 4.0 ± 1.5 10^-8 M) and guinea pig trachea (ED₅₀ = 8.6 ± 3.5 10^-8 M) are discussed in comparison with the corresponding activities of Substance P.     

Effect of neurotensin, substance P and TRH on the regulation of dopamine receptors in rat brain

Rats were exposed for one week to either neurotensin (4 micrograms/h), substance P (3.3 micrograms/h), thyrotropin-releasing hormone (5 micrograms/h), or saline administered intracerebroventricularly via mini osmotic-pumps and either haloperidol (2.5 mg/kg i.p., 2 X daily) or vehicle control. The peptide treatments by themselves did not alter [3H]spiroperidol binding in either the nucleus accumbens or the striatum. Neurotensin, however, augmented the increase in [3H]spiroperidol binding caused by the haloperidol treatment in both the nucleus accumbens and striatum.     

P物质多囊脂质体的制备工艺优化及相关性质考察

目的:优化P物质多囊脂质体的制备工艺,并考察其相关性质。方法:复乳法制备P物质多囊脂质体,以包封率为指标,磷脂与胆固醇的摩尔比(A)、超声乳化时间(B)、初乳与二相小的体积比(C)、辅助乳化剂的浓度(D)为因素进行正交试验设计,优化其制备条件;对优化条件所制脂质体进行包封率、粒径分布、体外释药性能、相转变温度和熔点的考察。结果:优化制备条件A为2:1,B为30 s,C为1:2,D为2%;所制多囊脂质体包封率可达85%,粒径分布为0.75～27.75μm,在生理盐水中释药t_(1/2)=11 h,可持续72 h释药,相转变温度为34℃,熔点为110℃。结论:所制P物质多囊脂质体包封率高,物理性能稳定,是一种较为理想的缓释制剂。     

Synthesis and biological activity of glycosylated analogs of the C-terminal hexapeptide and heptapeptide of Substance P

The synthesis of two glycosylated analogs of Substance P is described. The activity of the peptides was assayed on the isolated guinea-pig ileum and their degradation was studied using rat hypothalamus slices. While glycosylation noticeably enhances the solubility of the corresponding compounds, the β-glucopyranosyl moiety only slightly modifies the biological half-life and the bioactivity of the glycopeptides.     

Synthesis and biological activity of Substance P C-terminal hexapeptide and heptapeptide analogues

The analogues [Glu(OBzl)¹¹]SP_6–11 and [Glu(OBzl)¹¹]SP_5–11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH₃ group of Met¹¹ is replaced by the COOCH₂C₆H₅ group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.     

藿香正气液对大鼠P物质的影响

目的:观察藿香正气液对大鼠P物质(SP)的影响。方法:将大鼠随机分为实验(藿香正气液)组和对照(生理盐水)组,分别灌胃相应药物1h、6h后观察大鼠胃肠动力的变化,测定血浆、胃窦和空肠组织匀浆中SP的含量及观察胃窦、空肠组织中SP阳性产物的分布情况。结果:实验组用药后大鼠胃肠动力显著增强,以用药后1h作用更明显;与对照组比较,实验组血浆、胃窦及空肠组织匀浆中SP含量及胃窦和空肠组织中SP阳性产物含量明显增加(P<0.05或P<0.01)。结论:藿香正气液促胃动力作用可能与其对SP的影响有关。     

Synthesis of potent agonists of Substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of Substance P

The analogues [Glp⁶,Glu(OBzl)¹¹]SP_(6-11) and [Glp⁵,Glu(OBzl)¹¹]SP_(5-11)) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH₂C₆H₅ ester group has replaced the SCH₃ group of the Met¹¹ side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively. © Munksgaard 1995.     

Conformations of neurotensin in solution and in membrane environments studied by 2-D NMR spectroscopy

Two-dimensional HOHAHA and ROESY nuclear magnetic resonance techniques are used to obtain complete proton resonance assignments and to perform a conformational investigation of the neuropeptide neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) in aqueous solution, methanol, and membrane-mimetic [deuterated sodium dodecylsulfate (SDS)] environments. Results suggest the absence of discernible elements of secondary structure in water and methanol. ROESY spectra confirm that Lys-Pro and Arg-Pro peptide bonds are all-trans, but that a significant population of cis Arg-Pro bonds arises in aqueous solution, which increases in the environment of SDS micelles. The conformational ensemble of the peptide is observed to narrow as it becomes bound through its cationic mid-region to SDS micelles, with the accompanying advent of local extended structure. The overall results indicate the inherent conformational flexibility of neurotensin, and emphasize the environmental dependence of conformation in peptides of medium length.     

Specificity of antisera obtained to Substance P and its C-terminal hexapeptide

Synthetic fragments and analogs were used to characterize specificity of antisera toSP and SP_6–11. [Tyr⁸]SP and [Lys⁶]SP_6–11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP_7–11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP_6–11 by His or Gly showed that all but Glu⁶ take part in the structure of the antigenic determinant.     

Conformational study of vasoactive intestinal peptide by computational methods

The conformational profile of vasoactive intestinal peptide (VIP) was characterized using computational methods. The strategy devised included a close examination of the conformational profile of the first 11 residues fragment followed by a study that considered the compatibility of the different conformations found with a continuation of the polypeptide chain in a-helical conformation. Accordingly, a detailed analysis of the conformational preferences of the N-terminal fragment of VIP(1–11) was carried out within the framework of the molecular mechanics approach, using simulated annealing in an iterative fashion as the sampling technique. In a second step, low-energy structures of the fragment were fused to the remainder of the VIP chain in the form of two noninteracting α-helices, according to a model of the structure of the peptide proposed from NMR studies. After investigation for compatibility of each of the low-energy structures of VIP(1–11) with the two helical regions by energy minimization, only 5 of 35 structures were discarded. Analysis of the structures characterized indicates that most of the conformations of VIP(1–11), including the global minimum, can be described as bent conformations. Conformations exhibiting α-turns and ß-turns, previously proposed by NMR studies were also characterized. The conformational analysis also suggests that the common structural features found in VIP(1–11) should also be present in VIP. Finally, because of the sequence homology between VIP and Peptide T, and the fact that both are ligands of the CD4 receptor, both sets of low-energy conformations were compared for similarity. The relevance of these results as guidance of the design of new peptide analogs targeted to the CD4 receptor is also discussed.     

Cyclization of peptides on a solid support. Application to cyclic analogs of Substance P

The general conditions for cyclization of peptides on polymer matrix by disulfide bridge formation are reported. This procedure is based on attack of 3-nitro-2-pyridinesulfenyl group (Npys) by a thiol function. It has been used for synthesis of five cyclic analogs of Substance P.     

Substance P enteric neurons mediate non-colinergic transmission to the circular muscle of the guinea-pig intestine

Summary The sites of action and possible roles of substance P in contracting the circular muscle of the guinea-pig ileum were studied using two analogues of substance P that act as antagonists of some of its actions. These ared-Arg¹,d-Pro²,d-Trp^7,9, Leu¹¹-substance P andd-Pro²,d-Trp^7,9-substance P, referred to by the single letter amino acid codes for the substituting amino acids as (RPWWL)-SP and (PWW)-SP, respectively.Records of circular muscle activity were taken from strips of intestine free of mucosa and submucosa and from rings with all layers of intestine intact. Substance P was equally effective in contracting the circular muscle strips as it was in contracting the longitudinal muscle. The contractions of strips were not blocked by hyoscine (2×10^–6 M) or tetrodotoxin (6×10^–7 M), but were substantially reduced by (RPWWL)-SP (6.7×10^–6 M) or (PWW)-SP (2×10^–5 M). In contrast, contractions of the circular muscle of whole rings of intestine elicited by low concentrations of substance P (4×10^–7M) were blocked by hyoscine or tetrodotoxin but notreduced by the substance P antagonists in the concentrations referred to above. These observations indicate that the antagonists are effective at receptors for substance P on the muscle, but not at substance P receptors on enteric cholinergic nerves.Transmural stimulation of strips of circular muscle or of intestinal rings in the presence of hyoscine evoked contractions that were blocked by tetrodotoxin. These hyoscineresistant, nerve-mediated contractions could be elicited by single pulses in the strips. The contractions were reduced to less than 20% of original amplitude by (RPWWL)-SP (6.7×10^–6M).Reflex contractions of the circular muscle recorded on the oral side of a distension stimulus had a low-threshold, hyoscine-sensitive and a high-threshold, hyoscine-insensitive, component. The low threshold component was unaffected by the substance P antagonists whereas the high threshold component was depressed.It is concluded that substance P nerves are effective in transmitting to the circular muscle, that they are final nerves in non-cholinergic excitatory reflexes, and that the substance P antagonist analogues can be used to distinguish actions of substance P at neural and muscle receptors.