Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization.

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system.

Upregulation of Mas-related G Protein coupled receptor X2 in asthmatic lung mast cells and its activation by the novel neuropeptide hemokinin-1.

Hemokinin-1 (HK-1) is a novel neuropeptide produced by human bronchial cells and macrophages and causes contraction of human bronchi ex vivo. It is also generated by antigen/IgE-activated murine mast cells (MCs) and contributes to experimental chronic allergic airway inflammation via the activation of the neurokinin receptor-1 (NK-1R) expressed on murine MCs. We found elevated MC numbers in the lungs of individuals who died from asthma (asthma) when compared to lungs of individuals who died from other causes (non-asthma). Mas-related G Protein coupled receptor X2 (MRGPRX2) is a novel G-protein coupled receptor (GPCR) that is expressed predominantly on human MCs. We detected low level of MRGPRX2 in non-asthma lung MCs but its expression was significantly upregulated in asthma lung MCs. HK-1 caused degranulation in a human MC line (LAD2) and RBL-2H3 cells stably expressing MRGPRX2 and this response was resistant to inhibition by an NK-1R antagonist. However, knockdown of MRGPRX2 in LAD2 cells resulted in substantial inhibition of HK-1-induced degranulation. These findings suggest that while HK-1 contributes to the development of experimental asthma in mice via NK-1R on murine MCs the effect of this neuropeptide on human bronchoconstriction likely reflects the activation of MRGPRX2 on lung MCs. Thus, development of selective MRGPRX2 antagonists could serve as novel target for the modulation of asthma.

Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

Autocrine hemokinin-1 functions as an endogenous adjuvant for IgE-mediated mast cell inflammatory responses.

BACKGROUND: Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FcεRI cross-linking by antigen-IgE complexes (FcεRI-activated mast cells [FcεRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FcεRI-MC-mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. OBJECTIVE: We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC-mediated allergic disorders. METHODS: Bone marrow-derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R(-/-) mice were used to investigate the effects of NK1R signaling on FcεRI-MCs. BMMCs generated from Tac1(-/-) mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R(-/-), and c-Kit(W-sh/W-sh) mice reconstituted with WT or NK1R(-/-) BMMCs were used to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and on airway inflammation. RESULTS: FcεRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. CONCLUSIONS: FcεRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders.

Role of Spinal Cholecystokinin Octapeptide, Nociceptin/Orphanin FQ, and Hemokinin-1 in Diabetic Allodynia.

A complication of diabetes is neuropathic pain, which is difficult to control with medication. We have confirmed that neuropathic pain due to mechanical allodynia in diabetic mice is mediated by a characteristic neuropeptide in the spinal cord. We evaluated the strength of mechanical allodynia in mice using von Frey filaments. When mice were intravenously injected with streptozotocin, mechanical allodynia appeared 3 days later. Antibodies of representative neuropeptides were intrathecally (i.t.) administered to allodynia-induced mice 7 days after the intravenous administration of streptozotocin, and allodynia was reduced by anti-cholecystokinin octapeptide antibodies, anti-nociceptin/orphanin FQ antibodies, and anti-hemokinin-1 antibodies. In contrast, i.t.-administered anti-substance P antibodies, anti-somatostatin antibodies, and anti-angiotensin II antibodies did not affect streptozotocin-induced diabetic allodynia mice. Mechanical allodynia was attenuated by the i.t. administration of CCK-B receptor antagonists and ORL-1 receptor antagonists. The mRNA level of CCK-B receptors in streptozotocin-induced diabetic allodynia mice increased in the spinal cord, but not in the dorsal root ganglion. These results indicate that diabetic allodynia is caused by cholecystokinin octapeptide, nociceptin/orphanin FQ, and hemokinin-1 released from primary afferent neurons in the spinal cord that transmit pain to the brain via the spinal dorsal horn.

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.

Hemokinin-1 and substance P stimulate production of inflammatory cytokines and chemokines in human colonic mucosa via both NK1 and NK2 tachykinin receptors.

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 ß, RANTES, TNF-α, IL-1ß 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 NK1 and NK2 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).

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.

Substance P and Hemokinin 1 in Nasal Lavage Fluid of Patients with Chronic Sinusitis and Nasal Polyposis.

This pilot study was undertaken to isolate and quantify substance P (SP) and hemokinin 1 (HK-1) in the nasal lavage fluid of patients with chronic rhinosinusitis with nasal polyps to better elucidate the pathophysiology underlying this inflammatory process, which remains poorly understood. Mucus samples were collected from this introductory cohort of 10 patients diagnosed with chronic rhinosinusitis with nasal polyps at Tufts Medical Center (Boston, Massachusetts). Relative levels of SP and HK-1 were measured with enzyme-linked immunosorbent assay methods. Both inflammatory neuropeptides were found in detectable and comparable amounts in patient samples and in concentrations up to 100-fold those established in past literature. The presence of SP and HK-1 necessitates further investigation into their role in nasal polyposis and the potentiation of the chronic inflammation inherent to chronic rhinosinusitis. Downregulating these peptides could therefore provide novel treatment targets to manage this disease process.

Distribution of hemokinin-1 in the rat trigeminal ganglion and trigeminal sensory nuclear complex.

OBJECTIVE: A new mammalian tachykinin peptide encoded in a TAC4 gene was identified and designated as hemokinin-1 (HK-1). A representative of the tachykinin peptide family is substance P (SP), and the function of SP has been well characterized as a pain transmitter or modulator, while it is possible that HK-1 is involved in pruriceptive processing, but, as yet, the distribution of HK-1 peptide in the trigeminal sensory system is still unknown. Thus, the aim of the present study was to elucidate the distribution of HK-1, while comparing the expression of SP, in the trigeminal ganglion and trigeminal sensory nuclear complex. DESIGN: The trigeminal ganglion and the brain stem of male SD rats were used in the immunohistochemical study. Since the amino acid sequence in the carboxyl-terminal regions of HK-1 and SP is common, polyclonal antibodies of HK-1 and SP derived from 6 amino acids consisting of amino-terminal regions of these peptides were produced in guinea pig and rabbit, respectively. The immunohistochemical staining of HK-1 and SP was conducted using frozen sections of the trigeminal ganglion and brain stem in rats. RESULTS: Immunohistochemical studies revealed the expression of HK-1 in small- and medium-sized trigeminal ganglion neurons, in the paratrigeminal nucleus, and in lamina I of the trigeminal nucleus caudalis, while there was no immunoreactivity of HK-1 in the trigeminal nucleus principalis, trigeminal nucleus oralis, and trigeminal nucleus interpolaris. CONCLUSION: These findings indicate that HK-1 is a target molecule for treatment of itch in the orofaicial regions.

Mouse Hemokinin-1 Decapeptide Subjected to a Brain-specific Post-translational Modification.

BACKGROUND/AIM: The tachykinin mouse hemokinin-1, expressed by the mouse Tac4 gene, produces either analgesia or nociception, interacting with the neurokinin 1 receptor. TAC4 precursor processing is not identical to the processing of the TAC1 precursor, for the release of substance P (amidated undecapeptide). The characterization of the mouse hemokinin-1 sequence was required. MATERIALS AND METHODS: We developed anti-tachykinin-specific antibodies for the immunoaffinity purification of tachykinins. RESULTS: Using MALDI-ToF, we identified mouse hemokinin-1 as an amidated decapeptide expressed in murine brain and periphery. Furthermore, we interestingly observed an additional mass peak corresponding to acetylated mouse hemokinin-1 and this post-translational modification is brain-specific, not detected in the periphery. CONCLUSION: We suggest that the N-terminal acetylation of the peptide provides greater potency for ligand-receptor interactions during neural cell signaling.

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.

Changes in the disposition of substance P in the rostral ventromedial medulla after inflammatory injury in the rat.

This study examined whether peripheral inflammatory injury increases the levels or changes the disposition of substance P (SubP) in the rostral ventromedial medulla (RVM), which serves as a central relay in bulbospinal pathways of pain modulation. Enzyme immunoassay and reverse transcriptase quantitative polymerase chain reaction were used to measure SubP protein and transcript, respectively, in tissue homogenates prepared from the RVM and the periaqueductal gray (PAG) and cuneiform nuclei of rats that had received an intraplantar injection of saline or complete Freund's adjuvant (CFA). Matrix-Assisted Laser Desorption/Ionization Time of Flight analysis confirmed that the RVM does not contain hemokinin-1 (HK-1), which can confound measurements of SubP because it is recognized equally well by commercial antibodies for SubP. Levels of SubP protein in the RVM were unchanged four hours, four days and two weeks after injection of CFA. Tac1 transcripts were similarly unchanged in the RVM four days or two weeks after CFA. In contrast, the density of SubP immunoreactive processes in the RVM increased 2-fold within four hours and 2.7-fold four days after CFA injection; it was unchanged at two weeks. SubP-immunoreactive processes in the RVM include axon terminals of neurons located in the PAG and cuneiform nucleus. SubP content in homogenates of the PAG and cuneiform nucleus was significantly increased four days after CFA, but not at four hours or two weeks. Tac1 transcripts in homogenates of these nuclei were unchanged four days and two weeks after CFA. These findings suggest that there is an increased mobilization of SubP within processes in the RVM shortly after injury accompanied by an increased synthesis of SubP in neurons that project to the RVM. These findings are consonant with the hypothesis that an increase in SubP release in the RVM contributes to the hyperalgesia that develops after peripheral inflammatory injury.

Neurokinin-1 receptor mediated breast cancer cell migration by increased expression of MMP-2 and MMP-14.

Breast cancer (BC) is a common reason of cancer-associated death in female. To develop novel strategy of therapeutics, it is crucial to comprehensively understand the receptor status of BC cells on the surface and inner, because chemical messengers can bind the receptors and promote tumorigenesis. Compared with normal and benign samples, BC cell lines and malignant biopsies showed higher expression of neurokinin-1 receptor (NK1). In current work, we examined the role and mechanism of NK1 receptor signaling in BC cell migration. Human hemokinin-1 (hHK-1) was the peripheral agonist of NK1 receptor. Our results showed that by activating NK1 receptor, hHK-1 promoted the migration of BC cells. Gelatin zymography and WB experiment showed that hHK-1 enhanced the levels of MMP-2 and MMP-14; inhibition of these two MMPs blocked hHK-1-induced cell migration. We further explored the underlying mechanism. hHK-1 incuced the phosphorylation of ERK1/2, JNK and Akt through PKC or PKA pathway. The phosphorylation of these kinases further regulated the activation of transcriptional factor AP-1 and NF-κB. Inhibition of AP-1 and NF-κB reduced the up-regulation of MMP-2 and MMP-14 by hHK-1. Taken together, we showed NK1 receptor was an important regulator of human BC cell migration and a potential target for BC treatment.

Human hemokinin-1 promotes migration of melanoma cells and increases MMP-2 and MT1-MMP expression by activating tumor cell NK1 receptors.

Receptors and their regulatory peptides are aberrantly expressed in tumors, suggesting a potential tumor therapy target. Human hemokinin-1 (hHK-1) is a tachykinin peptide ligand of the neurokinin-1 (NK1) receptor which is overexpressed in melanoma and other tumor tissues. Here, we investigated the role of hHK-1 and the NK1 receptor in melanoma cell migration. NK1 receptor expression was associated with melanoma metastatic potential. Treatment with hHK-1 significantly enhanced A375 and B16F10 melanoma cell migration and an NK1 receptor antagonist L732138 blocked this effect. MMP-2 and MT1-MMP expression were up-regulated in hHK-1-treated melanoma cells and cell signaling data suggested that hHK-1 induced phosphorylation of ERK1/2, JNK and p38 by way of PKC or PKA. Kinase activation led to increased MMP-2 and MT1-MMP expression and melanoma cell migration induced by hHK-1. Thus, hHK-1 and the NK1 receptor are critical to melanoma cell migration and each may be a promising chemotherapeutic target.

Hemokinin-1 mediates pruriceptive processing in the rat spinal cord.

Hemokinin-1 (HK-1) is a new mammalian tachykinin peptide consisting of the amino acid sequence similar to substance P (SP). Although the function of SP, a representative tachykinin peptide, has been well established in the pain system, that of HK-1 has not yet been elucidated. [Leu(11)]-SP had an antagonistic effect on SP-induced scratching behavior, suggesting that [Leu(11)]-HK-1 may also attenuate the induction of scratching behavior by HK-1. Thus, the effects of a pretreatment with [Leu(11)]-HK-1 were evaluated to clarify the function of HK-1. The intrathecal administration of [Leu(11)]-HK-1 attenuated the induction of scratching by HK-1, but not SP, while [Leu(11)]-SP reduced the induction of scratching by SP, but not HK-1. These results indicated that [Leu(11)]-HK-1 may be a more specific antagonist of HK-1-preferred receptors and [Leu(11)]-SP has an antagonistic effect on the SP-preferred receptor, the neurokinin 1 receptor. In the formalin test for examining noxious response, the intrathecal administration of [Leu(11)]-SP, but not [Leu(11)]-HK-1, reduced the number of flinchings and c-Fos-positive cells in the spinal dorsal horn following formalin injection into the plantar region of the hind paw. These results indicated that SP, but not HK-1, is involved in nociceptive processing at the spinal level. To evaluate the involvement of HK-1 and SP in pruritic processing, the effect of [Leu(11)]-HK-1 and [Leu(11)]-SP on the induction of scratching behavior and c-Fos expression by serotonin (5-HT) and histamine was evaluated. The increased induction of scratching behavior and c-Fos expression by 5-HT and histamine was markedly attenuated by pretreatment with both [Leu(11)]-HK-1 and [Leu(11)]-SP, suggesting that HK-1 and SP may be involved in pruritic processing. These results indicate that HK-1 is involved in pruritic processing and [Leu(11)]-HK-1 is a valuable tool for clarifying the mechanisms underlying pruritic processing.

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

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