Substance P Research & Studies

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

The wound healing process requires enough blood to supply nutrients and various growth factors to the wound area. However, chronic wounds such as diabetic skin ulcers have limited regeneration due to a lack of cellular and molecular signals because of a deficient blood flow. Mesenchymal stem cells (MSCs) are known to provide various factors, including growth factors, cytokines, and angiogenic mediators. Although MSCs have great therapeutic potential, their transplantation has many obstacles, including the time required to culture the cells, the invasiveness of the procedure, and limited stem cell sources. In this study, we induced a diabetic 1 model in rats aged 7 weeks by injecting streptozotocin and citrate buffer solution. After confirming that diabetes was induced in the rats, we created critical sized wounds on the dorsal area of the rats and then injected hydrogels. We performed the experiments with four groups (defect model for the control, self-assembled peptides (SAPs), SAP with soluble substance P, and SAP conjugated with substance P) to treat the wound defect. Tissues were harvested at 1, 2, and 3 weeks after injection and examined for the wound closure, histological analysis, quantitative real-time polymerase chain reaction analysis, and quantification of collagen deposits to investigate stem cell recruitment and full recovery of wounds at an accelerated time period. As our results show, the wounds treated with SAP and substance P exhibited significantly accelerated wound closure, enhanced collagen deposition, and increased angiogenesis. Furthermore, we confirmed the ability of SAP with substance P to promote the recruitment and homing of cells by immunofluorescence staining of a MSC marker. In addition, it was observed that substance P remained in the wound area up to 3 weeks after the injection of SAP with substance P. It is believed that the endogenous MSCs mobilized by substance P had therapeutic effects through their proper differentiation and release of paracrine factors into the wound sites. In conclusion, this study shows that SAP with substance P can promote wound healing to enhance skin regeneration without cell transplantation in a diabetic model.

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.

Formation of mature active neuropeptides such as substance P (SP) from their glycine extended precursors entails alpha-amidation of peptide precursors by the sequential enzymatic action of peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL). We reported that these two enzymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidation of peptides occurs in endothelial cells and that these peptides are critically involved in the overall regulation of cardiovascular function. In this study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precursor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulating nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PBA) and PGL (5-acetamido-2,4-diketo-6-phenyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 34, and 45%, respectively. They also reduced the production of NO induced by SP-Gly by 63, 68, and 69%, respectively, but had no effect on NO production in response to either SP or acetylcholine. SP and SP-Gly also were equipotent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endothelium dependent and inhibited by the NOS antagonist L-nitroarginine methyl ester (L-NAME), but it was not affected by inhibition of prostaglandin synthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelaxing actions of SP-Gly, whereas responses to SP were not affected. A cumulative infusion of PBA into the femoral artery of rabbits, at final concentrations of 2.4, 24, and 240 microM for 20 min each, increased the vascular resistance (VR), indicating the tonic production of vasodilating amidated peptide(s). This effect was maximum at 60 min after infusion (20.5 +/- 4.7 vs. 8.2 +/- 0.7 mm Hg/ml/min; p < 0.05). These results suggest that endothelial cells can produce mature SP from its SP-Gly precursor and that a product of peptide alpha-amidation tonically stimulates endothelial cell NO release to control vascular tone.

The membrane-associated conformation of substance P (RPKPQQFFGLM-NH2) has been previously proposed to be the NK1-receptor-active conformation. In this work, NMR methods are applied to explore the orientation and dynamics of substance P at lipid surfaces for which the peptide's three-dimensional structure had been previously determined. Here the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS) micelles has been found to cause sequence specific changes in the acid- and base-catalyzed amide proton exchange rates relative to the solution state values. On binding of substance P to SDS micelles, the FFG portion showed the largest decreases in the base-catalyzed amide exchange rates. Similar sequence-specific changes in substance P are observed in the presence of DPC micelles, albeit at much weaker levels due to fast exchange between free and bound forms of the peptide. These differences are attributed to the location of the amide protons either in the surface double layer (via electrostatic effect) or inserted into the polar head group region of the micelles (via low dielectric). The sequence-specific effects of micelle association were also observed in the homonuclear nonselective spin-lattice relaxation time; these, in combination with spin-spin relaxation times, were used to calculate correlation times for the backbone amide protons. These data combined with paramagnetic broadening observations on peptide protons in the presence of spin-labeled lipids yield a detailed model of the interaction of substance P with lipid surfaces.

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Animal studies suggest that the neuropeptides, substance P and vasoactive intestinal peptide (VIP), may influence carotid body chemoreceptor activity and that substance P may take part in the carotid body response to hypoxia. The effects of these peptides on resting ventilation and on ventilatory responses to hypoxia and to hypercapnia have been investigated in six normal humans. Infusions of substance P (1 pmol.kg-1.min-1) and of VIP (6 pmol.kg-1.min-1) were compared with placebo and with nitroprusside (5 micrograms.kg-1.min-1) as a control for the hypotensive action of the peptides. Both peptides caused significantly less hypotension than nitroprusside. Substance P and nitroprusside caused significantly greater increases in ventilation and in the hypoxic ventilatory response than VIP. No changes were seen in hypercapnic sensitivity. The stimulation of ventilation and the differential effects on ventilatory chemosensitivity that accompanied hypotension are consistent either with stimulation of carotid body chemoreceptor activity or with an interaction with peripheral chemoreceptor input to the respiratory center, as is seen in animals. The similar cardiovascular but different ventilatory effects of the peptides suggest that substance P may also stimulate the carotid body in a manner independent of the effect of hypotension. This is consistent with a role of substance P in the hypoxic ventilatory response in humans.

Primary afferent nerve terminals located in the mammalian gut wall may play a role in region-specific modulation of gastrointestinal motility. In the present study, we sought to characterize the effect of neuropeptides released from these afferents by capsaicin (CAP) on contractile activity of smooth muscle from the distal rabbit colon. CAP caused a release of acetylcholine and immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) from the muscle coat. CAP caused a dose-dependent transient stimulation of longitudinal muscle contractions, followed by prolonged inhibition of spontaneous but not stimulated contractile activity. The initial stimulation was abolished by the SP antagonist spantide and by atropine. The inhibitory effect was reduced by repeated exposure of muscle to CGRP. The effect of CGRP on spontaneous contractions differed between longitudinal and circular muscle. In longitudinal muscle, a stimulation was preceded by a transient inhibition, whereas in circular muscle, only inhibition was seen. Both effects were resistant to tetrodotoxin. Repeated exposure of circular but not longitudinal muscle to CGRP resulted in a disappearance of the peptide's inhibitory effect. Exogenously applied CGRP was only a weak antagonist of contractions stimulated by SP and bethanechol. These findings suggest that in the rabbit colon at least the following two neuropeptides are released from CAP-sensitive nerve fibers: a neurokinin peptide from nerve terminals located within the myenteric plexus and CGRP from terminals probably located within the circular muscle layer.

The multiple and diverse roles played by neuropeptide Y, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide and other biologically active peptides in the cardiovascular system are considered. A model of the vascular neuroeffector junction is described, which illustrates the interactions of peptidergic and nonpeptidergic transmitters that are possible at pre- and postjunctional sites. The effects of peptides on specific endothelial receptors are also described, which highlights the ability of these agents to act as dual regulators of vascular tone at both adventitial and intimal surfaces, following local release from nerves, or from endothelial cells themselves. Changes in expression of vascular neuropeptides that occur during development and aging in some disease situations and following nerve lesion are discussed.

Recent research has disclosed that neurotransmitters and neuropeptides released within the autonomic nervous system exert homeostatic control of nasal secretion. Although cholinergic and adrenergic influences have long been thought to be the predominant mechanisms, the nonadrenergic, noncholinergic responses may have more suitable, longer-lasting effects. Peptides from sensory nerves, such as calcitonin gene related peptide, substance P, and neurokinin A, may participate in axon response-mediated vasodilation and plasma extravasation. Substance P and gastrin releasing peptide may induce glandular secretion. Defensive responses to local mucosal injury may be amplified by axon response, which initiates these vascular and glandular reactions. Cholinergic effects are primarily responsible for mediating parasympathetic reflexes, but vasoactive intestinal peptide may regulate acetylcholine release, augment glandular secretory responses, and have a vasodilatory effect. In the sympathetic nervous system, neuropeptide Y probably functions as a long-acting vasoconstrictor. Integration of sympathetic and parasympathetic influence may regulate the normal nasal cycle, and sensory and parasympathetic defensive reflexes may respond to epithelial and mast cell stimulation. It is possible, then, that the pathophysiology of vasomotor rhinitis involves an exaggeration of these neural influences.

High levels of substance-P are present in the plasma of patients with carcinoid tumours and some thyrotoxic conditions. The majority of the substance-P in the blood plasma was shown, by immunoassay, to be associated with high molecular-weight material in a complex that could be dissociated by repeated gel-filtration. Smaller amounts of an intermediate molecular-weight (about 65,000 Da) complex were also detected. Chemical crosslinking with glutaraldehyde was used to show that the radioactively-labelled derivative [125I]Tyr-8-substance-P was able to bind to the high-Mr fraction of human plasma and also to human serum albumin. Binding to serum albumin was also demonstrated by equilibrium gel-filtration. Substance-P added to human plasma from a thyrotoxic subject, which contained high endogenous levels of the tachykinin (980 pg/mL), was rapidly degraded during incubation at 37 degrees, whereas the endogenous substance-P was considerably more stable. These results suggest that the binding of substance-P to blood plasma components may play an important role in protecting it against degradation. Furthermore, immunoassay techniques involving prior extraction, which fail to detect the bound substance-P, will give inaccurate measurements of the levels of this peptide in plasma.

The brain is both the source and the recipient of peptide signals. The question is: Do endogenous, blood-borne peptide molecules influence brain function? Brain regions with the tight capillaries of the blood-brain barrier (BBB) extract low but measurable amounts of labeled peptide molecules from an intracarotid bolus injection. In the rat, the extraction fractions of beta-casomorphin-5, DesGlyNH2-arginine-vasopressin, arginine-vasopressin, lysine-vasopressin, oxytocin, gonadoliberin, substance P, and beta-endorphin, studied in this laboratory, range from 0.5% (substance P) to 2.4% (arginine-vasopressin). Extraction varies little among the 15 examined brain regions. As shown for arginine-vasopressin, the extracted peptides may be bound in part to specific binding sites located on the luminal membrane of the tight endothelial cells. Transport of peptide molecules across the BBB cannot be ruled out, but it is unlikely that endogenous peptides pass the BBB in physiologically significant amounts. In contrast, in brain regions with leaky capillaries, e.g., selected circumventricular organs including the pineal gland, neurohypophysis, and choroid plexus, the peptide fraction extracted approaches that of water. Within the circumventricular organs, the peptide molecules actually reach the cellular elements of the tissue. However, no studies definitively show that peptides reach neurons in the deeper layers of the brain. On the other hand, blood-borne peptides influence the BBB permeability by altering the transport of essential substances. The effect may be mediated by specific peptide binding sites located at the luminal membrane of the endothelium. It is possible that the effect of peptides on the BBB is necessary for proper brain function.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of substance P in human plasma at 37 degrees C resulted in rapid conversion to des (Arg1-Pro2) substance P (fragment 3-11) and to des (Arg1-Pro2-Lys3-Pro4) substance P (fragment 5-11). The metabolites were purified by high-performance liquid chromatography and identified by sequence analysis. These data are consistent with the hypothesis that substance P is metabolized by enzyme(s) with the specificity of dipeptidyl aminopeptidase IV (EC 3.1.14.5). Analysis by high-performance liquid chromatography of plasma extracts following intravenous infusion of Substance P (300-350 nmoles) into anaesthetized rats showed that the peptide was cleared from the circulation within 1-2 minutes. No circulating metabolites could be identified.

Prolyl endopeptidase (E.C. 3.4.21.26) an enzyme previously called post proline cleaving enzyme, TRH-deamidase or kininase B, may play a role in neuropeptide metabolism. This enzyme, highly active in brain and other tissues, catabolizes proline-containing peptides such as substance P, neurotensin, luteinizing hormone-releasing hormone, thyrotropin releasing hormone, bradykinin and angiotensin II. The structure of beta-neo-endorphin suggests that this opioid peptide is formed by the action of prolyl endopeptidase on a precursor of higher molecular weight. Formation of two biologically active fragments of substance P also requires the action of this enzyme. This review summarizes the current knowledge of the biochemistry of this enzyme, and its potential significance for neuropeptide physiology and pharmacology.

Substance P, somatostatin, VIP, CCK, angiotensin II, and bombesin have all been localized by immunohistochemical or radioimmunological means in neurons of sensory ganglia or in the dorsal horn of the spinal cord. Most of these neuropeptides have electrophysiological effects on spinal neurons and for substance P and somatostatin, these effects have been associated with particular sensory modalities. Newer investigations using the compound capsaicin are consistent with the hypothesis that substance P is an important neurochemical mediator of certain kinds of noxious peripheral stimuli. The newly described substance P antagonists promise to be important pharmacological tools for investigation of the long-neglected neurochemical bases of sensory neuron function. Elaboration of the roles of these sensory neuropeptides will no doubt shed light on many disease states in which there seems to be sensory neuron involvement.

The ability of various related peptides and substance P analogues to compete for the binding of 3H-labelled substance P to rat brain membranes corresponds with their known biological activities, providing a simple model for studies of peptide receptors in the central nervous system. In salivary gland and brain slices substance P and related peptides stimulate the rate of incorporation of phosphatidylinositol, offering an alternative biochemical model for substance P receptor studies. Two types of receptor may be responsible for the actions of substance P on peripheral tissues: the SP-P type, where all tachykinins are approximately equally active, and the SP-E type, where eledoisin and kassinin are more potent than the other tachykinins. Alkyl esters of substance P appear to act as selective SP-P agonists.

We studied the actions of substance P, bombesin, vasoactive intestinal peptide (VIP), and the octapeptide of cholecystokinin (CCK-8-S) on the release of somatostatin, insulin, and glucagon from the isolated perfused pancreatico-duodenal canine preparation. Substance P at concentrations ranging from 0.2-5.0 nM stimulated the secretion of somatostatin, insulin, and glucagon in a dose-dependent manner. However, the responses evoked by substance P were modified by the prevailing glucose level; higher somatostatin and insulin and lower glucagon responses were obtained at the high glucose concentration of 8.3 mM rather than at the low glucose concentration of 2.8 mM. At a glucose concentration of 5.5 mM, somatostatin release was above the prestimulation level in response to 1 nM substance P (89 +/- 15%; P less than 0.01), VIP (49 +/- 7%; P less than 0.01), or CCK-8-S (99 +/- 21%; P less than 0.01); bombesin was without effect (16 +/- 14; P = NS). Insulin release was enhanced by substance P (150 +/- 45%; P less than 0.05), bombesin (162 +/- 56%; P less than 0.05), VIP (44 +/- 5%; P less than 0.01), and CCK-8-S (190 +/- 17%; P less than 0.001). Furthermore, a significant release of glucagon was evoked by 1 nM substance P (501 +/- 158%; P less than 0.05), bombesin (30 +/- 10%; P less than 0.05), VIP (43 +/- 8%; P less than 0.01), or CCK-8-S (140 +/- 19%; P less than 0.001).

This paper reviews recent evidence that a number of small peptides found in the brain are active in the central nervous system and behaviorally. Attention is focused on MSH/ACTH 4-10, alpha- and beta-MSH, and the prohormone beta-LPH, as they produce a syndrome of yawning and stretching. Studies with substance P and mainly with MIF-I are also reviewed. It is shown that substance P is an excitatory transmitter or modulator in the dorsal spinal cord with that MIF-I has antiparkinson properties. It is concluded that many polypeptides have direct actions on the central nervous system independent of their neuroendocrine properties.