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).     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

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.     

Post-translational processing of preprotachykinins. Isolation of protachykinin-(1-37)-peptide from human adrenal-medullary phaeochromocytoma tissue.

The biosynthetic precursors of the mammalian tachykinins, alpha-, beta-and gamma-preprotachykinins, contain a common N-terminal region of 74 amino acids. A polyclonal antiserum was raised against a synthetic peptide representing N-tyrosylated beta-preprotachykinin-(48-56)-peptide as predicted from the nucleotide sequence of cloned DNA complementary to human beta-preprotachykinin mRNA. By using this antiserum in radioimmunoassay, a single immunoreactive peptide was identified in an extract of a human pheochromocytoma that produced substance P and neurokinin A. Partial microsequencing and determination of the amino acid composition of the peptide indicated identity with preprotachykinin-(20-56)-peptide. Thus the data demonstrate that the Ala19-Glu20 bond in preprotachykinin is the site of cleavage of the signal peptide.     

Biosynthesis of tachykinins (substance P, neurokinin A and neuropeptide K) in neurons of the guinea pig myenteric plexus

Incubation of the myenteric plexus-containing longitudinal muscle layer of guinea pig small intestine with [³⁵S]methionine for up to 9 h resulted in a progressive increase in the incorporation of radioactivity into newly synthesized substance P, neurokinin A and neuropeptide K. The radiolabelled peptides were isolated from tissue extracts by immunoprecipitation using regionally-specific antisera and purified by reverse-phase high performance liquid chromatography. Incorporation of radioactivity into the tachykinins was abolished by cycloheximide and was not observed when [³⁵S]cysteine was substituted for [³⁵S]methionine. The method may be used to study the regulation of biosynthesis and posttranslational modification of protachykinins in the gut.     

cDNA sequence of human beta-preprotachykinin, the common precursor to substance P and neurokinin A

The nucleotide sequence of cDNA encoding the human substance P precursor, beta-preprotachykinin (beta-PPT), has been determined. The source of mRNA was a human laryngeal carcinoid tumour that contained a high concentration of immunoreactive substance P. The human beta-PPT polypeptide is 129 amino acids long and contains regions encoding substance P and neurokinin A, each flanked by basic amino acid residues. Residues 72-107 of the human beta-PPT polypeptide encode the sequence of neuropeptide K, an N-terminally extended form of neurokinin A recently isolated from porcine brain.     

Neuropeptide K: a major tachykinin in plasma and tumor tissues from carcinoid patients

Evidence is presented for the presence of an entire family of tachykinin-immunoreactive peptides in plasma and tumor tissues from patients with carcinoid tumors. The peptides include in addition to substance P and neurokinin A; neurokinin B, an eledoisin like peptide and neuropeptide K--a 36 amino acid long tachykinin which contains neurokinin A at its C-terminus. Neuropeptide K seems to be the tachykinin which is present in highest concentrations in plasma as well as in acetic acid extracts of tumor tissues. It is highly biologically active, and may therefore contribute to the clinical symptoms of carcinoid tumors.     

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.     

Effect of kassinin, neurokinin A and neurokinin B on drinking behaviour in the pigeon

Intracerebroventricular (i.c.v.) injection of kassinin produced a prompt and copious drinking response at doses of 10-1000 ng/pigeon, in the absence of other behavioural alterations or of changes in core temperature. Neurokinin A and B evoked drinking, but they were respectively 10 and 100 times less potent than kassinin. Intraperitoneal injection of kassinin elicited drinking, but at doses about 1000 X larger than the i.c.v. ones. The angiotensin antagonist [Sar1, Leu8]angiotensin II did not reduce drinking induced by i.c.v. kassinin, suggesting that its effect is not due to interaction with the central renin-angiotensin system. Moreover, the effect is apparently independent of the mechanisms controlling hypovolaemic and hyperosmotic thirst since exact additivity was found in the dipsogenic response when i.c.v. kassinin was administered in the presence of a hypovolaemic (subcutaneous (s.c.), polyethylene glycol) or hyperosmotic (s.c. hypertonic NaCl) dipsogenic stimulus. The present findings show that kassinin, neurokinin A and B share with the tachykinins already tested (eledoisin, physalaemin, substance P) a common dipsogenic action in pigeons. However, marked differences exist in their dipsogenic potency. This order of potency, eledoisin = kassinin = physalaemin greater than neurokinin A = substance P greater than neurokinin B, is not consistent with the tachykinin receptor subtypes so far proposed.     

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.     

Antisera raised against eledoisin and kassinin detect elevated levels of immunoreactive material in plasma and tumor tissues from patients with carcinoid tumors

Radioimmunoassays based on antisera raised against the tachykinins eledoisin (antiserum E7) and kassinin (antiserum K12) were used to measure the concentration of tachykinin-like immunoreactivity (TKLI) in plasma from 52 healthy subjects. 65 patients with carcinoid tumors (of which 46 had symptoms of both flushing and diarrhoea), and 6 patients with endocrine pancreatic tumors. The antisera did not crossreact with substance P (SP). Elevated concentrations of TKLI, as compared with healthy subjects, were found in 75% of the carcinoid patients, but in none of the patients with pancreatic tumors. Tumor metastases from 8 of the carcinoid patients all contained TKLI. Ion-exchange chromatography of plasma samples and tumor tissue extracts indicated the presence of several immunoreactive molecular forms. The elution patterns of the immunoreactivity detected by antisera E7 and K12 were similar, indicating that the same molecular species are measured by these antisera. None of the components coeluted with synthetic SP. One of the immunoreactive components in carcinoid tumor extracts coeluted with synthetic NKA. The major immunoreactive components in plasma from the patients eluted in a position different from that of all currently known mammalian tachykinins. Tachykinin immunoreactive material detected in tumor tissue and plasma of patients with carcinoid tumor may play a role in the symptomatology of the carcinoid syndrome.     

Demonstration and Distribution of Kassinin-Like Material (Substance K) in the Rat Central Nervous System

Antiserum was raised against kassinin in rabbits. Cross-reactivity with other tachykinins was determined; these included substance K (100%) and substance P (0.1%). Peptides extracted from rat brain and synthetic tachykinins were chromatographed by reverse-phase HPLC. The major peak of kassinin-like material eluted at a time different from that of synthetic kassinin, eledoisin, physalaemin, neurokinin beta, and substance P but coeluted with substance K. Measurement of kassinin-like material in macrodissected and microdissected brain regions indicated that the distribution of kassinin-like material was similar to that of substance P.     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

Neurokinin A, neurokinin B and neuropeptide K in the rabbit iris: a study comparing different extraction methods

Neurogenic inflammation in the rabbit eye is thought to be partly mediated by tachykinins released from trigeminal sensory nerve fibres. In the present study we have investigated the occurrence of neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK) and related immunoreactive components in the rabbit iris-ciliary complex using neutral and different types of acidic media for extraction, reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). The immunoreactive material detected with an antiserum reacting almost equally well with NKA, NKB and NPK consisted mainly of NKA, and small amounts of NPK but almost no NKB. Acidic media seemed to be more effective than neutral media for extraction of NKA and NPK. Acid extraction yielded also an NKA-immunoreactive component which eluted immediately before NKA while neutral extracts, on the other hand, contained a component which appeared behind NKA, i.e. in the position of NKA-(3-10) and NKA-(4-10). The present results indicate that NKA but not NKB may play a role in neurogenic inflammation in the rabbit eye.     

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).     

Membrane-assisted molecular mechanism of neurokinin receptor subtype selection.

Based on the observed membrane structures of substance P, physalaemin, and eledoisin, preferred conformations, orientations and accumulations of 13 mammalian neurokinins and non-mammalian tachykinins were estimated and compared with pharmacologic and selective binding data taken from the literature. Principal site affinities and relative affinities supported the view that neurokinins bind to three principal mammalian sites: the NK-1 (preferring substance P), the NK-2 (preferring neurokinin A), and the NK-3 site (preferring neurokinin B). Strong hydrophobic membrane interaction of the C-terminal message segment as a perpendicularly oriented alpha-helical domain correlated with NK-1 selection. Electrostatic accumulation of the peptide at the anionic fixed charge layer of the membrane without hydrophobic interaction through a helix correlated with NK-2 preference. Electrostatic repulsion by the anionic fixed charge layer correlated with NK-3 selection. Thus, neurokinin receptor selection is guided by the same principles as opioid receptor selection. Membrane catalysis of specific agonist--receptor interactions may prove to be a quite general phenomenon, and the membrane structure of a peptide more important for its structure--activity relationship than its crystal structure or its mixture of conformers in solution or in vacuo.     

Ontogeny of the novel tachykinins neurokinin A, neurokinin B and neuropeptide K in the rat central nervous system

Neurokinin A, neurokinin B and neuropeptide K content has been measured in several regions of the rat central nervous system at different stages of postnatal development. For this, we have employed a combination of HPLC separation and radioimmunoassay detection using a neurokinin A antiserum which also recognizes neurokinin B and neuropeptide K. All 3 tachykinins were detectable during postnatal development in the various regions studied (hypothalamus, striatum, substantia nigra, cerebral cortex and spinal cord). Interestingly, a general increase in the tachykinin concentrations was observed during the second week of life. Some of these concentrations reached values on postnatal day 15 which far exceeded those observed in the adult. After day 15 most areas showed a slow decline in their tachykinin content until adult values were finally achieved. The developmental profiles obtained for these tachykinins are in good agreement with previous studies on the ontogeny of substance P and its receptors and support the view that tachykinins may play an important role in the organization and maturation of the developing central nervous system.     

Immunochemical characterisation of tachykinin immunoreactivity in the nervous system of the garden snail,Helix aspersa

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-tenninal structural characteristics similar to mammalian SP.     

Substance-P-related and neurokinin-A-related peptides from the brain of the cod and trout.

An extract of the brain of the rainbow trout, Oncorhynchus mykiss contained high concentrations of both neurokinin A-like immunoreactivity (corresponding to 90 pmol mammalian neurokinin A/g wet tissue) and substance-P-like immunoreactivity (corresponding to 50 pmol mammalian substance P/g wet tissue) measured by radioimmunoassay using antisera directed against the C-terminal regions of the mammalian peptides. In contrast, an extract of the Atlantic cod. Gadus morhua contained only neurokinin-A-like immunoreactivity (151 pmol/g). This apparent paradox was resolved by determination of the primary structures of the fish tachykinins. Trout substance P (Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-MetNH2) has the same amino acid sequence in its C-terminal region as that in the corresponding region of mammalian substance P. Cod substance P (Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-MetNH2), however, contains a substitution at position 8 (Phe----Ile) that abolishes reactivity with the antiserum to substance P but permits reactivity with the antiserum to neurokinin A. The amino acid sequence of cod and trout neurokinin A is the same (His-Lys-Ile-Asn-Ser-Phe-Val-Gly-Leu-MetNH2) and shows two substitutions (Thr3----Ile and Asp4----Asn) compared with mammalian neurokinin A. The data indicate that nervous tissue of teleost fish contain tachykinins that are analogous to the peptides found in mammalian tissues.     

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.