Neurokinin B in a human pheochromocytoma measured with a specific radioimmunoassay

An N-terminally directed antiserum to neurokinin B was raised in rabbits using an immunogen prepared by coupling the free-SH group of neurokinin B extended from its C-terminus by a cysteine residue (NKB-Cys) to an -NH2 group on human serum albumin using a heterobifunctional cross-linking reagent. In radioimmunoassay with 125I-Bolton-Hunter-labelled NKB-Cys as tracer, the antiserum showed no cross-reactivity with other tachykinins. An extract of a human pheochromocytoma, previously shown to contain peptides derived from preprotachykinin A, contained NKB-LI (13 pmol/g wet weight). The retention time of tumor neurokinin on reversed-phase HPLC was the same as that of synthetic neurokinin B. Peptides with the retention times of substance P, neurokinin A, neurokinin A (3-10)-peptide and neuropeptide K were also identified in the tumor extract. NKB-LI was not detected in extracts of a further nine pheochromocytomas or in five carcinoid tumors that expressed the preprotachykinin A gene.     

Characterization of the C-terminal flanking peptide of human beta-preprotachykinin

The nucleotide sequence of cDNA encoding the common biosynthetic precursor of substance P, neurokinin A and neuropeptide K (beta-preprotachykinin) predicts that, in the human, the precursor contains a C-terminal flanking peptide of 19 amino acid residues [beta-preprotachykinin(111-129)-peptide]. Using an antiserum raised against synthetic human beta-preprotachykinin(117-126)-peptide in radioimmunoassay, we have demonstrated that an extract of a human neuroendocrine tumor of the adrenal medulla contained approximately equimolar concentrations of C-terminal preprotachykinin immunoreactivity (C-PPT-IR), substance P and neurokinin A. The C-terminal preprotachykinin flanking peptide was purified to homogeneity and its primary structure was determined. The amino acid sequence of the peptide, Ala-Leu-Asn-Ser-Val-Ala-Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, indicates identity with beta-preprotachykinin(111-126)-peptide. The data suggest that the C-terminal flanking peptide, like the tachykinins, is packed into secretory storage vesicles but the Arg127-Arg128-Arg129 residues in human beta-preprotachykinin are removed from the peptide by the action of endogenous processing enzyme(s).     

Measurement and partial characterization of the multiple forms of neurokinin A-like immunoreactivity in carcinoid tumours

An antiserum raised against neurokinin A has been used to demonstrate storage and release of neurokinin A-like immunoreactivity by carcinoid tumours. The antiserum showed reactivity towards members of the tachykinin family of polypeptides in the order: neurokinin A greater than eledoisin greater than neurokinin B greater than kassinin greater than substance P greater than physalaemin but the magnitude of the cross-reactivity with substance P and physalaemin was less than 1% of that of neurokinin A. A sensitive (IC50 238 fmol/ml; minimum detectable concentration, 9 fmol/ml) radioimmunoassay was set up using this antiserum. Extracts of metastatic tumour tissue from four patients with a primary carcinoid tumour in the midgut contained both neurokinin A-like immunoreactivity (NKA-LI) and substance P-like immunoreactivity (SP-LI). The concentrations (pmol/g wet weight) of NKA-LI and SP-LI in the tumours were: patient A 210, 201; patient B 2276, 6849; patient C 1198, 834 and patient D 424, 379. Analysis of the tumour extracts by reverse phase HPLC indicated that the NKA-LI was heterogeneous. Under two different conditions of chromatography, one component was eluted with the same retention time as neurokinin A. Two further components were more hydrophobic than neurokinin A but were not eluted with the retention time of neurokinin B. Analysis of these components by gel filtration indicated a molecular weight in the 3000-4000 range suggesting that they may be related to neuropeptide K, an N-terminally extended form of neurokinin A. NKA-LI and SP-LI were undetectable in the plasma of patients A and D but were elevated in patient B (NKA-LI 1005 +/- 114; SP-LI 345 +/- 85 fmol/ml) and patient C (NKA-LI 80 +/- 31; SP-LI 21 +/- 13 fmol/ml).     

Ranakinin: A Novel NK1 Tachykinin Receptor Agonist Isolated with Neurokinin B from the Brain of the Frog Rana ridibunda

An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P-like immunoreactivity, measured with an antiserum directed against the COOH-terminal region of mammalian substance P and neurokinin B-like immunoreactivity, measured with an antiserum directed against the NH2-terminus of neurokinin B. The primary structure of the substance P-related peptide (ranakinin) was established as: Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin B-related peptide was established as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. This amino acid sequence is the same as that of mammalian neurokinin B. Ranakinin was equipotent with substance P and [Sar9,Met(O2)11]substance P in inhibiting the binding of 125I-Bolton-Hunter-[Sar9,Met(O2)11]substance P, a selective radioligand for the NK1 receptor, to binding sites in rat submandibular gland membranes (IC50 1.6 +/- 0.3 nM; n = 5). It is concluded that ranakinin is a preferred agonist for the mammalian NK1 tachykinin receptor subtype.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Occurrence and effects of multiple tachykinins; substance P, neurokinin A and neuropeptide K in human lower airways

In the present work we have studied the occurrence of different tachykinins (substance P (SP), neurokinin A (NKA) and neuropeptide K (NPK)) in human distal bronchi and pulmonary arteries by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). We have also compared the biological effects of different tachykinins on isolated human bronchi and pulmonary arteries in vitro. The concentration of immunoreactive SP using antiserum SP2 in the pulmonary arteries was higher (1.34 +/- 0.15 pmol/g) than in the bronchi (0.56 +/- 0.05 pmol/g). The contents of other tachykinins than SP measured using antiserum K12 was on the other hand considerably higher in the bronchi (0.33 +/- 0.14 pmol/g) than in pulmonary arteries (0.13 +/- 0.02 pmol/g). Immunoreactive materials corresponding to SP, NKA and NPK were identified in bronchial extracts by RIA combined with HPLC, which also indicated the presence of an eledoisin (ELE)-like component. In vitro studies showed that NKA was the most potent of the tachykinins as a bronchoconstrictor agent, being several hundred-fold more active than SP, acetylcholine and histamine. NPK had an intermediate potency. The bronchoconstrictor effect of NKA was unaffected by atropine, mepyramine and cimetidine. The tachykinins SP and NKA had on the other hand, a rather equal potency in inducing relaxation of serotonin precontracted pulmonary arteries. In conclusion, multiple tachykinins are present in lower airways of man. These peptides exert different biological activities whereby NKA is a very active bronchoconstrictor agent compared to SP while both NKA and SP have rather similar relaxatory activities of vascular smooth muscle.     

Carassin: A Tachykinin That Is Structurally Related to Neuropeptide-γ from the Brain of the Goldfish

A 21-amino-acid residue tachykinin-related peptide, carassin, was isolated in pure form from an extract of the brain of the goldfish, Carrassius auratus, by reversed-phase HPLC. The primary structure of the peptide was established as the following: Ser-Pro-Ala-Asn-Ala-Gln-Ile-Thr-Arg-Lys-Arg-His-Lys-Ile-Asn- Ser-Phe-Val-Gly-Leu-Met.NH2. This amino acid sequence is the same length as and shows structural similarity (57% homology) to the mammalian tachykinin, neuropeptide-gamma, which is a product of the posttranslational processing of gamma-preprotachykinin. The mammalian tachykinins, substance P and neurokinin B, were not detected in the extract by using specific antisera directed against the NH2-termini of the peptides, but an antiserum directed against the COOH-terminal region of substance P did detect a low concentration of immunoreactive material.     

Projections and actions of tachykininergic, cholinergic, and serotonergic neurones in the intestine of the Atlantic cod

The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine. Received: 6 March 1997 / Accepted: 15 September 1997     

Tachykinin production in granulomas of murine schistosomiasis mansoni

Preprotachykinins, the products of one gene, are the precursor molecules of three mammalian tachykinins called substance P (SP), substance K (SK), and neuropeptide K. An additional mammalian tachykinin, neurokinin B, has also been described. SP and possibly other tachykinins may modulate immunologic responses. Granulomas that form around parasite ova in murine schistosomiasis were examined for tachykinins. Tachykinins were extracted from granulomas by boiling or with detergent. Extracts examined by RIA and HPLC contained only immunoreactive SP. Granulomas were dispersed with collagenase and cultured in vitro for up to 4 h. Only immunoreactive SP appeared in the culture medium. SP immunoreactivity localized solely to granuloma eosinophils as demonstrated by a sensitive immunohistochemical technique. An antiserum that recognized SK, neuropeptide K, and neurokinin B, but which possessed low reactivity to SP, also stained these cells. Only prior absorption of each antiserum with the appropriate synthetic neuropeptide would abrogate the immunostaining. This suggested that tachykinins other than SP were present within these cells. However, results of in situ hybridization experiments intimated that eosinophils produced predominantly preprotachykinin mRNAs which encode SP but are devoid of the SK/neuropeptide K sequence. It is concluded that granuloma eosinophils make predominantly SP in deference to other tachykinins, and that tachykinins other than SP are unlikely to be important in the regulation of the early granulomatous response of murine schistosomiasis.     

Isolation and characterization of neurokinin A, neurokinin A(3-10) and neurokinin A(4-10) from a neutral water extract of a metastatic ileal carcinoid tumour

A metastasis to the right liver lobe of an argyrophil/argentaffin midgut carcinoid tumour in a patient with the classical carcinoid syndrome was examined for the presence of tachykinins other than substance P, using a specific antiserum. The extract was initially purified using SepPak cartridges, and subsequently subjected to cation-exchange chromatography on SP Sephadex C-25 which separated the immunoreactive material into two main components (components I and II). Both were further purified by anion-exchange chromatography on DEAE-Sephadex A-25, and by reverse-phase fast protein liquid chromatography. Component II was identified as neurokinin A by its immunochemical and chromatographic properties and amino acid sequence analysis. Component I consisted of two molecular forms which were identified as neurokinin A(3-10) and neurokinin A(4-10) by amino acid sequence analysis. The tumour tissue contained only small amounts of the eledoisin-like peptide that has earlier been demonstrated in mammalian tissues. Although this component behaved like the nonmammalian peptide eledoisin on reverse-phase HPLC and on reverse-phase ion-pair chromatography, eledoisin-specific antiserum E2 indicated that eledoisin-like peptide is not identical to eledoisin. Neurokinin A in carcinoid tumours has an N-terminal heterogeneity; this multiplicity constitutes a further support for the hypothesis that carcinoid tumours produce a number of tachykinins which may be present in different relative amounts in individual patients and may contribute to the individual differences in symptomatology.     

Tachykinin immunoreactivity in the European common frog, Rana temporaria: localization, quantification and chromatographic characterization.

1. Tachykinin immunoreactivity has been localized, quantified and chromatographically-characterized in the brain, stomach, intestine and skin of Rana temporaria. 2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium. 3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain. 4. Chromatographic analysis of both frog tachykinins revealed that they possessed different physico-chemical properties than their mammalian counterparts.     

Neurokinin B- and substance P-like immunoreactivity are co-localized in enteric nerves of rat ileum

The tachykinins (TKs) substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) have conserved C-terminal sequences and mediate similar physiological responses by activating neurokinin receptors found on neural and smooth muscle cells. Many enteric nerves express preprotachykinin A (PPT A) mRNA and synthesize SP and NKA. However, it is unclear if NKB is synthesized in enteric neurons as many antibodies developed against NKB also recognize other TKs. Therefore, the cellular distribution of NKB-like-immunoreactivity (NKB-ir) in rat ileum was examined using selective antisera raised against either synthetic Cys10-NKB or peptide 2 (P2), a non-tachykinergic peptide sequence in NKB precursor protein. NKB-ir and P2-ir had a similar distribution in varicose nerve fibers in submucosal and myenteric ganglia and almost all ganglia contained immunoreactive nerves. Few submucosal or myenteric neuronal somata contained strong immunoreactivity. Preabsorption of NKB or P2 antisera with their respective cognate peptides, but not with other TK peptides, abolished specific immunostaining. Finally, co-localization of NKB-/P2-ir with SP-ir suggested that most NKB-/P2-ir nerve fibers contain SP-ir, but some SP-ir nerves do not contain detectable NKB-/P2-ir. These results indicate that PPT B products P2 and NKB are localized in a subpopulation of enteric nerves containing TKs encoded by PPT A. Stimulation of these nerves may release NKB to activate local neurokinin receptors.     

A novel radioimmunoassay for neuromedin K. I. Absence of neuromedin K-like immunoreactivity in guinea pig ileum and urinary bladder. II. Heterogeneity of tachykinins in guinea pig tissues

A novel and highly specific radioimmunoassay for the tachykinin peptide neuromedin K (NMK, also known as neurokinin beta, neurokinin B) has been developed and used to determine the distribution of this peptide in extracts of guinea pig tissues. In addition to immunoreactive components coeluting with the 3 mammalian tachykinins, substance P (SP), substance K (SK) and NMK, analyses using reverse-phase HPLC revealed immunoreactive peaks coeluting with the C-terminal octapeptide of SK (SK-(3-10], an N-terminally extended form of SK (gamma-preprotachykinin-(72-92)amide), and a yet unidentified peak eluting before NMK in the extracts of guinea pig brain and spinal cord. In contrast to the other tachykinins, SP and SK, which were present in high concentrations in extracts of all peripheral and central tissues examined, NMK-like immunoreactivity was detected only in extracts of central tissues. NMK-like immunoreactivity was not detected in extracts of terminal ileum and urinary bladder.     

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.     

Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P

Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse-phase HPLC. Using an antiserum specific for the C-terminal hexapeptide amide of substance P, levels of 3.43 +/- 0.33 ng g-1 and 12.45 +/- 0.76 ng g-1 (mean +/- SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross-reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (less than 0.1%) were 12.46 +/- 0.47 ng g-1 and 7.20 +/- 0.37 ng g-1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse-phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co-eluting with the respective sulphoxides was higher in acidified ethanol extracts, and substance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse-phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scyliorhinin I and II: two novel tachykinins from dogfish gut

Two peptides with tachykinin-like ability to contract longitudinal muscle from the guinea pig ileum were isolated from the intestine of the common dogfish, Scyliorhinus caniculus. The amino acid sequence of scyliorhinin I was established as Ala-Lys-Phe-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2 and this peptide cross-reacted with antisera directed against the C-terminal region fo substance P. The amino acid sequence of scyliorhinin II was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met- NH2 and this peptide cross-reacted with antisera directed against the C-terminal region of neurokinin A. The mammalian peptides substance P and neurokinin A were absent from the dogfish intestinal tissue.     

Establishment of highly specific radioimmunoassays for neurokinin A and neurokinin B and determination of tissue distribution of these peptides in rat central nervous system

Highly specific radioimmunoassays (RIAs) for neurokinin A (NKA) and neurokinin B (NKB) were developed. Antisera were produced by the procedure which involved immunization with NKA or NKB, both conjugated with keyhole limpet hemocyanin, and treatments with a tolerogenic conjugate of kassinin and a copolymer of D-glutamic acid and D-lysine (D-GL) to inhibit the production of cross-reactive antibodies against common C-terminal region of tachykinins. Cross-reactivities of anti-NKA antiserum (R704), thus produced, with NKB, kassinin, eledoisin were 12.6%, 10.6% and 11.5%, respectively. This was in sharp contrast with those of antiserum obtained from the rabbit not treated with kassinin-D-GL, these values corresponding to 129.0%, 42.5% and 94.4%, respectively. The cross-reactivities of R704 with substance P and physalaemin were 0.3% and 1.5%, respectively. This antiserum also bound 35.6% of neuropeptide K which contains NKA at its C-terminal. More importantly, anti-NKB antiserum (R707) obtained by the above tolerizing regimen was highly specific for NKB and the cross-reactivities with NKA, neuropeptide K, kassinin and other tachykinins were all less than 0.001%. RIAs using these specific antisera allowed us to measure directly NKA and NKB in tissue extracts without their fractionation by chromatography prior to RIAs. Measurements of immunoreactive NKA and NKB in different rat brain regions and spinal cord revealed that they are present with various ratios (NKA/NKB: 1.1-9.9) depending on the region.     

Distribution of neurokinin A-like and neurokinin B-like immunoreactivity in human peripheral tissues.

Using specific radioimmunoassay and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of the two peptides in human peripheral tissues were studied. Both NKA-like immunoreactivity (NKA-LI) and NKB-like immunoreactivity (NKB-LI) were present in the walls of the gut and gall bladder and in the pancreas. In the gut, the values for NKA-LI were 0.56-35.73 pmol/g wet weight, while those in pancreas and gall bladder were 0.64-0.68 and 0.36 pmol/g wet weight, respectively. The values of NKB-LI were 0.45-2.66 pmol/g wet weight in the gut, 0.93-1.65 pmol/g wet weight in the pancreas, and 0.30 pmol/g wet weight in the gall bladder. The immunocytochemical reactivity to both peptides was localized to ganglia of the submucosal and myenteric nerve plexuses in the gut wall, and to neurons in the muscle layer and mucosa of the gut wall. Weak but positive NKA-LI appeared in nerve cells of the pancreas, while NKB-LI was not detectable in the pancreas. Conversely, in the gall bladder wall, NKA-LI was undetectable while a very faint NKB-LI was found in the muscle layer. The localization of NKA corresponded closely to that of NKB in the tissues although the relative concentrations of the peptides varied from organ to organ.     

Growth-inhibitory properties of vasoactive intestinal polypeptide

It has recently been demonstrated that several neuropeptides can affect cell growth. The mammalian tachykinins substance P and neurokinin A, which are present in peripheral sensory neurons, stimulate growth of cultured connective tissue cells. Substance P-like immunoreactivity has been demonstrated in neuroblastoma cell lines. Neuroblastoma cells also produce other neuropeptides, among them vasoactive intestinal polypeptide (VIP). We report here that VIP is a potent inhibitor of serum-induced DNA synthesis in cultured smooth muscle cells (SMC), whereas no growth-inhibition was seen in SMC exposed to neurokinin A, calcitonin-gene related peptide, neuropeptide Y, somatostatin, or cholecystokinin. The growth-inhibitory effect of VIP was closely related to its ability to induce formation of cyclic AMP. Our results raise the possibility that peptides released by neurons, endocrine cells, as well as by transformed cells, may not only function as mitogens but also as inhibitory modulators of cell growth.     

Tachykinins and tachykinin receptors: a growing family

The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as excitatory neurotransmitters. Currently, the concept that tachykinins act exclusively as neuropeptides is being challenged, since the best known members of the family, substance P, neurokinin A and neurokinin B, are also present in non-neuronal cells and in non-innervated tissues. Moreover, the recently cloned mammalian tachykinins hemokinin-1 and endokinins are primarily expressed in non-neuronal cells, suggesting a widespread distribution and important role for these peptides as intercellular signaling molecules. The biological actions of tachykinins are mediated through three types of receptors denoted NK(1), NK(2) and NK(3) that belong to the family of G protein-coupled receptors. The identification of additional tachykinins has reopened the debate of whether more tachykinin receptors exist. In this review, we summarize the current knowledge of tachykinins and their receptors.