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There is increasing focus on the involvement of tachykinins in immune and inflammatory responses. Hemokinin-1 (HK-1) is a recently identified tachykinin that originates primarily from immune cells, and has structural similarities to substance P (SP), found mainly in neurons. However, there are species differences in HK-1, and the role of HK-1 in humans, particularly the intestine, has received minimal attention. The aim of this study was to investigate the inflammatory role of human HK-1 in the human colon. The effects of HK-1 and SP were compared on the production of multiple inflammatory cytokines and chemokines from human colonic mucosal explants. Data generated by Procarta multiplex assay and QuantiGene assay demonstrated that 4 h incubation with HK-1 (0.1 μM) significantly stimulated transcript expression and release of MCP-1, MIP-1α and β, RANTES, TNF-α, IL-1β and IL-6 from the mucosa. SP (0.1 μM) had comparable actions, but had no effect on MCP-1 or RANTES. These effects were inhibited separately by tachykinin NK 1 and NK 2 receptor antagonists SR140333 and SR48968 (both 0.1 μM), suggesting that these responses were mediated by both NK1 and NK2 receptors. In conclusion, these data support a novel inflammatory role for HK-1 in human colon, signaling via NK1 and NK2 receptors (and possibly other tachykinin-preferring receptors) to regulate the release of a broad spectrum of proinflammatory mediators. The study suggests that along with SP, HK-1 is also a proinflammatory mediator, likely involved in colonic inflammation, including inflammatory bowel disease (IBD)., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The present study focused on the interactive pain regulation of endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) or endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on chimeric peptide MCRT (YPFPFRTic-NH2, based on YPFP-NH2 and PFRTic-NH2) at the supraspinal level in mice. Results demonstrated that the co-injection of nanomolar EKA/B and MCRT showed moderate regulation, whereas 30 pmol EKA/B had no effect on MCRT. The combination of EKC/D and MCRT produced enhanced antinociception, which was nearly equal to the sum of the mathematical values of single EKC/D and MCRT. Mechanism studies revealed that pre-injected naloxone attenuated the combination significantly compared with the equivalent analgesic effects of EKC/D alone, suggesting that EKC/D and MCRT might act on two totally independent pathways. Moreover, based on the above results and previous reports, we made two reasonable hypotheses to explain the cocktail-induced analgesia, which may potentially pave the way to explore the respective regulatory mechanisms of EKA/B, EKC/D, and MCRT and to better understand the complicated pain regulation of NK1 and μ opioid receptors, as follows: (1) MCRT and endomorphin-1 possibly activated different μ subtypes; and (2) picomolar EKA/B might motivate the endogenous NPFF system after NK1 activation.

Background and Purpose: Peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P (SP), neurokinin A (NKA) and B (NKB)., Experimental Approach: The following in vitro assays were used: calcium mobilization in cells expressing human recombinant NK receptors, BRET studies of G-protein - NK1 receptor interaction, guinea pig ileum and rat urinary bladder bioassays. Nociceptive behavioural response experiments were performed in mice following intrathecal injection of PWT2-SP., Key Results: In calcium mobilization studies, PWT tachykinin derivatives behaved as full agonists at NK receptors with a selectivity profile similar to that of the natural peptides. NK receptor antagonists display similar potency values when tested against PWT2 derivatives and natural peptides. In BRET and bioassay experiments PWT2-SP mimicked the effects of SP with similar potency, maximal effects and sensitivity to aprepitant. After intrathecal administration in mice, PWT2-SP mimicked the nociceptive effects of SP, but with higher potency and a longer-lasting action. Aprepitant counteracted the effects of PWT2-SP in vivo., Conclusions and Implications: The present study has shown that the PWT technology can be successfully applied to the peptide sequence of tachykinins to generate tetrabranched derivatives characterized with a pharmacological profile similar to the native peptides. In vivo, PWT2-SP displayed higher potency and a marked prolongation of action, compared with SP., (© 2014 The British Pharmacological Society.)

Our previous studies have shown that an active fragment of human tachykinins (hHK-1(4-11)) produced an opioid-independent analgesia after intracerebroventricular (i.c.v.) injection in mice, which has been markedly enhanced by a δ OR antagonist, naltrindole hydrochloride (NTI). In this study, we have further characterized the in vivo analgesia after i.c.v. injection of hHK-1(4-11) in mouse model. Our qRT-PCR results showed that the mRNA levels of several ligands and receptors (e.g. PPT-A, PPT-C, KOR, PDYN and PENK) have not changed significantly. Furthermore, neither transcription nor expression of NK1 receptor, MOR and POMC have changed noticeably. In contrast, both mRNA and protein levels of DOR have been up-regulated significantly, indicating that the enhanced expression of δ opioid receptor negatively modulates the analgesia induced by i.c.v. injection of hHK-1(4-11). Additionally, the combinatorial data from our previous and present experiments strongly suggest that the discriminable distribution sites in the central nervous system between hHK-1(4-11) and r/mHK-1 may be attributed to their discriminable analgesic effects. Altogether, our findings will not only contribute to the understanding of the complicated mechanisms regarding the nociceptive modulation of hemokinin-1 as well as its active fragments at supraspinal level, but may also lead to novel pharmacological interventions.

Hemokinin-1 is a peptide encoded by Pptc, which belongs to the family of mammalian tachykinins. Our previous results showed that rat/mouse hemokinin-1 (r/m HK-1) produced striking analgesia after intracerebroventricular (i.c.v.) injection in mice, and the analgesia could be blocked by the NK1 receptor antagonist and the opioid receptor antagonist, respectively. However, the precise distribution sites and the molecular mechanism involved in the analgesic effect after i.c.v. administration of r/m HK-1 are needed to be further investigated deeply. Using the fluorescence labeling method, our present results directly showed that r/m HK-1 peptides were mainly distributed at the ventricular walls and several juxta-ventricular structures for the first time. Our results showed that the mRNA expressions of NK1 receptor, PPT-A, PPT-C, KOR, PDYN, DOR and PENK were not changed markedly, as well as the protein expression of NK1 receptor was hardly changed. However, both the transcripts and proteins of MOR and POMC were up-regulated significantly, indicating that the analgesic effect induced by i.c.v. administration of r/m HK-1 is related to the activation of NK1 receptor first, then it is related to the release of endogenous proopiomelanocortin, as well as the increased expression level of μ opioid receptor. These results should facilitate further the analysis of the analgesia of r/m HK-1 in the central nerval system in acute pain and may open novel pharmacological interventions., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to investigate the potential of intranasal immunization with non-ionic surfactant vesicles (NISV) containing either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine reach peptides (DTK) for induction of protective immunity against genital herpes infection in mice. NISV were prepared by lipid film hydration method. The mean diameter of vesicles was around 390 nm for DTK-containing NISV (DTK-NISV) and 320 nm for gB1s-containing NISV (gB1s-NISV). The encapsulation efficiency of the molecules was comprised between 57% and 70%. After 7-14 day NISV maintained stable dimensions and a drug encapsulation higher than 48%. We showed that intranasal immunization with gB1s-NISV induces gB-specific IgG antibody and lymphoproliferative responses, whereas vaccination with DTK-NISV was not able to generate a gB-specific immune response. Our results indicate that vaccination of BALB/c mice with gB1s-NISV induced Th1 responses, as characterized by increased titre of IG2a in plasma and IFN-production in CD4+ splenic cells. Intranasal immunization with gB1s-NISV could elicit 90% (almost complete) protection against a heterologous lethal vaginal challenge with herpes simplex virus type 2. These data may have implications for the development of a mucosal vaccine against genital herpes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Endokinins designated from the human TAC4 gene consist of endokinin A, endokinin B, endokinin C (EKC) and endokinin D (EKD). EKC/D is a peptide using the common carboxyl-terminal in EKC and EKD and consists of 12 amino acids, and exerts antagonistic effects on the induction of scratching behavior by substance P (SP). Some of SP-preferring receptor antagonists have several d-tryptophan (d-Trp); however, the pharmacological effect of EKC/D-derived peptides with d-Trp remains to be solved. Therefore, to clarify the pharmacological characteristics of EKC/D-derived peptides, effects of pretreatment with these peptides on SP-induced scratching and thermal hyperalgesia, formalin-induced flinching and carrageenan-induced inflammation were evaluated. Intrathecal administration of [d-Trp(8)]-EKC/D and [d-Trp(10)]-EKC/D showed a markedly long inhibitory effect, at least 14 h, whereas the antagonistic effects of [d-Trp(8,10)]-EKC/D and EKC/D without d-Trp disappeared after 1h. Furthermore, the inhibitory effect of [d-Trp(10)]-EKC/D-derived peptides was dependent on the number of amino acids from the amino-terminus, and the more numerous the amino acids, the more marked the antagonistic effect. Thus, these results indicate that the effective duration of EKC/D-derived peptides is dependent on the number of d-Trp in the carboxyl-terminal region and the amino-terminal region regulates the antagonistic effect of EKC/D., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Endokinins are four novel human tachykinins, including endokinins A (EKA), B (EKB), C (EKC), and D (EKD). Endokinin A/B (EKA/B) is the common C-terminal decapeptide in EKA and EKB, while endokinin C/D (EKC/D) is the common C-terminal duodecapeptide in EKC and EKD. In this study, we attempted to investigate the interactions between EKA/B, EKC/D, and endomorphin-1 (EM-1) on the depressor effect at peripheral level. The effects of EKA/B produced a U-shaped curve. The maximal effect was caused by 10 nmol/kg. EKC/D and EM-1 showed a dose-dependent relationship. Co-administration of EKA/B (0.1, 1, 10 nmol/kg) with EM-1 produced effects similar to those of EKA/B alone but slightly lower. Co-injection of EKA/B (100 nmol/kg) with EM-1 caused an effect stronger than any separate injection. Co-administration of EKC/D (10 nmol/kg) with EM-1 (30 nmol/kg) caused a depressor effect, which was one of the tradeoffs of EM-1 and EKC/D. Mechanism studies showed that SR140333B could block the depressor effects of EKA/B, EKC/D, EM-1, EKA/B+EM-1, and EKC/D+EM-1; SR48968C could block EM-1, EKA/B, EKC/D, and EKC/D+EM-1 and partially block EKA/B+EM-1; SR142801 could block EM-1, EKC/D, and EKC/D+EM-1 and partially block EKA/B and EKA/B+EM-1; naloxone could block EM-1, EKC/D, and EKC/D+EM-1 and partially block EKA/B and EKA/B+EM-1. Pretreatment with NG-nitro-l-arginine methyl ester partially decreased depressor intensity and half-recovery time of EKA/B and EKC/D., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Endokinins, encoded by the human preprotachykinin C (PPT-C)/TAC4 gene, are peptides that consist of endokinin A (EKA), B (EKB), C (EKC) and D (EKD) and belong to the tachykinin family. Intrathecal injection of EKC/D (using the common carboxyl-terminal duodecapeptide in EKC and EKD) markedly attenuated the induction of thermal hyperalgesia and scratching behavior by intrathecal administration of substance P (SP), indicating that EKC/D has an antagonistic effect on the neurokinin 1 receptor (NK1R), SP-preferring receptor, at the spinal level; however, the pharmacological function of EKC/D at the periphery is not yet understood. Therefore, to clarify the effect of EKC/D on the peripheral tissue, the effect of subcutaneous injection of EKC/D on carrageenan-induced inflammation was examined. Subcutaneous injection of EKC/D attenuated an increase in paw volume following carrageenan-induced inflammation in a dose-dependent manner. Indeed, the increased paw volume was significantly decreased 40 min after treatment with 10(-4) M (10 nmol) and 10(-3) M (100 nmol) EKC/D (100 microl/rat). Similarly, injection of NK1R antagonists such as L-703,606 and Spantide I (10(-3) M) attenuated the increased paw volume following inflammation. Furthermore, the reduced withdrawal latency evoked by inflammation following subcutaneous injection of carrageenan was also dose-dependently attenuated by EKC/D administration. These results indicate that subcutaneous injection of EKC/D elicits an anti-inflammatory effect on carrageenan-induced inflammation., (Copyright 2010. Published by Elsevier Inc.)

Hemokinin-1 is a novel mammalian tachykinin cloned from mouse bone marrow. At present, pharmacological profile and physiological role of hemokinin-1 are still unclear. In the present study, we found that intrathecal (i.t.) administration of hemokinin-1 (0.00625-1.6 nmol) induced nociceptive responses consisting of scratching, biting and licking, which resemble substance P-induced behavioral responses in mice. The behaviors evoked by low-dose of hemokinin-1 (0.0125 nmol) were dose-dependently inhibited by i.t. co-administration of CP-99,994, a non-peptidic tachykinin NK(1) receptor antagonist, whereas high-dose of hemokinin-1 (0.1 nmol)-induced behaviors were not affected. Moreover, sendide, a peptidic tachykinin NK(1) receptor antagonist, failed to reduce the behavioral responses of both low- and high-dose of hemokinin-1. In contrast, substance P-induced behaviors were completely suppressed by both CP-99,994 and sendide. These results suggest that hemokinin-1 plays an important role in pain transmission at spinal cord. Moreover, the mechanism of hemokinin-1-induced nociceptive behaviors may be dose-dependent, and distinct from substance P-induced nociceptive behaviors., (Copyright 2010 Elsevier Inc. All rights reserved.)

Two tachykinin peptides, substance P (SP) and hemokinin-1 (HK-1), and three transient receptor potential (TRP) channels, TRPV1, TRPA1 and TRPM8, are similarly localized in the spinal dorsal horn and dorsal root ganglion, suggesting that TRP channels may be related or modulated by these tachykinin peptides. Thus, to clarify whether the responses of TRP channels are modulated by SP or HK-1, the effects of pretreatment with SP or HK-1 on the induction of scratching behavior by TRP channel agonists were examined. Pretreatment with SP or HK-1 enhanced the induction of scratching behavior by resiniferatoxin, a TRPV1 agonist, whereas scratching behavior induced by menthol, a TRPM8 agonist, was suppressed by pretreatment with these peptides. On the other hand, pretreatment with SP, but not HK-1, suppressed the induction of scratching behavior by cinnamaldehyde, a TRPA1 agonist. Taken together, the present results indicate that SP or HK-1 differentially modulated the response of TRPV1, TRPA1 or TRPM8 channel., ((c) 2009. Published by Elsevier Ltd. All rights reserved.)

Endokinins are novel tachykinins encoded on the human TAC4 and consist of Endokinin A (EKA), B (EKB), C (EKC) and D (EKD). To date, the function of Endokinins in pain processing was not fully understood. Therefore the aim of this study was to investigate the effects of Endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) and Endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on pain modulation at supraspinal level in mice. Intracerebroventricular (i.c.v.) administration of EKA/B (1, 3, 12, 20nmol/mouse) dose dependently induced potent analgesic effect. This effect could be fully antagonized by SR140333B but not SR48968C or SR142801. Naloxone could also block the analgesic effect, suggesting that this analgesic effect is related to opioid receptors. However, i.c.v. administration of EKA/B (10, 30, 100pmol/mouse) caused hyperalgesic effect significantly, with a "U" shape curve. Interestingly, the hyperalgesic effect induced by EKA/B could be attenuated by SR140333B, SR142801 but not SR48968C. I.c.v. administration of EKC/D (1, 3, 12, 20nmol/mouse) also dose dependently induced analgesic effect, which could not be blocked by SR48968C or SR142801 or naloxone. But to our astonishment, it could be significantly enhanced by SR140333B. More interestingly, the hyperalgesic effect induced by EKA/B could be significantly attenuated by EKC/D. In addition, the analgesic effect induced by co-administration of EKA/B and EKC/D was much less stronger than the effect of either EKA/B or EKC/D.

The central mechanisms that mediate neuropeptide K (NPK) associated anorexia are poorly understood in any species, and information in this area of avian biology is totally lacking. Thus, the effects of intracerebroventricular NPK treatment were studied in Cobb-500 chicks (Gallus gallus). In Experiment 1, NPK caused decreased feed intake, but did not affect water intake or whole blood glucose concentration. In Experiment 2, NPK-treated chicks had increased c-Fos immunoreactivity in the parvicellular division of the paraventricular nucleus and arcuate nucleus. The lateral hypothalamus, ventromedial hypothalamus, dorsomedial hypothalamus, periventricular nucleus, magnocellular division of the paraventricular nucleus, and the superchiasmatic nucleus were not affected by NPK treatment. In Experiment 3, the number of feed pecks, exploratory pecks, jumps, escape attempts, and distance moved were decreased, while time spent standing was increased. None of the NPK-treated chicks sat or entered deep rest. In Experiment 4, blockage of corticotrophin releasing factor receptors did not affect NPK-induced anorexia. Thus, we conclude that NPK is a regulator of chick appetite and the effects may be mediated directly in the arcuate nucleus and parvicellular division of the paraventricular nucleus.

Objective: To assess role of hydroxyl radials in the ozone-induced upper airway hyper-responsiveness to tachykinins., Methods: A prospective, controlled, animal model (n = 96) was performed. Half of them exposed to air (A-group, placebo) and the other half exposed to 3 ppm ozone (O-group) for 2 h. Two hours post air/ozone exposure, animals were anesthetized and equally randomized to be pretreated with one of the three treatments, including saline vehicle, dimethylthiourea (DMTU; 500 mg/kg m, a hydroxyl radical scavenger), or phosphoramidon (Phos; 2 mg/kg, an inhibitor for neutral endopeptidase). Ten minutes after pretreatment, half of the animals in each group were i.v. injected with capsaicin (2 microg/kg), and the other half were i.v. injected with substance P (10 microg/kg) to produce Evans blue dye extravasation., Results: Nasal exudative response to capsaicin or substance P in O-group was found to be significantly greater than that in A-group. This ozone-induced nasal airway hyperresponsiveness was largely prevented by DMTU. Phosphoramidon produced a similar nasal airway hyperresponsiveness in the A-group, but failed to alter ozone-induced nasal airway hyperresponsiveness in O-group. In sharp contrast, only substance P, but not capsaicin, produced a laryngeal exudative response in the A-group, which was similar to that in the O-group. The laryngeal exudative response to substance P was not significantly affected by DMTU or Phos., Conclusion: In the guinea-pig model, hydroxyl radicals play a vital role in the development of ozone-induced nasal airway hyperresponsiveness to tachykinins. It is possibly mediated through the suppressive action of ozone on the tachykinin degradation.

Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4-11) are mammalian tachykinin peptides encoded by the recently identified TAC4 gene in human, and the biological functions of these peptides have not been well investigated. In the present study, an attempt has been made to investigate the effects and mechanisms of action of h HK-1 and h HK-1(4-11) in pain modulation at the supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of h HK-1 (0.3, 1, 3 and 6 nmol/mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the NK(1) receptor antagonist SR140333, but not by the NK(2) receptor antagonist SR48968, indicating that the analgesic effect induced by i.c.v. h HK-1 is mediated through the activation of NK(1) receptors. Interestingly, naloxone, beta-funaltrexamine and naloxonazine, but not naltrindole and nor-binaltorphimine, could also block the analgesic effect markedly, suggesting that this effect is related to descending mu opioidergic neurons (primary mu(1) subtype). Human HK-1(4-11) could also induce a dose- and time-dependent analgesic effect after i.c.v. administration, however, the potency of analgesia was less than h HK-1. Surprisingly, SR140333 could not modify this analgesic effect, suggesting that this effect is not mediated through the NK(1) receptors like h HK-1. SR48968 could modestly enhance the analgesic effect induced by h HK-1(4-11), indicating that a small amount of h HK-1(4-11) may bind to NK(2) receptors. Furthermore, none of the opioid receptor (OR) antagonists could markedly block the analgesia of h HK-1(4-11), suggesting that the analgesic effect is not mediated through the descending opioidergic neurons. Blocking of delta ORs significantly enhanced the analgesia, indicating that delta OR is a negatively modulatory factor in the analgesic effect of h HK-1(4-11). It is striking that bicuculline (a competitive antagonist at GABA(A) receptors) effectively blocked the analgesia induced by h HK-1(4-11), suggesting that this analgesic effect is mediated through the descending inhibitory GABAergic neurons. The novel mechanism involved in the analgesic effect of h HK-1(4-11), which is different from that of h HK-1, may pave the way for a new strategy for the investigation and control of pain.

Desensitization is induced by the repeated administration of high doses of substance P (SP) or hemokinin-1 (HK-1). However, little information is available about the mechanisms involved in the induction of desensitization by these peptides. Thus, to characterize this desensitization, we examined the dose-dependent effect of these peptides, the effect of pretreatment with neurokinin 1(NK1) receptor antagonists, and the effect of pretreatment with inhibitors of protein kinases such as protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin kinase II (CaMKII) and mitogen-activated protein kinase kinase (MEK). The number of scratchings induced by 10(-3)M SP or HK-1 decreased following pretreatment with 10(-11)-10(-3)M SP or HK-1 with a marked reduction at 10(-3) and 10(-6)M SP or HK-1. The effect of NK1 receptor antagonists on desensitization induced by pretreatment with 10(-6)M SP was marked, whereas there was little effect of pretreatment with these antagonists on 10(-6)M HK-1-induced desensitization. Additionally, 10(-6)M SP- and HK-1-induced desensitization was attenuated by pretreatment with PKA, PKC and MEK inhibitors, except a CaMKII inhibitor that inhibited SP-induced desensitization. These results indicate that the receptor and kinases involved in HK-1-induced desensitization are partially different from those of SP.

We have recently reported that rat/mouse hemokinin-1 (r/m HK-1), a mammalian tachykinin, produced dose- and time-related antinociceptive effects at the supraspinal level via activating NK(1) receptors. Moreover, r/m HK-1 remarkably enhanced both the antinociceptive extent and duration of morphine administered at the peripheral and supraspinal level through a convergence of pharmacological effects of opioid-responsive neurons. Pethidine hydrochloride is an important narcotic analgesic, which acts as an opiate agonist and has pharmacological effects similar to morphine. To improve our knowledge of the pharmacology of pethidine, the aim of the present study was to investigate the relationship between the nociception of r/m HK and pethidine by comparing it with that of r/m HK-1 and morphine. Our data showed that r/m HK-1 remarkably enhanced the antinociceptive extent of pethidine administered at the peripheral level, but not at the supraspinal level. These antinociceptive effects were blocked by prior treatment with the classical opioid receptor antagonist naloxone, indicating that the potentiated analgesic effect is mediated by opioid-responsive neurons. Differences in the antinociceptive activity of pethidine and morphine in combination with r/m HK-1, arise because there are differences in the physicochemical and pharmacokinetic properties of pethidine and morphine, particularly their lipophilicity. Our results may pave the way for a new strategy for the control of pain and may provide a clinical strategy to enable selection of either opioid as a priority.

The brains of teleost fish contain members of the tachykinin family that are the products of orthologous genes expressed in mammalian nervous tissues, but little is known regarding the physiological effects of these peptides in their species of origin. The present study compares the central actions of trout neuropeptide gamma (NPgamma), substance P (SP) and neurokinin A (NKA) (5-250 pmol) on ventilatory and cardiovascular parameters in the unanesthetized rainbow trout Oncorhynchus mykiss. Intracerebroventricular (ICV) injection of NPgamma evoked a dose-dependent elevation of the ventilation rate (f(V)) but a reduction of the ventilation amplitude (V(AMP)) that was caused by a reduction of the magnitude of the adduction phase of the ventilatory signal. The net effect of NPgamma was to produce an hypoventilatory response since the total ventilation (V(TOT)) was significantly reduced. The minimum effective dose for a significant effect of NPgamma on f(V) and V(AMP) was 50 pmol. SP evoked a significant elevation of f(V), a concomitant depression of V(AMP), and a resultant decrease in V(TOT) but only at the highest dose (250 pmol). NKA was without action on f(V) but significantly decreased V(AMP) at only the highest dose tested. In this case also, the net effect of NKA was to reduce V(TOT). When injected centrally, none of the three peptides, at any dose tested, produced changes in heart rate or mean dorsal aortic blood pressure (P(DA)). Intra-arterial injection of the three tachykinins (250 pmol) produced a significant (P<0.05) increase in P(DA), but only SP and NKA induced concomitant bradycardia. None of the three peptides produced any change in f(V) or V(AMP). In conclusion, our results demonstrate that centrally injected tachykinins, particularly NPgamma, produce a strong hypoventilatory response in a teleost fish and so suggest that endogenous tachykinins may be differentially implicated in neuroregulatory control of ventilation.

Some novel tachykinin peptides exhibiting homology with known members of the tachykinin family have been recently reported; however, little is known about the function of these peptides. Repeated intrathecal administration of substance P (SP) causes desensitization by binding SP to neurokinin 1 (NK1) receptor. Thus, to clarify the characteristics of the receptors involved in these novel peptides, we investigated whether desensitization is induced by intrathecal administration of these peptides in rats since desensitization is induced by binding these peptides to the receptor. Intrathecal administration of 10(-3) M hemokinin-1 (HK-1) and 10(-3) M decapeptide common in the carboxyl-terminal region of endokinin A and endokinin B (EKA/B) as well as SP evoked scratching behavior. When each peptide was administered twice with an interval of 15 min, remarkable desensitization of scratching behavior was produced. Furthermore, the first administration of EKA/B or SP produced clear cross-desensitization to SP, EKA/B and HK-1, whereas the first administration of HK-1 demonstrated weak cross-desensitization to EKA/B and SP. These results suggest that EKA/B and SP may bind to both the NK1 receptor and HK-1-preferred receptor, and HK-1 may preferentially bind to its preferred receptor.

Rat/mouse hemokinin 1 (r/m HK-1) is a mammalian tachykinin peptide whose biological functions are not fully understood. Our recent report showed that i.c.v. administration of r/m HK-1 could produce dose- and time-related antinociceptive effect at nanomole concentration, and naloxone significantly antagonized this effect. Thus, we provide indirect evidence favoring a role of NK1 supraspinal receptors in the inhibitory control of descending pain pathways, a role that seems to partially involve the activation of the endogenous opioid systems. Based on this report, the present study was conducted to further investigate the direct functional interaction between supraspinal tachykinin (r/m HK-1) and opioid systems. The results demonstrate that i.c.v. administration of r/m HK-1 (5 nmol/kg) could significantly potentiate the antinociceptive effects of morphine which was injected at peripheral and supraspinal level. These antinociceptive effects were blocked by prior treatment with the classical opioid receptors antagonist naloxone, indicating that the potentiated analgesic response is mediated by opioid-responsive neurons. Consistent with previous biochemical data, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on the ability of r/m HK-1 to release endogenous opioid peptides. We suggest that there may be a cascade amplification mechanism in pain modulation when the two agents were co-administrated. The synergistic analgesic relationship of morphine and r/m HK-1 established here supports the hypothesis that supraspinal tachykinin and peripheral and central opioid systems have a direct functional interaction in the modulation of local nociceptive responses.

Hemokinin-1 (HK-1) is a new peptide described as a member of the tachykinin family. HK-1 has biological effects similar to substance P (SP), a representative of the tachykinin family, following central administration. However, the biological function of HK-1 at the spinal level has not been well characterized. Thus, we investigated the effect of intrathecal administration of HK-1 by comparing it with that of SP. Intrathecal administration of HK-1 as well as SP at 10(-3) M caused pain-related behavior such as scratching. The scratching by HK-1 administration was inhibited by pretreatment with an antagonist of substance P receptor. In addition, SP (10(-8)-10(-6) M) decreased the latency of the withdrawal response of the hind paw to noxious thermal stimulation 20-30 min after intrathecal administration, whereas administration of HK-1 had little effect on this response. These results suggest that there may exist a proper receptor related to HK-1.

More information is needed on the physiological role of the tachykinins (TKs), especially neurokinin3-receptor (NK3) agonists, in the pancreas. In this paper we investigated and compared the effect of PG-KII (10(-9) to 10(-6) M), a natural NK3-receptor agonist, with that of the known secretagogues substance P (10(-9) to 10(-6)M), caerulein (10(-11) to 10(-8) M) and carbachol (10(-8) to 10(-5) M), on amylase secretion from dispersed pancreatic acini of the guinea pig and rat. PG-KII (10(-7) M) significantly increased basal amylase release from guinea pig pancreatic acini (from 5.4+/-0.9% to 11.3+/-0.5%, P < 0.05) but left basal release in the rat unchanged (6.5+/-0.5%). The stimulant effect of PG-KII on guinea pig acini was significantly reduced by the NK3-receptor antagonist, SR 142801 (5 x 10(-7) M), and left unchanged by the NK1-receptor antagonist, SR 140333 (5 x 10(-7) M). Conversely, substance P (10(-7) M) significantly stimulated amylase secretion from rat and guinea pig acini (12.6+/-0.6% and 12.1+/-0.7%, P < 0.05). This stimulated effect of substance P was antagonized by the NK1--receptor antagonist (5 x 10(-7) M), but not by the NK3-receptor antagonist (5 x 10(-7) M). The PG-KII- and substance P-evoked maximal responses were lower than those evoked by caerulein (10(-9) M) (guinea pig, 19.1+/-1.3%; rat, 1802+/-0.9%, P < 0.01) and carbachol (10(-5) M) (guinea pig, 23.3+/-1.2%; rat, 24.0+/-1.1%, P < 0.01). The inhibitors of phospholipase C U-73122 (10(-5) M), phospholipase A2 quinacrine (10(-5)M), and protein tyrosine kinase genistein (10(-4) M), partly but significantly inhibited PG-KII, as well as carbachol-stimulated amylase release. Coincubation of PG-KII 10(-7) M with submaximal doses of caerulein (10(-11) to 10(-10) M) and carbachol (10(-7) to 10(-6) M) had an additive effect on amylase release. Pre-incubation with PG-KII (10(-7) M) for 30 min significantly reduced the subsequent amylase response to PG-KII, whereas pre-incubation with caerulein 10(-10) M or carbachol 10(-6) M did not. These findings suggest that PG-KII directly contributes to pancreatic exocrine secretion by interacting with acinar NK3 receptors of the guinea pig but not of the rat. PG-KII signal transduction involves the intracellular phospholipase C, phospholipase A2 and protein tyrosine kinase pathways. The NK3 receptor system cooperates with the other known secretagogues in regulating guinea pig exocrine pancreatic secretion and undergoes rapid homologous desensitization.

Hemokinin-1 (HK-1) is a recently described mouse tachykinin peptide whose biological functions are not fully understood. To date, a unique receptor for HK-1 has not been identified. Recent studies suggest HK-1 may have a role in immunological functions, but there has been little characterization of HK-1's effects in the central nervous system (CNS). In the present studies, we confirm that HK-1 is an endogenous agonist at all of the known tachykinin receptors, and is selective for the NK1 receptor over the NK2 and NK3 subtypes. CHO cells transfected with the human NK1 receptor released intracellular calcium in response to HK-1. In addition, HK-1 competed with substance P (SP) for binding to mouse NK1 and human NK1 receptors. In vivo central administration of HK-1 to gerbils and mice induced foot-tapping and scratching behaviors, respectively, similar to those observed following central administration of SP or the NK1 receptor agonist, GR-73632. Furthermore, these behavioral effects were blocked by the selective NK1 receptor antagonist, MK-869. Finally, a comprehensive expression analysis of HK-1 demonstrated that HK-1 mRNA is much more broadly expressed than previously reported with expression observed in many brain regions. Together these data demonstrate that HK-1 is a functional agonist at NK1 receptors and suggest that HK-1 may function both centrally and peripherally.

The tachykinins, including substance P, neurokinin A and neurokinin B, are a mammalian peptide family that have documented motor, sensory and circulatory neurotransmitter functions in the gut. Little is known about their action on the exocrine pancreas. In this study we investigated the effects of PG-KII, a natural NK3-tachykinin receptor agonist, and senktide, a synthetic NK3-tachykinin receptor agonist, on amylase release from isolated pancreatic lobules of the guinea pig in comparison with the secretagogues carbachol, caerulein and substance P and the depolarizing agent KCl. When added to incubation flasks at various concentrations (from 10(-10) to 10(-6)M), PG-KII and senktide both caused a dose-dependent increase in amylase release from pancreatic lobules. PG-KII and senktide elicited a lower maximal response (7.5+/-0.8 and 8.1+/-0.6% of the total lobular amylase content) than carbachol (34.4+/-3.9%), caerulein (26.5+/-2.8%) and KCl (22.5+/-3.8%). Whereas atropine left PG-KII and senktide-stimulated secretion unaffected, the non peptide NK3 receptor antagonist SR 142801 significantly reduced the stimulant effect of PG-KII and senktide. PG-KII (10(-7)M) also slightly though significantly increased the response to lower concentrations of caerulein (10(-11) and 10(-10)M) and carbachol (10(-7) and 10(-6)M). These findings show that PG-KII and senktide are weak stimulants of exocrine pancreatic secretion that act directly on the acinar cells through NK3 receptors, without cholinergic involvement. We suggest also that the tachykininergic NK3 receptor system cooperates with the other known secretagogues in the control of pancreatic exocrine secretion.

1. Scald injury in Sv129+C57BL/6 mice induced a temperature and time dependent oedema formation as calculated by the extravascular accumulation of [(125)I]-albumin. Oedema formation was suppressed in NK(1) knockout mice compared to wildtypes at 10 (P<0.01) and 30 min (P<0.001). However, at 60 min a similar degree of extravasation was observed in the two groups. 2. Kinin B(1) (des-Arg(10) Hoe 140; 1 micromol kg(-1)) and B(2) (Hoe 140; 100 nmol kg(-1)) antagonists caused an inhibition of oedema in wildtype mice at 10 and 30 min (P<0.001), but not at 60 min or at 30 min in NK(1) receptor knockout mice. 3. The inhibition of thermic oedema by des-Arg(10) Hoe 140 was reversed by des-Arg(9) bradykinin (0.1 micromol kg(-1); P<0.01) and also observed with a second B(1) receptor antagonist (des-Arg(9) Leu(8) bradykinin; 3 micromol kg(-1); P<0.01). Furthermore des-Arg(10) Hoe 140 had no effect on capsaicin (200 microg ear(-1)) ear oedema, but this was significantly reduced with Hoe 140 (P<0.05). 4. Scalding induced a large neutrophil accumulation at 4 h, as assessed by myeloperoxidase assay (P<0.001). This was not suppressed by NK(1) receptor deletion or kinin antagonists. 5. These results confirm an essential role for the NK(1) receptor in mediating the early, but not the delayed phase of oedema formation or neutrophil accumulation in response to scalding. The results also demonstrate a pivotal link between the kinins and sensory nerves in the microvascular response to burn injury, and for the first time show a rapid involvement of the B(1) receptor in murine skin.

Objectives: In order to determine the active sites for salivation of various tachykinins, the regulatory roles of the N-terminal portion of various newly-synthesized tachykinins were studied after i.p. injection of rats using the submandibular glands as model organs., Methods: N-shortened oligopeptides from kassinin, eledoisin, neurokinins A (NKA) and NKB were synthesized by the multipin peptide synthesis method. Amino acids were eliminated one by one to form octa- to undeca-peptides adjoining the inactive or less active heptapeptides and various heptapeptides, in which an amino acid in position 8 (Xaa8), numbering as in an undecapeptide, was replaced with Tyr, Phe, Ile or Val., Results: The N-terminal amino acids in positions 1 to 4 could be activators or inhibitors, depending on whether the C-terminal heptapeptide was inactive or less active. The Xaa8 residue, in combination with amino acids in positions 5 and 6, seemed to be very important in determining the sialogogic activity of a heptapeptide. The discrimination between NKA and NKB appeared due to the N-terminal amino acid sequence in positions I to 4 including Phe or Ser in position 6., Conclusions: It is concluded that the N-terminal amino acids in positions I to 4 serve as either activators or inhibitors depending upon the sialogogic activity of the C-terminal heptapeptide, in which particular amino acids in positions 5, 6 and 8 regulate its activity.

We investigated and compared the effects of two amphibian tachykinins, the NK1 receptor agonist PG-SPI and the NK3 receptor agonist PG-KII, and the mammalian tachykinins substance P, neurokinin A and neurokinin B on the reaction time to a painful radiant heat stimulus (tail-flick test in rats) after intracerebroventricular injection. PG-SPI (1, 10 and 20 microg) and PG-KII (1, 5 and 10 microg) significantly increased the reaction time. Substance P (10 microg) injected intracerebroventricularly induced antinociception, whereas neurokinin A and neurokinin B did not. Like analgesia evoked by exogenous substance P, PG-SPI-evoked analgesia was blocked by pretreatment with naloxone. Naloxone left PG-KII antinociception unchanged, but the NK3 receptor selective antagonist markedly reduced it. These findings suggest NK1 and NK3 tachykinin receptor system involvement in supraspinal analgesia in rats.

The mechanisms involved in tachykinin-induced neurokinin-1 (NK(1)) receptor-mediated edema formation have been studied in anesthetized wild-type and NK(1) knockout mice. Intradermally injected substance P (30-300 pmol), NK(1) agonists septide (3-30 pmol) and GR-73632 (3-30 pmol), and the mast cell-degranulating agent, compound 48/80 induced dose-dependent edema in wild-type skin, measured by the accumulation of intravenously injected (125)I-labeled albumin. Septide was 3-10x more potent than substance P. The tachykinins were inactive in knockout mice, but compound 48/80 induced a significantly greater edema (P < 0.05) than that observed in paired wild-type mice. Capsaicin (which releases endogenous neuropeptides) and exogenous tachykinins induced edema formation, which was reduced by the mast cell amine histamine H(1) antagonist mepyramine (P < 0.05). These findings confirm that tachykinins mediate edema formation via the NK(1) receptor and provide direct evidence that the septide-sensitive binding site is on the NK(1) receptor. Furthermore, results suggest that edema induced by the tachykinins, although totally dependent on NK(1) receptor-mediated mechanism, contains a mast cell-dependent component. The evidence is in keeping with an NK(1) receptor on mast cells.

The effects of lateral ventricular injections of succinyl-[Asp6, N-Me-Phe8]-substance P (SENK; 25, 100, 200 ng), a tachykinin NK3 receptor agonist, and [Sar9, Met(O2)11]-substance P (Sar Met; 100, 200 ng), an NK1 receptor agonist, on normal (gastric fistula closed) and sham drinking (gastric fistula open) of hypertonic NaCl by sodium-deficient rats were compared. Intraventricular injections of Sar Met had no effect on NaCl intake in either condition. Injections of 100 ng and 200 ng SENK caused an equal suppression of NaCl intake in the 2 fistula conditions. The latency to drink was not affected, but the initial lick rate was significantly lower and decayed more rapidly after 100 ng SENK than after saline or 25 ng SENK. The results show that (a) the tachykinin subtypes are not equally involved in the control of need-induced salt intake; (b) negative feedback from the stomach and distal gastrointestinal tract is not required for intraventricular injections of SENK to suppress sodium appetite; (c) the activation of NK3 receptors decreases the oral excitatory influence of hypertonic NaCl in sodium-deficient rats.

The inhibitory action of the natural selective tachykinin NK3 receptor agonist, PG-KII, (pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2), on colonic propulsion was studied in rats after central administration. Intracerebroventricular injection of PG-KII (0.1, 1, 10 and 100 ng/rat) produced a dose-related inhibition of colonic propulsion, measured as the increase in the mean expulsion time of a 5-mm glass bead placed in the distal colon. At the same doses as PG-KII, the selective tachykinin NK3 receptor agonist, senktide, (succ-[Asp6-MePhe8] substance P-(6-11)), induced a similar dose-related inhibition. Conversely, substance P (0.1, 1 and 10 microg/rat), a tachykinin NK1-preferring receptor agonist, had weaker antipropulsive effects, neurokinin A (0.1, 1 and 10 microg/rat), a tachykinin NK2-preferring receptor agonist, at the highest dose used only slightly inhibited colonic propulsion and neurokinin B (0.1, 1 and 10 microg/rat), a tachykinin NK3-preferring receptor agonist, left propulsion unchanged. Pretreatment with the selective tachykinin NK3 receptor antagonist, 3-indolycarbonyl-Hyp-Phg-N(me)-Bzl, referred as to R820 (6.2 microg/rat), prevented PG-KII-induced colonic antipropulsion, whereas the tachykinin NK1 receptor antagonist, (S)-1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pi peridin-3-yl] ethyl)-4-phenyl-1-azoniabicyclo[2.2.2] octane chloride, referred to as SR 140,333 (1 microg/rat), and the tachykinin NK2 receptor antagonist, ([Tyr5,D-Trp6,8,9, Arg10] neurokinin A-(4-10)), referred to as Men 10,376 (5 microg/rat), left it unchanged. These findings show that of the tachykinins tested, PG-KII and senktide are the most potent central inhibitors of colonic propulsion in the rat, suggesting that the central tachykinin NK3 receptor system plays an inhibitory role in modulating colonic transit. As well as confirming the selectivity of PG-KII for tachykinin NK3 receptors, we show that PG-KII provides useful information about the physiological role of central tachykinin NK3 receptors and that glass bead expulsion test is a reliable non-invasive in vivo method for evaluating the tachykinin NK3 receptor selectivity of new synthetic or natural tachykinins.

Repeated oesophageal acidification is a definitive feature of gastro-oesophageal reflux disease, which in turn is caused by relaxation of the lower oesophageal sphincter (LOS). This study in anaesthetised ferrets investigates the reflex pathways involved in effects of oesophageal acidification on motor function of the LOS, with particular focus on the role of tachykinins. LOS pressure was monitored with a perfused micromanometric sleeve assembly. Oesophageal acidification reduced LOS pressure by 48 +/- 5% until washout with saline. This reduction became larger with repeated tests, and was unaffected in amplitude by acute bilateral vagotomy, although the response became slower in onset. Intra-oesophageal capsaicin (0.5% solution) caused a 68 +/- 17% decrease in LOS pressure which remained unchanged with repeated tests. The NK-1 receptor antagonist CP96,345 (1-5 mg/kg intravenous (i.v.) blocked the post-vagotomy LOS responses to both intra-luminal acid and capsaicin. Close intra-arterial (i.a.) injections of capsaicin (1-100 micrograms) gut induced LOS relaxation which was neither vagally nor NK-1 receptor-mediated. Substance P or the selective NK-1 receptor agonist [Sar9, Met(O2)11] substance P (25-500 ng close i.a.) caused a biphasic LOS response, consisting of initial brief contraction followed by prolonged, dose-dependent relaxation. Tetrodotoxin (10 micrograms/kg close i.a.) changed the biphasic response to substance P to excitation only. The neurokinin-1 (NK-1) receptor antagonist CP96,345 (0.3-10 mg/kg i.v.) dose-dependently reduced the inhibitory response to substance P. The excitatory phase of the response to substance P was larger and prolonged after guanethidine (5 mg/kg, i.v.), or propranolol (1 mg/kg, i.v.). L-NAME (100 mg/kg i.v.) reduced the inhibitory phase. The selective NK-2 receptor agonist [beta-Ala8] neurokinin A(4-10) caused LOS excitation only. These data indicate that intra-oesophageal acid causes substance P release from extrinsic afferent nerve endings which activates local inhibitory pathways to the LOS via NK-1 receptors.

1. The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [beta-Ala8]NKA (4-10) and [MePhe7]neurokinin B (NKB) at T13 thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function. 2. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9 +/- 0.8; SP value 0.7 +/- 0.3 micromol min(-1), P<0.01) as well as the renal excretion of water (aCSF value 48.9 +/- 5.8; SP value 14.5 +/- 4.0 microl min(-1), P<0.01) and potassium (aCSF value 4.8 +/- 0.6; SP value 1.5 +/- 0.6 micromol min(-1), P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (-18.8 +/- 3.8 mmHg at 1 min and +6.3 +/- 2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes. 3. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2 +/- 5.1; NKA value 11.3 +/- 4.2 microl min(-1), P<0.05), sodium (aCSF value 1.7 +/- 0.8; NKA value 0.4 +/- 0.2 micromol min(-1), P<0.05) and potassium (aCSF value 4.1 +/- 0.7; NKA value 1.5 +/- 0.4 micromol min(-1), P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK2 ([beta-Ala8]NKA(4-10)) and NK3 ([MePhe7]NKB) receptors were devoid of renal effects. 4. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK1 receptors and the sympathetic renal nerve.

Intracerebroventricular (i.c.v.) injection of tachykinins (TKs) inhibits ethanol intake and angiotensin II-induced water intake; the effects are apparently mediated by NK-3 and NK-1 receptors, respectively. The present study evaluated the effect of the TK PG-KII, a novel kassinin-like peptide isolated from the skin of the Australian frog Pseudophryne güntheri, in these in vivo tests for central activity. PG-KII, given by i.c.v. injection, potently inhibited alcohol intake in genetically selected alcohol-preferring rats, being about 3 times more potent than the selective NK-3 receptor agonist NH2-SENK. The dose of 100 ng/rat, that markedly inhibited ethanol intake, did not inhibit food intake and prandial drinking in food deprived rats, providing evidence that the effect of PG-KII on ethanol intake is behaviorally selective. The effect on ethanol intake was inhibited by i.c.v. injection of the NK-3 receptor antagonist R820, but was not modified by the NK-1 receptor antagonist SR 140333. PG-KII inhibited drinking induced by angiotensin II only at doses of 300 or 1000 ng/rat, being about 5 times less potent than the selective NK-1 receptor agonist [Sar9, Met(O2)11] substance P. These doses of PG-KII produced also marked increase in competing behaviors, such as grooming and locomotion. The dose of 1000 ng/rat evoked a general inhibition of the ingestive behavior, reducing also food intake. The i.c.v. injection of the NK-1 receptor antagonist SR 140,333 only slightly inhibited the effect of PG-KII on angiotensin II-induced drinking, while it markedly reduced that of [Sar9, Met(O2)11] substance P. These findings, in accordance with those of previous studies, indicate that PG-KII is endowed with marked activity at central NK-3 receptors, and low activity at NK-1 receptors.

Intrathecal (i.t.) administration of morphine in the spinal subarachnoid space of mice produced a severe hindlimb scratching followed by biting and licking. The onset of the scratching behaviour was observed 60-70 s after i.t. injection of morphine (60 and 90 nmol), and had a duration of 3-4 min. The morphine-induced behaviour was increased additively by i.t. co-administration of substance P (SP). This characteristic behavioural response was inhibited dose-dependently by i.t. co-administration of the tachykinin NK-1 receptor antagonists, sendide and CP-96,345. Significant antagonistic effects of SP (1-7), a putative antagonist for NK-1 receptors and [D-Phe7, D-His9]SP (6-11), a selective antagonist for SP receptors, were observed against the morphine-induced behaviour. Pretreatment with i.t. SP antiserum and i.t. capsaicin resulted in reduction of the response to morphine. I.t. administration of somatostatin (SOM) antiserum, cysteamine, a relatively selective depletor of SOM and cyclo-SOM, a SOM receptor antagonist, produced no inhibitory effect on the morphine-induced behaviour. These results demonstrate that a spinal system of neurones containing SP may be involved in elicitation of the behavioural episode following i.t. injection of morphine in mice.

Tachykinin control of gut blood flow (measured by pulsed Doppler technique), dorsal aortic pressure, and heart rate were studied in unrestrained spiny dogfish Squalus acanthias injected with the elasmobranch tachykinins scyliorhinin I and II (SCY I and SCY II), the trout tachykinins substance P (SP), and neurokinin A (NKA). Effects on somatic vasculature were measured by in vitro perfusion of the isolated tail. SCY I and trout SP produced hypotension due to a general vasodilation. This caused a transient increase in mesenteric blood flow and a prolonged increase in celiac blood flow. SCY II caused an initial hypertension induced by a general vasoconstriction, followed eventually by an elevated flow in both gut arteries due to dilation of the vascular beds. Trout NKA evoked a short-lasting increase in celiac blood flow due to a decrease in vascular resistance, a late decrease in mesenteric flow due to vasoconstriction, and no effect on the somatic vasculature. None of the peptides affected heart rate. The study demonstrates a significant vasoactive function of fish tachykinins in the vascular system of an elasmobranch species and, in addition, the occurrence of tachykinin receptor subtypes. Immunohistochemistry revealed a NKA/SCY II-like peptide in nerve fibers innervating many vessels, including the celiac and the mesenteric arteries, the gastrointestinal canal, and the heart.

1. In the conscious rat, cardiovascular responses to intrathecally (i.t.) administered neuropeptide gamma (NP gamma) were studied prior to and after the i.t. pretreatment with selective antagonists at NK1 ((+/-)-CP 96345 and RP 67580), NK2 (SR 48968) and NK3 (R 486) receptors. Pretreatment with a mixture of peptidase inhibitors (phosphoramidon, captopril, bacitracin, phenanthroline) was also tested to ascertain whether or not the effect of NP gamma was mediated by a metabolite. The involvement of peripheral catecholamines was examined with intravenous injection of alpha-adrenoceptor (phentolamine) and beta-adrenoceptor (propranolol) antagonists. 2. NP gamma (0.078-78 nmol) induced dose-dependent increases in heart rate (HR) and mean arterial blood pressure (MAP). The highest dose of 78 nmol did not induce an increase of MAP greater than that with 7.8 nmol but was preceded by a transient decrease of MAP (1-3 min). No desensitization was observed when three injections of 7.8 nmol NP gamma were given at 90 min intervals. 3. Cardiovascular and behavioural (biting/scratching) effects evoked by 0.78 nmol NP gamma were significantly reduced by the NK1 antagonists, (+/-)-CP 96345 (65 nmol) or RP 67580 (7.8 and 78 nmol). However, cardiovascular responses to NP gamma were not affected by (+/-)-CP 96345 (6.5 nmol), SR 48968 (7.8 and 78 nmol) or R 486 (25 nmol). Pretreatment with peptidase inhibitors significantly enhanced the cardiovascular and behavioural responses to NP gamma. 4. The pressor response to 7.8 nmol NP gamma was converted to a vasodepressor response by pretreatment with phentolamine (2 mg kg-1, i.v.) while the chronotropic response was markedly reduced by propranolol (2 mg kg-1, i.v.). 5. These results suggest that the cardiovascular responses to i.t. NP gamma are mediated by NK1 receptors in the spinal cord leading to the peripheral release of catecholamines from sympathetic fibres or the adrenal medulla. It is unlikely that the spinal action of NP gamma results from its metabolic conversion into neurokinin A or another major metabolite.

1. The cardiovascular and behavioural effects elicited by the intracerebroventricular (i.c.v.) administration of neuropeptide gamma (NP gamma) in the conscious rat were assessed before and 5 min after i.c.v. pretreatment with antagonists selective for NK1 (RP 67,580), NK2 (SR 48,968) and NK3 (R 820) receptors. In addition, the central effects of NP gamma before and after desensitization of the NK1 and NK2 receptors with high doses of substance P (SP) and neurokinin A (NKA) were compared. 2. Intracerebroventricular injection of NP gamma (10-780 pmol) evoked dose- and time-dependent increases in mean arterial blood pressure (MAP), heart rate (HR), face washing, head scratching, grooming and wet-dog shake behaviours. Similar injection of vehicle or 1 pmol NP gamma had no significant effect on those parameters. 3. The cardiovascular and behavioural responses elicited by NP gamma (25 pmol) were significantly and dose-dependently reduced by pretreatment with 650 pmol and 6.5 nmol of SR 48,968. No inhibition of NP gamma responses was observed when 6.5 nmol of RP 67,580 was used in a similar study. Moreover, the prior co-administration of SR 48,968 (6.5 nmol) and RP 67,580 (6.5 nmol) with or without R 820 (6.5 nmol) did not reduce further the central effects of NP gamma and significant residual responses (30-50%) remained. 4. No tachyphylaxis to NP gamma-induced cardiovascular and behavioural changes was observed when two consecutive injections of 25 pmol NP gamma were given 24 h apart. 5. Simultaneous NK1 and NK2 receptor desensitization reduced significantly central effects mediated by 25 pmol NP gamma. However, significant residual responses persisted as seen after pretreatment with SR 48,968. 6. The results suggest that the central effects of NP gamma are mediated partly by NK2 receptors and by another putative tachykinin receptor subtype (NP gamma receptor?) that appears to be different from NK1 and NK3 receptors.

We have investigated the effects of actinomycin D on mouse ear oedema induced by capsaicin, neuropeptides, and established inflammatory mediators. Actinomycin D (0.5 mg/kg, i.v.) significantly (P < 0.01) inhibited ear oedema induced by topical application of capsaicin, while adriamycin (6.0 mg/kg, i.v.) and cycloheximide (6.0 mg/kg, i.v.) had no effect on oedema. The ear oedema induced by intradermal injection of neuropeptides such as mammalian tachykinins, calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP), was markedly (P < 0.05, P < 0.01 or P < 0.001) suppressed by actinomycin D. The drug was also effective (P < 0.01 or P < 0.001) in inhibiting bradykinin (BK)- and compound 48/80-induced ear oedema, but did not inhibit oedema induced by histamine, 5-HT, leukotriene C4 (LTC4), and platelet activating factor (PAF) at a dose of 1 mg/kg. In mast cell-deficient W/WV mice, actinomycin D (1.0 mg/kg, i.v.) failed to inhibit substance P (SP)-induced ear oedema whereas spantide (0.5 mg/kg, i.v.) was an effective (P < 0.01) inhibitor of oedema formation. Furthermore, actinomycin D (10-100 microM) dose-dependently prevented histamine release from rat peritoneal mast cells evoked by SP, compound 48/80, and the ionophore A23182, respectively. These results strongly suggest that an inhibitory effect of actinomycin D on neurogenic inflammation is due primarily to the prevention of mast cell activation mediated by neuropeptides, rather than an interaction with DNA or receptors of neuropeptides.

1. The effects on micturition of RP 67,580, a selective NK1 receptor antagonist, and SR 48,968, a highly, potent antagonist at NK2 receptor sites, given intrathecally (i.t.) or intra-arterially (i.a.) near the bladder, were investigated in unanaesthetized rats with and without bladder outlet obstruction. 2. In normal rats, RP 67,580, given i.t. in doses of 2 and 20 nmol per rat, decreased micturition pressure, but did not change other cystometric parameters. After 20 nmol of RP 67,580, dribbling incontinence due to retention was observed in 1 out of 7 animals. This effect was reversible. I.t. RP 67,580 in a dose of 2 nmol, had no effect on hyperactivity induced by intravesically instilled capsaicin. 3. In animals with bladder hypertrophy secondary to outflow obstruction, RP 67,580, given i.t. in a dose of 2 nmol per rat, decreased the micturition pressure, but had no effect on other cystometric parameters. After 20 nmol, dribbling incontinence due to retention was observed in 5 out of 7 animals. 4. RP 67,580, given i.a. in a dose of 4 nmol, had little effect on the cystometric parameters investigated, both in normal animals and rats with bladder hypertrophy. 5. SR 48,968, given i.t. in doses of 2 and 20 nmol per rat, had no clear-cut effects on the micturition pattern in normal rats, or rats with bladder hypertrophy. However, the drug reduced capsaicin-induced bladder hyperactivity. When given i.a. in a dose of 4 nmol, SR 48,968 had no effect on cystometric parameters in normal rats or rats with bladder hypertrophy. 6. The effects of both RP 67,580 and SR 48,968 were stereoselective, their enantiomers (RP 68,651 and SR 48,965) being inactive.7. These results thus suggest that at the spinal level there is a tachykinin involvement (via NK,receptors) in the micturition reflex induced by bladder filling, both in normal rats, and, more clearly, in animals with bladder hypertrophy secondary to outflow obstruction. The bladder response to filling was not influenced by blockade of vesical NKI and NK2 receptors. On the other hand, the bladder hyperactivity evoked by intravesical capsaicin seems to involve NK2 receptors both at the bladder and spinal levels.

Tachykinins are potent bronchoconstrictors. Here we have used novel highly selective agonists and antagonists to characterize tachykinin receptors mediating bronchoconstriction and hypotension in anaesthetized, paralysed guinea-pigs. Total lung resistance (RL) and dynamic lung compliance (Cdyn) were measured using an on-line, breath-by-breath method to analyse lung mechanics. The effects of iv injections of neuropeptide gamma (NP gamma), [Sar9,Met(O2)11]-SP (Sar-SP) (NK-1 agonist), [Lys5,MeLeu9,Nle10]-NKA(4-10) (Lys-NKA) (NK-2 agonist) and senktide (NK-3 agonist) were compared, before and after iv injection of the NK-1 antagonist CP 96345 or the NK-2 antagonists MDL 29913 and SR 48968. NP gamma, Sar-SP and Lys-NKA, but not senktide, increased RL and decreased Cdyn, accompanied by a fall in blood pressure (BP). The potency for increase of RL was Lys-NKA > NP gamma congruent to Sar-SP, for reduction of Cdyn was Lys-NKA = Sar-SP > NP gamma, and for fall in BP was Sar-SP > NP gamma >> Lys-NKA. CP 96345 (50 nmol/kg & 200 nmol/kg) significantly antagonized the changes in RL, Cdyn and BP induced by Sar-SP, and the fall in BP (but not changes in lung mechanics) in response to NP gamma, but did not alter any responses to Lys-NKA. In contrast, MDL 29913 (200 nmol/kg & 10 mumol/kg) weakly and SR 48968 (100 nmol/kg) potently inhibited the effects on RL and Cdyn induced by Lys-NKA and NP gamma. Our results show that both NK-1 and NK-2 receptors are represented in guinea-pig airways. MDL 29913 is a weak NK-2 antagonist in guinea-pig airways compared to SR 48968. In addition, NK-1 but not NK-2 receptors appear to be important in mediating the hypotensive response to these tachykinins. In spite of its ability to cause a fall in BP via NK-1 receptors, NP gamma exerted its effects on RL and Cdyn solely via NK-2 receptors.

1. The effects of intracerebroventricular (i.c.v.) injection of selective and potent NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 486, [Trp7, beta-Ala8]NKA(4-10)) receptor antagonists were assessed on the cardiovascular and behavioural responses elicited by the i.c.v. injection of substance P (SP), neurokinin A (NKA) or [MePhe7]neurokinin B ([MePhe7]NKB) in the conscious freely moving rat. 2. SP, NKA and [MePhe7]NKB (5-650 pmol) evoked dose-dependent increases in mean arterial blood pressure (MAP) and heart rate (HR) with the rank order of potency SP > NKA > [MePhe7]NKB. The cardiovascular responses were accompanied by excessive face washing, grooming and wet dog shakes. 3. The cardiovascular effects and face washing behaviour induced by SP (25 pmol) were significantly reduced by the pre-injection (i.c.v., 5 min earlier) of RP 67580 (6.5 nmol). However, this antagonist failed to affect the central effects of 25 pmol NKA or [MePhe7]NKB. 4. The cardiovascular and behavioural responses (except for wet dog shakes) elicited by NKA (25 pmol) were significantly reduced by 6.5 nmol SR 48968. However, the latter antagonist had no effect on the SP or [MePhe7]NKB-mediated responses. 5. Both cardiovascular and behavioural effects produced by either SP or NKA (25 pmol) were completely abolished when rats were pretreated with a combination of RP 67580 (6.5 nmol) and SR 48968 (6.5 nmol), yet this combination of antagonists failed to modify the central effects of [MePhe7]NKB. 6. R 486 (6.5 nmol) inhibited the cardiovascular effects as well as wet dog shakes produced by [MePhe7]NKB, but it was inactive against the responses induced by either SP or NKA. 7. None of the tachykinin receptor antagonists or agonists caused motor impairment or respiratory distress. All antagonists blocked in a reversible manner and were devoid of intrinsic activity except R486 (6.5 nmol) which produced a transient increase of MAP and HR.8. These results suggest that the central effects of SP, NKA and [MePhe7]NKB are primarily mediated by central NK1, NK2 and NK3 receptors, respectively. However, a minor activation of NK2 receptors bySP and NK1 receptors by NKA was seen during blockade of both receptors. This study therefore supports the existence of functional NK1, NK2 and NK3 receptors in the adult rat brain.

Responses to the administration of graduated doses of tachykinins were examined in various vascular beds of the dog. Among the tachykinins, all vascular beds studied demonstrated that the greatest vasodilation responses was produced by substance P. However, each tachykinin demonstrated a specific pattern of response. With substance P the greatest sensitivity was seen in the superior mesenteric artery (SMA), with neurokinin B in the celiac axis, and with neurokinin B a prolonged vasodilation response of the SMA compared to the other two vessels was seen. The response to substance P in the SMA of the rat was considerably less than that of the dog.

Scyliorhinin-I amide (SCY-I) (selective for NK-1 and NK-2 receptors) or scyliorhinin-II amide (SCY-II) (selective for NK-3 receptors) were injected either spinally (i.t.; intrathecally) or supraspinally (i.c.v.; intracerebroventricularly) to mice. Following i.c.v. administration, SCI-I and SCY-II produced potent, dose-related reciprocal hindlimb scratching about equipotently (ED50 = 0.05 and 0.08 nmol, respectively). However, following i.t. administration, only SCY-I elicited greater than 50% response (ED50 = 0.07 nmol). Reciprocal hindlimb scratching is a behavioral response that has not been associated previously with neurokinins. These results might provide the first functional in vivo correlate for the differential localization of neurokinin receptor types within the mammalian central nervous system.

The present study evaluated the sensitivity of spontaneously hypertensive (SHR) and of Wistar Kyoto (WKY) rats to the hypotensive effect of tachykinins (TKs). Eledoisin, substance P, and the NK-1-selective agonist [Sar9,Met(O2)11]substance P evoked a smaller hypotensive response in SHR than in WKY rats. The hypotensive effect of NKA was slightly smaller in SHR, but no significant strain difference was observed. The NK-2-selective agonist [beta Ala8]NKA(4-10) was a very weak hypotensive agent in WKY rats, while being completely inactive in SHR. The NK-3-selective agonists [Asp5,6,MePhe8]substance P(5-11) and [MePhe7]NKB did not modify blood pressure in both strains. Heart rate was essentially unmodified following the NK-3 agonists, while it was increased after injection of substance P, [Sar9,Met(O2)11]substance P, and neurokinin A, the increase being greater in WKY than in SHR. Surprisingly, eledoisin increased heart rate in SHR, but not in WKY rats, despite the greater hypotensive effect elicited in the latter strain. The present results confirm that the hypotensive effect of peripheral TKs is mediated by NK-1 receptors and show that SHR are less sensitive than WKY rats to this effect.

The tachykinins are a group of structurally related peptides found in the rat hypothalamus and anterior pituitary. We have evaluated the effects of four tachykinins on LH release in male rats. In intact male rats, intracerebroventricular (icv) injection of neurokinin A (NKA), neuropeptide K (NPK), and neuropeptide-gamma (NP gamma) elicited dose-related, transient increases in plasma LH. Substance P (SP) was ineffective under these conditions. A further examination showed that in vitro incubation with either NPK or NP gamma of hemipituitaries from intact but not castrated male rats promoted release of LH into the medium, thereby revealing that the excitatory effects of tachykinins in intact male rats may, in part, be a result of stimulation of LH release directly from the anterior pituitary. On the other hand, the effects of these four tachykinins on LH release were different in castrated rats. Intracerebroventricular injection of NPK, NKA, and NP gamma as well as SP, which was ineffective in intact male rats, evoked a long-lasting suppression of LH release. Comparatively, NPK was the most effective tachykinin in eliciting LH responses in both of these tests involving different endocrine environments. We next evaluated the possibility that the inhibitory effects of tachykinins (NPK) may be mediated by activation of inhibitory endogenous opioid peptides. The results showed that iv infusion of the opiate receptor antagonist naloxone, to block the possible inhibitory effects of endogenous opioid peptides, only partially counteracted the suppressive effects of icv NPK on plasma LH levels. Thus, in addition to revealing the diverse effects of structurally related tachykinins on LH release, the results of these investigations showed specifically that the NK-2 receptor agonists NPK, NP gamma, and NKA stimulated LH release in intact rats, in part, by a direct action at the level of the pituitary, whereas the NK-1 receptor agonist SP was inactive under these conditions. These findings imply a paracrine/autocrine mode of excitatory action on LH release involving pituitary NK-2 receptor subtypes. On the other hand, in castrated rats, all four tachykinins readily suppressed LH release by a central action involving, in part, an activation of hypothalamic opioid systems.

Repeated sodium depletions produce a persistent, enhanced need-free salt intake in the rat, particularly in the female. The neurochemical mechanisms underlying the phenomenon are still unknown. The present studies evaluated the effect on the enhanced need-free salt intake of the female rat (1) of pharmacological interference with the natriorexigenic hormones angiotensin II and aldosterone and (2) of the central injection of the tachykinin peptides, which are endowed with antinatriorexic activity. The need-free salt intake of the female rat is not modified by treatment with the angiotensin-converting enzyme inhibitor captopril or by the aldosterone receptor antagonist RU-28318. On the other hand, the behavior is highly sensitive to the inhibitory effect of central tachykinins, suggesting the possibility that need-free salt intake might be linked to modification (down-regulation) of the inhibitory tachykininergic system.

Central injection of the tachykinins (TKs) neurokinin A (NKA), eledoisin (ELE), and kassinin (KASS) produced long-lasting inhibition (up to 6 h) of drinking induced by subcutaneous hypertonic NaCl, while substance P (SP) and physalaemin (PHYS) evoked short-lasting effects. The hypothesis that water retention or increased Na+ excretion by the kidney (induced by TKs) may reduce the need for water ingestion was tested. The results obtained both in urine collection experiments and in nephrectomized rats showed that the duration of the effect of NKA, ELE, and KASS is not due to water retention or increased Na+ excretion by the kidney. The effect of NKA, but not that of ELE and KASS, was shortened by nephrectomy, even though NKA did not modify urine volume or Na+ excretion. Indomethacin pretreatment, like nephrectomy, reduced the duration of the NKA effect, suggesting that renal prostaglandins are involved in it. On the other hand, the long-lasting effect of the three TKs cannot be easily explained in terms of slow metabolic degradation, particularly for NKA. Alternatively, it might be hypothesized that these TKs produce a modification of osmoreceptor function lasting well beyond the life of the peptide, and/or that they produce Na+ loss through emunctories different from the kidney.

Tachykinins, a family of biologically active related peptides, are found in variable amounts in the rat hypothalamus. We assessed the effects of five tachykinins, substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NP gamma), and neurokinin B (NKB), on LH release in different experimental model systems in ovariectomized rats. In the first series of experiments rats were ovariectomized and implanted with permanent cannulae in the third cerebroventricle of the rat brain. Two weeks later, the effects of intracerebroventricular injection of 0.5 or 1.25 nm various tachykinins on LH release were studied. The results showed that whereas SP, NKA, and NKB were ineffective, and NP gamma was marginally effective, NPK produced a long-lasting suppression of LH release. NPK decreased LH release in a dose- and time-related fashion. Similarly, in the second series of experiments, whereas SP and NKA were inactive, NPK completely suppressed the LH surge induced by progesterone in estrogen-primed ovariectomized rats. In the third series of experiments we observed that NK-2 receptor agonist [Nle10]NKA4-10, and not NK-1 receptor agonist [Sar9,Met(O2)11]SP, suppressed both the release of LH in vivo and basal and KCl-induced hypothalamic LHRH release in vitro. These results show that NPK is the most effective tachykinin in suppressing LH release, and the inhibitory response is mediated by hypothalamic NK-2 receptors. These findings are in accord with the hypothesis that NPK may serve as a hypothalamic inhibitory neurotransmitter/neuromodulator of LHRH secretion.

Neurokinins (NK) are a group of peptides that share a common C-terminal and play an important role in control of the motor functions of the mammalian gut. Neurokinin receptors such as NK1, NK2 are certainly present in any segment of gut, whereas the NK3 receptors are probably present in the ileum of the Guinea pig and duodenum of the rat. We measured gastrointestinal responses with natural NK and their very specific agonists to determine the probable receptors in the stomach and small intestine of Sprague-Dawley rats. After overnight fasting, the rats were intubated with a catheter to feed saline liquid meal that contained 10% charcoal. Simultaneously, various doses of NK ranged selected from 10(-10) and 10(-7) mol kg-1 included substance P (SP), neurokinin A (NKA), neurokinin B (NKB), septide, [Nle10]-NKA4-10, senktide, and vehicle were intraperitoneally injected. At 15 min after being fed test meal, the rats were sacrificed. Then we removed entire gut including the stomach to measure the total length of small intestine and transit length of the charcoal, from which the transit ratio was calculated. In comparison with ratios of rats treated with vehicle, the inhibited transit ratios for charcoal were seen among SP at 10(-10) mol kg-1, NKA at 10(-10) and 10(-7) mol kg-1, [Nle10]-NKA4-10 at 10(-7) mol kg-1, and senktide at all doses except 10(-8) mol kg-1. Enhanced transit ratios were seen for septide at the doses 10(-8) and 10(-7) mol kg-1. Likewise the mean total intestinal lengths of rats if they received various treatments of peptides except that rats treated with NKB had somewhat diminished length than those of vehicle-treated rats. Some natural NK and their very specific receptor agonists mainly inhibited rat gastrointestinal charcoal transits. We suggest the probable presence of NK1, NK2 and NK3 receptors in the small intestine and stomach including pylorus of the rat.