Virokinin,a bioactive peptide of the tachykinin family,is released from the fusion protein of bovine respiratory syncytial virus

Tachykinins, an evolutionary conserved family of peptide hormones in both invertebrates and vertebrates, are produced by neuronal cells as inactive preprotachykinins that are post-translationally processed into different neuropeptides such as substance P, neurokinin A, and neurokinin B. We show here that furin-mediated cleavage of the bovine respiratory syncytial virus fusion protein results in the release of a peptide that is converted into a biologically active tachykinin (virokinin) by additional post-translational modifications. An antibody directed to substance P cross-reacted with the C terminus of mature virokinin that contains a classical tachykinin motif. The cellular enzymes involved in the C-terminal maturation of virokinin were found to be present in many established cell lines. Virokinin is secreted by virus-infected cells and was found to act on the tachykinin receptor 1 (TACR1), leading to rapid desensitization of this G protein-coupled receptor as shown by TACR1-green fluorescent protein conjugate translocation from the cell surface to endosomes and by co-internalization of the receptor with beta-arrestin 1-green fluorescent protein conjugates. In vitro experiments with isolated circular muscle from guinea pig stomach indicated that virokinin is capable of inducing smooth muscle contraction by acting on the tachykinin receptor 3. Tachykinins and their cognate receptors are present in the mammalian respiratory tract, where they have potent effects on local inflammatory and immune processes. The viral tachykinin-like peptide represents a novel form of molecular mimicry, which may benefit the virus by affecting the host immune response.     

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord.

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.     

Identification and cDNA sequence of delta-preprotachykinin, a fourth splicing variant of the rat substance P precursor.

The neuropeptides substance P and neurokinin A are synthesised from a family of precursor polypeptides encoded by the preprotachykinin A (PPT) gene. In addition to a mRNA (beta-PPT) containing all 7 exons of the gene, alternatively spliced mRNAs lacking either exon 4 (gamma-PPT) or exon 6 (alpha-PPT) have been identified. We have determined the sequences of cDNA clones encoding four variants of PPT mRNA from rat dorsal root ganglion (DRG), including a novel mRNA species (delta-PPT) in which both exons 4 and 6 are absent. The sequence of delta-PPT predicts the existence of a novel tachykinin precursor polypeptide.     

Mapping of tachykinins in the cat spinal cord

Using an indirect immunoperoxidase technique, the location of cell bodies and fibers containing substance P, neurokinin A and neurokinin B was studied in the cat spinal cord. The former two neuropeptides showed a widespread distribution throughout the whole spinal cord, whereas the distribution of neurokinin B was more restricted. Neurokinin A-immunoreactive structures showed a more widespread distribution and a higher density than the immunoreactive structures observed to contain substance P. In the cat spinal cord, we observed cell bodies containing neurokinin A, but no cell bodies containing neurokinin B or substance P were found. These cell bodies were located in laminae V (sacral 1 and 2 levels), VI (sacral 1 and 3), VII (lumbar 7, sacral 1 and 3, caudal 1) and X (sacral 1). Laminae I and II showed the highest density of immunoreactive fibers for each of the three tachykinins studied, being in general lamina IV who showed the lowest number of immunoreactive fibers containing substance P, neurokinin A or B. The anatomical distribution of the three tachykinins studied in the cat spinal cord indicates that the neuropeptides could be involved in the neurotransmission and/or in the neuromodulation of nociceptive information, as well as in autonomic and affective responses to pain. Moreover, the involvement of substance P, neurokinin A or B in other functions unrelated to the transmission of pain is also possible (autonomic and motor functions). The distribution of the neuropeptides studied in the cat is compared with the location of the same neuropeptides in the spinal cord of other species. The possible origin of the tachykinergic fibers in the cat spinal cord is also discussed.     

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.     

Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.     

Neurokinin A and B.

The chemical features, pharmacological and biochemical characteristics of novel mammalian tachykinin peptides, neurokinin A and neurokinin B are described and are compared with those of substance P, a representative of tachykinin family.     

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.     

Neurokinin-immunoreactivity in human neuroblastomas. Evidence for selective expression of the preprotachykinin (PPT) II gene.

Factors regulating differentiation of the peripheral nervous system (PNS) have been widely studied in neuroblastomas which are tumors of the PNS. Five neuroblastomas were investigated for their content of tachykinin neuropeptides, which arise from two distinct genes which appear differentially expressed in the PNS. Radioimmunoassay and column chromatography revealed large amounts of neurokinin B in three of these tumors and the absence of substance P, neurokinin A, neuropeptide K and neuropeptide gamma from all five tumors. This suggests that neuroblastomas can selectively express the preprotachykinin (PPT) II gene and that they may be valuable for investigating the factors involved in the regulation of these two structurally-related neuropeptide genes.     

Effects of supraspinal administration of PG-SPI and PG-KII, two amphibian tachykinin peptides, on nociception in the rat

We investigated and compared the effects of two amphibian tachykinins, the NK1 receptor agonist PG-SPI and the NK3 receptor agonist PG-KII, and the mammalian tachykinins substance P, neurokinin A and neurokinin B on the reaction time to a painful radiant heat stimulus (tail-flick test in rats) after intracerebroventricular injection. PG-SPI (1, 10 and 20 microg) and PG-KII (1, 5 and 10 microg) significantly increased the reaction time. Substance P (10 microg) injected intracerebroventricularly induced antinociception, whereas neurokinin A and neurokinin B did not. Like analgesia evoked by exogenous substance P, PG-SPI-evoked analgesia was blocked by pretreatment with naloxone. Naloxone left PG-KII antinociception unchanged, but the NK3 receptor selective antagonist markedly reduced it. These findings suggest NK1 and NK3 tachykinin receptor system involvement in supraspinal analgesia in rats.     

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.     

Canine myenteric, deep muscular, and submucosal plexus preparations of purified nerve varicosities: content and chromatographic forms of certain neuropeptides

Partially purified nerve varicosities prepared from canine small intestinal myenteric, deep muscular and submucosal plexuses were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P, Leu-enkephalin, Met-enkephalin, vasoactive intestinal polypeptide (VIP) and neurokinin A, but did not contain detectable amounts of neurokinin B. In all three plexus preparations, VIP was present in the highest concentration. In contrast to other species, GRP and the enkephalins were found to be present in relatively high concentrations in the submucosal plexus and GRP was present in low concentrations in the deep muscular plexus. Equal concentrations of substance P and neurokinin A were found in the myenteric and deep muscular plexus preparations but greater concentrations of substance P relative to neurokinin A were found in the submucosal plexus preparations. On reverse phase HPLC, a major peak of immunoreactivity occurred at the retention times of standard preparations for all six neuropeptides measured. Significant heterogeneity was found for GRP- and VIP-like immunoreactivity, especially in the submucosal plexus preparations. These partially purified canine small intestine nerve varicosity preparations may prove of value in studying release mechanisms for, and the posttranslational processing of, neuropeptides.     

Differentiation of multiple neurokinin receptors in the guinea pig ileum

We have studied the selectivity and competitiveness of three neurokinin antagonists and atropine against substance P, neurokinin A, and neurokinin B. DPDTNLE-NB, [D-Pro2, D-Trp6,8, Nle10]-neurokinin B is a competitive antagonist of neurokinin B (pA2 = 5.5), but not substance P or neurokinin A. DPDT-SP ([D-Pro2,Trp7,9]-substance P), competitively blocks substance P (pA2 = 6.9) and neurokinin B (pA2 = 6.8), but not neurokinin A. Spantide ([D-Arg1, D-Trp7,9, Leu11]-substance P) competitively blocks substance P (pA2 = 6.7) and at a log unit higher concentration blocks neurokinin A (pA2 = 5.8), but does not block neurokinin B. Atropine is a competitive antagonist of neurokinin B (pA2 = 9.0) at ten times the concentration needed to block acetylcholine (pA2 = 10.1), but does not inhibit the other neurokinins. These results support the hypothesis of multiple neurokinin receptors in the guinea pig ileum and indicate that the site of neurokinin B, but not substance P or neurokinin A is predominantly on intramural neurons. This indirect stimulation appears to be dependent on the release of acetylcholine. Neurokinin B also has activity on smooth muscle receptors since the contractile response could not be completely antagonized by atropine. There appear to be two smooth muscle neurokinin receptors on the basis of results obtained with DPDT-SP and spantide, one predominantly responsive to substance P and the other to neurokinin A. Only spantide appeared to have any effect on the neurokinin A receptor and that was at a much higher concentration than that needed to block substance P.     

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues,and in bone and dental hard tissue cells

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.     

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).     

The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.     

Effect of sensory neuropeptides on canine bronchial and pulmonary vessels in vitro

Sensory neuropeptides may be important in the noncholinergic component of parasympathetic vasodilation in the tracheobronchial circulation. We studied the effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and calcitonin gene-related peptide (CGRP) on the isolated canine bronchial artery and used pulmonary artery and vein of similar size for comparison. CGRP (10pM-300nM) was a potent relaxant of the bronchial and pulmonary arteries, and the pulmonary vein with equal potency in all vessels. SP in low concentrations (10pM-100nM) caused vasodilation of the precontracted bronchial artery and in high concentration (10-100 microM) contracted the vessel from resting tone. SP also relaxed the pulmonary artery and vein. NKA and NKB caused relaxation in all three vessels. All of the vascular effects of the sensory neuropeptides were concentration-dependent. The order of potency of the neuropeptides in the bronchial and pulmonary artery was SP greater than NKA greater than CGRP greater than NKB. In the pulmonary vein NKB caused a much larger relaxation than SP and NKA but it was less potent than either NKA or CGRP. Capsaicin (1 microM) caused a large contraction of the bronchial artery, similar in magnitude to the contraction caused by high dose of SP. Neuropeptide Y was also studied and found to cause no consistent constriction of any of the vessels studied. In conclusion, CGRP is a universal dilator of the bronchial and pulmonary blood vessels. SP and NKA exert their main effect on arterial vasomotor tone, whereas NKB is the only tachykinin producing marked dilation of the pulmonary vein.     

Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, alpha-, beta-, and gamma-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants

The rat preprotachykinin I gene mRNA is alternatively spliced to yield three different mRNA species differing in their protein coding regions. We have produced recombinant vaccinia viruses expressing alpha-, beta-, and gamma-preprotachykinin to examine the tachykinin-related peptides produced upon post-translational processing of each individual precursor. Infection of BSC-40 or AtT-20 cell lines with a beta-preprotachykinin-encoding vaccinia virus recombinant results in the expression of the precursor protein. The pro-form (signal peptide removed) can be immunoprecipitated from extracts of infected cells. Infected cells of both types secrete into the culture medium a product(s) which reacts in radioimmunoassay with an antiserum shown to recognize precursor as well as mature substance P. Infected AtT-20, but not BSC-40, cells secrete into the culture medium a processed form(s) of beta-preprotachykinin which reacts in radioimmunoassay with an anti-serum which recognizes the amidated carboxyl terminus of substance P. The molecular nature of the tachykinin products produced in and secreted from AtT-20 cells infected with alpha-, beta-, and gamma-preprotachykinin-encoding recombinants was analyzed by combined high performance liquid chromatography and radioimmunoassay. Peptides were identified based on comigration with synthetic standards and antisera cross-reactivity. We determined that alpha-preprotachykinin is processed to the mature undecapeptide, substance P. beta-Preprotachykinin was processed into multiple products, including substance P, neurokinin A, neurokinin A(3-10), and neuropeptide K. gamma-Preprotachykinin was processed into substance P, neurokinin A, neurokinin A(3-10), and neuropeptide gamma. These five tachykinin peptide products were all routed through the regulated secretory pathway and were secreted into the medium in a cAMP-stimulatable fashion. Since all of these peptides have been shown to be biologically active, it is important to consider the biological consequences of their co-secretion in vivo.     

Comparison of responses by striatonigral substance P and neurokinin A systems to methamphetamine treatment

Multiple administrations of high doses of methamphetamine (METH) previously have been shown to significantly elevate the concentrations of substance P-like immunoreactivity in CNS regions associated with the basal ganglia. Recently, another tachykinin, neurokinin A (NKA), has been found to be closely associated with substance P (SP). While both neuropeptides exert comparable effects when locally injected, there are significant differences in their potencies apparently based on the relative concentrations of their unique receptors. Due to the controversy which has arisen as to their respective roles within the basal ganglia, we have evaluated and compared the responses of the striatal and nigral SP and NKA systems to METH treatment. We observed that multiple high doses of this stimulant increased the nigral and striatal concentrations of both neuropeptides in an identical fashion. Our observation that METH treatment did not alter the relative concentrations of SP and NKA suggests that responses of both transmitter systems, associated with the basal ganglia, parallel each other and are sensitive to the same regulatory mechanisms.