Development of Neuropeptide Hemokinin-1 Analogues with Antimicrobial and Wound-Healing Activity.

Wound healing is a complex process that can be delayed in some pathological conditions, such as infection and diabetes. Following skin injury, the neuropeptide substance P (SP) is released from peripheral neurons to promote wound healing by multiple mechanisms. Human hemokinin-1 (hHK-1) has been identified as an SP-like tachykinin peptide. Surprisingly, hHK-1 shares similar structural features with antimicrobial peptides (AMPs), but it does not display efficient antimicrobial activity. Therefore, a series of hHK-1 analogues were designed and synthesized. Among these analogues, AH-4 was found to display the greatest antimicrobial activity against a broad spectrum of bacteria. Furthermore, AH-4 rapidly killed bacteria by membrane disruption, similar to most AMPs. More importantly, AH-4 showed favorable healing activity in all tested mouse full-thickness excisional wound models. Overall, this study suggests that the neuropeptide hHK-1 can be used as a desirable template for developing promising therapeutics with multiple functions for wound healing.

Regional differences of tachykinin effects on smooth muscle and pacemaker potentials of the stomach, duodenum, ileum and colon of an emetic model, the house musk shrews.

BACKGROUND AND AIMS: The contractile effects of tachykinins on the gastrointestinal tract are well-known, but how they modulate slow-waves, particularly in species capable of emesis, remains largely unknown. We aimed to elucidate the effects of tachykinins on myoelectric and contractile activity of isolated gastrointestinal tissues of the Suncus murinus. METHODS: The effects of substance P (SP), neurokinin (NK)A, NKB and selective NK1 (CP122,721, CP99,994), NK2 (SR48,968, GR159,897) and NK3 (SB218,795, SB222,200) receptor antagonists on isolated stomach, duodenum, ileum and colon segments were studied. Mechanical contractile activity was recorded using isometric force displacement transducers. Electrical pacemaker activity was recorded using a microelectrode array. RESULTS: Compared with NKA, SP induced larger contractions in stomach tissue and smaller contractions in intestinal segments, where oscillation magnitudes increased in intestinal segments, but not the stomach. CP122,721 and GR159,897 inhibited electrical field stimulation-induced contractions of the stomach, ileum and colon. NKB and NK3 had minor effects on contractile activity. The inhibitory potencies of SP and NKA on the peristaltic frequency of the colon and ileum, respectively, were correlated with those on electrical pacemaker frequency. SP, NKA and NKB inhibited pacemaker activity of the duodenum and ileum, but increased that of the stomach and colon. SP elicited a dose-dependent contradictive pacemaker frequency response in the colon. CONCLUSION: This study revealed distinct effects of tachykinins on the mechanical and electrical properties of the stomach and colon vs. the proximal intestine, providing a unique aspect on neuromuscular correlation in terms of the effects of tachykinin on peristaltic and pacemaker activity in gastrointestinal-related symptoms.

Pharmacological importance of TG12 from tachykinin and its toxicological behavior against multidrug-resistant bacteria Klebsiella pneumonia.

Development of antimicrobial drugs against multidrug-resistant (MDR) bacteria is a great focus in recent years. TG12, a short peptide molecule used in this study was screened from tachykinin (Tac) protein of an established teleost Channa striatus (Cs) transcriptome. Tachykinin cDNA has 345 coding sequence, that denotes a protein contained 115 amino acids; in which a short peptide (TG12) was identified at 83-94. Tachykinin mRNA upregulated in C. striatus treated with Aeromonas hydrophila and Escherichia coli lipopolysaccharide (LPS). The mRNA up-regulation was studied using real-time PCR. The up-regulation tachykinin mRNA pattern confirmed the immune involvement of tachykinin in C. striatus during infection. Further, the identified peptide, TG12 was synthesized and its toxicity was demonstrated in hemolytic and cytotoxic assays using human erythrocytes and human dermal fibroblast cells, respectively. The toxicity study exhibited that the toxicity of TG12 was similar to negative control, phosphate buffer saline (PBS). Moreover, the antibiogram of TG12 was active against Klebsiella pneumonia ATCC 27736, a major MDR bacterial pathogen. Further, the antimicrobial activity of TG12 against pathogenic bacteria was screened using minimum inhibitory concentration (MIC) and anti-biofilm assays, altogether TG12 showed potential activity against K. pneumonia. Fluorescence assisted cell sorter flow cytometer analysis (FACS) and field emission scanning electron microscopy (FESEM) was carried on TG12 with K. pneumonia; the results showed that TG12 significantly reduced K. pneumonia viability as well as TG12 disrupt its membrane. In conclusion, TG12 of CsTac is potentially involved in the antibacterial immune mechanisms, which has a prospectus efficiency in pharma industry against MDR strains, especially K. pneumonia.

Endokinin A/B stimulates rat gastric motility through myogenic NK1 receptors located in the fundus.

Endokinin A/B (EKA/B), the common C-terminal decapeptide in endokinins A and B, is a preferred ligand of the NK1 receptor and regulates pain and itch. The study focused on the effects of EKA/B on rat gastric motility in vivo and in vitro. Gastric emptying was measured to evaluate gastric motility in vivo. Intragastric pressure and the contraction of gastric muscle strips were measured to evaluate gastric motility in vitro. Moreover, various neural blocking agents and neurokinin receptor antagonists were applied to explore the mechanisms. TAC4 and TACR1 mRNAs were expressed throughout rat stomach. EKA/B promoted gastric emptying by intraperitoneal injection in vivo. Correspondingly, EKA/B also increased intragastric pressure in vitro. Additionally, EKA/B contracted the gastric muscle strips from the fundus but not from the corpus or antrum. Further studies revealed that the contraction induced by EKA/B on muscle strips from the fundus could be significantly reduced by NK1 receptor antagonist SR140333 but not by NK2 receptor antagonist, NK3 receptor antagonist, or the neural blocking agents used. Our results suggested that EKA/B might stimulate gastric motility mainly through the direct activation of myogenic NK1 receptors located in the fundus.

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells.

Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.

Pharmacological characteristics of hemokinin-1-derived peptides in rat pruriceptive processing.

Hemokinin-1 (HK-1) is a member of mammalian tachykinin peptide family, and [Leu11]-HK-1 has an antagonistic effect on HK-1. The attenuation of pruritogen-induced scratching behavior by pretreatment with [Leu11]-HK-1 indicates the involvement of HK-1 in pruriceptive processing. However, it remains unclear whether the intrathecal or intranasal administration of HK-1-derived peptides, such as [D-Trp7,9]-[Leu11]-HK-1 or [D-Trp7]-[Leu11]-HK-1, elicits the effects different from [Leu11]-HK-1. The induction of scratching by intrathecal administration of HK-1 was attenuated 30 min, 4 h and 24 h after pretreatment with [Leu11]-HK-1, [D-Trp7,9]-[Leu11]-HK-1 and [D-Trp7]-[Leu11]-HK-1 or [D-Trp9]-[Leu11]-HK-1, respectively. Similarly, the scratching induced by subcutaneous injection of pruritogens as chloroquine and histamine was ameliorated 30 min and 24 h after pretreatment with [Leu11]-HK-1 and these three HK-1-derived peptides, respectively. Moreover, the effective minimum concentrations of intrathecal administrations of [D-Trp9]-[Leu11]-HK-1 on scratching induced by chloroquine and histamine were 10-6 M, while the effective minimum concentrations of intranasal administration of this peptide on scratching induced by chloroquine and histamine were 10-5 M and 10-4 M, respectively. Thus, the present results indicate that the intrathecal administration of HK-1-derived peptides with D-Trp extends its effective time on scratching induced by intrathecal administration of HK-1 and pruritogens such as chloroquine and histamine. Similarly, the induction of scratching by pruritogens was attenuated by intranasal administration of HK-1-derived peptide, although the effective minimum concentration of this peptide was slightly lower than that of intrathecal administration, indicating that intranasal administration is an effective tool for carrying peptides into the brain.

Novel Biology of Tachykinins in Gonadotropin-Releasing Hormone Secretion.

The tachykinin family of peptides, composed of the neurokinins A and B (NKA, NKB) and substance P are involved in the central control of gonadotropin-releasing hormone (GnRH) release through a variety of neuronal circuitries that mediate the activation of Kiss1 neurons and the synchronization of their activity within the arcuate nucleus. The major outcome of this role is the precise regulation of the pulsatile pattern of GnRH release. In addition, tachykinins are involved in the maturation of the reproductive axis by determining the optimal timing of puberty onset, as well as in the timing of the preovulatory luteinizing hormone surge in females. Therefore, the action of tachykinins in reproduction appears to extend to all the critical aspects required for the successful attainment and maintenance of fertility. In this review, we summarize the latest advances in our understanding of the biology of tachykinins in the control of GnRH release, addressing the existing controversies, open questions, and future perspectives.

The anorexigenic effect of neuropeptide K in chicks involves the paraventricular nucleus and arcuate nucleus of the hypothalamus.

Neuropeptide K (NPK) induces satiety in birds and mammals. We demonstrated that in birds this effect was associated with the hypothalamus, but beyond this little is known in any species regarding the central mechanism of action. Thus, this study was designed to identify hypothalamic molecular mechanisms associated with the food intake-inhibiting effects of NPK in chicks. In Experiment 1, intracerebroventricular (ICV) injection of 1.0 and 3.0 nmol of NPK reduced food intake and we identified an effective dose for microinjection. In Experiment 2, food intake was reduced when NPK was microinjected into the PVN. In Experiment 3, whole hypothalamus was collected from chicks at 1 h post-ICV NPK injection. The abundance of corticotropin-releasing factor (CRF) and agouti-related peptide (AgRP) mRNA was reduced in NPK-injected chicks. In Experiment 4, within the isolated paraventricular nucleus (PVN) there was less CRF mRNA, and within the arcuate nucleus (ARC) there was less AgRP mRNA, in NPK- than vehicle-treated chicks at 1 h post-injection. We conclude that there are first order neurons for NPK that reside within the PVN, and the anorexigenic effect of NPK is associated with a decrease in AgRP in the ARC.

Hypothalamic mechanisms associated with neuropeptide K-induced anorexia in Japanese quail (Coturnix japonica).

Central administration of neuropeptide K (NPK), a 36-amino acid peptide, is associated with anorexigenic effects in rodents and chickens. The mechanisms underlying the potent anorexigenic effects of NPK are still poorly understood. Thus, the aim of the present study was to identify the hypothalamic nuclei and neuropeptides that mediate anorexic effects of NPK in 7 day-old Japanese quail (Coturnix japonica). After a 6 h fast, intracerebroventricular (ICV) injection of NPK decreased food and water intake for 180 min post-injection. Quail injected with NPK had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), lateral hypothalamus, and paraventricular nucleus (PVN) compared to the birds that were injected with the vehicle. In the ARC of NPK-injected quail, there was decreased neuropeptide Y (NPY), NPY receptor sub-type 1, and agouti-related peptide mRNA, and increased CART, POMC, and neurokinin receptor 1 mRNA. NPK-injected quail expressed greater amounts of corticotropin-releasing factor (CRF), CRF receptor sub-type 2, melanocortin receptors 3 and 4, and urocortin 3 mRNA in the PVN. In conclusion, results provide insights into understanding NPK-induced changes in hypothalamic physiology and feeding behavior, and suggest that the anorexigenic effects of NPK involve the ARC and PVN, with increased CRF and melanocortin and reduced NPY signaling.

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee ( Apis mellifera) Brain: Effect of Aggressiveness.

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.

Naturally Occurring Missense MRGPRX2 Variants Display Loss of Function Phenotype for Mast Cell Degranulation in Response to Substance P, Hemokinin-1, Human ß-Defensin-3, and Icatibant.

Human mast cells (MCs) express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR X2 (MRGPRX2). Activation of this receptor by a diverse group of cationic ligands such as neuropeptides, host defense peptides, and Food and Drug Administration-approved drugs contributes to chronic inflammatory diseases and pseudoallergic drug reactions. For most GPCRs, the extracellular (ECL) domains and their associated transmembrane (TM) domains display the greatest structural diversity and are responsible for binding different ligands. The goal of the current study was to determine if naturally occurring missense variants within MRGPRX2's ECL/TM domains contribute to gain or loss of function phenotype for MC degranulation in response to neuropeptides (substance P and hemokinin-1), a host defense peptide (human ß-defensin-3) and a Food and Drug Administration-approved cationic drug (bradykinin B2 receptor antagonist, icatibant). We have identified eight missense variants within MRGPRX2's ECL/TM domains from publicly available exome-sequencing databases. We investigated the ability of MRGPRX2 ligands to induce degranulation in rat basophilic leukemia-2H3 cells individually expressing these naturally occurring MRGPRX2 missense variants. Using stable and transient transfections, we found that all variants express in rat basophilic leukemia cells. However, four natural MRGPRX2 variants, G165E (rs141744602), D184H (rs372988289), W243R (rs150365137), and H259Y (rs140862085) failed to respond to any of the ligands tested. Thus, diverse MRGPRX2 ligands use common sites on the receptor to induce MC degranulation. These findings have important clinical implications for MRGPRX2 and MC-mediated pseudoallergy and chronic inflammatory diseases.

MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH2, regulates the depressor effects induced by endokinin A/B.

The interactions of the chimeric peptide MCRT (YPFPFRTic-NH2), based on morphiceptin and neuropeptide FF derivative PFRTic-NH2, on the effects of endokinin A/B (EKA/B) on mean arterial blood pressure of the urethane-anaesthetized rat have been investigated in the absence and presence of tachykinin receptor antagonists, naloxone and NO synthase inhibitors. While MCRT produced dose dependent decreases in mean arterial pressure, in its presence only a small but statistically insignificant decreases in the magnitude and the time course of the depressor effect of EKA/B (10nmol/kg) were observed. MCRT had little influence on the depressor effect of EKA/B (1 nmol/kg), but strongly potentiated that of EKA/B (100nmol/kg). The tachykinin NK1 receptor antagonist SR140333B (1mg/kg) and the NK3 antagonist SR142891 (2.79mg/kg) both reduced the hypotensive effects of EKA/B and MCRT alone and blocked those of the two peptides in combination. The NK2 antagonist GR159897 (4mg/kg) partially blocked the depressor effects of EKA/B and MCRT alone. Naloxone (2mg/kg) completely blocked the depressor effect of MCTR, but partially blocked that of EKA/B. The NO synthase inhibitor l-NAME (50mg/kg) partially blocked the depressor effects of EKA/B, MCRT, and EKA/B + MCRT. These results could help to better understand the role of tachykinin receptors, opioid receptors and neuropeptide FF receptors in cardiovascular system.

Pain regulation of endokinin A/B or endokinin C/D on chimeric peptide MCRT in mice.

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.

Involvement of spinal glutamate in nociceptive behavior induced by intrathecal administration of hemokinin-1 in mice.

The most recently identified tachykinin, hemokinin-1, was cloned from mouse bone marrow. While several studies indicated that hemokinin-1 is involved in pain and inflammation, the physiological functions of hemokinin-1 are not fully understood. Our previous research demonstrated that the intrathecal (i.t.) administration of hemokinin-1 (0.00625-1.6 nmol) dose-dependently induced nociceptive behaviors, consisting of scratching, biting and licking in mice, which are very similar with the nociceptive behaviors induced by the i.t. administration of substance P. Low-dose (0.0125 nmol) hemokinin-1-induced nociceptive behavior was inhibited by a specific NK1 receptor antagonist; however, high-dose (0.1 nmol) hemokinin-1-induced nociceptive behavior was not affected. In the present study, we found that the nociceptive behaviors induced by hemokinin-1 (0.1 nmol) were inhibited by the i.t. co-administration of MK-801 or D-APV, which are NMDA receptor antagonists. Moreover, we measured glutamate in the extracellular fluid of the mouse spinal cord using microdialysis. The i.t. administration of hemokinin-1 produced a significant increase in glutamate in the spinal cord, which was significantly reduced by co-administration with NMDA receptor antagonists. These results suggest that hemokinin-1-induced nociceptive behaviors may be mediated by the NMDA receptor in the spinal cord.

Tachykinin-1 in the central nervous system regulates adiposity in rodents.

Ghrelin is a circulating hormone that targets the central nervous system to regulate feeding and adiposity. The best-characterized neural system that mediates the effects of ghrelin on energy balance involves the activation of neuropeptide Y/agouti-related peptide neurons, expressed exclusively in the arcuate nucleus of the hypothalamus. However, ghrelin receptors are expressed in other neuronal populations involved in the control of energy balance. We combined laser capture microdissection of several nuclei of the central nervous system expressing the ghrelin receptor (GH secretagoge receptor) with microarray gene expression analysis to identify additional neuronal systems involved in the control of central nervous system-ghrelin action. We identified tachykinin-1 (Tac1) as a gene negatively regulated by ghrelin in the hypothalamus. Furthermore, we identified neuropeptide k as the TAC1-derived peptide with more prominent activity, inducing negative energy balance when delivered directly into the brain. Conversely, loss of Tac1 expression enhances the effectiveness of ghrelin promoting fat mass gain both in male and in female mice and increases the susceptibility to diet-induced obesity in ovariectomized mice. Taken together, our data demonstrate a role TAC1 in the control energy balance by regulating the levels of adiposity in response to ghrelin administration and to changes in the status of the gonadal function.

Pharmacological characterization of tachykinin tetrabranched derivatives.

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.

Hemokinin-1(4-11)-induced analgesia selectively up-regulates δ-opioid receptor expression in mice.

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.

Bronchoconstriction induced by increasing airway temperature in ovalbumin-sensitized rats: role of tachykinins.

This study was carried out to determine the effect of allergic inflammation on the airway response to increasing airway temperature. Our results showed the following: 1) In Brown-Norway rats actively sensitized by ovalbumin (Ova), isocapnic hyperventilation with humidified warm air (HWA) for 2 min raised tracheal temperature (Ttr) from 33.4 ± 0.6°C to 40.6 ± 0.1°C, which induced an immediate and sustained (>10 min) increase in total pulmonary resistance (Rl) from 0.128 ± 0.004 to 0.212 ± 0.013 cmH2O·ml(-1)·s (n = 6, P < 0.01). In sharp contrast, the HWA challenge caused the same increase in Ttr but did not generate any increase in Rl in control rats. 2) The increase in Rl in sensitized rats was reproducible when the same HWA challenge was repeated 60-90 min later. 3) This bronchoconstrictive effect was temperature dependent: a slightly smaller increase in peak Ttr (39.6 ± 0.2°C) generated a significant but smaller increase in Rl in sensitized rats. 4) The HWA-induced bronchoconstriction was not generated by the humidity delivered by the HWA challenge alone, because the same water content delivered by saline aerosol at room temperature had no effect. 5) The HWA-evoked increase in Rl in sensitized rats was not blocked by atropine but was completely prevented by pretreatment either with a combination of neurokinin (NK)-1 and NK-2 antagonists or with formoterol, a ß2 agonist, before the HWA challenge. This study showed that increasing airway temperature evoked a pronounced and reversible increase in airway resistance in sensitized rats and that tachykinins released from the vagal bronchopulmonary C-fiber endings were primarily responsible.

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms.

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure-function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.

Hemokinin-1 competitively inhibits substance P-induced stimulation of osteoclast formation and function.

Hemokinin-1 (HK-1) is a novel member of the tachykinin family that is encoded by preprotachykinin 4 (TAC4) and shares the neurokinin-1 receptor (NK1-R) with substance P (SP). Although HK-1 is thought to be an endogenous peripheral SP-like endocrine or paracrine molecule in locations where SP is not expressed, neither the distribution of HK-1 in the maxillofacial area nor the role HK-1 in bone tissue have been examined. In this study, we investigated the distribution of HK-1 in trigeminal ganglion (TG) and maxillary bone, and assessed the expression of HK-1 during osteoclast differentiation. In vivo, rat molars were loaded for 5 days using the Waldo method. In vitro, rat osteoclast-like cells were induced from bone marrow cells. HK-1 distribution and expression were examined by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, HK-1 was localized in rat TG neurons; however, the number of HK-1-positive neurons was less than that of SP-positive neurons. In the maxillary bone, nerve fibers, blood vessels, and osteocytes were immunopositive for HK-1. Furthermore, HK-1-positive immunoreactivity was found in osteoclasts on the pressure side. In vitro, PCR showed that TAC4 and NK1-R mRNA was expressed in osteoclasts as well as in bone marrow cells. Although SP (10⁻7 M) treatment led to an increased number of osteoclasts, HK-1 (10⁻7 M) treatment did not. The numbers of biotin-labeled HK-1 peptides bound osteoclasts significantly decreased upon incubation with unlabeled SP and biotin-labeled HK-1 compared with biotin-labeled HK-1 alone. These results suggest that HK-1 may not stimulate the differentiation and function of osteoclasts. SP-stimulated osteoclast formation is competitively regulated by peripheral HK-1 through NK1-Rs.