Effects of substance P and its fragments in physiological and pathological pain

It has been shown that substance P and its fragments can produce under certain conditions an analgetic effect on both physiological and pathological pain (i.e. on pain syndrome of spinal origin). The data obtained give evidence that prolonged hypoalgesia is caused by the injection of substance P and its fragments to nucleus raphe dorsal--a structure of the antinociceptive system. This analgetic effect can be initiated by the activation of the antinociceptive system influenced by substance P and or its fragments.     

I. P. Pavlov on animal experiments]

The paper is dedicated to the 150th anniversary of academician I.P. Pavlov's birthday. Pavlov's points of view on vivisection are analyzed and are compared with the modern conception of ethics of carrying out experiments on animals. It is discovered that Pavlov's points of view are in accordance with the key positions of modern requirements to ethics of experimenting on animals which are adopted in the world practice.     

Immunochemical analysis of substance P using its synthetic fragments and analogs

Synthetic fragments and analogs were used to characterize specificity of antisera to substance P. Both, the C-terminal hexapeptide and the pentapeptide completely inhibited binding of 125I-[Tyr8]substance P by these antisera, showing the antigenic identity with substance P. Synthetic fragments shorter than peptide (7-11) did not react with anti-substance P antisera in this system. Substitution of amino acids in different positions in the fragments (6-11) or (7-11) by histidine or glycine revealed that all five amino-acid residues take part in a structure of the antigenic determinant.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Effects of intranasally administered substance P in parkinsonian syndrome]

The effect of intranasal substance P injection on parkinsonian syndrome and the generator of pathologically enhanced excitation (GPEE) in caudate nuclei (CN) was investigated. MPTP or reserpine administration in old rats induced oligokinesia, rigidity and tremor followed by the high amplitude slow and rapid waves in both CN. The bilateral intranasal injection of substance P (25 micrograms/kg) resulted in an increase in motor activity and almost completely abolished the rigidity and tremor. The reduction of extrapyramidal symptoms was considered as a result of the inhibition of GPEE in CN. The possibility of substance P entry from nasal cavity into the brain was discussed. The changes of the substance P balance in nigrostriatal system was suggested to be on of the pathogenetic links of parkinsonian syndrome.     

Effects of aspartate,substance P,and serotonin on neuronal activity in the central gray substance:In vitro experiments

Effects of aspartate (2 · 10^–5 M), substance P (10^–7–10^–8 M), and serotonin (5-hydroxytryptamine, 5-HT; 5 · 10^–5 M) on the background activity of neurons in the central gray substance (CGS) were studied on slices of the rat midbrain. Aspartate and substance P (transmitters of nociceptive signals), and 5-HT (modulator of transmission of nociceptive influences) were found either to facilitate or to depress the activity of CGS neurons. The predominant effect of substance P or 5-HT applications to neurons of the dorsal CGS part was facilitation, and to neurons of the ventral CGS part, inhibition. The effects of aspartate application on studied CGS neurons were of varying nature, but inhibitory effects were found to prevail.The findings support our earlier hypothesis that assigned the studied neurons to spontaneously discharging inhibitory CGS interneurons, which control the activity of efferent CGS neurons. The role of tested substances in the regulation of CGS neuronal activity and the antinociceptive CGS effects is discussed.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 354–362, September–October, 1993.     

Conversion of substance P to C-terminal fragments in human plasma

Substance P is rapidly converted by enzyme(s) in human plasma to des-[Arg1Pro2]-substance P (fragment 3-11) and to des-[Arg1Pro2Lys3Pro4]-substance P (fragment 5-11). These metabolites were isolated by HPLC and partially sequenced. No evidence was obtained for deamidation of substance P in plasma or for the formation of the N-terminal tetrapeptide [Arg-Pro-Lys-Pro]. The data suggest that substance P is metabolized in human plasma by an enzyme with the specificity of dipeptidyl-aminopeptidase IV. Consistent with this hypothesis, the rate of degradation of substance P measured with an antibody directed against the N-terminal region is 2-3-fold greater than measured with a C-terminally directed antibody. The degrading activity of plasma was purified 522-fold and was eluted from a gel filtration column in the molecular weight zone 150 000-170 000 and from a chromatofocusing column in the pH range 4.5 to 5.5.     

I. P. Pavlov on vivisection and the modern ethical requirements for animal experiments]

It is shown that Pavlov's opinion is in agreement with the modern requirements for experiments in living animals: use of living animals in experiments is recommended only in the cases when there is no alternative and scientific-practical significance of the planned experiments is justified; minimization (as much as possible) of discomfort, distress and pain of animals under study without sacrifice of the quality of scientific research; use of appropriate number of animals necessary for obtaining reliable results which are adequate to a given experimental situation and general state of the animals inadmissibility of using extra number of animals; drawing up a protocol concerning the experimental procedure and results; reflection in the protocol the techniques of sedation, anesthesia, and euthanasia; constant care of the improvement of the theoretical and practical qualification of a researcher in the ethical questions and experimental technique. Pavlov's views led his time and at present they agree with modern concepts on the experimental ethics.     

Comparative study of substance P and its fragments: analgesic properties, effect on behavior and monoaminergic processes

The effect of substance P (SP) and of its fragments 5-11, 8-11, 9-11, 10-11 administered into the brain ventricles in doses of 5, 25 and 50 nM on the behavior and content of biogenic monoamines of the rat brain was studied. The analgetic properties of the substances under consideration and those of fragment SP 10-11 in doses of 5, 25, 50 and 100 nM were also subjected to examination. It was found that SP and fragment 5-11 stimulate and enhance the locomotor activity in rats, while fragments 8-11 and 9-11 provoke hypoactivity. The substances under study increase the serotonin and dopamine turnover, whereas SP and fragment 8-11 lower the serotonin content as well. After administration of SP and fragment 5-11 analgesia was seen to transform to hyperalgesia depending on the dose. Fragments 8-11 and 9-11 produce analgetic effect. It is suggested that both SP fragments and the whole SP molecule can influence the neurochemical process that regulate behavior and pain perception.     

Antinociceptive and Met-enkephalin releasing effects of tachykinins and substance P fragments

Substance P (SP), physalaemin, SP4-11, SP5-11 and the SP5-11 analog DiMe-C7 induce an antinociceptive effect in rats after intraventricular administration. Other tachykinins and the N-terminal fragments of SP are inactive. All antinociceptive peptides increase the Met-enkephalin efflux from slices of rat periaqueductal gray matter and their antinociceptive potency is correlated with their capacity to release Met-enkephalin. The results, discussed in the light of current theories on different tachykinin receptors, suggest that the SP-P receptor subtype may be involved in the control of noxious stimulation elicited by SP at supraspinal levels.     

Intrathecal substance P augments morphine-induced antinociception: Possible relevance in the production of substance P N-terminal fragments

The present study sought to examine the mechanism of substance P to modulate the antinociceptive action of intrathecal (i.t.) morphine in paw-licking/biting response evoked by subcutaneous injection of capsaicin into the plantar surface of the hindpaw in mice. The i.t. injection of morphine inhibited capsaicin-induced licking/biting response in a dose-dependent manner. Substance P (25 and 50 pmol) injected i.t. alone did not alter capsaicin-induced nociception, whereas substance P at a higher dose of 100 pmol significantly reduced the capsaicin response. Western blots showed the constitutive expression of endopeptidase-24.11 in the dorsal and ventral parts of lumbar spinal cord of mice. The N-terminal fragment of substance P (1–7), which is known as a major product of substance P by endopeptidase-24.11, was more effective than substance P on capsaicin-induced nociception. Combination treatment with substance P (50 pmol) and morphine at a subthreshold dose enhanced the antinociceptive effect of morphine. The enhanced effect of the combination of substance P with morphine was reduced significantly by co-administration of phosphoramidon, an inhibitor of endopeptidase-24.11. Administration of d-isomer of substance P (1–7), [d-Pro², d-Phe⁷]substance P (1–7), an inhibitor of [³H] substance P (1–7) binding, or antisera against substance P (1–7) reversed the enhanced antinociceptive effect by co-administration of substance P and morphine. Taken together these data suggest that morphine-induced antinociception may be enhanced through substance P (1–7) formed by the enzymatic degradation of i.t. injected substance P in the spinal cord.     

Effects of substance P on acetylcholinesterase activity

The effects of substance P on acetylcholinesterase activity have been examined. The neuropeptide produced a significant increase in the activity of the enzyme in rat cerebral cortex. Pretreatment of rats with either actinomycin-D or cycloheximide did not fully abolish the substance P-mediated stimulation of cerebral acetylcholinesterase. Substance P increased the enzyme activity in rat brain slices; moreover, substance P increased the activity of electric eel acetylcholinesterase in in vitro experiments. These observations indicate that substance P produces an induction of acetylcholinesterase in cerebral cortex of rats and in addition indicate that a direct action on the enzyme takes place.     

Conformational study of the neurotensin and substance P fragments: Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro

The conformational behaviour of the basic hydrophilic Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro peptides, neurotensin (NT) and Substance P fragments, has been taken up by semi-empirical calculations. The presence of two Pro residues prevents these peptides from giving any folded structure (alpha helix, beta turn . . .). In both peptides the most stable conformations are essentially relative to more or less stretched structures; structures involving one or more residues in a gamma turn form are often encountered in Pro-Arg-Arg-Pro peptide while mixed structures involving residues in very different conformations are found for the Arg-Pro-Lys-Pro-peptide. In both peptides, positively charged Lys and Arg side-chains most often point in opposite directions. The Pro-Arg-Arg-Pro peptide is part of the active NT (7-13) fragment where both Arg residues are necessary to the activity. A tentative study shows that the hydrophilic tetrapeptide induces NT (7-13) stretched conformations.     

Failure of tachykinins including substance P and its fragments to influence proteoglycan and protein synthesis in bovine chondrocytes in vitro.

Adult bovine articular chondrocytes were exposed to substance P, neurokinins A and B or substance P fragments, SP1-4, SP1-6 and SP7-11 in vitro. Proteoglycan synthesis was assessed by measuring proteoglycans which were released into the culture medium or incorporated into the cell layer. The intact tachykinins or substance P fragments had no direct effect on proteoglycan synthesis. Nor was total protein production affected. Gel chromatography, under dissociative conditions, revealed that sulphated proteoglycans detected in the medium or cell layer following treatment of chondrocytes with substance P, contained proteoglycans of similar molecular weight to those produced by cells exposed only to diluent controls. Therefore, we conclude that the acceleration of arthritis by substance P does not appear to be mediated through an effect on chondrocyte synthetic function.     

Effects of T-activin in viral myocarditis in animal experiments

The efficacy of immunomodulating agent T-activin has been studied in adult BALB/C mice infected with Coxsackie B1 virus. Optical, electron microscopic and immunological tests have shown that T-activin protects myocardium and modulates immune disorders in mice with viral myocarditis.     

Effects of substance P on experimental parkinsonian syndrome

The aim of this study was to investigate the effect of substance P (SP) on parkinsonian syndrome and the generator of pathologically enhanced excitation (GPEE) in the caudate nuclei (CN). Repeated i. p. administration of MPTP in 12 month rats induced oligokinesia and rigidity followed by the high amplitude slow and rapid waves in both CN. The changes of electrical activity in CN were more prominent than in the sensorimotor cortex. The bilateral intracaudate injection of SP (5 micrograms) resulted in an increase in motor activity and almost completely abolished the rigidity. The reduction of extrapyramidal symptoms was considered as a result of the inhibition of GPEE. The changes of the SP balance in nigro-striatal system was suggested to be one of the pathogenetic links of parkinsonian syndrome.     

An NMR study of the conformations of N-terminal substance P fragments and antagonists

Three N-terminal fragments of the neurotransmitter Substance P as well as two antagonist heptapeptides containing D-amino-acid residues were studied using different 1D and 2D NMR techniques. Total nonexchangeable 1H-NMR assignments were carried out in D2O and the NH protons were assigned in H2O by means of COSY experiments. The spectral data indicates that there are no preferred conformations for the backbone. The N-terminal tetrapeptide SP1-4-OH exists as a mixture of cis/trans isomers and this effect was studied as a function of pH.     

Radioimmunoassay of substance P and its stability in tissue

A sensitive and specific radioimmunoassay for substance P has been developed and its full characterisation is described. The assay was used to investigate the stability of substance P in tissue in order to establish the optimal conditions of tissue storage for maximal recovery of the peptide. The results indicate unexpected post-mortem changes in tissue substance P content and suggests its existence in at least two separate pools, which differ in their susceptibility to degradation. These results highlight the need for careful consideration of both extraction technique and assay characteristics in radioimmunological investigations of substance P.