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Background: We previously reported upregulation of expression of Mas-related G protein-coupled receptor X2 (MRGPRX2) on mast cells (MCs) in the skin of patients with severe chronic spontaneous urticaria (CSU). Serum levels of substance P (SP) were reportedly significantly elevated, in correlation with the severity of CSU. Hemokinin-1 (HK-1) reportedly induced histamine release from LAD2 cells via MRGPRX2. We aimed to investigate HK-1's role in CSU., Methods: The concentrations of HK-1 and SP were measured using ELISAs. Skin- and synovium-derived cultured MCs were generated by culturing dispersed skin and synovial cells, respectively, with stem cell factor. MRGPRX2 expression in the MCs was reduced using a lentiviral shRNA silencing technique., Results: Anti-SP Ab used in the SP ELISA showed 100% cross-reactivity to HK-1, but anti-HK-1 Ab showed 0% cross-reactivity to SP. The serum level of HK-1 was significantly lower in patients with CSU (n = 151) than in non-atopic healthy control (NC) subjects (n = 114). The EC 50 of histamine release from MCs induced by HK-1 (5056 nM) was 12-fold higher than by SP (426 nM). Brief pretreatment of MCs with HK-1 at concentrations of 3.0-10 μM significantly reduced histamine release by 0.1 μM SP. However, brief incubation of MCs with HK-1 did not elicit rapid MRGPRX2 internalization., Conclusions: In NC subjects, high HK-1 concentrations may desensitize MGRPRX2-mediated MC activation, thereby preventing MC degranulation by SP., (Copyright © 2021 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Objective: Hemokinin-1, the newest tachykinin encoded by the preprotachykinin C (Tac4) gene, is predominatly produced by immune cells. Similarly to substance P, it has the greatest affinity to the tachykinin NK1 receptor, but has different binding site and signaling mechanisms. Furthermore, several recent data indicate the existence of a not yet identified own receptor and divergent non-NK1-mediated actions. Since there is no information on its functions in the airways, we investigated its role in endotoxin-induced pulmonary inflammation., Methods: Acute pneumonitis was induced in Tac4 gene-deleted (Tac4(-/-)) mice compared to C57Bl/6 wildtypes by intranasal E. coli lipopolysaccharide (LPS). Airway responsiveness to inhaled carbachol was measured with unrestrained whole body plethysmography 24h later. Semiquantitative histopathological scoring was performed; reactive oxygen species (ROS) production was measured with luminol bioluminescence, myeloperoxidase activity with spectrophotometry, and inflammatory cytokines with Luminex., Results: All inflammatory parameters, such as histopathological alterations (perivascular edema, neutrophil/macrophage accumulation, goblet cell hyperplasia), myeloperoxidase activity, ROS production, as well as interleukin-1beta, interleukin-6, tumor necrosis factor alpha, monocyte chemoattractant protein-1 and keratinocyte chemoattractant concentrations were significantly diminished in the lung of Tac4(-/-) mice. However, bronchial hyperreactivity similarly developed in both groups. Interestingly, in LPS-treated Tac4(-/-) mouse lungs, bronchus-associated, large, follicle-like lymphoid structures developed., Conclusions: We provide the first evidence that hemokinin-1 plays a crucial pro-inflammatory role in the lung by increasing inflammatory cell activities, and might also be a specific regulator of lymphocyte functions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Incorporation of molecular adjuvants into DNA vaccines is often used to improve the induction of immune responses, but few approaches aim to specifically activate B cells for an enhanced humoral response only. Hemokinin-1 (HK-1) is a factor that activates B cells for proliferation, survival, differentiation into plasma cells, and Ab production. Therefore, we investigated if it may be used as a molecular adjuvant for DNA vaccines to elicit strong humoral and memory responses. The HK-1 coding sequence was sub-cloned as single or triple copies in-frame downstream of S2 HBsAg in the proVAX/S2 construct. Compared to mice immunized with proVAX/S2 or proVAX/S2-HK-1, proVAX/S2-3HK-1 induced a higher level of IgG production, a higher percentage of differentiated antibody-secreting plasma cells, and a higher level of T-cell proliferation. Furthermore, a higher proportion of B cells had the B220(+)CD27(+) phenotype in these groups, and specific antigen re-challenge induced a higher level of total IgG production 60 d after the last immunization, suggesting that the use of HK-1 as an adjuvant promoted immunological memory. Taken together, these results suggest that using HK-1 as an adjuvant molecule could enhance the immunogenicity of HBsAg DNA vaccines, and result in stronger humoral and memory responses. Therefore, HK-1 may lead to the development of a novel humoral-biased molecular adjuvant for an HBsAg DNA vaccine against hepatitis B infection.

Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. In mice, acute RSV infection causes airway hyperresponsiveness (AHR), inflammation, and mucus hypersecretion. Infected cells induce complement activation, producing the anaphylatoxin C3a. In this paper, we show RSV-infected wild-type mice produce Th17 cytokines, a response not previously associated with viral infections. Mice deficient in the C3aR fail to develop AHR following acute RSV infection, and production of Th17 cytokines was significantly attenuated. Tachykinin production also has been implicated in RSV pathophysiology, and tachykinin receptor-null mice were similarly protected from developing AHR. These animals were also deficient in production of Th17 cytokines. Tachykinin release was absent in mice deficient in C3aR, whereas C3a levels were unchanged in tachykinin receptor-null animals. Thus, our data reveal a crucial sequence following acute RSV infection where initial C3a production causes tachykinin release, followed by activation of the IL-17A pathway. Deficiency of either receptor affords protection from AHR, identifying two potential therapeutic targets.

Objective: This study aimed to determine the effect of hydrogen sulfide (H2S) on Toll-like receptor 4 (TLR4)-mediated innate immune signaling in acute pancreatitis (AP) via substance P., Methods: Male Swiss mice were treated with hourly intraperitoneal injections of cerulein (50 μg/kg) for 10 hours. dl-propargylglycine ([PAG] 100 mg/kg, intraperitoneally), an inhibitor of H2S formation, was administered 1 hour after the induction of AP. Pancreatic acinar cells from male preprotachykinin-A gene-knockout mice (PPTA) and their wild-type counterparts were incubated with or without cerulein (10 M for 60 minutes). To better understand the effect of H2S in inflammation, acinar cells were stimulated with cerulein after addition of H2S donor, sodium hydrosulfide. In addition, cerulein-treated pancreatic acinar cells were pretreated with PAG (30 μM) for 1 hour., Results: The H2S inhibitor PAG eliminated TLR4, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and nuclear factor-κB (NF-κB) levels in in vitro and in vivo models of cerulein-induced AP. PPTA gene deletion reduced TLR4, myeloid differentiation factor 88, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and NF-κB in cerulein-treated pancreatic acinar cells, whereas administration of sodium hydrosulfide resulted in a further rise in TLR4 and NF-κB levels in cerulein-treated pancreatic acinar cells., Conclusion: The present findings show for the first time that in AP, H2S may up-regulate the TLR4 pathway and NF-κB via substance P.

Hemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4(-/-) mice exhibit an increase of CD19(+)CD117(+)HSA(+)BP.1(-) "fraction B" pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4(-/-) bone marrow, sorted "fraction B" pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.

Hemokinin 1 (HK-1) is a substance P-like tachykinin peptide predominantly expressed in non-neuronal tissues. In addition to a prominent function in lymphoid development, recent studies indicate a potential role for HK-1 in immunoregulation. The current study was focused on its action on mature B cells. Despite the negligible effect on its own, HK-1 exhibited a profound influence on B cell activation elicited by several classical signals, including LPS stimulation, BCR cross-linking, and CD40 ligation. Cells therefore showed enhanced proliferation, survival, and CD80/86 expression, and produced more IgM with a higher frequency of Ab-forming cells. Biochemical analysis revealed that HK-1 alone was sufficient to induce the activation of MAPKs and the expression of Blimp-1 and Xbp-1 in B cells. Nevertheless, costimulation with a known B cell activator resulted in much enhanced phosphorylation of MAPKs and transcriptional activation of Blimp-1 and Xbp-1. Overall, these data support that HK-1 provides an important costimulatory signal for B cell activation, possibly through synergistic activation of the MAPK pathway and induction of transcription factors critical for plasmacytic differentiation.

During the course of polymicrobial sepsis, a range of pro- and antiinflammatory cytokines are produced by the host immune system. Successful recovery from sepsis involves striking a balance between these counteracting cytokines. We herein investigated the circulating cytokine profiles in preprotachykinin-A knockout (PPTA(-/-)) mice, which have been found to be protected significantly against microbial sepsis, by employing multiplexed bead-based suspension arrays for the measurement of 18 plasma cytokines. Four sets of PPTA(-/-) and wild-type mice, each with six mice, were subjected to cecal ligation and puncture-induced sepsis or a sham procedure and were killed at 1, 5, 8 and 24 h post surgery. The cytokine profiles revealed, rather interestingly, that both pro- and antiinflammatory cytokines were elevated in the knockout group in response to a septic challenge. The higher systemic levels of both pro- and antiinflammatory cytokines in PPTA(-/-) septic mice was similar to the increase that we observed earlier in lung tissue of PPTA(-/-) mice after induction of sepsis. Thus, elevated levels of both pro- and antiinflammatory mediators may act simultaneously and help to resolve the infectious assault at the early stages of sepsis without excessively damaging the host tissue in PPTA(-/-) mice. In addition, our results underline the importance of comprehensive clinical analysis of multiple biomarkers to provide a better prognostic tool.

The mouse/rat hemokinin-1 (m/rHK-1) was discovered nearly 9 years ago. This molecule is a peptide comprising 11 amino acids. The m/rHK-1 was found to be mainly expressed in central immune organs like bone marrow, and was proven to have lymphopoietic roles in B and T lymphocyte development. m/rHK-1was also reported to have analgesic roles in rat spinal cord, in addition to other functions such as relaxing activity on coronary artery. Unlike its analogues SP, NKA, and NKB, m/rHK-1 does not express in the nervous system. To further study the distribution and function of m/rHK-1, we carried out conventional immunization and cell fusion procedures to acquire the hybridomas secreting specific monoclonal antibodies to m/rHK-1. In the 17 positive clones obtained, three antibodies named 1B12, 2B4, and 4G5 were shown representative in cross-reactivity against m/rHK-1 and its analogues by indirect ELISA, competitive indirect ELISA, and immunofluorescence assays. Among the three clones, the 2B4 monoclonal antibody appeared to be the high-titered and specific clone to m/rHK-1. Monoclonal antibodies to m/rHK-1 will function as good tools in the physiological study of m/rHK-1 in the near future.

Objective: A new antibody, active against the common tachykinin (TK) C-terminal, was used to study TK expression in patients with endocrine tumors and a possible association between plasma-TK levels and symptoms of diarrhea and flush in patients with metastasizing ileocecal serotonin-producing carcinoid tumors (MSPCs)., Method: TK, serotonin and chromogranin A (CgA) immunoreactivity (IR) was studied by immunohistochemistry in tissue samples from 33 midgut carcinoids and 72 other endocrine tumors. Circulating TK (P-TK) and urinary-5 hydroxyindoleacetic acid (U-5HIAA) concentrations were measured in 42 patients with MSPCs before treatment and related to symptoms in patients with the carcinoid syndrome. Circulating CgA concentrations were also measured in 39 out of the 42 patients., Results: All MSPCs displayed serotonin and strong TK expression. TK-IR was also seen in all serotonin-producing lung and appendix carcinoids. None of the other tumors examined contained TK-IR cells. Concentrations of P-TK, P-CgA, and U-5HIAA were elevated in patients experiencing daily episodes of either flush or diarrhea, when compared with patients experiencing occasional or none of these symptoms. In a Spearman partial rank test, the correlation of P-TK with daily diarrhea was independent of both U-5HIAA and CgA levels., Conclusion: We found that TK synthesis occurs in serotonin-IR tumors and that P-TK levels are significantly correlated with symptoms of flush and diarrhea in patients with MSPCs. This is, to our knowledge, the first report demonstrating an independent correlation of P-TKs with carcinoid diarrhea, a symptom that is customarily regarded as serotonin mediated. Further investigations may present opportunities for new therapeutic possibilities.

Substance P (SP) is a neuropeptide that is released from sensory nerves and several types of immune cells. It is involved in the transmission of pain and has a number of pro-inflammatory effects. Like other neuropeptides, SP is derived from a large precursor peptide, protachykinin A (PTA). Alternative splicing results in the production of four distinct PTA molecules that all contain the sequence of SP and a common N-terminal region consisting of 37 amino acids. We have developed a sandwich immunoassay using antibodies against the N-terminal part of PTA. Here we demonstrate that N-terminal PTA immunoreactivity is present in human circulation and cerebrospinal fluid (CSF). The concentration was about 90 times higher in CSF than in EDTA-plasma. Analytical reversed phase HPLC revealed that NT-PTA 1-37 is the main immunoreactivity in human circulation and CSF. Moreover, compared to the low in vitro stability of SP of less than 12 min, NT-PTA immunoreactivity is absolutely stable in EDTA-plasma and CSF for more than 48 h. As NT-PTA 1-37 is produced in stoichiometric amounts and is theoretically co-released with SP, we suggest the measurement of NT-PTA immunoreactivity as surrogate molecule for the release of bioactive SP.

Hematopoiesis is the process by which immune and blood cells are produced from a finite number of relatively few hematopoietic stem cells (HSCs). In adults, hematopoiesis occurs in the adult bone marrow (BM), with the support of stromal cells. This support partly occurs through the production of hematopoietic regulators belonging to the families of cytokines and neuropeptides/neurotransmitters, which mediate their actions through specific receptors. Thus, stromal cells could be central to the neural-hematopoietic-immune axis. This study focuses on Tac1, which encodes hematopoietic regulators belonging to the tachykinin family of neuropeptides. We examined post-transcriptional regulation of Tac1 in BM stroma. Since this gene is inducible in stroma, we selected cytokines with varying hematopoietic effects: stimulator Stem Cell Factor (SCF), broad-acting IL-11 and suppressive TGF-beta1. RNA shift with Tac1 mRNA and cytoplasmic extracts from IL-11 and SCF-stimulated stroma showed RNA shift after 15min at 37 degrees C, whereas a shift was detected with extracts from TGF-beta1-stimulated stroma after 5min at room temperature. Another level of post-transcriptional regulation was observed by the detection of miRNAs that interact with the 3' untranslated region of Tac1 mRNA. In summary, this study showed that cytokine induced miRNA downregulation and RNA-binding protein(s) are involved in post-transcriptional regulation of Tac1 in BM stroma. The broad categories of cytokines as hematopoietic stimulators or inhibitors might depend on the avidity of RNA-binding protein(s) for Tac1 mRNA, as well as the ability to degrade or stabilize the specific miRNAs.

Background: Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs)., Methods: An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays., Results: The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, alpha-NGF, TSP2). In the absence of NK1R, the matrix Nkx2-5_02 had a predominant participation driving 8 transcripts, which includes those involved in cancer (EYA1, Trail, HSF1, and ELK-1), smooth-to-skeletal muscle trans-differentiation, and Z01, a tight-junction protein, expression. Electrophoretic mobility shift assays confirmed that, in the mouse urinary bladder, activation of NK1R by substance P (SP) induces both NKx-2.5 and NF-kappaB translocations., Conclusion: This is the first report describing a role for Nkx2.5 in the urinary tract. As Nkx2.5 is the unique discriminator of NK1R-modulated inflammation, it can be imagined that in the near future, new based therapies selective for controlling Nkx2.5 activity in the urinary tract may be used in the treatment in a number of bladder disorders.

Stromal cell-derived growth factor-1alpha (SDF-1alpha) is a member of the CXC chemokines and interacts with the G protein, seven-transmembrane CXCR4 receptor. SDF-1alpha acts as a chemoattractant for immune and hemopoietic cells. The Tac1 gene encodes peptides belonging to the tachykinin family with substance P being the predominant member. Both SDF-1alpha and Tac1 peptides are relevant hemopoietic regulators. This study investigated the effects of SDF-1alpha on Tac1 expression in the major hemopoietic supporting cells, the bone marrow stroma, and addresses the consequence to hemopoiesis. Reporter gene assays with the 5' flanking region of Tac1 showed a bell-shaped effect of SDF-1alpha on luciferase activity with 20 ng/ml SDF-1alpha acting as stimulator, whereas 50 and 100 ng/ml SDF-1alpha acted as inhibitors. Gel shift assays and transfection with wild-type and mutant IkappaB indicate NF-kappaB as a mediator in the repressive effects at 50 and 100 ng/ml SDF-1alpha. Northern analyses and ELISA showed correlations among reporter gene activities, mRNA (beta-preprotachykinin I), and protein levels for substance P. Of relevance is the novel finding by long-term culture-initiating cell assays that showed an indirect effect of SDF-1alpha on hemopoiesis through substance P production. The results also showed neurokinin 1 and not neurokinin 2 as the relevant receptor. Another crucial finding is that substance P does not regulate the production of SDF-1alpha in stroma. The studies indicate that SDF-1alpha levels above baseline production in bone marrow stroma induce the production of substance P to stimulate hemopoiesis. Substance P, however, does not act as autocrine stimulator to induce the production of SDF-1alpha. This study adds SDF-1alpha as a mediator within the neural-immune-hemopoietic axis.

Background: Allergic airway inflammation has been shown to induce pro-inflammatory neuropeptides such as tachykinin peptides substance P (SP) and neurokinin A (NKA) together with related peptide like calcitonin gene-related peptide (CGRP) in nodose sensory neurons innervating guinea-pig airways., Objective: The present study was designed to examine the effects of allergen sensitization and challenge on the SP/NKA expression in the jugular-nodose ganglion neurons innervating the murine airways., Methods: Using retrograde neuronal tracing technique in combination with double-labelling immunohistochemistry, the expression of SP/NKA was investigated in a murine model of allergic airway inflammation., Results: Allergic airway inflammation was found to induce the expression of SP/NKA (13.2+/-1.43% vs. 5.8+/-0.37%, P<0.01) in large-diameter (>20 microm) vagal sensory neurons retrograde labelled with Fast blue dye from the main stem bronchi., Conclusion: Based on the induction of tachykinins in airway-specific large-sized jugular-nodose ganglia neurons by allergic airway inflammation, the present study suggests that allergen sensitization and challenge may lead to de novo induction of tachykinins in neurons. This may partly contribute to the pathogenesis of airways diseases such as allergic airway inflammation.

Objective: The purpose of this study was to investigate the effects of progesterone and the most commonly prescribed synthetic progestogen, norethisterone, on regional immune-like reactivity of neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA) and neurotensin (NT) in brains of female ovariectomized estradiol-substituted rats., Results: Norethisterone+estradiol-treated rats had 44% lower SP levels compared with estradiol-only-treated in frontal cortex and 20% lower NKA levels in comparison with progesterone+estradiol-treated in frontal cortex. Progesterone+estradiol-treated rats had 66% lower SP levels in striatum in comparison with both estradiol-only-treated and norethisterone+estradiol-treated. No significant results were found for NPY and NT., Conclusion: Progesterone and the synthetic progestogen, norethisterone, have different effects on SP- and NKA-like immunoreactivity in rat cortex and striatum. The effects of NET on SP- and NKA-like immunoreactivity in frontal cortex may contribute to the mood effects ascribed to this progestogen in clinical usage.

Background: Changes in levels of substance P and substance P-binding sites have been implicated in schizophrenia. However, no studies have used receptor-specific antibodies to directly investigate the substance P (neurokinin 1) receptor in schizophrenia., Methods: We used an antibody directed against the human neurokinin-1 receptor to compare the distribution of neurokinin-1 receptors in the prefrontal cortices from six subjects with schizophrenia and six control subjects, matched for age, gender, and postmortem interval., Results: In control tissue, dots of neurokinin-1 receptor immunoreactivity were observed in layer I to upper/mid layer III only. In contrast, dots of neurokinin-1 receptor immunoreactivity were observed in all layers of the prefrontal cortex in subjects with schizophrenia, and the density of dots was significantly greater than in control subjects., Conclusions: This is the first report of increased neurokinin-1 receptor immunoreactivity in the prefrontal cortex in subjects with schizophrenia. These changes may have implications for understanding the pathophysiology of the prefrontal cortex in schizophrenia and for the treatment of this disorder.

We report here the molecular cloning of a newly identified preprotachykinin gene, Pptc, which specifies the sequence for a new preprotachykinin protein and bioactive peptide designated hemokinin 1 (HK-1). PPT-C mRNA was detected primarily in hematopoietic cells in contrast to the previously described Ppta and Pptb genes, which are predominantly expressed in neuronal tissues. HK-1 has several biological activities that are similar to the most studied tachykinin, substance P, such as induction of plasma extravasation and mast cell degranulation. However, HK-1 also has properties that are indicative of a critical role in mouse B cell development. HK-1 stimulated the proliferation of interleukin 7-expanded B cell precursors, whereas substance P had no effect. HK-1, but not substance P, promoted the survival of freshly isolated bone marrow B lineage cells or cultured, lipopolysaccharide-stimulated pre-B cells. N-acetyl-L-trytophan-3,5-bistrifluromethyl benzyl ester, a tachykinin receptor antagonist, increased apoptosis of these cells and in vivo administration of this antagonist led to specific reductions of the B220lowCD43 population (the pre-B cell compartment) in the bone marrow and the IgMhighIgDlow population (the newly generated B cells) in the spleen. Thus, HK-1 may be an autocrine factor that is important for the survival of B cell precursors at a critical phase of development.

Off-axis illumination elicits lateral inhibition at the primary visual synapse in crustacea and insects. The evidence suggests that the inhibitory action is presynaptic (i.e., on the photoreceptor terminal) and that the amacrine neurons of the lamina ganglionaris (the first synaptic layer) may be part of the inhibitory pathway. The neurotransmitters and the synaptic mechanisms are unknown. We show by immunocytochemistry that GABA and a tachykinin-related peptide (TRP) are localized in the amacrine neurons of the crayfish lamina ganglionaris. Indirect evidence suggests that GABA and TRP may be colocalized in these neurons. The extensive processes of the amacrine neurons occupy lamina layers containing the terminals of photoreceptors. Application of exogenous GABA and TRP to photoreceptor terminals produces a short-latency, dose-dependent hyperpolarization with a decay time constant on the order of a few seconds. TRP also exhibits actions that evolve over several minutes. These include a reduction of the receptor potential (and the light-elicited current) by approximately 40% and potentiation of the action of GABA by approximately 100%. The mechanisms of TRP action in crayfish are not known, but a plausible pathway is a TRP-dependent elevation of intracellular Ca(2+) that reduces photoreceptor sensitivity in arthropods. Although the mechanisms are not established, the results indicate that in crayfish photoreceptors TRP displays actions on two time scales and can exert profound modulatory control over cell function.

The modifying effects of tachykinins substance P, neurokinin A and neurokinin B on immunoglobulin production were analyzed in an in vitro culture system. Purified human T- and B-cells were stimulated with TGFbeta2 and IL-5 to induce preferential IgA production. Neuropeptides had the following effects. (1) The levels of IgA and IgG4 production were enhanced by IL-5 and TGFbeta2; IgA levels remained constant or were slightly augmented by neuropeptides, whereas IgG4 was further augmented. (2) IL-5 and TGFbeta2 did not alter IgG3 production, but neuropeptides stimulated secretion of this subclass. (3) IgG1 and IgM production were inhibited by IL-5 and TGFbeta2. This effect was prevented by neuropeptides. (4) Other isotypes including IgG2 and IgE remained unaffected. Except for IgM, these effects were blocked by specific receptor antagonists indicating specificity. The tachykinin receptor NK-1 mRNA was detected in B- and T-cells, whereas NK-3 mRNA was only present in T- and B-cell coculture following activation. Furthermore, neuropeptide effects depended on cytokine co-stimulation and the presence of T-cells. These results suggest that neuropeptides are potent modifiers of preferential IgA synthesis.

Culex pipiens and Aedes aegypti mosquitoes were fed on C3H/HeJ mice and systemic cytokine production was quantified from stimulated lymphocytes harvested four to ten days after feeding. Mosquito feeding on C3H/HeJ mice significantly down regulated IFN gamma production seven to ten days post feeding by Cx. pipiens and seven days after Ae aegypti feeding. Th2 cytokines, IL-4 and IL-10, were significantly up regulated 4-7 days after Cx. pipiens and Ae. aegypti feeding. The immunosuppressive effect of Cx. pipiens feeding on systemic cytokine production was not evident in congenic flavivirus resistant (C3H/RV) mice, as systemic IFN gamma and IL-2 were significantly up regulated at days 7 and 10, correlating with a significant decrease in IL-4 10 days after feeding by Cx. pipiens mosquitoes. Inoculation of 5-1000 ng of sialokinin-I into C3H/HeJ mice mimicked the effect of Ae. aegypti feeding by down regulating Th1 cytokines and significantly up regulating Th2 cytokines four days post inoculation. Injections of sialokinin-II resulted in only moderate effects on IFN gamma and IL-4 production seven and ten days after injection. Thus natural feeding by two arbovirus vectors had a profound T cell modulatory effect in vivo in virus susceptible animals which was not demonstrated in the flavivirus resistant host. Moreover, sialokinin-I and sialokinin-II mimicked the effect of mosquito feeding by modulating the host T cell response. These results may lend new insight into specific aspects of the role of the mosquito vector in potentiating virus transmission in the mammalian host.

We have characterized preprotachykinin (PPT-A) gene transcript splicing products and identified a fourth isoform of PPT-A mRNA transcript in human peripheral blood-isolated monocytes and PBL. Using RT-PCR, Southern blot analysis and nucleotide sequencing analysis, we have identified the four isoforms of PPT-A transcripts (alpha, beta, gamma and delta) in human peripheral blood-isolated monocytes and PBL. The delta-PPT transcript present in the immune cells lacks exons 4 and 6. The sequences of exons 3, 5 and 7 of delta-PPT transcript completely match those of beta-PPT transcript. The alpha-PPT and beta-PPT sequences in these cells are identical to those obtained by Tan and Too (GenBank accession number U37539) and Harmar et al. (Genbank accession number X54469), but differ by a single nucleotide from another entry by Chiwakata et al. (Genbank accession number M68906). In comparison to this latter sequence, there was a C-->T change at amino acid position 87 (CCT-->CTT) which may result in a Pro to Leu change. Identification of the new SP mRNA transcript in both human CNS and immune cells supports the concept of an important biological link between CNS and immune system.

Trigeminal sensory neurons innervate the nasal cavity and may release substance P (SP) upon exposure to inhaled irritants. The purpose of this study was to determine if silica dust, an occupational irritant causing inflammation, activates sensory neurons supplying the nasal cavity. Male Fischer 344 rats were placed in inhalation chambers and exposed daily to 2 mg/m3 of fresh silica (average diameter 1 microm) for 6 mo. Following exposure, the trigeminal ganglia (TG) were removed and prepared for SP immunocytochemistry and for preprotachykinin (PPT) autoradiographic in situ hybridization. The SP-like immunofluorescence in TG neurons was subjectively categorized as high, moderate, or low (background) intensity. In situ hybridization autoradiographs were quantified on the basis of grain density using digital imaging analysis. The SP immunoreactivity and PPT mRNA expression in the TG neurons were significantly increased after silica inhalation. The proportion of highly positive SP-immunoreactive neurons shifted from 1.30 +/- 0.58% in controls to 11.30 +/- 1.15% after silica treatment. The neurons exhibiting high grain density for PPT mRNA increased from 1.50 +/- 0.87% in controls to 11.67 +/- 0.58% in the silica group. Thus, inhalation of silica causes upper airway irritation resulting in increased levels of immunoreactive neuronal SP and PPT mRNA. These findings suggest that silica activates sensory pathways that may be involved in nasal inflammation.

Tachykinins, found in sensory nerves, have effects in the airways which suggest that they may contribute to the pathogenesis of asthma. We aimed to find evidence for tachykinin involvement in the immediate airway response to allergen in a sheep model of experimental asthma. Twenty-four sheep were actively sensitized to Ascaris suum, then challenged with nebulized Ascaris extract in a dose-response fashion. Change in lung resistance (RL) in response to challenge was measured. Responder sheep (those with an increase in RL of > or = 100% over baseline) that had reproducible responses over three challenges were identified (n = 4 sheep) and a PC100 (number of breaths of extract required to induce a 100% increase in RL) was determined. The effect of the neutral endopeptidase inhibitor phosphoramidon, the NK-1 receptor-specific antagonist CP 96, 345 and capsaicin desensitization on the RL response to Ascaris challenge was then assessed. Administration of phosphoramidon before Ascaris decreased the PC100 to 31 +/- 7% of the PC100 seen with Ascaris alone (P < 0.05), whereas CP 96,345 and capsaicin desensitization increased the PC100 to 285 +/- 41% and 555 +/- 93% respectively (P < 0.05 for both). These findings suggest that endogenous tachykinins are released in response to allergen challenge and that they contribute to the immediate increase in RL.

Four tachykinin-related peptides, locustatachykinin 1-4 (LomTK 1-4) are distributed in interneurons throughout the central nervous system of the locust Locusta migratoria and may have important roles as neurotransmitters or neuromodulators. In search of the central actions of LomTKs, we analyzed the response of the efferent dorsal unpaired median (DUM) neurons in the locust metathoracic ganglion. Immunocytochemistry, using an antiserum against LomTK 1, combined with intracellular filling of efferent DUM neurons with Lucifer yellow, revealed that LomTK-immunoreactive fibers are in close proximity to dendritic arborizations of the DUM neurons. Hence, LomTKs may act on DUM neurons by releasing locally in the metathoracic ganglion. Intracellular recordings were made from somata of DUM neurons, and LomTKs were either bath-applied to an isolated metathoracic ganglion or pressure-ejected onto the DUM neuron soma. LomTK 1 at concentrations of 0.1 mM-0.1 microM caused a relatively slow, reversible depolarization with a subsequent increase in the frequency of action potential firing. Amino-terminally truncated forms of LomTK 1 were applied to DUM neurons. The heptapeptide [3-9]-LomTK 1 had a substantially reduced activity, and bioactivity was lost after further truncation. Spantide 1, an antagonist of mammalian tachykinin receptors, reversibly blocked the effect of LomTK 1. The effect of LomTK 1 was clearly reduced in the presence of GDP-beta-S, a stable analog of GDP that inactivates G-proteins. The action of LomTK 1 was potentiated by both IBMX and theophylline, two cyclic AMP (cAMP) phosphodiesterase inhibitors. The action of LomTK 1 was mimicked by pressure-ejecting 8-bromo-cAMP, a membrane permeable analog of cAMP, and by forskolin, an adenylate cyclase activator. Furthermore, cAMPS, a blocker of protein kinase A activity, reduced the effect of LomTK 1. These findings indicate that cAMP is involved in mediating DUM neuron depolarization.

Tachykinin (TK) peptides, which include substance P, neurokinin A, two neurokinin A-related peptides and neurokinin B, are widely present in the nervous system, including the retina, where they act as neurotransmitters/modulators as well as growth factors. In the present study, we investigated the maturation of TK-immunoreactive (IR) cells in the rabbit retina with the aim of further contributing to the knowledge of the development of transmitter-identified retinal cell populations. In the adult retina, the pattern of TK immunostaining is consistent with the presence of TK peptides in amacrine, displaced amacrine, interplexiform and ganglion cells. In the newborn retina, intensely immunostained TK-IR somata are located in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL). They are characterized by an oval-shaped cell body originating a single process without ramifications. TK-IR processes are occasionally observed in the IPL and in the outer plexiform layer (OPL). Long TK-IR fiber bundles are observed in the ganglion cell axon layer. TK-IR profiles resembling small somata are rarely observed in the INL adjacent to the OPL. At postnatal day (PND) 2, some TK-IR cells display more complex morphologic features, including processes with secondary ramifications. Long TK-IR processes in the IPL are often seen to terminate with growth cones. Between PND 6 and PND 11 (eye opening), there is a dramatic increase in the number of immunolabeled processes with growth cones both in the IPL and in the OPL and the mature lamination of TK-IR fibers in laminae 1, 3 and 5 of the IPL is established. TK-IR cells attain mature morphological characteristics and the rare, putative TK-IR somata in the distal INL are no longer observed. After eye opening, growth cones are not present and the pattern typical of the adult is reached. These observations indicate that the development of TK-IR cells can be divided into an early phase (from birth to PND 6) in which these cells establish their morphological characteristics, and a later phase (from PND 6 to eye opening) in which they are involved in active growth of their processes and likely in synapse formation. Since TK peptides are thought to play neurotrophic actions in the developing nervous system and they are consistently present in the retina throughout postnatal development, they may also act as growth factors during retinal maturation.

Tachykinins are a family of peptides that may be present in and secreted from carcinoid tumours of mid-gut origin. They are likely to play a role in the pathogenesis of, e.g. the flush, dyspnoea and valvular heart disease seen in the carcinoid syndrome. Since tachykinins are secreted from the tumour into the circulation in bursts, coinciding with flushing attacks, and have short half-lives, we anticipated that analysis of 24-h urine excretion of immunoreactive tachykinin metabolites might prove to be a more sensitive and stable parameter for monitoring than tachykinin-like immunoreactivity in plasma. The study included 48 patients hospitalized for treatment of advanced carcinoid tumours and 32 healthy controls. The urine excretion of tachykinin-like immunoreactive metabolites in the carcinoid patients (median 27.5 pmol 24 h-1, interquartile range (IQR) 8.5-51.0 pmol 24 h-1) was significantly (p<0.001) higher than that in the 32 healthy subjects (median 3.0 pmol 24 h-1, IQR 0.9-4.20 pmol 24 h-1). Of the patients, 38 (79%) had elevated 24-h urine excretion of tachykinin-like immunoreactive metabolites while 31 (64%) had elevated plasma concentrations of tachykinin-like immunoreactive metabolites. Of the patients, 27 (56%) had elevated concentrations of tachykinin-like immunoreactive metabolites both in plasma and urine, 12 (25%) had elevated concentrations only in urine excretion, 3 (6%) had elevated concentrations of only plasma tachykinin-like immunoreactive metabolites and 7 (14%) had elevation of neither plasma nor urine concentrations. Analysis by means of different column chromatographic techniques indicated that the immunoreactive material was heterogeneous, with some components co-eluting with oxidized neurokinin A (NKA) and neuropeptide K (NPK). The urine tachykinin-like immunoreactivity correlates well with that of plasma, but is a slightly more sensitive indicator of elevated tachykinin-like immunoreactivity, probably since levels of urine tachykinin-like immunoreactive metabolites reflect the overall amount of the latter secreted into the circulation during 24 h.

An antiserum raised to the locust neuropeptide locustatachykinin I (LomTK I) was used for analysis of the distribution of tachykinin-related peptide in the cockroach Leucophaea maderae. Extracts of dissected brains, suboesophageal ganglia, thoracic ganglia and midguts were separated by high performance liquid chromatography and the fractions analysed in enzyme-linked immunosorbent assay with use of the LomTK antiserum. Each of the tissues was found to contain LomTK-like immunoreactive (LomTK-LI) components with retention times corresponding approximately to synthetic LomTK I and II and callitachykinins I and II. The LomTK antiserum was also used for immunocytochemical mapping of peptide in the nervous system and intestine of L. maderae. A large number of LomTK-LI interneurons were detected in the proto-, deuto- and tritocerebrum of the brain and in the suboesophaegeal ganglion. The immunoreactive neurons supply processes to most parts of the brain: the central body, protocerebral bridge, mushroom body calyces, antennal lobes, optic lobe and most regions of the non-glomerular neuropil. A few protocerebral neurons send LomTK-LI processes to the glandular lobe of the corpora cardiaca. In each of the thoracic ganglia there are six LomTK-LI interneurons and in each of the unfused abdominal ones there are two interneurons. The fused terminal ganglion contains some additional cell bodies in the posterior neuromers. LomTK-LI cell bodies were detected in the frontal ganglion and fibres were seen in this ganglion as well as in the hypocerebral ganglion. The frontal ganglion supplies LomTK-LI processes to the muscle layer of the pharynx. The muscle layer of the midgut is innervated by LomTK-LI fibres from the stomatogastric system (oesophageal nerve and associated ganglia). Additionally the midgut contains numerous LomTK-LI endocrine cells. A number of the pharyngeal dilator muscles were also found to be innervated by LomTK-LI fibres, probably derived from cell bodies in the suboesophageal ganglion. All the LomTK-LI neurons of the central nervous system appear to be interneurons, suggesting a neuromodulatory role of the endogenous tachykinins. The tachykinin-like peptides from peripheral ganglia may be involved in the control of foregut and midgut contractility and possibly the peptide of the endocrine cells in the midgut has additional actions related to intestinal function.

Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of different tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay with various C-terminally directed tachykinin antisera and a newly developed N-terminally directed substance P (SP)-antiserum (SPN 1) were used. Antiserum SPN 1 fully reacts with SP-analogues modified at the C-terminal end (SP free acid and SP-Gly-Lys) and also (77%) with SP(1-9) but not with C-terminal SP-fragments lacking 2 or more N-terminal amino acids. The highest levels of SP-like immunoreactivity (LI) and neurokinin A (NKA)-LI were measured after combined water and acetic acid extraction procedures. Also when measuring cholecystokinin-like immunoreactivity the highest level was obtained following this extraction procedure. RP-HPLC revealed a major component of SP-LI at the position of synthetic SP irrespectively of the extraction method and if the C- or N-terminally directed antiserum was used. Neutral water extracts contained a late eluting component detected with the C-terminally, but not with the N-terminally, directed antiserum. Acid and alkaline extracts, in contrast, contained components which could be detected with the N-terminally, but not with the C-terminally, directed SP-antiserum. Immunoreactive components eluting at the position of NKA and NKB were found in all types of extracts with NKA-, kassinin- and eledoisin-antisera. The NKB- and neuropeptide K (NPK)-components were more prominent in acid than in neutral and alkaline extracts. In conclusion, the present results indicate that rat spinal cord may contain molecular forms of tachykinin-like immunoreactivity in addition to those previously described and illustrate the importance of the choice of extraction method in immunochemical studies. Combined extraction in water and acetic acid appears to be a suitable method when the content of peptides with different chemical properties are to be measured in a tissue sample.

The localization of tachykinin-immunoreactivity in the cat visual cortex (area 17) was investigated using immunohistochemical methods. Strong laminar specificity was observed, with immunoreactivity highest in layer V, followed by layers I, VI, II and III, and the lowest density in layer IV. Most of the immunoreactive product was localized in neuronal processes. A few immunopositive cell bodies were also present. The immunopositive neurons were non-pyramidal, multipolar, or bipolar in shape, and mostly found in layer V. There were particularly dense immunopositive fibers and varicosities around somata in layer V. These may represent tachykinin-containing presynaptic terminals (boutons). The results provide anatomical evidence that tachykinins may primarily affect layer V neurons in the cat visual cortex.

Anatomical relationships between tachykinin-containing terminals and neurons of the medial preoptic area that innervate the arcuate nucleus were studied using silver staining of the retrograde tracer wheat germ agglutinin-apoperoxidase-gold (WGA-ApoHRP-gold) complex injected in the arcuate nucleus and pre-embedding immunocytochemistry for neurokinin A (NKA). At the histological level, retrogradely labeled cells not stained for NKA were seen to be surrounded by numerous NKA-immunopositive punctate profiles, in particular in the dorsal part of the medial preoptic area. At the ultrastructural level, retrogradely labeled cell bodies and dendritic profiles displayed highly electron-dense silver particle accumulations over the cytoplasm. The were seen in synaptic contact with one or several NKA-immunoreactive axon terminals containing small clear vesicles and dense-cored vesicles. Such synapses were either symmetrical or asymmetrical. The occurrence of synaptic contacts between tachykinin terminals and cells innervating the arcuate nucleus in the medial preoptic region provides a morphological support for a tachykinergic regulation of preoptic afferences to the arcuate nucleus. These results suggest that tachykinins are implicated in the indirect control of neuronal activity in the arcuate nucleus notably via the preoptic area. Consequently, tachykinins are potentially able to regulate indirectly numerous neuroendocrine events involving the tuberoinfundibular system.

An antiserum raised against locustatachykinin I, one of four myotropic peptides that have been isolated from the locust brain and corpora cardiaca, was characterized by enzyme-linked immunosorbent assay (ELISA) and used for immunocytochemical detection of neurons and endocrine cells in the nervous system and intestine of the blowfly Calliphora vomitoria. The ELISA characterization indicated that the antiserum recognizes the common C-terminus sequence of the locustatachykinins I-III. Hence, the cross reaction with locustatachykinin IV is less, and in competitive ELISAs no cross reaction was detected with a series of vertebrate tachykinins tested. It was also shown that the antiserum recognized material in extracts of blowfly heads, as measured in ELISA. In high-performance liquid chromatography the extracted locustatachykinin-like immunoreactive (LomTK-LI) material eluted in two different ranges. A fairly large number of LomTK-LI neurons was detected in the blowfly brain and thoracicoabdominal ganglion. A total of about 160 LomTK-LI neurons was seen in the proto-, deuto-, and tritocerebrum and subesophageal ganglion. Immunoreactive processes from these neurons could be traced in many neuropil regions of the brain: superior and dorsomedian protocerebrum, optic tubercle, fan-shaped body and ventral bodies of the central complex, all the glomeruli of the antennal lobes, and tritocerebral and subesophageal neuropil. No immunoreactivity was seen in the mushroom bodies or the optic lobes. In the fused thoracicoabdominal ganglion, 46 LomTK-LI neurons could be resolved. The less evolved larval nervous system was also investigated to obtain additional information on the morphology and projections of immunoreactive neurons. In neither the larval nor the adult nervous systems could we identify any efferent or afferent immunoreactive axons or neurosecretory cells. The widespread distribution of LomTK-LI material in interneurons suggests an important role of the native peptide(s) as a neurotransmitter or neuromodulator within the central nervous system. Additionally a regulatory function in the intestine is indicated by the presence of immunoreactivity in endocrine cells of the midgut.

We examined the mechanisms of bradykinin-induced airway microvascular leakage in guinea pig airways by measuring extravasation of Evans blue dye. Animals were pretreated with propranolol (1 mg/kg, intravenous) and atropine (1 mg/kg, intravenous) to block the beta-adrenergic and muscarinic responses, respectively. Bradykinin (250 nmol) instillation into airways significantly increased the leakage of dye in the trachea, main bronchi, and intrapulmonary airways to the same degree. The bradykinin B2-receptor antagonist HOE140 (500 nmol/kg, intravenous) did not alter basal leakage but almost completely inhibited bradykinin-mediated leakage. By contrast, the neurokinin NK1 antagonist FK888 (10 mg/kg, intravenous) partially inhibited bradykinin-induced leakage in trachea (p < 0.01) and main bronchi (p < 0.01), but had no significant effect on intrapulmonary airways. Indomethacin (5 mg/kg, intravenous) had no effect on the plasma leakage after instilled bradykinin. We concluded that the airway inflammatory response to bradykinin administered directly into the airways is mediated by bradykinin B2 receptors and partially mediated by tachykinin release from sensory nerve terminals, whereas cyclooxygenase products have no important role in the response. In the central airways, the contribution of sensory neuropeptides to the bradykinin response is greater than that caused by direct stimulation of the B2 receptor on the endothelium at the postcapillary venule of the bronchial circulation. In contrast, in the peripheral airways, the contribution of direct B2-receptor stimulation on the airway vasculature is greater than that involving sensory neuropeptides.

Strong connections with sexually dimorphic nuclei suggest that the ventral premammillary nucleus (PMv) may be involved in the mediation of reproductive behavior. Steroid autoradiography and immunohistochemistry were used to show that: (1) there is a sex difference in the numbers of PMv neurons that contain tachykinin peptides, (ii) dihydrotestosterone concentrating cells are densely distribution in the PMv and about a quarter of these also contain immunoreactive tachykinin and, (iii) size of the immunoreactive tachykinin population does not respond to alterations in levels of gonadal steroids in adulthood. Thus the PMv appears to make a contribution to the regulation of sexual behavior through androgen-concentrating, tachykinin-containing pathways that are anatomically distinct from estrogen receptive circuitry.

This study examines the contribution of tachykinins to the processes of inflammation. Neurokinin A (NKA), neurokinin B (NKB) and eledoisin (E) but not kassinin (K) have similar effects to substance P (SP) in priming neutrophils for increased superoxide anion (O2-) production in response to formyl-methionyl-leucyl-phenylalanine (FMLP). This similarity in activity may be due to the carboxy amino acid terminal end of these tachykinins being highly conserved. This was confirmed by demonstrating that SP fragment 7-11 (SP7-11) had the same priming effect as the whole molecule, whereas, the amino end fragment 1-4 (SP1-4) inhibited the response to FMLP. The priming effect of tachykinins was not confined to a single stimulus, such as FMLP, since NKA, NKB and SP also enhanced O2- production stimulated by platelet-activating factor (PAF), an important mediator of inflammation but a weak stimulus of O2- production on its own. In addition, all the tachykinins studied increased neutrophil antibody-dependent cell-mediated cytotoxicity (ADCC) towards opsonized target cells. In contrast to their effects on FMLP-induced O2- production, both SP fragments, SP1-4 and SP7-11, stimulated neutrophil ADCC and had a synergistic effect when used together.

In a study to test the hypothesis that defects in the metabolism of neuropeptides might be a contributing factor to human anterior pituitary tumor formation, the proenkephalin A, proopiomelanocortin (POMC), and tachykinin systems, which produce methionine enkephalin (ME), beta-endorphin (BE), and substance P (SP), respectively, were measured in patients who had a wide variety of pituitary tumors. Mass spectrometry was used to optimize the level of molecular specificity of the ME and BE analytical measurements, and radioimmunoassay was used to measure SP-like immunoreactivity (SP-li). Compared to data obtained from pituitaries from post-mortem controls, the non-secreting tumors contained a significantly lower amount of the POMC neuropeptide, BE. The lower ME level was not significant. However, two adrenocorticotrophic hormone (ACTH)-secreting tumors contained ME, BE, and SP-li amounts that were much higher than both the controls and nonsecreting tumors. These data suggest that a hypometabolism of the POMC precursor may be operating in non-secreting tumors, and that a hypermetabolism of the proenkephalin A, POMC, and tachykinin precursors may be operating in two ACTH-secreting tumors. These data demonstrate that mass spectrometry plays a critical role in the study of human pituitary tumors.

1. Circumoesophageal ganglia and foot muscle of the garden snail, Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE). 2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum. 3. SP and NKA radioimmunoassays were performed on extracts of circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum. 4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide. 5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-terminal structural characteristics similar to mammalian SP.

When testing a large number of antisera against tachykinins of various vertebrates and one insect (the cockroach Leucophaea maderae) we found three distinct populations of tachykinin-immunoreactive neurons in the blowfly: (1) one recognized by antisera against substance P, (2) another by antisera against the frog peptide kassinin and (3) a third with antisera raised against the cockroach peptide Leucokinin I. As a comparison tests on the cockroach Leucophaea showed that only antisera against neurokinin A (NKA) and leucokinin I gave immunostaining, RIA and immunocytochemical displacement tests demonstrated that the each of the three listed types of antisera was specific for the corresponding antigenic peptide and showed virtually no cross reactivity with the other tachykinin peptides. By immunocytochemistry we have mapped the three populations of tachykinin-immunoreactive neurons in the blowfly CNS. Two constitute unique sets of interneurons that were previously not detected with other antisera, the third, recognized by antisera against substance P, is a subpopulation of the FMRFamide immunoreactive neurons. The leucokinin immunoreactive material in the blowfly seems to be chemically different from that in Leucophaea and also the sets of neurons immunolabeled in the two insect species are to some extent different. The preliminary results presented here indicate the presence of multiple forms of tachykinin-like peptides in the blowfly and that these are distinct from those in the cockroach. From the immunocytochemistry it appears that the insect tachykinins may be involved in a variety of regulatory functions in the CNS and in some cases possibly act as neurohormones.

Neuro-immunology is becoming an increasingly important discipline of immunology. This review has examined the immunomodulatory function of one group of neuropeptides, the TK, particularly SP and NKA. These peptides are localized in primary afferent nerves which have been shown to innervate several immune organs. In addition, binding sites for the TK have been demonstrated in thymus, spleen and lymph node. Several immune cell types also express neurokinin receptors including human circulating lymphocytes with binding to the Th/i class predominating, murine T and B cells, a human T lymphoblastoid cell line, human monocytes, rabbit polymorphonuclear leucocytes and guinea-pig macrophages. The apposition of nerves with immune cells and receptors for neuropeptides thus produces an environment for interaction between the nervous and immune systems. Studies in vitro and, more recently, in vivo have examined how the TK regulate immune cell responses. The TK stimulate proliferation of T cells, enhance mitogen-induced release of cytokines including IFN-gamma, TNF-alpha, IL-1 and IL-6 from mononuclear cells and macrophages, enhance immunoglobulin secretion and affect cellular chemotaxis and phagocytosis. Studies in vivo have shown a role for TK in lymphocyte recirculation of sheep lymph nodes, reversal of stress-induced thymic involution and Ig production in both rat and mouse. Many of these effects appear to be mediated via NK-2 type receptors. To date, most of the work has involved studies in vitro, but the results from these are now being validated by studies in vivo where both the immune system and neuropeptides are able to interact at many anatomical sites. The complexities of the immune and the nervous systems mean that only a small number of potential interactions has been examined. Future studies can be expected to amplify these observations, especially with respect to the understanding of inflammatory and immune diseases in humans.

Light microscopic (LM) immunoenzyme and electron microscopic (EM) immunogold cytochemistry were used to demonstrate the presence and subcellular distribution of tachykinin (substance P)-like immunoreactivity in Merkel cells of pig skin. Merkel cells of sinus hair follicles were strongly immunoreactive for tachykinins. In contrast, tachykinin-like immunoreactivity was absent from or very weak in epidermal Merkel cells while subepidermal and some intraepidermal nerve fibres were clearly immunopositive for tachykinins. Postembedding immunogold cytochemistry on the EM level revealed that tachykinin-like immunoreactivity was confined to the secretory granules in Merkel cells. Tachykinin immunoreactivity was clearly absent from the axon of the Merkel cell-axon complex. The selective presence of tachykinin immunoreactivity in secretory granules strongly suggests that tachykinins are synthesized in cutaneous Merkel cells, at least of pig. This is a further indication for the concept that the Merkel cell is a member of the diffuse neuroendocrine system. However, the functional role of tachykinins like substance P and neurokinin A and of other peptides present in Merkel cells remains enigmatic.

Tachykinins and CGRP label two distinct populations of neurons innervating the digestive system: intrinsic and extrinsic, afferents. The bulk of SP/tachykinin innervation originates from intrinsic neurons, even though a minor component of this innervation derives from afferent neurons, which are mostly located in dorsal root ganglia. Afferent SP/tachykinin fibers are mainly confined to a perivascular location and to the submocosa in the gut, but are distributed also to the hepatobiliary pathway and pancreas. On the contrary, the extrinsic CGRP-containing afferents form a major component of the sensory innervation of the alimentary tract, including the rich CGRP innervation of the esophagus, stomach, hepatobiliary tract, pancreas, and vasculature, as well as a portion of non-vascular fibers distributed to the intestinal wall. Tachykinin and CGRP immunoreactivities appear to be colocalized in a population of nerve fibers, which are likely to be extrinsic, afferent, since colocalization of these peptide immunoreactivities has not been reported in intrinsic neurons. The presence of SP/NKA-encoding transcripts in the enteric nervous system and sensory ganglia and the lack of hybridization signal with RNA probes complementary to NKB mRNA indicate that the PPT I gene, but not the PPT II gene, is transcribed in these structures. This observation, along with receptor binding sites and radioimmunoassay data, which have failed to detect NKB receptor binding sites or immunoreactivity (Eysselein et al., 1990; Maggio, 1988; Mantyh et al., 1988; 1989) in the intestine of several mammals, is consistent with a differential expression of the two PPT genes in the periphery and in the central nervous system (Brecha et al., 1989; Warden and Young, 1988). A differential expression of the tachykinin-encoding genes, the existence of multiple tachykinin receptor subtypes (Mantyh et al., 1988; 1989), and the findings that tachykinins can be differentiated on the basis of the potency of their activities (Galligan et al., 1987; Maggio, 1988), support the possibility that each tachykinin is expressed in separate, and perhaps functionally distinct neuronal systems. alpha- and beta-CGRP genes also are differentially expressed according to the neuronal populations: alpha-CGRP mRNA is the most prominent form in sensory ganglia, and beta-CGRP mRNA is the only form detected in enteric neurons (Mulderry et al., 1988; Sternini and Anderson, 1990). In addition, distinct distributions of mRNAs generated from the two CGRP genes have been reported in the central nervous system (Amara et al., 1985). The differential expression patterns of alpha- and beta-CGRP mRNAs are consistent with a differential regulation of the alpha- and beta-CGRP genes.(ABSTRACT TRUNCATED AT 400 WORDS)

The concentrations of tachykinins in rat spinal cord and dorsal root ganglia (DRGs) were measured using a combination of high performance liquid chromatography (HPLC) and radioimmunoassays (RIAs). Substance P-like immunoreactivity (SPLI) was found to be significantly higher than either substance K-like immunoreactivity (SKLI) or neuromedin K-like immunoreactivity (NMKLI) in both tissues. In the spinal cord, the concentration of SKLI was comparable to that of NMKLI. In DRGs, NMKLI is present at concentrations much lower than those of SKLI or SPLI. In addition to immunoreactive components co-eluting with the three mammalian tachykinins SP, SK and NMK, analyses using reverse-phase HPLC revealed an immunoreactive peak co-eluting with the C-terminal octapeptide of SK (SK3-10), and a yet to be identified peak eluting before SK. This study also demonstrates the use of a novel and highly specific RIA for NMK to measure NMKLI without the need of reverse-phase HPLC.

The origin of tachykinin- and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibres in the guinea pig carotid body and carotid sinus was determined by retrograde labelling of the carotid sinus nerve with Fluoro-gold and immunohistochemical double staining with fluorescein- and rhodamine-conjugated second antisera. Fluoro-gold-labelled perikarya with characteristic features of primary sensory neurones were numerous in the glossopharyngeal (petrosal) ganglion and occurred rarely in the closely attached superior vagal (jugular) ganglion. An efferent pathway from the brainstem could not be detected. Co-existence of tachykinin- and CGRP-LI was observed in 25-47% of labelled sensory neurones; less than 1% of Fluoro-gold-containing perikarya were exclusively stained by CGRP antiserum. Co-existence of tachykinin- and CGRP-LI was also demonstrated in nerve fibres of the carotid body and carotid sinus. Somatostatin-, cholecystokinin- and dynorphin-LI did not co-exist with tachykinin-LI in these fibres. Thus, tachykinin/CGRP-LI fibres in the carotid presso- and chemoreceptive areas exhibit a peptide pattern being generally characteristic for sensory fibres supplying great vessels in the guinea pig. In view of the present findings doubt is raised as to a primary involvement of these fibres in presso- or chemoreception, although a modulatory influence on these specific functions appears to be likely.

The N-terminal hexa- or pentapeptide sequences of the three mammalian tachykinins substance P, neurokinin A, and neurokinin B have been synthesized by the conventional solid-phase procedure with 6-aminocaproyl-S-(acetamidomethyl)cysteine as a C-terminal spacer and attachment function. A fourth sequence, with an additional N-terminal 6-aminocaproyl residue on the substance P-hapten sequence, was cyclized N- to C-terminally. For this purpose, a four-level protection scheme has been applied: BOC-TFA for N-terminal protection and cleavage; TFA-stable but HF-labile anchoring function and side-chain protection; S-acetamidomethyl for semipermanent thiol protection. The side chain amino function of Lys was protected with NO2Z, stable against HF but readily cleaved with hydrogenation. The hapten sequences were coupled to maleimidated BSA, after the Acm group was removed by mercury/hydrogen sulfide treatment. Mice immunized with the three linear hapten sequences produced sera that were specific in enzyme-linked immunosorbant assay for the presented hapten and the respective tachykinin but displayed no crossreactivity at all toward the other haptens nor to one of the other tachykinins. It is concluded that this approach produced antisera, specific and selective for its respective mammalian tachykinins.

The mutilated foot rat is a mutant with autosomal recessive sensory neuropathy and frequent mutilation of the hindlimbs. Decreased numbers of dorsal root ganglion cells and diminished sensitivity to painful stimuli are characteristics of these animals. By use of immunocytochemistry, changes in the distributions of peptides involved in sensory and/or autonomic regulation, i.e., calcitonin gene-related peptide (CGRP), tachykinins, enkephalin and neuropeptide Y in spinal cord, dorsal root ganglia and skin of these animals, were studied. In comparison with normal litter-mate controls, the dorsal horn of mutilated foot rats contained substantially fewer CGRP- and tachykinin-immunoreactive fibres but more fibres immunoreactive for enkephalin. Many enkephalin-immunoreactive cell bodies were also found in the dorsal horn of the mutants, by contrast none were visible in control animals. Neuropeptide Y immunoreactivity was, however, unchanged in the spinal cord of the mutants. In the dorsal root ganglia of the mutants, the number of CGRP- or tachykinin-immunoreactive cells and their proportion to total neuronal numbers were significantly less in comparison with normal controls. The diameter range of CGRP- and tachykinin-immunoreactive cells shifted from small (15-25 microns) to medium size (25-45 microns) as revealed by frequency distribution histograms. The skin from the affected fore- and hindlimbs of the mutant rats, in keeping with fewer CGRP- and tachykinin-immunoreactive cells in the dorsal root ganglia, contained substantially less fibres immunoreactive for CGRP and tachykinins; a difference that was not seen in skin of unaffected areas (whiskers and snout). By contrast, neuropeptide Y-immunoreactive fibres showed a normal distribution around blood vessels and sweat glands of mutilated foot rats. The data suggest that diminished pain perception in the mutilated foot rat is related to loss of peptide-containing sensory neurones. Furthermore, the intraspinal increase of enkephalinergic neurons in the dorsal horn, concomitant with the decreased number of primary sensory neurones, may also play a contributory rôle in reducing pain thresholds.

The aim of the present study was to evaluate the possible presence of immunoreactive neuropeptide-gamma (irNPY) in human placenta. Acidic extracts of human placental tissue collected at term pregnancy contained high irNPY concentrations. The extracted irNPY eluted from HPLC with the same retention time as synthetic NPY. The presence of the peptide in placental cells was confirmed by immunohistochemical findings showing numerous cells of the cytotrophoblast layer positively staining for NPY. Further supporting local production of the peptide, primary cultures of human placental cells released irNPY into the culture medium and the addition of high K+ concentrations increased the release of the peptide. The finding of irNPY in human placenta stimulated the characterization of binding sites of NPY in the same tissue. Using autoradiographic techniques we showed specific binding of [125I]NPY in human placental tissue. The binding of [125I]NPY to the placental receptors was saturable and widely distributed within the placental tissue. Finally, the addition of NPY to the medium of cultured placental cells increased the release of immunoreactive CRF, suggesting a possible role of NPY in placental hormone production. The effect of NPY was dose related and augmented by the addition of norepinephrine (10 nM). These results showed that human placenta produces and secretes irNPY and that NPY receptors are present in placental tissue. Moreover, the evidence that NPY stimulated the release of immunoreactive CRF from cultured placental cells suggests an action of NPY in placental hormonogenesis.

Permeability changes in the guinea-pig skin following intradermal (i.d.) injection of tachykinin agonists or antigen were monitored through the extravasation of 99mTc-labelled human serum albumin and blood flow changes through the accumulation of 51Cr-labelled microspheres. A variety of synthetic and natural tachykinins, including substance P and neurokinins A and B, were shown to be potent inducers of permeability changes. Neurokinins A and B, but not substance P, were also shown to be apparent vasoconstrictor agents. Permeability responses in sensitized guinea pigs to i.d. injection of antigen and substance P, but not histamine, were abolished by pretreatment with the tachykinin antagonists [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-substance P and [D-Pro2, D-Trp7,9]-substance P. Interpretation of such results was complicated by the fact that such antagonists may in themselves induce mast cell activation. Depletion of substance P containing neurons by pretreatment of guinea pigs with capsaicin also produced significant inhibition of antigen-induced permeability changes. These results indicate a possible role for tachykinins, such as substance P, in cutaneous anaphylaxis in the guinea pig.