Substance P Fragments Research Articles

The amino-terminal heptapeptide of the algesic substance P provides analgesic effect in relieving chronic neuropathic pain

Of painful conditions, somatic pain of acute nociceptive origin can be effectively managed clinically, while neuropathic pain of chronic neuropathy origin is difficult to control. For molecules involved in pain sensation, substance P (SP) is algesic, exacerbating painful sensation, while its amino-terminal fragment, heptapeptide SP(1-7), confers biological activities different from its full-length parent neuropeptide precursor. We previously demonstrated SP(1-7) interaction with pain processing to alleviate chronic pain. Here we evaluated SP(1-7) and its C-terminal amidated analogue SP(1-7)amide, together with SP and opioid agonist DAMGO. We tested mouse behaviors of both acute somatic pain in tail-flick latency assay, and neuropathic pain in sciatic nerve injury model of chronic constriction injury (CCI). DAMGO produced dose-dependent analgesia for somatic pain as expected, so did both SP(1-7) and its analogue SP(1-7)amide, while SP yielded the opposite effect of algesia, in a phenomenon we termed ‘contrintus’, meaning ‘opposite from within’ to denote that two peptides of the same origin (SP and its metabolic fragment SP(1-7)) produced opposite effects. In CCI model, DAMGO showed a general reduction in allodynia sensitivity for both nerve-injured and normal paws, without selective effect for neuropathic pain, consistent with clinical observation that opioids are less effective for chronic neuropathic pain. On the other hand, both SP(1-7) and SP(1-7)amide displayed dose-dependent anti-allodynia effect that is selective for neuropathic pain. These findings suggest that SP(1-7) and its analogue may be useful for developing pharmaceuticals to treat neuropathic pain.

Targeting Neuropathic Pain: Pathobiology, Current Treatment and Peptidomimetics as a New Therapeutic Opportunity.

There is a huge need for pharmaceutical agents for the treatment of chronic Neuropathic Pain (NP), a complex condition where patients can suffer from either hyperalgesia or allodynia originating from central or peripheral nerve injuries. To date, the therapeutic guidelines include the use of tricyclic antidepressants, serotonin-noradrenaline reuptake inhibitors and anticonvulsants, beside the use of natural compounds and non-pharmacological options. Unfortunately, these drugs suffer from limited efficacy and serious dose-dependent adverse effects. In the last decades, the heptapeptide SP1-7, the major bioactive metabolite produced by Substance P (SP) cleavage, has been extensively investigated as a potential target for the development of novel peptidomimetic molecules to treat NP. Although the physiological effects of this SP fragment have been studied in detail, the mechanism behind its action is not fully clarified and the target for SP1-7 has not been identified yet. Nevertheless, specific binding sites for the heptapeptide have been found in brain and spinal cord of both mouse and rats. Several Structure-Affinity Relationship (SAR) studies on SP1-7 and some of its synthetic analogues have been carried out aiming to developing more metabolically stable and effective small molecule SP1-7-related amides that could be used as research tools for a better understanding of the SP1-7 system and, in a longer perspective, as potential therapeutic agents for future treatment of NP.

Enhanced cell selectivity of hybrid peptides with potential antimicrobial activity and immunomodulatory effect

BackgroundHybridization is a useful strategy to bond the advantages of different peptides into novel constructions. We designed a series of AMPs based on the structures of a synthetic AMP KFA3 and a naturally-occurred host defense peptide substance P (SP) to obtain peptides retaining the high antibacterial activity of KFA3 and the immunomodulatory activity and low cytotoxicity of SP. MethodsTwo repeats of KFA and different C terminal fragments of SP were hybridized, generating a series of novel AMPs (KFSP1–8). The antibacterial activities, host cell toxicity and immunomodulation were measured. The antibacterial mechanisms were investigated. ResultsHybrid peptides KFSP1–4 exerted substantial antibacterial activities against Gram-negative bacteria of standard strains and clinical drug-resistant isolates including E.coli, A.baumannii and P.aeruginosa, while showing little toxicity towards host cells. Compared with KFA3, moderate reduction in α-helix content and the interruption in α-helix continuality were indicated in CD spectra analysis and secondary-structure simulation in these peptides. Membrane permeabilization combined with time-kill studies and FITC-labeled imaging, indicated a selective membrane interaction of KFSP1 with bacteria cell membranes. By specially activating NK1 receptor, the hybrid peptides kept the ability of SP to induce intracellular calcium release and ERK1/2 phosphorylation, but unable to stimulate NF-κB phosphorylation. KFSP1 facilitated the survival of mouse macrophage RAW264.7, directly interacting with LPS and inhibiting the LPS-induced NF-κB phosphorylation and TNF-α expression. ConclusionHybridization is a useful strategy to bond the advantages of different peptides. KFSP1 and its analogs are worth of advanced efforts to explore their potential applications as novel antimicrobial agents.

Cerebrospinal fluid levels of substance P (SP) N-terminal fragment SP1–7 in patients with neuropathic pain

Abstract Aims Neuropathic pain is a complex and painful condition, which is difficult to treat and causes a lot of suffering. The substance P (SP) system is well known to be involved in nociceptive signaling and it has previously been shown that the cerebrospinal fluid (CSF) level of SP is decreased in neuropathic pain. In this study we analyzed CSF from chronic neuropathic pain patients for the levels of SP1–7, an N-terminal fragment of SP with the ability to alleviate thermal as well as mechanical hypersensitivity in different animal models of chronic neuropathic pain, e.g. [1,2]. Methods CSF was collected from 11 neuropathic pain patients, treated with SCS, who had refrained from using their spinal cord stimulator for 48h. Control CSF was collected from 11 patients without any known neurological disorder, who underwent minor surgery under spinal anesthesia. The CSF samples were analyzed for the levels of SP1–7 using radioimmunoassay. Results The results revealed a decrease in the level of SP1–7 compared to controls. We believe that the lower level ofSP1–7 most likely is a consequence of reduced amount of its precursor SP in the neuropathic pain patients. Conclusions Our results indicate that the SP system is changed in patients with neuropathic pain and that SP-related peptides, including SP1–7, might serve as biological markers for the patho-physiology of chronic neuropathic pain.

Substance P and the neurokinin-1 receptor regulate electroencephalogram non-rapid eye movement sleep slow-wave activity locally

The neuropeptide substance P is an excitatory neurotransmitter produced by various cells including neurons and microglia that is involved in regulating inflammation and cerebral blood flow—functions that affect sleep and slow-wave activity (SWA). Substance P is the major ligand for the neurokinin-1 receptor (NK-1R), which is found throughout the brain including the cortex. The NK-1R is found on sleep-active cortical neurons expressing neuronal nitric oxide synthase whose activity is associated with SWA. We determined the effects of local cortical administration of a NK-1R agonist (substance P-fragment 1, 7) and a NK-1R antagonist (CP96345) on sleep and SWA in mice. The NK-1R agonist significantly enhanced SWA for several hours when applied locally to the cortex of the ipsilateral hemisphere as the electroencephalogram (EEG) electrode but not after application to the contralateral hemisphere when compared to saline vehicle control injections. In addition, a significant compensatory reduction in SWA was found after the NK-1R agonist-induced enhancements in SWA. Conversely, injections of the NK-1R antagonist into the cortex of the ipsilateral hemisphere of the EEG electrode attenuated SWA compared to vehicle injections but this effect was not found after injections of the NK-1R antagonist into contralateral hemisphere as the EEG electrode. Non-rapid eye movement sleep and rapid eye movement sleep duration responses after NK-1R agonist and antagonist injections were not significantly different from the responses to the vehicle. Our findings indicate that the substance P and the NK-1R are involved in regulating SWA locally.

An amino-terminal fragment of hemokinin-1 has an inhibitory effect on pruritic processing in rats

Hemokinin-1 (HK-1) is a peptide encoded by the preprotachykinin gene, TAC-4, and shares the hydrophobic carboxyl-terminal (C-terminal) region common to mammalian tachykinin peptides, such as substance P (SP). It is generally believed that C-terminal fragments of SP elicit an excitatory effect, while pretreatment with amino-terminal (N-terminal) fragments of SP inhibits the function of SP; however, there is no available information on HK-1. Therefore, to clarify the characteristics of C-terminal and N-terminal fragments of HK-1, HK-1 was divided into HK-1 (1–5) as the N-terminal fragment and HK-1 (6–11) as the C-terminal fragment based on the similarity of amino acids between HK-1 and SP. Intrathecal administration of HK-1 (6–11) induced scratching behavior similar to HK-1, while HK-1 (1–5) hardly induced scratching. Pretreatment with HK-1 (1–5), however, attenuated scratching induced by HK-1 and SP, whereas pretreatment with SP (1–5) attenuated SP-induced scratching, but not HK-1. Furthermore, intrathecal administration of HK-1 (1–5) and SP (1–5) markedly attenuated the induction of flinching and enhancement of c-Fos expression in the spinal cord following the intradermal administration of formalin, a noxious stimulant, while pretreatment with HK-1 (1–5), but not SP (1–5), markedly attenuated the induction of scratching behavior by subcutaneous administration of pruritic agents, such as serotonin or histamine. Taken together, these findings indicate that HK-1 (1–5) suppresses pruritic and nociceptive processing, while SP (1–5) suppresses nociceptive processing. Therefore, it is suggested that HK-1 (1–5) may be a useful tool for revealing pruritic processing and HK-1 may play a crucial role in pruritic processing.

Proteolysis Controls Endogenous Substance P Levels

Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP 1–9-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.

Synthesis, biological activity and structure–activity relationship of endomorphin-1/substance P derivatives

Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2–5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP8–11 (2) or SP7–11 (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent μ-opioid receptor binding affinity (Kiμ=3.87±0.51nM) with dose-dependent agonist activity in the nanomolar range (IC50=45±13nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1–5 in the active site of μ-opioid receptors. The docking scores were in agreement with the Kiμ values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.

Investigation of the metabolic biotransformation of substance P in liver microsomes by liquid chromatography quadrupole ion trap mass spectrometry

Substance P (SP) belongs to the tachykinin family and plays an essential role in pain transmission and in neurogenic inflammation. It can be detected in the central and peripheral nervous systems. The objectives of this study were to establish SP metabolic stability in liver microsomes in three species (rat, mouse and human), and identify and characterize SP metabolites by LC-MS/MS. Endogenous peptide metabolism is not well documented and this is particularly true for neuropeptides participating in neurogenic inflammation. In vitro, T(1/2) results in pooled liver microsomes were 9.2, 5.6 and 18.6 min for rat, mouse and human liver microsomes, respectively. Five major SP metabolites were identified and quantified, including C-terminal SP fragments SP(3-11) , SP(5-11) , SP(6-11) , SP(8-11) as well as N-terminal fragment SP(1-7) . The results suggest significant differences between species in SP metabolite concentrations. Consequently, the metabolic profile of each species is distinctive and may have a significant impact on biomolecular mechanisms involved in specific pathophysiological changes.

The effects of substance P fragments on breast cancer cells.

295 Background: Substance P (SP), a neuropeptide, is known to induce tumor cell proliferation. In contrast with intact peptide, the fragments of SP are suggested to inhibit the growth of various cancer cells. The aim of the present study was to determine cytotoxic effects of physiological fragments of SP either alone or in combination with radiotherapy on mouse breast cancer cells. Methods: In this study, we tested the physiological fragments of SP such as SP (4-11), SP (6-11) and SP (1-7). Dose-response and time-course studies were carried out with various concentrations (100-0.001 nM) of SP fragments and the intact peptide. 4T1 mouse breast cancer cell lines were used in this study. The cytotoxic effect of SP fragments alone or in combination with radiotherapy was determined via WST-1 assay. Changes in substance P amounts in cells and in mediums determined by SP EIA kit. Results: SP(4-11) and SP(6-11), but not SP(1-7), inhibited the proliferation of breast cancer cells and potentiated antitumor effects of radiotherapy. Moreover, the intact peptide alone did not alter the proliferation rate of 4T1 cells and the cytotoxic effects of the fragments were not inhibited by SP. Conclusions: These results demonstrate that combined treatment with 2 fragments of SP (4-11 and 6-11) and radiotherapy induce cytotoxic effects. These data may provide the basis for a strategy, in which it is possible to use SP fragments and radiotherapy together to improve the efficiency of the independent therapies.

The amino-terminal region of hemokinin-1 regulates the induction of thermal hyperalgesia in rats

It is known that intrathecal administration of substance P (SP) induces thermal hyperalgesia, whereas hemokinin-1 (HK-1), a member of the same tachykinin family as SP, hardly induces thermal hyperalgesia; however, the underlying mechanism remains to be elucidated. Therefore, we aimed to clarify which amino acid of these peptides contributes to the induction of thermal hyperalgesia. When two chimera peptides between the N-terminal region of SP and the C-terminal region of HK-1, and vice versa, SP (1-5)/HK-1 and HK-1 (1-5)/SP, were intrathecally administered, SP (1-5)/HK-1 induced thermal hyperalgesia whereas HK-1 (1-5)/SP had hardly any effect; furthermore, thermal hyperalgesia was induced by only C-terminal fragments of HK-1 and SP. These findings indicate that the N-terminal region of HK-1 is involved in the non-induction of thermal hyperalgesia. Next, we synthesized and intrathecally administered these chimera peptides in which part of the N-terminal region of HK-1 was replaced with that of SP, and vice versa, and all synthesized peptides induced thermal hyperalgesia. Both SP (1-2)/HK-1 and HK-1 (1-4)/SP certainly induced thermal hyperalgesia, although HK-1 and HK-1 (1-5)/SP had hardly any effect; therefore, it is probable that Ser at the 2nd position and Arg at the 5th position of HK-1 may be involved in the non-induction of thermal hyperalgesia. Furthermore, peptides in which amino acid at the 3rd and/or 4th positions of HK-1 was replaced with that of SP were synthesized. Intrathecal administration of HK-1 (1-2,4-5)/SP, but not HK-1 (1-2,5)/SP and HK-1 (1-3,5)/SP, hardly induced thermal hyperalgesia. These findings indicate that three amino acids, Ser, Thr and Arg at the 2nd, 4th and 5th positions of HK-1, respectively, regulate the induction of thermal hyperalgesia by HK-1.

The C-terminal amidated analogue of the substance P (SP) fragment SP 1–7 attenuates the expression of naloxone-precipitated withdrawal in morphine dependent rats

We previously demonstrated that intracerebroventricular (i.c.v.) administration of the substance P (SP) aminoterminal fragment SP 1–7 attenuates the expression of morphine withdrawal in the male rat. In this study we have used a synthetic analogue of this peptide, i.e. the SP 1–7 amide showing higher binding potency than the native heptapeptide, in a similar experimental set-up. Thus, Wistar male rats were made tolerant to morphine by daily injections of the opiate during 8 days. Following peptide administration (i.c.v.) and a subsequent naloxone challenge a variety of physical syndromes of withdrawal were recorded. We observed that the SP 1–7 amide potently and dose-dependently reduced several signs of reaction to morphine withdrawal. Interestingly, the effect of the peptide amide was significantly attenuated by the addition of the sigma agonist (+)-SKF-10047. We conclude that the SP 1–7 amide mimics the effect of the native SP fragment and that the mechanisms for its action involve a sigma receptor site.

Endomorphins interact with the substance P (SP) aminoterminal SP 1–7 binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP 1–7 in the rat spinal cord. This site appeared very specific for SP 1–7 as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP 1–7 from this site. In the present work using a [ 3H]-labeled derivative of the heptapeptide we have identified and characterized [ 3H]-SP 1–7 binding in the rat ventral tegmental area (VTA). Similarly to the [ 3H]-SP 1–7 binding in the spinal cord the affinity of unlabeled SP 1–7 to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the μ-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP 1–7 site was 4–5 times weaker than that for SP 1–7 but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP 1–7 binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.

Small peptides mimicking substance P (1–7) and encompassing a C-terminal amide functionality

Some of the biological effects demonstrated after administration of substance P (SP) in vivo can indirectly be attributed to the fragmentation of the undecapeptide to its N-terminal bioactive fragment SP(1-7). This heptapeptide (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is a major bioactive metabolite from SP that frequently exerts similar biological effects as the parent peptide but also, in several cases, completely opposite actions. Specific binding sites for the heptapeptide SP(1-7) that are separate from the SP preferred NK receptors have been identified. In this study we demonstrate that (a) the C-terminal part of the SP metabolite SP(1-7) is most important for binding as deduced from an Ala scan and that a replacement of Phe(7) for Ala is deleterious, (b) truncation of the N-terminal amino acid residues of SP(1-7) delivers peptides with retained binding activity, although with somewhat lower binding affinities than SP(1-7) and (c) a C-terminal amide group as a replacement for the terminal carboxy group of SP(1-7) and for all of the truncated ligands synthesized affords approximately 5-10-fold improvements of the binding affinities.

Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP 1–7 in the rat spinal cord

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the μ-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP 1–7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP 1–7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP 1–7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar 9, Met(O 2) 11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP 1–7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP 1–7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP 1–7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPγS assay. This suggests that the identified SP 1–7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.

The substance P (SP) heptapeptide fragment SP 1–7 alters the density of dopamine receptors in rat brain mesocorticolimbic structures during morphine withdrawal

The aminoterminal fragment of substance P (SP), SP 1–7, has been suggested to modulate the expression of opiate tolerance and withdrawal behaviors in rodents. However, the mechanism of this effect is not yet clarified. Using a rat model we have previously demonstrated that SP 1–7 affects dopamine transmission and the expression of the dopamine D2-receptor gene transcript in the nucleus accumbens during naloxone precipitated morphine withdrawal. In the present study, we have applied autoradiography to investigate the effect of the heptapeptide on the binding of dopamine D1- and D2-receptors in mesocorticolimbic brain areas of male rats during morphine withdrawal. Morphine dependent animals were treated with an injection of SP 1–7 into the ventral tegmental area prior to naloxone challenge. The result indicated that the SP fragment elicited a significant decrease in specific binding to D1-like receptors in the caudate putamen, nucleus accumbens shell, nucleus accumbens core, substantia nigra and medial globus pallidus. Radioligand binding to dopamine D2-like receptors was also altered by SP 1–7. The heptapeptide induced a decreased density of these sites in the ventral tegmental area but an increased binding in the substantia nigra and the frontal cortex. The observed alterations in the D1- and D2-like receptor density could reflect activations in dopamine pathways associated with the above-mentioned brain regions. The result provides further evidence for the modulatory effect of SP 1–7 on dopamine systems during opioid withdrawal, suggesting the possible role for the heptapeptide to regulate morphine withdrawal reactions.

Possible role of substance P in the ischemia-reperfusion injury in the isolated rabbit lung.

The origin of the endothelial damage leading to the ischemia-reperfusion injury after lung transplantation has not been elucidated. We postulated that neurotransmitters released during the preservation of the donor lung might explain this vascular derangement. Thus, in isolated rabbit lungs preserved over 24 hours, we evaluated the release of acetylcholine (ACh) and substance P (SP), the activity of their major degrading enzymes, acetylcholinesterase (AChE) and neutral endopeptidase (NEP), and changes in the capillary permeability. Both neurotransmitters showed the highest release rate in the first 15 minutes, followed by a sharp exponential decrement at 1, 6, 12 and 24 hours. AChE and NEP activities showed no variation at these time intervals. Basal capillary permeability significantly increased (P<0.01) after 24 hours preservation with saline. This increased permeability was avoided (P<0.01) by the SP fragment 4-11 (an SP receptors antagonist), but not by atropine. These results suggest for the first time a pathogenic role of SP in the ischemia-reperfusion injury, and thus the potential usefulness of SP antagonists as additives in the lung preservation solutions should be explored.

Acetylcholine and tachykinins involvement in the caffeine-induced biphasic change in intracellular Ca2+ in bovine airway smooth muscle.

1. Caffeine has been widely used as a pharmacological tool to evaluate Ca(2+) release from the sarcoplasmic reticulum in isolated smooth muscle cells. However, in nervous tissue this drug also causes neurotransmitters release, which might cause additional effects when smooth muscle strips are evaluated. To assess this last possibility, simultaneous measurements of contraction and cytosolic Ca(2+) concentration (using Fura-2/AM) were carried out in bovine airway smooth muscle strips during caffeine stimulation. 2. A first stimulation (S1, n=11) with caffeine (10 mM) induced a biphasic change in cytosolic Ca(2+), which consisted of a transient Ca(2+) peak (254+/-40 nM, X+/-SEM) followed by a plateau (92+/-13 nM), and a transient contraction (204.72+/-31.56 mg tension mg tissue(-1)). A second caffeine stimulation (S2) produced a similar response but these parameters had a different magnitude. The S2/S1 ratios for these parameters were 0.69+/-0.02, 0.83+/-0.06 and 1.01+/-0.03, respectively. Addition of omega-conotoxin GVIA (1 micro M) and tetrodotoxin (3.1 micro M) before S2 significantly diminished these S2/S1 ratios (0.26+/-0.05, 0.26+/-0.09 and 0.64+/-0.11, respectively, n=5, P<0.05), implicating the neurotransmitters release involvement in the response to caffeine. A similar effect (P<0.01) was observed with atropine (1 micro M, n=4), the fragment 4-11 of substance P (SP) (an SP receptor antagonist, 10 micro M, n=5), and with both substances (n=4). 3. We discarded a direct effect of omega-conotoxin GVIA (1 micro M) plus tetrodotoxin (3.1 micro M) or of atropine (1 micro M) plus SP fragment 4-11 on smooth muscle cells because they did not modify caffeine responses in isolated tracheal myocytes. 4. We confirmed by HPLC that caffeine increased the release of acetylcholine (from 0.43+/-0.19 to 2.07+/-0.56 nM mg tissue(-1), P<0.02) in bovine airway smooth muscle strips. Detection of substance P by ELISA was not statistically different after caffeine stimulation (geometric means before and after caffeine, 0.69 vs. 1.97 pg ml(-1) mg tissue(-1), respectively, P=0.053). 5. We concluded that acetylcholine and tachykinins release are involved in the caffeine-induced biphasic changes in cytosolic Ca(2+) concentration.

Substance P induces activation, adherence and migration of equine eosinophils.

The tachykinin, substance P (SP), affects eosinophil function by direct and indirect mechanisms and has been shown to cause equine eosinophils to adhere to vascular endothelium and to release cytokines that increase cell adherence. The aim of this study was to determine whether SP could act directly on equine eosinophils in vitro. Eosinophil activation was also compared in cells from normal ponies and those with insect hypersensitivity as SP may be released in the skin of hypersensitive animals. SP caused equine eosinophils to adhere, migrate and produce superoxide, although high concentrations were required to produce these effects [10 +/- 2% adherence, 45 +/- 20 cells/0.3 mm2 and 48 +/- 7 nmol (of reduced cytochrome C)/106 cells, respectively, at 3 x 10-4 m]. That the 7-11, but not the 1-7, amino acid fragment of SP caused superoxide production, suggested the effects of SP were receptor mediated. Eosinophils from hypersensitive ponies produced more superoxide in response to SP, but not phorbol myristate acetate or histamine, over the concentration range tested when compared with cells from normal ponies. The data obtained in this study suggest that although SP can directly activate equine eosinophils, in view of the high concentrations required, such actions may be of less relevance physiologically than other SP-mediated effects.

Neuropeptide conversion to bioactive fragments--an important pathway in neuromodulation.

Biosynthetic pathways for the formation of neuroactive peptides and the processes for their inactivation include several enzymatic steps. In addition to enzymatic processing and degradation, several neuropeptides have been shown to undergo enzymatic conversion to fragments with retained or modified biological activity. This has most clearly been demonstrated for e.g. opioid peptides, tachykinins, calcitonin gene-related peptide (CGRP) as well as for peptides belonging to the renin-angiotensin system. Sometimes the released fragment shares the activity of the parent compound. However, in many cases the conversion reaction is linked to a change in the receptor activation profile, i.e. the generated fragment acts on and stimulates a receptor not recognized by the parent peptide. This review will describe the characteristics of certain neuropeptide fragments having the ability to modify the biological action of the peptide from which they are derived. Focus will be directed to the tachykinins, the opioid peptides, angiotensins as well as to CGRP, bradykinin and nociceptin. The kappa opioid receptor selective opioid peptide, dynorphin, recognized for its ability to produce dysphoria, is converted to the delta opioid receptor agonist Leu-enkephalin, with euphoric properties. The tachykinins, typified by substance P (SP), is converted to the bioactive fragment SP(1-7), a heptapeptide mimicking some but opposing other effects of the parent peptide. The bioactive angiotensin II, known to bind to and stimulate the AT-1 and AT-2 receptors, is converted to angiotensin IV (i.e. angiotensin 3-8) with preference for the AT-4 sites or to angiotensin (1-7), not recognized by any of these receptors. Both angiotensin IV and angiotensin (1-7) are biologically active. For example angiotensin (1-7) retains some of the actions ascribed for angiotensin II but is shown to counteract others. Thus, it is obvious that the activity of many neuroactive peptides is modulated by bioactive fragments, which are formed by the action of a variety of peptidases. This phenomenon appears to represent an important regulatory mechanism that modulates many neuropeptide systems but is generally not acknowledged.