Involvement of substance P and the NK-1 receptor in human pathology

The peptide substance P (SP) shows a widespread distribution in both the central and peripheral nervous systems, but it is also present in cells not belonging to the nervous system (immune cells, liver, lung, placenta, etc.). SP is located in all body fluids, such as blood, cerebrospinal fluid, breast milk, etc. i.e. it is ubiquitous in human body. After binding to the neurokinin-1 (NK-1) receptor, SP regulates many pathophysiological functions in the central nervous system, such as emotional behavior, stress, depression, anxiety, emesis, vomiting, migraine, alcohol addiction, seizures and neurodegeneration. SP has been also implicated in pain, inflammation, hepatitis, hepatotoxicity, cholestasis, pruritus, myocarditis, bronchiolitis, abortus, bacteria and viral infection (e.g., HIV infection) and it plays an important role in cancer (e.g., tumor cell proliferation, antiapoptotic effects in tumor cells, angiogenesis, migration of tumor cells for invasion, infiltration and metastasis). This means that the SP/NK-1 receptor system is involved in the molecular bases of many human pathologies. Thus, knowledge of this system is the key for a better understanding and hence a better management of many human diseases. In this review, we update the involvement of the SP/NK-1 receptor system in the physiopathology of the above-mentioned pathologies and we suggest valuable future therapeutic interventions involving the use of NK-1 receptor antagonists, particularly in the treatment of emesis, depression, cancer, neural degeneration, inflammatory bowel disease, viral infection and pruritus, in which that system is upregulated.     

Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. -tetrahydroisoquinoleic acid (Tic), -fluorenylglycine (Flg), -diphenylalanine (Dip), the diastereoisomers of -1-indanylglycine (Ing) and -benz[ƒ]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (Δ^ZPhe, Δ^EPhe, Δ^ZNal, Δ^ENal) and (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S₇ and S₈ subsites, corresponding to residues Phe⁷ and Phe⁸ of substance P. According to the binding potencies of these substituted-SP analogues, the S₇ binding subsite is smaller than the S₈ subsite: the S₇ subsite accepts only one aromatic nucleus, while the S₈ can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S₈ binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu⁶, Pro⁹]SP(6–11), discrepancies are observed between affinity (K_i) and activity (EC₅₀) values for IPs production. While a weak correlation between K_i and EC₅₀ values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K_i) and their potencies (EC₅₀) for cAMP production (r = 0.97). The high potency (EC₅₀) observed for ‘septide-like’ molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC₅₀ values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

According to the binding potencies of constrained analogues of phenylalanine, the S₇ binding subsite of human NK-1 receptor is small, whereas the S₈, which can accommodate three coplanar nuclei, might probably reside in the extracellular loop. The discrepancies observed between affinity (K_i) and activity (EC₅₀) values for IPs production are not an artefact of CHO cells since a good correlation was found between EC₅₀ for PI hydrolysis and those measured in guinea pig ileum bioassay.     

Self-association of the neuroregulatory peptide substance P and its C-terminal sequences

Self-association of substance P and its C-terminal partial peptide sequences was studied by CD, quasi-electric light scattering, and sedimentation experiments. CD spectra of these peptides are strongly influenced by self-association. They exhibit strong characteristic negative ellipticities, suggesting the formation of a presumably B-type ordered structure. The tendency to form multimers depends on chain length and constitution and has its maximum at the octapeptide (SP 8). The peptide multimers have a broad distribution of sizes in the range of 30- and 800-nm diameter. Subdivision of this distribution into two size classes gives mean diameters of 60–100 nm (predominating)/200–800 nm for substance P and 30–50 nm/200–800 nm for SP 8 multimers.     

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.     

Effect of substance P, neurokinin A and a selective agonist of tachykinin receptors of the NK-2 type on bile metabolism

Based on the experiments on rats, changes of the level of bile secretion and chemical composition of bile following application of substance P, neurokinin A, and selective agonist of the NK-2 tachykinins receptors neurokinin A Fragment (4-10), are interpreted. These results demonstrate that tachykinins (SP, NKA, and NKA Fragment (4-10)) are true holeretics and affect various enzyme systems in the hepatocyte. The alterations of the coefficients of bile confirm that.     

Evaluation of serum level of substance P and tissue distribution of NK-1 receptor in colorectal cancer

Colorectal cancer (CRC) is known as the most common form of malignancies in the world and its occurrence is annually increasing. Due to the relatively high death rates in patients, finding better diagnostic and prognostic factors are required. Substance P (SP) belongs to the tachykinin family that acts as an immunomodulator by binding to the neurokinin-1 receptor (NK1R). The interaction of SP with NK1R might be involved in tumor cell proliferation, angiogenesis, and migration. Hence, this study was aimed to evaluate the serum SP level and tissue distribution of NK1Rs in CRC. Also, we assessed the relationship between tissue distribution of NK1R and some different tumor characteristics, including tumor size, and lymph node status. Recruiting 38 patients primarily diagnosed with CRC, the tissue distribution of NK1R was immunohistochemically evaluated in tumor tissues and their adjacent normal tissue. The serum level of SP was measured using an ELISA method in both cases and healthy control group. The SP value was significantly increased in the serum of patients in comparison with the healthy group (p?=?0.001). Tumor tissues expressed a higher number of NK1R than adjacent normal tissues (p?=?0.01) considering both the percentage of stained cells and intensity of staining. However, there was not any statistically significant relevance between NK1R distribution and tumor characteristics. The SP/NK1R system is involved in tumorigenesis of CRC, and might be suggested as a potent prognostic or diagnostic factor, or a new target in the treatment of CRC.

     

Internalization of [3H]substance P analogues in NK-1 receptor transfected CHO cells

The internalization of [3H]propionyl[Met(O2)11]SP(7-11) which binds one binding site and of [3H][Pro9]SP which binds the two binding sites associated with the NK-1 receptor has been examined in CHO cells. The quantity of [3H][Pro9]SP measured inside the cytoplasm in kinetic experiments is fully temperature-dependent. In contrast, [3H]propionyl[Met(O2)11]SP(7-11) internalization reaches the same extent whatever the temperature, although the rate slowed down with lower temperature. The extent of internalization of [3H][Pro(9)]SP relative to the total specific bound is biphasic, when the extent of internalization of [3H]propionyl[Met(O2)11]SP(7-11) remains constant. For [3H][Pro9]SP, a high-affinity high-yield component inhibited in the presence of propionyl[Met(O2)11]SP(7-11) and a low-affinity low-yield component in the internalization process could be determined. Saturation studies show that [3H][Pro9]SP-binding parameters are insensitive to both phenylarsine oxide and monensin treatment, whereas [3H]propionyl[Met(O2)11]SP(7-11) maximal binding is decreased in both cases. Altogether, these data suggest that the two radiolabeled peptides should not follow the same internalization pathway.     

A novel bioassay for the NK-2 neurokinin receptor: the guinea pig gallbladder

Although three neurokinin receptors (NK-1, NK-2, NK-3) have been identified by radioligand binding assays, only the NK-1 and NK-3 types have been found in smooth muscle bioassays. In this study, evidence is presented demonstrating functional NK-2 type receptors in the guinea pig gallbladder (GPGB). The potencies of the following neurokinins were determined in the GPGB and the guinea pig ileum (GPI): substance P (SP), physalaemin (PH), eledoisin (EL), substance K (SK) and kassinin (KA). ED50 values were determined by linear regression analysis of the dose-related increases in the force generated by each peptide. In the GPI, the rank order of potency was SP = PH = EL greater than SK = KA, indicating NK-1 selectivity. In the GPGB, the relative potencies were SK greater than KA greater than EL much greater than PH greater than SP, which is similar to that reported for the NK-2 receptor in radioligand binding assays. These findings demonstrate the NK-2 receptor tissue selectivity of the GPGB.     

Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.     

The two NK-1 binding sites are distinguished by one radiolabelled substance P analogue

Two non-stoichiometric binding sites had previously been characterized for the NK-1 receptor using two different types of radiolabelled analogues of substance P. However, the question remained on their eventual conformational interconversion induced or not by the ligand. In this study, kinetic, saturation, and competition studies using [3H]propionyl[Pro(9)]SP demonstrate the existence of two independent binding components in CHO cells transfected with the human NK-1 receptor, with K(d) values of 0.040 nM ( approximately 20% of total sites) and 5.9 nM ( approximately 80% of total sites) that correspond to those of the two previously described binding sites. These two binding sites do not seem to interconvert since the minor one can be selectively extinguished in saturation studies in the presence of a SP analogue specific of this binding site.     

Novel potent substance P and neurokinin A receptor antagonists. Conception,synthesis and biological evaluation of indolizine derivatives

Exploration of SAR around dual NK(1)/NK(2) antagonist Cbz-Gly-Leu-Trp-OBzl(CF(3))(2) and its derivatives disclosed the essential requirements for more potent dual NK(1)/NK(2) binding. We report here the synthesis and the biological properties of a novel series of indolizine including pharmacophoric elements.     

Human substance P receptor lacking the C-terminal domain remains competent to desensitize and internalize

Substance P receptor (SPR) and its naturally occurring splice-variant, lacking the C-terminal tail, are found in brain and spinal cord. Whether C-terminally truncated SPR desensitizes like full-length SPR is controversial. We used a multivaried approach to determine whether human SPR (hSPR) and a C-terminally truncated mutant, hSPRDelta325, differ in their desensitization and internalization. In HEK-293 cells expressing either hSPRDelta325 or hSPR, SP-induced desensitization of the two receptors was similar when measured by inositol triphosphate accumulation or by transient translocation of coexpressed PKCbetaII-GFP to the plasma membrane. Moreover, translocation of beta-arrestin 1 or 2-GFP (betaarr1-GFP or betaarr2-GFP) to the plasma membrane, and receptor internalization were also similar. However, hSPR and hSPRDelta325 differ in their phosphorylation and in their ability to form beta-arrestin-containing endocytic vesicles. Unlike hSPR, hSPRDelta325 is not phosphorylated to a detectable level in intact HEK293 cells, and whereas hSPR forms vesicles containing either betaarr1-GFP or betaarr2-GFP, hSPRDelta325 does not form any vesicles with betaarr1-GFP, and forms fewer vesicles with betaarr2-GFP. We conclude that truncated hSPR undergoes agonist-dependent desensitization and internalization without detectable receptor phosphorylation.     

Arg3]substance P and neurokinin A from chicken small intestine

Using antisera specific for NH2-terminal and COOH-terminal regions of substance P and for the COOH-terminal region of neurokinin A, peptides with tachykinin-like immunoreactivity were isolated from extracts of chicken small intestine. The peptide Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 differs from human substance P by substitution of the lysyl residue by an arginyl residue at position 3. Synthetic [Arg3]substance P showed identical chromatographic and immunochemical properties to chicken substance P and was equipotent with substance P in contracting the guinea pig ileum. A second peptide His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 isolated from the extracts is identical to human neurokinin A. A third peptide was immunoreactive towards the COOH-terminally directed anti-serum to substance P only but was not characterized structurally in this study.     

The role of substance P in the marginal division of the neostriatum in learning and memory is mediated through the neurokinin 1 receptor in rats

Substance P (SP) is a neuropeptide that plays an important role in inflammation, respiration, pain, aggression, anxiety, and learning and memory mainly through its high affinity neurokinin 1 receptor (NK1R). The marginal division (MrD) is a pan-shaped subdivision in the caudomedial margin of the neostriatum in the mammalian brain and is known to be involved in learning and memory. We studied the expression of SP, NK1R and NK1R mRNA in the rat striatum by immunohistochemistry, immunofluorescence and in situ hybridization, and found that the levels of SP, NK1R protein and NK1R mRNA were high in the cell bodies, fibers and terminals of neurons in the neostriatum, especially in the MrD. Knocking down NK1R activity in the MrD by using an antisense oligonucleotide against NK1R mRNA inhibited learning and memory in a Y-maze behavioral test. Our results show that NK1R mediates the role of SP in the MrD in learning and memory.     

Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)*neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.     

Tracheal smooth muscle responses to substance P and neurokinin A in the piglet.

The tachykinins substance P (SP) and neurokinin A (NKA) have been shown to induce airway smooth muscle contraction in mature animals, and the enzyme neutral endopeptidase (NEP) modulates this effect. We evaluated maturation of SP- and NKA-induced tracheal smooth muscle contraction and modulation of their effects by NEP in anesthetized, paralyzed, and artificially ventilated piglets less than 4 days, 2-3 wk, and 10 wk of age. Tracheal smooth muscle tension was measured in vivo from an open tracheal segment by use of a force transducer. Intravenous SP caused a dose-dependent increase in tracheal tension in all three age groups; however, the response in less than 4-day-old piglets was significantly weaker than in 2- to 3- and 10-wk-old piglets. NKA caused a dose-dependent increase in tracheal tension only in 2- to 3- and 10-wk-old piglets. The response of tracheal tension to NKA was weaker than the response to SP in all age groups. Atropine (2 mg/kg) significantly diminished the responses of tracheal tension to SP and NKA, indicating a cholinergic contribution to these responses at all ages. Intravenous thiorphan, a known NEP inhibitor, potentiated the effects of SP only in 2- to 3- and 10-wk-old piglets and did not affect the response of tracheal tension to NKA at any age. Biochemical analyses demonstrated a significant increase in tracheal NEP activity in comparably aged piglets over the first 10 wk of life.(ABSTRACT TRUNCATED AT 250 WORDS)     

A constitutively active form of neurokinin 1 receptor and neurokinin 1 receptor-mediated apoptosis in glioblastomas

Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P increases Akt phosphorylation by 2.5-fold, with an EC₅₀ of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with substance P reduces apoptosis by 53 ± 1% ( p  <   0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16% ( p  <   0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase. Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and phosphatidyl-3-kinase, a partial involvement of epidermal growth factor receptor, and no involvement of mitogen-activated protein/extracellular signal-related kinase. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis.     

Functional neurokinin and NMDA receptor activity in an animal naturally lacking substance P: the naked mole-rat

Naked mole-rats are extremely unusual among mammals in that their cutaneous C-fibers lack the neuropeptide Substance P (SP). In other mammals, SP plays an important role in nociception: it is released from C-fibers onto spinal neurons where it facilitates NMDA receptor activity and causes sensitization that can last for minutes, hours or days. In the present study, we tested the effects of intrathecal application of: 1) SP, 2) an SP antagonist (GR-82334), and 3) an NMDA antagonist (APV) on heat-evoked foot withdrawal. In the naked mole-rat, at a high enough concentration, application of SP caused a large, immediate, and long-lasting sensitization of foot withdrawal latency that was transiently reversed by application of either antagonist. However, neither SP nor NMDA antagonists had an effect when administered alone to na?ve animals. In contrast, both antagonists induced an increase in basal withdrawal latency in mice. These results indicate that spinal neurons in naked mole-rats have functional SP and NMDA receptors, but that these receptors do not participate in heat-evoked foot withdrawal unless SP is experimentally introduced. We propose that the natural lack of SP in naked mole-rat C-fibers may have resulted during adaptation to living in a chronically high carbon dioxide, high ammonia environment that, in other mammals, would stimulate C-fibers and evoke nocifensive behavior.     

Effect of aging on the substance P receptor, NK-1, in the spinal cord of rats with peripheral nerve injury

Substance P (SP) levels in the spinal cords of very old rats are less than the levels in younger rats (Bergman et al., 1996). After injury to a peripheral nerve in young rats, immunoreactivity (ir) to the SP receptor, NK-1 (neurokinin-1), increases in the spinal cord ipsilateral to the injury and the increases are correlated with the development of thermal hyperalgesia (Goff et al., 1998). Thus we postulated that aged rats might display an increased sensitivity to thermal stimulation before peripheral nerve injury and that they might respond differently to injury than do younger rats. To test this hypothesis, we used the Bennett and Xie model (1988) of chronic constriction injury (CCI) to the sciatic nerve to induce a neuropathic pain condition. We investigated the effect of age on changes in NK-1 ir in superficial layers of the dorsal horn and on numbers of NK ir cells in deeper laminae at the L4-L5 levels of the spinal cord after CCI. NK-1 receptors were tagged immunohistochemically and their distribution quantified by use of computer-assisted image analysis. NK-1 ir changes were related to alterations in thermal and tactile sensitivity that developed after CCI in young, mature and aged (4-6, 14-16, and 24-26 months) Fischer F344 BNF1 hybrid rats. No differences in thermal or tactile sensitivity of young and aged rats were seen in the absence of nerve injury. After injury, aged rats developed thermal hyperalgesia and tactile allodynia more slowly than did the younger rats. NK-1 receptor ir and numbers of NK-1 ir cells in the dorsal horn increased with time post-injury in all three groups. NK-1 ir increases were correlated with the development of thermal hyperalgesia in those rats that displayed hyperalgesia. However, some rats developed an increased threshold to thermal stimuli (analgesia) and that also was correlated with increases in NK-1 ir. Thus NK-1 ir extent, while correlated with thermal sensitivity in the absence of injury, is not a specific marker for disturbances in one particular sensory modality; rather it increases with peripheral nerve injury per se.