Characterization of neuropeptide K processing in rat spinal cord S9 fractions using high-resolution quadrupole-Orbitrap mass spectrometry.

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord, leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA was very short, resulting in half-lives of 1.9 and 2.2 min respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK1-26 , NKA, NKA2-10 , NKA3-10 , NKA5-10 and NKA6-10 , which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA, providing strong evidence that proprotein convertase is involved in C-terminal processing of NPK in the spinal cord, leading to the formation of NKA.

An Analysis of Neuropeptides at Nasal Contact Points of Patients With Secondary Headache.

OBJECTIVES: This prospective research study was designed to analyze the surgical outcomes and the intensity of substance P (SP), neurokinin A (NA), and calcitonin gene-related peptide (CGRP) in contact and noncontact nasal mucosa of patients with headache. METHODS: Twenty adults with secondary headache and correctible nasal obstruction were included in this study. The patients had nasal contact points between the nasal septum and the middle or inferior turbinates on nasal endoscopy and computed tomography scan. During surgical procedures, sample tissues were obtained from the nasal contact point and the noncontact area of the lateral nasal wall of these patients. Fluorescein staining intensity for antibodies against SP, NA, and CGRP was analyzed using image J software. Headaches were evaluated using a visual analog scale preoperatively and postoperatively. RESULTS: The differences between the preoperative and the postoperative 3rd month (P < 0.001) and 12th month (P < 0.001) visual analog scale scores were statistically significant. However, fluorescein staining intensity for SP (P = 0.631), NA (P = 0.546), and CGRP (P = 0.683) did not show statistically significant differences between the contact mucosa and the noncontact mucosa groups. CONCLUSIONS: Although in selected patients significant relief of headache can be obtained by surgery, there is no evidence from this study that SP, NA, and CGRP are responsible for the initiation of headache.

Anti-inflammatory properties of montelukast, a leukotriene receptor antagonist in patients with asthma and nasal polyposis.

BACKGROUND: Leukotrienes, especially LTC4, are important inflammatory mediators in allergic and nonallergic inflammation of the entire airways. Of particular interest are numerous theories regarding the pathogenesis of aspirin intolerance with subsequent hyperproduction of leukotrienes and inhibition of cyclooxygenase. OBJECTIVE: To examine the influence of the cysteinyl-leukotriene receptor antagonist montelukast on clinical symptoms and inflammatory markers in nasal lavage fluid in patients with bronchial asthma and nasal polyps, and determine its dependency on aspirin sensitization. METHODS: Twenty-four patients (7 women, 17 men; median age, 55.5 years) with nasal polyps and controlled asthma (n=12 with aspirin intolerance) were treated with 10 mg montelukast once daily for 6 weeks in a blinded, placebo-controlled fashion. The placebo phase was randomly assigned 4 weeks before (n=12) or after treatment (n=12). Symptom score, rhinoendoscopy, rhinomanometry, smears for eosinophils, and nasal lavages for the determination of different mediators were performed. RESULTS: Compared to placebo, there were significant improvements in the nasal symptom score and airflow limitation as well as a reduction in the inflammatory mediators in nasal lavage fluid after treatment. Furthermore, reduced eosinophils in nasal smears and peripheral blood were observed 2 and 6 weeks after treatment. CONCLUSION: Leukotriene 1 receptor blockade led to a significant decrease in eosinophil inflammation accompanied by a reduction in other mediators such as neurokinin A and substance P in the nasal lavage fluid of patients with nasal polyps and asthma, with or without aspirin intolerance.

Salivary and serum levels of substance P, neurokinin A and calcitonin gene related peptide in burning mouth syndrome.

BACKGROUND: Burning mouth syndrome (BMS) is an enigmatic condition with the aetiopathogenesis remaining largely obscure. However, a neuropathic basis for BMS continues to be an area of active clinical and research interest. AIM: It is becoming increasingly evident that certain oral disorders may be modulated by imbalances in certain neuropeptides such as substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) therefore we measured SP, NKA and CGRP in the saliva and sera of BMS patients as well as controls. SUBJECTS AND METHODS: Salivary and serum SP, NKA and CGRP were determined in the 26 female patients with burning mouth syndrome (age range 51-78, mean 65.69 yrs), and in the 22 female controls (age range 24-82, mean 49.72 yrs). Serum and salivary SP, NKA, CGRP levels were determined by commercial competitive enzyme immunoassay kits. Statistical analysis was performed by use of descriptive statistics and analysis of variance. RESULTS AND CONCLUSIONS: No significant differences in salivary SP, NKA and CGRP as well as serum SP and CGRP between BMS patients and controls could be found. However, significantly decreased serum neurokinin A (p<0.05) in BMS patients may reflect an inefficient dopaminergic system.

Comparison of Neurokinin A, Substance P, Interleukin 8, and Matrix Metalloproteinase-8 Changes in Pulp tissue and Gingival Crevicular Fluid Samples of Healthy and Symptomatic Irreversible Pulpitis Teeth.

INTRODUTION: The aim of this study was to compare levels of neurokinin A (NKA), substance P (SP), interleukin (IL)-8, and matrix metalloproteinase-8 (MMP-8) in pulp tissue and gingival crevicular fluid (GCF) samples of healthy and symptomatic irreversible pulpitis teeth. METHODS: Forty patients diagnosed with healthy and symptomatic irreversible pulpitis teeth were included in this study. NKA, SP, IL-8, and MMP-8 levels were measured using the enzyme-linked immunosorbent assay test after pulp and GCF samples were obtained from healthy (n = 20) and symptomatic irreversible pulpitis teeth (n = 20). GCF sampling of 40 teeth was repeated 1 week later. Routine root canal treatment procedures of the teeth were performed, and the treatment process was completed. As a control group, GCF samples were taken from the contralateral teeth in both groups. Statistical analysis was performed using dependent and independent t tests, analysis of variance, Kruskal-Wallis, Mann-Whitney U tests, and Pearson correlation analysis. RESULTS: Comparing the groups, all mediator levels were significantly higher in the pulp samples in the pulpitis group compared with the healthy group (NKA: P < .001, SP: P = .005, IL-8: P < .001, and MMP-8: P < .001). Likewise, in the pulpitis group, all mediator levels were significantly higher in the first GCF samples compared with the healthy group (NKA: P = .01, SP: P < .001, IL-8: P = .001, and MMP-8: P < .001). CONCLUSIONS: It was observed that NKA, SP, IL-8, and MMP-8 increased significantly in pulp tissue and GCF specimens of symptomatic irreversible pulpitis teeth compared with pulp tissue and GCF specimens of healthy teeth. Second, it was determined that NKA, SP, IL-8, and MMP-8 levels decreased significantly in GCF samples in teeth diagnosed with symptomatic irreversible pulpitis 1 week after the removal of inflamed pulp. Finally, SP, IL-8, and MMP-8 levels were found to be higher in pulp tissue samples of the patients with symptomatic irreversible pulpitis with higher pain scores than those with low pain scores.

Sputum neurokinin A in Egyptian asthmatic children and adolescents: relation to exacerbation severity.

BACKGROUND: Neurogenic inflammation may participate in the development and progression of bronchial asthma. The molecular mechanisms underlying neurogenic inflammation are orchestrated by a large number of neuropeptides including tachykinins such as neurokinin A (NKA) and substance P. Tachykinins are secreted from sensory airway nerves and inflammatory cells after allergens exposure. In clinical practice, assessment of airway inflammation is difficult. Therefore, detection of biological markers of airway inflammation in sputum might offer help for proper monitoring of asthma severity. AIM OF THE STUDY: We aimed to measure sputum NKA in relation to acute asthma exacerbations of varying severity. METHODS: Sputum NKA was measured by enzyme-linked immunosorbent assay in 24 children and adolescents during and after acute asthma exacerbation and 24 healthy matched controls. RESULTS: Sputum NKA was significantly higher in asthmatic patients during acute exacerbation than controls [217.5 (284) vs 10 (7) ng/ml, P < 0.001]. When patients with acute asthma exacerbation were followed-up till remission, sputum NKA levels decreased significantly, but they remained significantly higher than controls. Sputum NKA levels were significantly higher in severe than moderate and in moderate than mild exacerbations, and was negatively correlated to peak expiratory flow rate (r = -0.9, P < 0.001). Sputum NKA had significant positive correlations to eosinophil counts in blood and sputum (r = 0.6, P < 0.001 and r = 0.7, P < 0.001 respectively). CONCLUSIONS: Sputum NKA is up-regulated during acute asthma exacerbation and it positively correlates to its severity. Thus, NKA may aid in objective classification of the exacerbation severity. In addition, NKA may be a target for new asthma therapy.

A novel, conformation-specific allosteric inhibitor of the tachykinin NK2 receptor (NK2R) with functionally selective properties.

The orthosteric agonist neurokinin A (NKA) interacts with the tachykinin NK2 receptors (NK2Rs) via an apparent sequential binding process, which stabilizes the receptor in at least two different active conformations (A1L and A2L). The A1L conformation exhibits fast NKA dissociation kinetics and triggers intracellular calcium elevation; the A2L conformation exhibits slow NKA dissociation kinetics and triggers cAMP production. The new compound LPI805 is a partial and noncompetitive inhibitor of NKA binding to NK2Rs. Analysis of NKA dissociation in the presence of LPI805 suggests that LPI805 decreases the number of NKA-NK2R complexes in A2L conformation while increasing those in the A1L conformation. Analysis of signaling pathways of NK2Rs shows that LPI805 dramatically inhibits the NKA-induced cAMP response while slightly enhancing the NKA-induced calcium response. Analysis of NKA association kinetics reveals that LPI805 promotes strong and specific destabilization of the NKA-NK2R complexes in the A2L conformation whereas access of NKA to the A1L conformations is unchanged. Thus, to our knowledge, LPI805 is the first example of a conformation-specific allosteric antagonist of a G-protein-coupled receptor. This work establishes the use of allosteric modulators in order to promote functional selectivity on certain agonist-receptor interactions.

Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation.

Substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) have potent proinflammatory effects in the airways. They are released from sensory nerve endings originating in jugular and dorsal root ganglia. However, the major sensory supply to the airways originates from the nodose ganglion. In this study, we evaluated changes in neuropeptide biosynthesis in the sensory airway innervation of ovalbumin-sensitized and -challenged guinea pigs at the mRNA and peptide level. In the airways, a three- to fourfold increase of SP, NKA, and CGRP, was seen 24 h following allergen challenge. Whereas no evidence of local tachykinin biosynthesis was found 12 h after challenge, increased levels of preprotachykinin (PPT)-A mRNA (encoding SP and NKA) were found in nodose ganglia. Quantitative in situ hybridization indicated that this increase could be accounted for by de novo induction of PPT-A mRNA in nodose ganglion neurons. Quantitative immunohistochemistry showed that 24 h after challenge, the number of tachykinin-immunoreactive nodose ganglion neurons had increased by 25%. Their projection to the airways was shown. Changes in other sensory ganglia innervating the airways were not evident. These findings suggest that an induction of sensory neuropeptides in nodose ganglion neurons is crucially involved in the increase of airway hyperreactivity in the late response to allergen challenge.

Distribution of neuroendocrine cells in the small and large intestines of the one-humped camel (Camelus dromedarius).

The distribution and relative frequency of neuroendocrine cells in the small and large intestines of one-humped camel were studied using antisera against 5-hydroxytryptamine (5-HT), cholecystokinin (CCK-8), somatostatin (SOM), peptide tyrosine tyrosine (PYY), gastric inhibitory polypeptide (GIP), neuronal nitric oxide synthase (nNOS), gastrin releasing peptide (GRP), substance P (SP), and neurokinin A (NKA). Among these cell types, CCK-8 immunoreactive (IR) cells were uniformly distributed in the mucosa, while others showed varied distribution in the villi or crypts of the small intestine. Immunoreactive cells like 5HT, CCK-8, and SOM showed peak density in the villi and crypts of the small intestine and in the colonic glands of the large intestine, while cells containing SP were discerned predominately in the crypts. 5-HT, CCK-8 and SOM cells were mainly flask-shaped and of the open-variety, while PYY and SP immunoreactive cells were mainly rounded or basket-shaped and of the closed variety. Basically the distribution pattern of the endocrine cells in the duodenum, jejunum and colon of the one-humped camel is similar to that of other mammals. Finally, the distribution of these bioactive agents may give clues as to how these agents aid in the function of the intestinal tract of this desert animal.

Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata.

OBJECTIVE: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium. DESIGN: Laboratory study. SETTING: University research laboratories and academic hospital. PATIENT(S): Women undergoing hysterectomy for symptomatic leiomyomas. INTERVENTION(S): Quantitative polymerase chain reaction, immunohistochemistry and Western blot. MAIN OUTCOME MEASURE(S): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium. RESULT(S): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells. CONCLUSION(S): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors.

Tachykinin neuropeptides in cerebellar granule neurons: an immunocytochemical study.

We previously demonstrated that exogenously administered neurokinin A and neurokinin B, but not substance P, increased the sensitivity of cultured cerebellar granule neurons (CGNs) to glutamate. In the present study, the presence of tachykinin neuropeptides in CGNs was tested by confocal-based immunofluorescence. We found that neurokinin A and neurokinin B are present in CGNs but absent in astrocytes while substance P is abundant in astrocytes but absent in CGNs. It is postulated that the different localization of tachykinin neuropeptides in CGNs and astroglial cells has a physiological role in the modulation of excitatory transmission.

[Effects of Suspending Moxibustion at "Dazhui" (GV 14) on Neurogenic Inflammation in Asthma Rats].

OBJECTIVE: To observe the effect of suspending-moxibustion stimulation of "Dazhui" (GV 14) with different quantities on the levels of nerve growth factor(NGF) , substance P(SP) , calcitonin gene-related peptide (CGRP), neurokinin A (NKA) , neurokinin B (NKB) and phosphorylated extracellular signal-regulated kinases (pERK) in the bronchoalveolar lavage fluid (BALF) of asthma rats, so as to analyze its mechanisms underlying improving asthma. METHODS: Sixty male SD rats were randomly divided into six groups: blank control, model, 15 min-moxibustion (15 min-moxi), 30 min-moxi, 60 min-moxi and 90 min-moxi (n = 10 rats in each group). The asthma model was established by intraperitoneal injection of suspension of egg protein, magaldrate, and inactivated Bacillus pertussis (on day 1 and 8), and inhaling the atomized ovalbumin saline (from day 15 on for 14 days). Mild moxibustion was conducted at "Dazhui" (GV 14) for 15 min, 30 min, 60 min and 90 min, respectively, once daily for 7 days. The levels of NGF, SP, CGRP, NKA, NKB, and pERK in the BALF were detected by ELISA (enzyme-linked immuno sorbent assay). RESULTS: The contents of NGF, SP, CGRP, NKA, NKB and pERK in the BALF in the model group were obviously higher than those in the blank control group (P < 0.01), suggesting an apparent inflammatory reaction in rats after modeling. Following moxibustion, the levels of NGF, SP, CGRP, NKA, NKB and pERK of the four treatment groups were significantly down-regulated compared with the model group (P < 0.01). The effect of 30 min-moxi group was obviously superior to that of 15 min-moxi group (P < 0.01), and those of 60 min-moxi and 90 min-moxi groups were markedly superior to those of 15 min-moxi and 30 min-moxi groups (P < 0.01) in down-regulating NGF, SP, CGRP, NKA, NKB, and pERK levels in the BALF. No significant differences were found between the 60 min-moxi and 90 min-moxi groups in down-regulating NGF, SP, CGRP, NKA, NKB, and pERK levels (P > 0.05). CONCLUSION: Suspending-moxibustion stimulation of GV 14 can down-regulate the contents of NGF, SP, CGRP, NKA, NKB, and pERK levels in the BALF in asthma rats, suggesting a relief of neurogenic inflammation reaction after moxibustion. The effect of moxibustion presents a time-dependant manner and peaks at 60 min.

Immunohistochemical distribution, biochemical characterization, and biological action of tachykinins in the frog adrenal gland.

The distribution of tachykinin-like immunoreactivity (LI) was studied in the adrenal gland of the frog Rana ridibunda using the immunofluorescence technique. A dense network of varicose fibers immunoreactive to both substance-P (SP) and neurokinin-A (NKA) was found in the adrenal tissue. In contrast, no positive fibers could be detected using antineurokinin-B (NKB) antibodies. At the electron microscope level, the immunogold technique revealed that tachykinin-LI was sequestered in dense core vesicles of 50-70 nm. Bilateral transection of either splanchnic or vagus nerves or total lesion of celiac sympathetic ganglion did not suppress tachykinin-LI. A combination of HPLC analysis and RIA detection was used to characterize tachykinin-LI in frog adrenal extracts. Two major peaks were resolved, which coeluted, respectively, with synthetic ranakinin, a novel tachykinin previously isolated from the frog brain, and [Leu3,Ile7]NKA previously isolated from the frog gut. No NKB could be detected in the extracts. The effects of various synthetic tachykinins on corticosteroid secretion were studied using perifused frog adrenal slices. For concentrations ranging from 10(-8)-10(-4) M, SP induced a dose-dependent stimulation of corticosterone and aldosterone release. A desensitization phenomenon was observed when iterative or prolonged infusions of SP were administered to the tissue. All mammalian or amphibian tachykinin-related peptides tested in our model also enhanced corticosteroid production. The effectiveness of the tachykinins tested was: [Pro7] NKB > NKA > ranakinin > [Pro9]SP > SP > kassinin > physalaemin > NKB > [Leu3,Ile7]NKA. SP also enhanced prostaglandin E2 and prostacyclin release in the effluent perifusate and the response preceded by 10-15 min the increase in corticosteroid output. Indomethacin (5 x 10(-6) M), a specific blocker of cyclooxygenase activity, totally suppressed SP-evoked steroid secretion. These data indicate that tachykinin-induced stimulation of steroidogenesis was mediated through activation of the arachidonic acid cascade. Taken together, our results show that the frog adrenal gland is innervated by a dense network of peptidergic fibers containing both ranakinin and [Leu3,Ile7]NKA, which, in vitro, stimulates corticosteroid secretion by adrenocortical cells through a prostaglandin-dependent mechanism. The present results support the view that tachykinins released by nerve fibers exert a neuroendocrine control on corticosteroid release in amphibians.

Immunohistochemical study of neuropeptides in vagal and glossopharyngeal afferent neurons in the rat.

The presence and distribution of multiple neuropeptides in vagal and glossopharyngeal afferent ganglia of the rat were studied using immunohistochemistry. Substance P-, calcitonin-gene related peptide-, cholecystokinin-, neurokinin A-, vasoactive intestinal polypeptide-, and somatostatin-immunoreactive neurons were detected in each visceral afferent ganglion. Neurotensin-immunoreactive cells were not observed. In the nodose ganglion (inferior ganglion of the vagus nerve) occasional immunoreactive cells were scattered throughout the main (caudal) portion of the ganglion with small clusters of cells seen in the rostral portion. The pattern of distribution of the various peptides in the nodose ganglion was similar, with the exception of vasoactive intestinal polypeptide-immunoreactive neurons which exhibited a more caudal distribution. The relative numbers of immunoreactive cells varied, with the greatest numbers being immunoreactive for substance P or vasoactive intestinal polypeptide, and the lowest numbers being immunoreactive for neurokinin A and somatostatin. A build-up of immunoreactivity for each of the peptides, except somatostatin and neurotensin, was detected in vagal nerve fibers of colchicine-injected ganglia. Numerous peptide-immunoreactive cells were also found in the petrosal (inferior ganglion of the glossopharyngeal nerve) and jugular (superior ganglion of the vagus nerve) ganglia. No specific intraganglionic distribution was noted although the relative numbers of cells which were immunoreactive for the different peptides varied considerably. Substance P and calcitonin-gene related peptide were found in large numbers of cells, cholecystokinin was seen in moderate numbers of cells, and neurokinin A, vasoactive intestinal polypeptide and somatostatin were seen in fewer cells. These data provide evidence for the presence and non-uniform distribution of multiple peptide neurotransmitters in vagal and glossopharyngeal afferent neurons. In general, relatively greater numbers of immunoreactive cells were located in the rostral compared with caudal nodose ganglion, and in the petrosal and jugular ganglia compared with the nodose ganglion. Thus, multiple neuropeptides may be involved as afferent neurotransmitters in the reflexes mediated by vagal and glossopharyngeal sensory nerves.

The effect of selective serotonergic neurotoxin treatment on tachykinin levels in the rat ventral spinal cord.

The levels of 5-hydroxytryptamine and tachykinin neuropeptides substance P, neurokinin A, neurokinin B and neuropeptide K were measured in the spinal cord of rats treated by intraventricular injection of the selective serotonergic neurotoxin 5,7-dihydroxytryptamine. The spinal cord levels of 5-hydroxytryptamine as measured by high performance liquid chromatography with electrochemical detection decreased by more than 90% in the ventral and dorsal cord compared to controls. The levels of substance P as measured by radioimmunoassay were significantly reduced (66%, P less than 0.01) in the ventral lumbar cord only. In this region, neurokinin A, neurokinin B and neuropeptide K levels were determined by combined high performance liquid chromatography and radioimmunoassay. The neurotoxin treatment also caused a significant reduction of neurokinin A (72% reduction, P less than 0.01) and a non-significant reduction of neuropeptide K, but virtually no change in the neurokinin B level. Immunohistochemical studies of the ventral lumbar cord of sham-operated animals showed immunoreactivity for 5-hydroxytryptamine as well as for substance P and neurokinin A in nerve fibres around motor neurons. In neurotoxin-treated rats this region was devoid of immunohistochemically detectable substance P- and neurokinin A-positive fibres and showed very sparse or no 5-hydroxytryptamine immunoreactivity. We conclude that among the tachykinins both neurokinin A and substance P, but probably not neurokinin B, co-exist with 5-hydroxytryptamine in nerve terminals in the rat ventral spinal cord.

Distribution of neurokinin B in rat spinal cord and peripheral tissues: comparison with neurokinin A and substance P and effects of neonatal capsaicin treatment.

In the present study, highly specific radioimmunoassays were developed and used to measure neurokinin B, neurokinin A and substance P in the rat spinal cord and various peripheral tissues. The results are as follows. (1) Neurokinin B and neurokinin A were distributed all along the rostrocaudal axis of the spinal cord, as is substance P, and were more concentrated in the dorsal than in the ventral region. (2) Substance P was more abundant in the central and peripheral nervous tissues than neurokinin A, while in certain peripheral organs, neurokinin A was more abundant than substance P. In the spinal cord, neurokinin B concentrations were lower than those of the other two tachykinins. (3) In contrast to neurokinin A and substance P, neurokinin B was not detected in any of the peripheral tissues examined. (4) Capsaicin treatment reduced by half neurokinin A and substance P concentrations in the dorsal region of the spinal cord, the dorsal root ganglia and the sciatic nerve, but was without effect on neurokinin B concentrations in the spinal cord. Neurokinin A, like substance P, may therefore have an important function in the transmission of sensory information, particularly in nociceptive transmission from the periphery to the spinal cord and in peripheral neurogenic inflammation. In contrast, since neurokinin B was not found in the sensory neurons, it is not likely to have these functions, but may perhaps control them.

Mapping of neurokinin-like immunoreactivity in the human brainstem.

BACKGROUND: Using an indirect immunoperoxidase technique, we have studied the distribution of immunoreactive fibers and cell bodies containing neurokinin in the adult human brainstem with no prior history of neurological or psychiatric disease. RESULTS: Clusters of immunoreactive cell bodies and high densities of neurokinin-immunoreactive fibers were located in the periaqueductal gray, the dorsal motor nucleus of the vagus and in the reticular formation of the medulla, pons and mesencephalon. Moreover, immunoreactive cell bodies were found in the inferior colliculus, the raphe obscurus, the nucleus prepositus hypoglossi, and in the midline of the anterior medulla oblongata. In general, immunoreactive fibers containing neurokinin were observed throughout the whole brainstem. In addition to the nuclei mentioned above, the highest densities of such immunoreactive fibers were located in the spinal trigeminal nucleus, the lateral reticular nucleus, the nucleus of the solitary tract, the superior colliculus, the substantia nigra, the nucleus ambiguus, the gracile nucleus, the cuneate nucleus, the motor hypoglossal nucleus, the medial and superior vestibular nuclei, the nucleus prepositus hypoglossi and the interpeduncular nucleus. CONCLUSION: The widespread distribution of immunoreactive structures containing neurokinin in the human brainstem indicates that neurokinin might be involved in several physiological mechanisms, acting as a neurotransmitter and/or neuromodulator.

Tachykinins in the pineal gland: effect of castration and ganglionectomy.

In this investigation, the presence of NKA-immunoreactive substances was determined in pineal glands from intact, castrated and castrated, testosterone-treated male rats. The effect of environmental light, melatonin treatment and superior cervical ganglionectomy on pineal NKA-immunoreactive substances was also investigated. The results obtained show that NKA is present in measurable amounts in the rat pineal, and NPK is probably also present, Orchidectomy was followed by an increase in the content of NKA-immunoreactive substances in the pineal gland. The replacement treatment with testosterone propionate in castrated rats blocked this effect. NKA-immunoreactive substances were not significantly different quantitatively in pineals from rats killed under light or under darkness. The removal of the superior cervical ganglia was followed by a significant increase in the NKA-immunoreactive substance content in the pineal gland of male rats. These results indicate that NKA and other tachykinins are present in the pineal gland of the male rat, and they seem to be regulated by gonadal hormones and the innervation originated from the superior cervical ganglia.

Substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract.

Immunoreactive substance P and neurokinin A were measured with radioimmunoassay in extracts of different segments of porcine gastrointestinal tract using C-terminally directed antisera. In all segments, the concentrations of substance P and neurokinin A were similar. The largest concentrations of both peptides were found in the mid-colon. By gel chromatography and reversed-phase high pressure liquid chromatography the immunoreactivity in extracts from ileum eluted as homogenous peptides at the positions of synthetic substance P and neurokinin A, respectively. No neurokinin B was found. By immunohistochemistry of porcine duodenum, jejunum, ileum and mid-colon, identical localization patterns were found for substance P and neurokinin A, and the two peptides demonstrated by double immunofluorescence to be colocalized in the enteric nervous system of the ileum. We conclude that the tachykinins substance P and neurokinin A are codistributed and colocalized in the procine gastrointestinal tract and suggest that the two peptides are produced from a common precursor, beta- and/or gamma-preprotachykinin, in the same neurons.

The peptidergic innervation of the human superficial temporal artery: immunohistochemistry, ultrastructure, and vasomotility.

The peptidergic innervation of the human superficial temporal artery was investigated by means of immunohistochemical, ultrastructural, and in vitro pharmacological techniques. A dense network of nerve fibers was found in the adventitia. The majority of the nerve fibers displayed immunoreactivity for tyrosine hydroxylase and neuropeptide Y (NPY). A moderate supply of perivascular nerve fibers displayed either acetylcholinesterase activity or immunoreactivity for vasoactive intestinal peptide (VIP), peptide histidine methionine-27 (PHM), and calcitonin gene-related peptide (CGRP). Only a few nerve fibers displayed substance P (SP), neurokinin A (NKA), and neuropeptide K (NPK) immunoreactivity. In double immunostained preparations, SP immunoreactivity was co-localized with NPK and CGRP in the same nerve fibers. Ultrastructural studies revealed the presence of numerous axon variocosities at the adventitial--medial border. NPY, VIP, and CGRP immunoreactivities occurred in the same type of large granular vesicles, but in morphological distinct nerve profiles. NPY had, in general, no direct vasoconstrictor effect. However, at a low concentration of NPY contractile response induced by NA (10(-7)-10(-6)M) was 9-15 times enhanced. The NPY-induced potentiation of the NA-induced contraction was not dependent on the presence of an intact endothelium. No significant difference was found between acetylcholine, VIP, and PHM in either potency or degree of relaxation. SP, NKA, and CGRP also acted as vasodilatory agents, with CGRP being more potent than the tachykinins. The response to SP, but not CGRP, was dependent on an intact endothelium. Pretreatment of the vessels with a low concentration of NPY did not change the responses to ACh, VIP, SP, or CGRP.