New insights into the antidepressant actions of substance P (NK1 receptor) antagonists

Considerable progress has been made in understanding the neural circuits involved the antidepressant and anxiolytic efficacy of substance P (NK, receptor) antagonists (SPAs). Progress has been hampered by species differences in the pharmacology of the NK1 receptor, and the availability of NK1R-/- mice has been a particularly useful resource in overcoming this difficulty. Using neuroanatomical, behavioural, and electrophysiological techniques, studies have now established that pharmacological blockade or deletion of the NK1 receptor produces an antidepressant and anxiolytic-like profile in a range of behavioural assays that is distinct from that of established drugs. There is evidence from focal injection studies that some of these effects may be mediated directly by blockade of NK, receptors in the amygdala and its projections to the hypothalamus, periaqueductal gray, and reticulopontine nucleus. Substance P and NK1 receptors are also intimately associated with ascending 5-HT and norepinephrine projections to the forebrain, and alterations in the function of these systems are also likely to be related to the antidepressant efficacy of SPAs. Unlike some established drugs, SPAs are generally well tolerated and do not induce sedation or motor impairment in preclinical species. These findings are consistent with a novel antidepressant mechanism of action of SPAs.     

Investigation of the effect of homocysteinylation of substance P on its binding to the NK1 receptor using molecular dynamics simulation

Substance P is a neurotransmitter or modulator in both the central and peripheral nervous systems. In this work, modifications of the lysine in SP by homocysteine and an acetyl group as well as the conformational dynamics of the native and modified SP peptides and their complexes with the NK1 receptor were studied via MD simulation. It was found that modifying SP stabilizes the peptide structure, but the modified SP peptides are less likely to bind to the NK1 receptor, so the resulting complexes are less stable. The RMSD of native SP (~0.33 nm) is about twice as large as that of the modified SP peptides (~0.18 nm), while the RMSD for the receptor complexed with native SP is ~0.3 nm, and that for the receptor complexed with either of the modified peptides is ~0.35 nm, which demonstrates the high stability of the modified SP peptides as well as the receptor complexed with native SP. Such behavior was also observed in other structural analyses. The binding free energies of the native and modified SP peptides with the NK1 receptor were also compared. The ΔG_bind values for the binding of homocysteinylated SP to the NK1 receptor and the binding of the acetylated SP and native SP to the NK1 receptor were ?38.89, ?64.46, and???264.52 kJ mol^?1, respectively. Modification of the lysine of SP decreases the binding affinity of the peptide to the NK1 receptor. In other words, homocysteinylation or acetylation of SP leads to weaker interactions of the peptide with the NK1 receptor compared to those between native SP and NK1. We propose that this phenomenon leads to increased levels of homocysteinylated SP in plasma in many diseases such as breast cancer.

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Graphical abstract Substance P (SP) is a neuropeptide which binds to the NK1 receptor. SP is of great pharmacological interest, as agonists and antagonists of SP can potentially be used to treat many chronic diseases. Therefore, in this work, the lysine (LYS) in SP was theoretically modified with a homocysteine or acetyl group to explore the effects of such a modification on the binding affinity of this peptide with the NK1 receptor and the structural dynamics of the resulting complex

     

N-terminal substance P fragments inhibit the spinally induced, NK 1 receptor mediated behavioural responses in mice.

N-terminal fragments of substance P (SP) were tested for antagonism against the aversive responses induced in mice by various tachykinin receptor agonists, somatostatin and bombesin. When co-administered with SP intrathecally, low doses (1.0-4.0 pmol) of SP (1-7) or SP (1-8) reduced the SP-induced behavioural responses of scratching, biting and licking. Aversive responses induced by two other neurokinin (NK) 1 receptor agonists, Septide and physalaemin, were also dose-dependently decreased by the simultaneous injection of small doses of SP (1-7) or SP (1-8). Aversive responses induced by 400 pmol of NK A were also significantly reduced by co-administration of SP (1-7) or SP (1-8). No significant effects of the N-terminal fragments were observed against the aversive responses elicited by NK A (300 pmol), eledoisin, NK B, somatostatin or bombesin. These results suggest that the behavioural antagonism produced by SP (1-7) and SP (1-8) may be limited to the NK 1 receptor at the spinal level in mice.     

The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway

Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.     

Substance P (NK1) receptor in relation to substance P innervation in rat duodenum after irradiation

It has previously been shown that high dose of irradiation to the rat abdomen leads to an increased level of substance P (SP) in the duodenum. In the present study the pattern of distribution of NK1 receptors (NK1-R) in rat duodenum after irradiation (5-30 Gy), was examined at the same time-point (7 days) after irradiation, comparisons being made with the distribution of SP-innervation. Immunohistochemical methods were used. In controls, NK1-R-like immunoreactivity (-LI) was detected in epithelial cells, in cells in the region of the intestinal cells of Cajal within the deep muscular plexus (ICC-DMP), in neuronal cells in the myenteric plexus, and variably in granulocytes in the mucosa. Irradiation with 5-10 Gy did not lead to obvious changes in the pattern of NK1-R-LI. After irradiation with the highest doses (25-30 Gy), the mucosa was often gravely damaged, displaying granulation tissue. No epithelial NK1-R-LI was detected in this tissue, but was present in less affected mucosa after these doses. In the region of the ICC-DMP, in the myenteric plexus, and in granulocytes, NK1-R-LI was detected also after high dose irradiation. However, the degree of NK1-R-LI in the region of the ICC-DMP was somewhat lower than seen in controls and after low doses. SP-immunoreactive nerve fibers were present in the regions where NK1-R-LI was detected. These findings support a suggestion that an increased level of SP after irradiation may contribute to the dose-dependent gastrointestinal adverse effects that occur after radiotherapy.     

A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains.

Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains.     

Modulation of basal and stress-induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L-822429

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.     

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.     

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.     

The LOX-1 scavenger receptor cytoplasmic domain contains a transplantable endocytic motif

Oxidized low-density lipoprotein particles is a pro-atherogenic factor implicated in atherosclerotic plaque formation. The LOX-1 scavenger receptor binds OxLDL and is linked to atherosclerotic plaque initiation and progression. We tested the hypothesis that the LOX-1 cytoplasmic domain contains a transplantable signal for membrane protein endocytosis. Structural modeling of the LOX-1 cytoplasmic domain reveals that a tripeptide motif (DDL) implicated in LOX-1 endocytosis is part of a curved β-pleated sheet structure. The two aspartic acid residues within this structural model are highly solvent-accessible enabling recognition by cytosolic factor(s). A triple alanine substitution of the DDL motif within the LOX-1 scavenger receptor substantially reduced endocytosis of OxLDL. Transplantation of the LOX-1 cytoplasmic domain into a transferrin receptor reporter molecule conferred efficient endocytosis on this hybrid protein. Mutation of the DDL motif within the hybrid LOX-1-TfR protein also substantially reduced receptor-mediated endocytosis. Thus a transplantable endocytic motif within the LOX-1 cytoplasmic domain is needed to ensure efficient internalization of pro-atherogenic OxLDL particles.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

The endocytic pathway followed by the keratinocyte growth factor receptor

Keratinocyte growth factor (KGF/FGF7) acts specifically on epithelial cells and regulates their proliferation and differentiation. It binds to and activates a receptor tyrosine kinase, the KGF receptor (KGFR), which is a splicing variant of the fibroblast growth factor receptor 2. The endocytic pathway followed by KGF and its receptor was analyzed here using immunofluorescence and confocal microscopy. After 10 min of internalization at 37 degrees C, both KGF and its receptor were localized in early endosomes, and after 30-60 min of endocytosis ligand and receptor were seen to reach perinuclear late endosomes and not the recycling endosomal compartment. Parallel western blot analysis revealed that KGFRs were tyrosine phosphorylated both at early and late steps of internalization, suggesting that KGF and KGFR remain associated in active complexes through the endocytic pathway. Pulse-chase experiments showed that the internalized KGFRs underwent degradation detectable at 1 h of endocytosis at 37 degrees C, indicating that KGFRs are functionally downregulated.     

Phosphoinositides and the endocytic pathway

Phosphorylation of the phosphatidylinositol headgroup generates seven varieties of phosphoinositide of which PtdIns(4,5)P₂, PtdIns3P and PtdIns (3,5)P₂ have established roles on the endocytic pathway. In this review, we discuss the enzymes responsible for generation and turnover of these lipids, which are keys to determining compartmental identity and the flux of material through the endocytic system. The enzymatic generation of lipids serves as an amplification mechanism through which a wide variety of effector molecules can be recruited.     

Are the proposed substance P receptor sub-types,substance P receptors?

Recently, a number of laboratories have postulated the existence of receptor sub-types for substance P. This review is intended to represent a critical appraisal of these reports. In the majority of cases, the evidence for the existence of receptor sub-types has been obtained from observed potency differences of agonists. The problems with this approach are discussed. In addition, information obtained through substance P antagonists, binding studies and investigations of second messenger systems is presented and discussed in relation to the above receptor subdivisions. It is concluded that the present results are consistent with the existence of three receptor sub-types; however, it is suggested that substance P is the natural agonist for only one of these receptors, and that substance K and tuftsin may be the transmitters for the other two receptor sub-types.     

Endospanins regulate a postinternalization step of the leptin receptor endocytic pathway

Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.