Pharmacological characterization of desensitization in scratching behavior induced by intrathecal administration of hemokinin-1 in the rat

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.     

Role of hemokinin-1 in health and disease

Hemokinin-1 (HK-1), the newest tachykinin encoded by the Tac4 gene was discovered in 2000. Its name differs from that of the other members of this peptide family due to its first demonstration in B lymphocytes. Since tachykinins are classically found in the nervous system, the significant expression of HK-1 in blood cells is a unique feature of this peptide. Due to its widespread distribution in the whole body, HK-1 is involved in different physiological and pathophysiological functions involving pain inflammation modulation, immune regulation, respiratory and endocrine functions, as well as tumor genesis. Furthermore, despite the great structural and immunological similarities to substance P (SP), the functions of HK-1 are often different or the opposite. They both have the highest affinity to the tachykinin NK1 receptor, but HK-1 is likely to have a distinct binding site and signalling pathways. Moreover, several actions of HK-1 different from SP have been suggested to be mediated via a presently not identified own receptor/target molecule. Therefore, it is very important to explore its effects at different levels and compare its characteristics with SP to get a deeper insight in the different cellular mechanisms.Since HK-1 has recently been in the focus of intensive research, in the present review we summarize the few clinical data and experimental results regarding HK-1 expression and function in different model systems obtained throughout the 16 years of its history. Synthesizing these findings help to understand the complexity of HK-1 actions and determine its biomarker values and/or drug development potentials.     

The role of the amino-terminal domain of tachykinins in neurokinin-1 receptor signaling and desensitization

Neurokinin A (NKA) has previously been shown to be a full agonist of the neurokinin-1 receptor (NK-1R) but is only able to cause partial homologous desensitization of the receptor compared to substance P (SP). NKA and SP share the same amino acid sequence at their C-terminal active site domains but differ in structure at their N-terminal domains. These observations have led to the proposal that the N-terminal domains of tachykinin peptides affect the desensitization but not the agonist activities of the peptides. Some of the preprotachykinin proteins contain SP and the NKA-like tachykinins neuropeptide K (NPK) and neuropeptide gamma (NPgamma), which contain NKA at their C-terminals and are N-terminally extended. In this study, the abilities of NKA, NPK, and NPgamma to stimulate NK-1R second messenger (IP(3)) signaling and rapid homologous desensitization of the NK-1R were examined. In addition, a similar analysis was performed using several nonmammalian tachykinin peptides in order to obtain additional insight into the role of the tachykinin N-terminal domain in these NK-1R functions. NPK and NPgamma were found, like NKA, to be full agonists of rat NK-1R IP(3) signaling but, unlike NKA, were also able to cause full rapid homologous desensitization of the receptor. The extended N-terminal domains of NPK and NPgamma thus increase the desensitization activities of these NKA-like peptides. Of the nonmammalian tachykinins tested, all were full agonists but kassinin and eledoisin had only partial homologous desensitization activity, suggesting that the N-terminal structures of these peptides also differentially affect agonist versus desensitization activities of the NK-1R.     

Pharmacology of MEN 11467: a potent new selective and orally- effective peptidomimetic tachykinin NK(1) receptor antagonist

We have investigated the pharmacological properties of MEN 11467, a novel partially retro-inverse peptidomimetic antagonist of tachykinin NK(1) receptors. MEN 11467 potently inhibits the binding of [(3)H] substance P (SP) to tachykinin NK(1) receptors in the IM9 limphoblastoid cell line (pK(i) = 9.4 +/- 0.1). MEN 11467 is highly specific for the human tachykinin NK(1) receptors, since it has negligible effects (pK(i) <6) on the binding of specific ligands to tachykinin NK(2) or NK(3) receptors and to a panel of 30 receptors ion channels unrelated to tachykinin receptors. The antagonism exerted by MEN 11467 at tachykinin NK(1) receptors is insurmountable in saturation binding experiments, both K(D) and B(max) of SP were significantly reduced by MEN 11467 (0.3-10 nM). In the guinea-pig isolated ileum, MEN 11467 (0.03-1 nM) produced a nonparallel rightward shift of the concentration-response curve to SP methylester with a concomitant reduction of the Emax to the agonist (pK(B) = 10.7 +/- 0.1). Moreover the antagonist activity of MEN 11467 was hardly reversible despite prolonged washout. In vivo, MEN 11467 produced a long lasting (> 2-3h) dose-dependent antagonism of bronchoconstriction induced by the selective tachykinin NK(1) receptor agonist, [Sar(9), Met(O(2))(11)]SP in anaesthetized guinea-pigs (ID(50)s' = 29+/-5, 31+/-12 and 670+/-270 microg/kg, after intravenous, intranasal and intraduodenal administration, respectively), without affecting bronchoconstriction induced by methacholine. After oral administration MEN 11467 produced a dose-dependent inhibition of plasma protein extravasation induced in guinea-pig bronchi by [Sar(9), Met(O(2))(11)] (ID(50) = 6.7 +/- 2 mg/kg) or by antigen challenge in sensitized animals (ID(50) = 1.3 mg/kg). After i.v. administration MEN 11467 weakly inhibited the GR 73632-induced foot tapping behaviour in gerbil (ED(50) = 2.96 +/- 2 mg/kg), indicating a poor ability to block central tachykinin NK(1) receptors. These results demonstrate that MEN 11467 is a potent, highly selective and orally effective insurmountable pseudopeptide antagonist of peripheral tachykinin NK(1) receptors with a long duration of action.     

Hemokinin-1 has Substance P-like function in U-251 MG astrocytoma cells: a pharmacological and functional study

Substance P (SP) triggers responses in astrocytoma cells, which are considered important for proliferation and neuroimmunomodulatory activity. In this study, we compared the effects of SP with those of the novel tachykinin Hemokinin-1 (HK-1) in the human astrocytoma cell line U-251 MG. We show that U-251 MG cells express high levels of Neurokinin-1 (NK-1) receptors. The binding affinities of 125I-SP and 125I-mHK-1 to these receptors were in a similar, subnanomolar range. HK-1 and SP stimulated Ca2+ mobilization and induced increased cytokine mRNA expression. A specific NK-1 receptor antagonist blocked the observed effects. We conclude that there are no qualitative differences in SP and HK-1-evoked responses, suggesting that both peptides act through NK-1 receptors in U-251 MG cells. Moreover, we show TAC4 mRNA expression in gliomas, indicating a possible involvement of HK-1 in glioma biology.     

Interaction of endokinin A/B and (Mpa6)-γ2-MSH-6–12 in pain regulation in mice

The present study focused on the interactive effects of (Mpa⁶)-γ2-MSH-6–12 (Mpa, spinal level) and endokinin A/B (EKA/B, supraspinal level) on pain regulation in mice. EKA/B (30 pmol) only weakened 100 pmol Mpa-induced hyperalgesia at 5 min, but could enhance it during 20–30 min. However, EKA/B (100 pmol) antagonized all dose levels of Mpa significantly at 5 min and blocked them completely at 10 min. EKA/B (3 nmol) co-injected with Mpa presented marked analgesia at 5 min and enduring hyperalgesia within 20–60 min. To investigate the underlying mechanisms between Mpa and EKA/B, SR140333B and SR142801 (NK₁ and NK₃ receptor antagonists, respectively) were utilized. SR140333B had no influence on Mpa, while SR142801 potentiated it during 20–30 min. Whereas, SR140333B and SR142801 could block the co-administration of Mpa and EKA/B (30 pmol) separately at 5 min and 30 min. These phenomena might attribute to that these two antagonists promoted the antagonism of EKA/B (30 pmol) at the early stage, while antagonized EKA/B preferentially in the latter period. SR140333B weakened the analgesia of EKA/B (3 nmol), but produced no effect on Mpa. However, SR140333B failed to affect the co-injection of Mpa and EKA/B, which implied that EKA/B cooperated with Mpa prior to SR140333B. These results could potentially help to better understand the interaction of NK and MrgC receptors in pain regulation in mice.     

Tachykinin receptors on human monocytes: their involvement in rheumatoid arthritis

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones. This study evaluated the effects of mammalian tachykinins and selective tachykinin agonists and antagonists on human monocytes isolated from healthy donors: SP, NKA and NKB all evoked a dose-dependent superoxide anion (O2-) production and the NK2 selective agonist [beta-Ala8]-NKA(4-10) induced a full response. The NK3 selective agonist senktide was inactive, while the NK1 selective agonists septide and [Sar9Met(O2)11]SP displayed some effects. These results indicate that NK2 and also some NK1 receptors are present in monocytes isolated from healthy donors. The role of tachykinin receptor activation in rheumatoid arthritis was also investigated, by measuring O2- production and TNF-alpha mRNA expression in monocytes isolated from rheumatoid patients. Tachykinins enhanced the expression of this cytokine in both control and rheumatoid monocytes and NK2 receptor stimulation was shown to trigger an enhanced respiratory burst in monocytes from rheumatoid patients. In conclusion, these results indicate that NK2 and NK1 receptors are present on human monocytes, the former being preferentially involved in rheumatoid arthritis.     

Autoradiographic localization and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues

Quantitative receptor autoradiography using several radiolabeled tachykinins was used to localize and characterize tachykinin peptide receptor binding sites in rat CNS and peripheral tissues. Autoradiographic localization and displacement experiments using several radiolabeled tachykinins indicate that in the rat there are at least 3 distinct tachykinin receptor binding sites. One of these is present in both the CNS and peripheral tissues, one is present only in the CNS, and one is present only in peripheral tissues. The first tachykinin receptor binding site, which is detectable in both the CNS and peripheral tissues, appears to prefer substance P (SP) as an endogenous ligand. Areas expressing high concentrations of this binding site include the medial septum, superior colliculus, inferior olive, inner plexiform layer of the retina, external muscle of the bladder, and the muscularis externa of the esophagus. The second type of tachykinin receptor binding site, which is detectable only in the CNS appears to prefer either neuromedin K (NK) and/or substance K (SK) as the endogenous ligand. This receptor binding site is labeled by Bolton-Hunter conjugates of NK, SK, eledoisin, or kassinin and is found in high concentrations in laminae 4 and 5 of the cerebral cortex, the ventral tegmental area, laminae 1 and 2 of the spinal cord, and the inner plexiform layer of the retina. The third type of tachykinin receptor binding site is detectable only in peripheral tissues and appears to prefer SK as the endogenous ligand. This receptor binding site is labeled by SK, eledoisin, or kassinin radioligands and tissues that express high concentrations include the muscularis mucosae of the esophagus, the circular muscle of the colon, and the external muscle of the bladder. These data suggest that SP receptors are expressed in the brain and peripheral tissues, NK receptors are expressed in the CNS, and SK receptors are expressed in peripheral tissue. These data fit well with radioimmunoassay data that suggest that, whereas in the CNS SP, SK and NK are present in high concentrations, in peripheral tissues only SP and SK are present in detectable concentrations. The present classification of tachykinin receptors places a lower limit on the number of mammalian tachykinin receptor types and provides a functional/morphological framework for exploring the diverse actions of tachykinin peptides in both the CNS and peripheral tissues.     

Substance P and neurokinin A induced desentization to cardiovascular and behavioral effects: evidence for the involvement of different tachykinin receptors

Desensitization and cross-desensitization to the cardiovascular and behavioral effects elicited by intracerebroventricular (i.c.v.) substance P (SP) and neurokinin A (NKA) injections were examined in conscious, freely moving rats. The cardiovascular responses to equimolar doses of both peptides were identical, however, the pattern of the behavioral responses differed. Relative to SP, NKA was weaker in eliciting hindquarter grooming but more effective in eliciting wet dog shakes. SP pretreatment (50 pmol) desensitized the cardiovascular and behavioral responses to both, subsequent injections of SP (50 pmol) as well as of NKA (50 or 500 pmol) injected 30 or 60 min after SP, indicating cross-desensitization. NKA pretreatment (50 pmol) partly reduced the cardiovascular but not the behavioral responses to subsequent equimolar doses of NKA. The cardiovascular responses to SP (50 pmol) were reduced only 30 min but not 60 min after pretreatment with a 10 times higher dose of NKA (500 pmol). Of all behavioral manifestations to i.c.v. SP, only hindquarter grooming was attenuated by pretreatment with either dose of NKA. The equal potency of SP and NKA in eliciting the cardiovascular effects but different pattern of behavioral responses to these peptides suggest an involvement of different types of tachykinin receptors in mediating the central effects of the two peptides. The fact that NKA induced cross-desensitization selectively to one type of behavioral manifestations elicited by SP, indicates the existence of two subtypes of SP (NK₁) receptors in the rat brain.     

In vitro characterization of the effects of rat/mouse hemokinin-1 on mouse colonic contractile activity: A comparison with substance P

Rat/mouse hemokinin-1 (r/m HK-1) has been identified as a member of the tachykinin family and its effect in colonic contractile activity remains unknown. We investigated the effects and mechanisms of actions of r/m HK-1 on the mouse colonic contractile activity in vitro by comparing it with that of substance P (SP). R/m HK-1 induced substantial contractions on the circular muscle of mouse colon. The maximal contractile responses to r/m HK-1 varied significantly among proximal-, mid- and distal-colon, suggesting that the action of r/m HK-1 was region-specific in mouse colon. The contractile response induced by r/m HK-1 is primarily via activation of tachykinin NK₁ receptors leading to activation of cholinergic excitatory pathways and with a minor contribution of NK₂ receptors, which may be on the smooth muscle itself. A direct action on colonic smooth muscles may be also involved. In contrast, SP induced biphasic colonic responses (contractile and relaxant responses) on the circular muscle, in which the contractile action of SP was equieffective with r/m HK-1. SP exerted its contractile effect predominantly through neural and muscular tachykinin NK₁ receptors, but unlike r/m HK-1 did not appear to act via NK₂ receptors. The relaxation induced by SP was largely due to release of nitric oxide (NO) produced via an action on neural NK₁ receptors. These results indicate that the receptors and the activation properties involved in r/m HK-1-induced mouse colonic contractile activity are different from those of SP.     

The N-terminal domain of substance P is required for complete homologous desensitization but not phosphorylation of the rat neurokinin-1 receptor

The agonist activity of substance P (SP) is a function of the C-terminal domain of the peptide. A C-terminal SP fragment (SP(6-11)) and analog (septide) and neurokinin A (NKA; a related tachykinin with a divergent N-terminal amino acid sequence) were found to be full neurokinin-1 receptor (NK-1R) agonists, but were not able to desensitize the receptor maximally as much as SP. Substance P caused 95.6 +/- 0.9% maximal desensitization of the NK-1R whereas SP(6-11), septide, and NKA(only)caused 74 +/- 3.5, 50.6 +/- 8, and 71.5 +/- 4.4% maximal desensitization, respectively (mean +/- SEM; P < 0.001 vs SP). When a series of SP C-terminal fragment peptides were tested for their NK-1R desensitizing activity, it was found that SP(5-11)and SP(6-11)caused significantly less maximal NK-1R desensitization than SP. SP N-terminal fragment peptides had no effect on the ability of SP(6-11)to compete with(3)H-SP binding, generate an IP(3)response, or cause NK-1R desensitization when tested with or without SP(6-11). SP, SP(6-11), septide, and NKA all maximally stimulated 8-9-fold increases in NK-1R phosphorylation. When attached to the C-terminal domain of SP responsible for NK-1R binding and agonism, the N-terminus of SP is responsible for 25-50% of homologous desensitization and this may occur via a mechanism other than NK-1R phosphorylation.     

NK(3) receptors in the feline nucleus tractus solitarius are not involved with the muscle pressor response

Isometric muscle contractions cause an increase in mean arterial pressure and heart rate. Previously, we showed that substance P (SP) is released from sites in the feline medial nucleus tractus solitarius (mNTS) in response to isometric muscle contractions, and that it most likely interacted with NK(1) tachykinin receptors at these sites. This study was undertaken to determine whether other tachykinin receptors in this area of the brainstem are involved with the muscle pressor response. Receptor autoradiography, using [(125)I]Bolton-Hunter SP and [(125)I] [MePhe(7)] neurokinin B to label NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) receptors in this region of the feline brainstem Injections of the specific NK(3) receptor antagonist, SR 142801 (0.1 to 10 microM) into the mNTS did not modify the pressor response or the heart rate response to isometric muscle contractions. Injection of SR142801 into the NTS prior to the injection of the NK(1) antagonist, GR82334 did not affect the action of GR82334 to attenuate the muscle pressor reflex. We conclude that NK(3) receptors in the NTS are not involved with the regulation of cardiovascular function during activation of the muscle pressor response.     

Differential expression of neurokinin B and hemokinin-1 in human immune cells

The tachykinins are a group of related peptides that are mainly synthesized in the central and peripheral nervous system, but are also present in peripheral non-neuronal cells. In humans, substance P (SP) is the most extensively studied tachykinin and is present, along with the NK-1 receptor, in several inflammatory and immune cells. The release of SP under the appropriate stimulus may act as a paracrine or autocrine signal that may help to initiate and/or propagate inflammation. In the present study we have determined the expression pattern of NKB and HK-1 mRNA in human lymphocytes, monocytes, neutrophils and eosinophils. In addition, we have detected for the first time the presence of NKB protein in these cellular types. These findings reinforce the suggestion that tachykinins play a central role in the pathophysiology of the inflammatory process.     

Functional substance P receptors on a human astrocytoma cell line (U-373 MG)

[125I]Bolton Hunter conjugate of substance P ([125I]BHSP) can bind to human astrocytoma membranes in a monophasic and saturable manner with a Kd of 0.57 +/- 0.17 nM and a Bmax of 67.8 +/- 5.5 fmol/mg protein. The rank order of potency of tachykinins and related analogues as inhibitors of [125I]BHSP binding to astrocytoma membranes and intact cells correlated with their relative abilities to stimulate uridine incorporation into nucleic acid. The observed specificity pattern conformed to that reported for the NK1 tachykinin receptor with SP much greater than eledoisin greater than neurokinin A greater than neurokinin B and [Glp6, L-Pro9]SP(6-11) much greater than [Glp6, D-Pro9]SP(6-11).     

Identification of mechanisms for duodenal contraction induced by tachykinins in the rat

Mechanisms for contractile effects of tachykinins on muscle strips of rat duodenum were studied using substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and neuropeptide K (NPK), and the tachykinin analogues SP methyl ester (SPME), Nle¹⁰-NKA(4–10) (NleNKA) and senktide (SENK) selective for neurokinin (NK)-1, NK-2 and NK-3 receptors, respectively. NK receptors responsible for smooth muscle contraction were identified using selective ligands to protect NK receptors combined with inactivation of residual receptors with N-ethylmaleimide. Tachykinins (10^?9 to 10^?5 M) caused dose-related contractions of the muscle strips. The order of potency of native tachykinins was NKA > NKB > SP > NPK in circular, and NKB > NKA > NPK > SP in longitudinal muscle, whereas that of selective tachykinin analogues was SENK > NleNKA > SPME. NKA, NleNKA and SENK were equieffective as acetylcholine, whereas SP, SPME, NKB and NPK were less effective. Spantide decreased the sensitivity to all tachykinin analogues. Atropine reduced the sensitivity to SENK only, whereas hexamethonium reduced the sensitivity to SENK and SPME, but not to NleNKA. Selective receptor protection with SPME, NleNKA or SENK protected contractions induced by SPME, NleNKA and SENK, respectively. Responses to tachykinin analogues were reduced in Ca²⁺-free medium. Thus, NKA is suggested to be the dominating tachykinin to stimulate contraction of the rat duodenum via NK receptors coupled to Ca²⁺-dependent signal transduction pathways. Of the receptors available, the NK-1 subtype involves a nicotinic transmission step, and the NK-3 subtype also a muscarinic step, whereas the NK-2 receptor subtype is not dependent on cholinergic mechanisms.     

Rat/mouse hemokinin-1, a mammalian tachykinin peptide, markedly potentiates the antinociceptive effects of morphine administered at the peripheral and supraspinal level

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.     

In vivo characterization of the effects of human hemokinin-1 and human hemokinin-1(4-11), mammalian tachykinin peptides, on the modulation of pain in mice

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 NK₁ receptor antagonist SR140333, but not by the NK₂ receptor antagonist SR48968, indicating that the analgesic effect induced by i.c.v. h HK-1 is mediated through the activation of NK₁ receptors. Interestingly, naloxone, β-funaltrexamine and naloxonazine, but not naltrindole and nor-binaltorphimine, could also block the analgesic effect markedly, suggesting that this effect is related to descending μ opioidergic neurons (primary μ₁ 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 NK₁ 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 NK₂ 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 δ ORs significantly enhanced the analgesia, indicating that δ 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.     

Tachykinin activation of human monocytes from patients with rheumatoid arthritis: in vitro and ex-vivo effects of cyclosporin A

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. We previously demonstrated that NK1 and NK2 receptors are present on human monocytes, SP and NKA inducing superoxide anion production and tumor necrosis factor-alpha (TNF-alpha) mRNA expression. NK2 receptor stimulation also triggered an enhanced respiratory burst in monocytes isolated from rheumatoid arthritis (RA) patients. This study was aimed to evaluate the in vitro and ex-vivo effects of cyclosporin A (CsA) on tachykinins-evoked TNF-alpha release from monocytes of healthy donors and RA patients. CsA (100 ng/ml) potently inhibited phorbol ester- and tachykinin-evoked TNF-alpha secretion. In RA patients treated with CsA (Sandimmun Neoral 2.5 mg/kg/day, a significant time-dependent reduction in TNF-alpha secretion from monocytes was measured. This may contribute to the CsA therapeutic activity in RA.     

Intrathecally administered big dynorphin,a prodynorphin-derived peptide,produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.