Characterization of tachykinin NK2 receptor in the anterior pituitary gland

Tachykinins are a family of bioactive peptides that interact with three subtypes of receptors: NK1, NK2 and NK3. Substance P has greater affinity for NK1, and neurokinin A (NKA) for NK2 receptor subtype. Although only NK1 receptor has been characterized in the anterior pituitary gland, some evidence suggests the existence of NK2 receptors in this gland. Therefore, we investigated the presence of NK2 receptors in the anterior pituitary gland of male rats by radioligand binding studies using labeled SR48968, a non peptidic specific antagonist. [3H]SR48968 specific binding to cultured anterior pituitary cells was time-dependent and saturable, but with a lower affinity than previously reported values for cells expressing NK2 receptors. Unlabeled NKA inhibited only partially [(3)H]SR48968 specific binding to whole anterior pituitary cells. Since SR48968 is a non polar molecule, we performed experiments to discriminate surface from intracellular binding sites. SR48968 exhibited both surface and intracellular specific binding. Analysis of the surface-bound ligand indicated that [3H]SR48968 binds to one class of receptor with high affinity. Neurokinin A completely displaced [3H]SR48968 surface specific binding fitting to a two-site/two-state model with high and low affinity. Additionally, immunocytochemical studies showed that the NK2 receptor is expressed at least in a subset of lactotropes. These results demonstrate the presence of NK2 receptors in the anterior pituitary gland and suggest that NKA actions in this gland are mediated, at least in part, by the NK2 receptor subtype.     

Role of the tachykinin NK1 receptor in a murine model of cigarette smoke-induced pulmonary inflammation

Background

The tachykinins, substance P and neurokinin A, present in sensory nerves and inflammatory cells such as macrophages and dendritic cells, are considered as pro-inflammatory agents. Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and increased tachykinin levels are recovered from the airways of COPD patients. The aim of our study was to clarify the involvement of the tachykinin NK₁ receptor, the preferential receptor for substance P, in cigarette smoke (CS)-induced pulmonary inflammation and emphysema in a mouse model of COPD.

Methods

Tachykinin NK₁ receptor knockout (NK₁-R^-/-) mice and their wild type controls (all in a mixed 129/sv-C57BL/6 background) were subjected to sub acute (4 weeks) or chronic (24 weeks) exposure to air or CS. 24 hours after the last exposure, pulmonary inflammation and development of emphysema were evaluated.

Results

Sub acute and chronic exposure to CS resulted in a substantial accumulation of inflammatory cells in the airways of both WT and NK₁-R^-/- mice. However, the CS-induced increase in macrophages and dendritic cells was significantly impaired in NK₁-R^-/- mice, compared to WT controls, and correlated with an attenuated release of MIP-3α/CCL20 and TGF-β1. Chronic exposure to CS resulted in development of pulmonary emphysema in WT mice. NK₁-R^-/- mice showed already enlarged airspaces upon air-exposure. Upon CS-exposure, the NK₁-R^-/- mice did not develop additional destruction of the lung parenchyma. Moreover, an impaired production of MMP-12 by alveolar macrophages upon CS-exposure was observed in these KO mice. In a pharmacological validation experiment using the NK₁ receptor antagonist RP 67580, we confirmed the protective effect of absence of the NK₁ receptor on CS-induced pulmonary inflammation.

Conclusion

These data suggest that the tachykinin NK₁ receptor is involved in the accumulation of macrophages and dendritic cells in the airways upon CS-exposure and in the development of smoking-induced emphysema. As both inflammation of the airways and parenchymal destruction are important characteristics of COPD, these findings may have implications in the future treatment of this devastating disease.     

Combined NK1 and NK2 tachykinin receptor antagonists: synthesis and structure-activity relationships of novel oxazolidine analogues

We report herein the synthesis and structure-activity relationships of a series of novel oxazolidine analogues with regards to NK1 and NK2 tachykinin receptor binding affinity. Among this series of oxazolidine analogues, some compounds exhibited excellent high binding affinities for both NK1 and NK2 receptors. In addition, we describe the inhibitory effect in vivo on SP-induced airway vascular hyperpermeability and NKA-induced bronchoconstriction in guinea pigs.     

Spinal actions of GR73632, a novel tachykinin NK1 receptor agonist.

Behavioral characterization of GR73632, a newly synthesized tachykinin NK1 receptor agonist, was examined in mice. Intrathecal (IT) injection of GR73632 in the spinal subarachnoid space of mice elicited a dose-dependent behavioral syndrome, consisting of scratching, biting and licking. The time course of the response to GR73632 was almost similar to that of substance P. GR73632 evoked much more licking and biting than did substance P, that in turn caused less scratching. GR73632 was approximately 200-fold more potent than substance P in inducing the characteristic behavioral response. The GR73632-induced behavioral response was inhibited by IT co-administration of CP-96,345, a non-peptide NK1 receptor antagonist, but not its inactive enantiomer CP-96,344. CP-96,345, co-injected IT with substance P, also inhibited the behavioral response to substance P. These results demonstrate that the scratching, biting and licking response induced by IT GR73632 may be mediated by the NK1 receptor in the spinal cord. These findings suggest that GR73632 may be useful as a tachykinin NK1 receptor agonist and also for evaluating spinal pharmacological activities of NK1 receptor antagonists.     

Central effects of selective NK1 and NK3 tachykinin receptor agonists on two models of experimentally-induced colitis in rats

Peripheral tachykinins (TKs) are believed to play a role in the pathogenesis of inflammatory bowel diseases (IBD). In this study we investigated changes induced by central administration of two natural TK receptor agonists, NK(1) (PG-SPI) and NK(3) (PG-KII), on trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced experimental colitis in rats. Colitis was induced by instilling a single intracolonic dose of TNBS 50 mgkg(-1) (0.5 ml in 50% ethanol) or by oral administration of 5% DSS for 7 days. Each group of rats was intracerebroventricularly injected daily with PG-SPI and PG-KII (0.5, 5, and 50 microgkg(-1)). On day 3, TNBS-treated animals were killed and the severity of gut inflammation was evaluated by measuring myeloperoxidase (MPO) activity, interleukin-1beta (IL-1beta) production and by scoring macroscopic and histologic colonic damage. DSS-treated animals were checked daily for the length of survival and for stool consistency and faecal blood. In the TNBS group, PG-SPI and PG-KII increased scores for the severity of colonic damage, stimulated the production of IL-1beta and increased granulocyte infiltration into the colon (MPO activity). In the DSS group, PG-SPI and PG-KII decreased the percentage of surviving animals, and increased the number of rats that developed loose stools and blood in the faeces and the MPO activity. These results indicate that centrally injected NK(1) and NK(3) tachykinin receptor agonists play a proinflammatory role in experimentally-induced colitis in rats.     

Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain

(2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA), which labels "septide-sensitive" but not classic NK(1) binding sites in peripheral tissues, was used to determine whether septide-sensitive binding sites are also present in the rat brain. Binding studies were performed in the presence of SR 48968 (NK(2) antagonist) and senktide (NK(3) agonist) because [(125)I]NKA also labels peripheral NK(2) binding sites and, as shown in this study, central NK(3) binding sites. [(125)I]NKA was found to label not only septide-sensitive binding sites but also a new subtype of NK(1) binding site distinct from classic NK(1) binding sites. Both subtypes of [(125)I]NKA binding sites were sensitive to tachykinin NK(1) antagonists and agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and neuropeptide gamma (NPgamma). However, compounds of the septide family such as substance P(6-11) [SP(6-11)] and propionyl-[Met(O(2))(11)]SP(7-11) and some NK(1) antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for the new NK(1)-sensitive sites than for the septide-sensitive sites. The hypothalamus and colliculi possess only this new subtype of NK(1) site, whereas both types of [(125)I]NKA binding sites were found in the amygdala and some other brain structures. These results not only explain the central effects of septide or SP(6-11), but also those of NKA, NPK, and NPgamma, which can be selectively blocked by NK(1) receptor antagonists.     

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.     

Characterization of the tachykinin NK2 receptor subtype in the rabbit pulmonary artery.

Contractile responses to neurokinin A (NKA), neuropeptide gamma(NP gamma), and the NK2 receptor-selective analogs [Lys5,MeLeu9,Nle10]NKA(4-10) and MDL 28,564 were determined in the endothelium-denuded rabbit pulmonary artery. Responses to NKA, NP gamma, and [Lys5,MeLeu9,Nle10]NKA(4-10) were antagonized by the NK2 receptor antagonist MDL 29,913, with pA2 values of 6.67, 6.46, and 7.32, respectively. Autoradiographic studies failed to demonstrate any specific binding sites for [125I]-iodohistidyl NKA (INKA) over the pulmonary artery. These data suggest the presence in rabbit pulmonary artery of an unusual "nonclassical" NK2 receptor subtype, which appears to lack affinity for INKA.     

3D-Quantitative structure-activity relationship and docking studies of the tachykinin NK3 receptor

The tachykinin NK(3) receptor (NK(3)R) is a novel drug target for schizophrenia and drug abuse. Since few non-peptide antagonists of this G protein-coupled receptor are available, we have initiated this study to gain a better understanding of the structure-activity relationships of NK(3) antagonist compounds. We developed a 3D comparative molecular similarity index analysis (CoMSIA) model that gave cross-validated PLS values with q(2) >0.5 which were validated using a test set. We also describe the development of a homology model of the NK(3)R. The model was then used to develop a pharmacophore for docked ligands. This pharmacophore showed two aromatic, two hydrogen donor and one acceptor/aromatic points. These data will be useful for future structure-based drug discovery of ligands for the NK(3)R.     

Localization of tachykinin binding sites (NK1, NK2, NK3 ligands) in the rat brain

A comparative autoradiographic analysis of the distribution of tachykinin binding sites was made on brain serial sections using several ligands. (1) 3H-SP, 125I-BHSP and 3H-physalaemin labeled identical binding sites (NK1 type). (2) 3H-NKB, 125I-BHE and 3H-eledoisin also labeled identical sites (NK3 type). (3) 125I-BHNKA preferentially labeled NK3 binding sites, the distribution of 125I-BHNKA binding sites being identical to that of 3H-NKB or 125I-BHE binding sites. (4) The distributions of 3H-SP and 3H-NKB binding sites were markedly different. (5) A very low density of labeling was found with 3H-NKA or 125I-NKA, and these binding sites were distributed only in areas rich in either 3H-SP or 3H-NKB binding sites. (6) Particular efforts were made to look for the presence of tachykinin binding sites in the substantia nigra, since this structure is particularly rich in SP and NKA and contains functional tachykinin receptors of the NK1 and NK2 types as suggested by physiological studies. Confirming previous reports, low or very low labeling was observed in the substantia nigra with 3H-SP or 125I-BHSP and 3H-NKB or 125I-BHE. Similar results were found with 3H-NKA, 125I-NKA or 125I-BHNKA. In conclusion, our data do not provide evidence yet for the existence of NK2 binding sites in the rat brain.     

Immunolocalization of full-length NK1 tachykinin receptors in human tumors.

Biological effects of substance P (SP) are mediated by the neurokinin-1 (NK1) receptor that exists as a full-length and as a carboxy-terminally truncated isoform in humans. Although NK1 receptor mRNA and binding sites have been detected in certain malignancies, little is known about the cellular and subcellular localization of NK1 receptor protein in human neoplastic tissues. We developed and characterized a novel anti-peptide antibody to the carboxy-terminal region of the human full-length NK1 receptor. Specificity of the antiserum was demonstrated by (1) detection of a broad band migrating at molecular mass 70,000-90,000 Da in Western blots of membranes from NK1-expressing tissues; (2) cell-surface staining of NK1-transfected cells; (3) translocation of NK1 receptor immunostaining after SP exposure; and (4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. Distribution of NK1 receptors was investigated in 72 formalin-fixed, paraffin-embedded human tumors showing that NK1 receptors were frequently expressed in glioblastomas and breast and pancreatic carcinomas. Immunoreactive NK1 receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumor cells. Development of this novel NK1 receptor antibody allows the efficient localization of NK1 receptor protein in human formalin-fixed, paraffin-embedded tissues. NK1 receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention using SP analogs.     

Peristalsis regulation by tachykinin NK1 receptors in the rabbit isolated distal colon

In the gastrointestinal tract, tachykinin NK1 receptors are widely distributed in a number of neuronal and nonneuronal cells involved in the control of gut motor activity. In particular, in the rabbit isolated distal colon, which is a suitable model system to investigate the contribution of tachykinins as noncholinergic excitatory transmitters, the influence of NK1 receptors in the regulation of peristalsis is not known. The selective NK1-receptor antagonists SR-140333 (0.3 and 1 nM) and MEN-10930 (0.3-10 nM) significantly enhanced the velocity of rabbit colonic propulsion to submaximal stimulation. The prokinetic effect of SR-140333 was prevented by N(omega)-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, indicating that NK1 receptors located on nitrergic innervation exert a functional inhibitory restraint on the circular muscle and probably on descending excitatory and inhibitory pathways during propulsion. Conversely, the selective NK1-receptor agonist septide (3-10 nM) significantly inhibited colonic propulsion. In the presence of L-NNA, the inhibitory effect of septide was reverted into a prokinetic effect, which is probably mediated by the activation of postjunctional excitatory NK1 receptors.     

SR 142801, The first potent non-peptide antagonist of the tachykinin NK3 receptor

SR 142801 is the first potent and selective non-peptide antagonist of the tachykinin NK₃ receptor. It inhibited [MePhe⁷]NKB binding to its receptor from various species, including humans. SR 142801 was a competitive antagonist of [MePhe⁷]NKB-mediated contractions of guinea-pig ileum and inhibited the acetylcholine release following the activation of the guinea-pig ileum tachykinin NK₃ receptor. In vivo, SR 142801 potently inhibited the turning behaviour induced by intrastriatal injection of senktide in gerbils, and appears as a powerful tool for investigation of the physiological and pathological role of NKB and its NK₃ receptor.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

Connectivity and orientation of the seven helical bundle in the tachykinin NK-1 receptor probed by zinc site engineering.

A high affinity, tridentate metal ion site has been constructed previously by His substitutions in an antagonist binding site located between transmembrane segment (TM)-V and TM-VI in the substance P NK-1 receptor. Here, an attempt is made to probe helix-helix interactions systematically in the NK-1 receptor by engineering of bis-His Zn(II) sites. His residues were introduced at selected positions individually and in combinations in the exterior segments of TM-II, III and V in both the wild-type background and after Ala substitution of naturally occurring His residues, and the increase in the affinity for Zn(II) was monitored in competition binding experiments with iodinated substance P or a tritiated non-peptide antagonist. In this way, two high affinity bis-His sites were constructed between position 193 in TM-V (Glu193, G1uV:01) and position 109 in TM-III (Asn1O9, AsnIII:05) as well as between the neighboring, naturally occurring His108 in TM-III (HisIII:04) and position 92 in TM-II (Tyr92, TyrII:24), respectively. Functionally, the coordination of zinc ions at these two sites blocked the receptor as it antagonized the substance P-induced increase in phosphatidylinositol turnover. It is concluded that the bis-His zinc sites from the central TM-III helix to TM-II and -V, respectively, together with the interconnected, previously constructed tridentate site between TM-V and -VI, constitute a basic network of distance constraints for the molecular models of receptors with seven transmembrane segments which, for example, strongly support an anti-clockwise orientation of the seven helical bundle as viewed from the extracellular space.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

Effect of chronic capsaicin treatment on tachykinin NK1 binding sites in the rat.

Binding sites for [125I]-Bolton-Hunter substance P (BHSP) were investigated in homogenates of rat submandibular gland, colon smooth muscle, and urinary bladder. In vehicle-treated animals, the equilibrium dissociation constant (KD) was similar for both submandibular gland (0.46 +/- 0.03 nM) and colon (0.57 +/- 0.04 nM), although the maximum density of binding sites (Bmax) was about six-fold higher in submandibular gland compared with colon. These binding parameters remained unchanged in capsaicin-pretreated animals (140 mg/kg IP). In contrast, capsaicin pretreatment reduced (p less than 0.05) the Bmax in urinary bladder by twenty-five percent (0.56 fmol/mg wet weight) when compared to vehicle-treated controls (0.73 fmol/mg wet weight), although the KD was unchanged (vehicle, 0.29 +/- 0.08 nM; capsaicin, 0.24 +/- 0.04 nM). These data demonstrate that the NK1 receptors in submandibular gland and colon smooth muscle are not associated with or dependent upon intact primary afferent sensory neurons. However, a minority of NK1 receptors in the urinary bladder were lost after capsaicin, indicating that these receptors are located on sensory terminals, or may be dependent on growth factors or other chemicals released from these nerves.     

Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.     

Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity.     

Antidepressant-type effect of the NK3 tachykinin receptor agonist aminosenktide in mouse lines differing in endogenous opioid system activity.

The influence of the tachykinin NK3 receptor agonist, aminosenktide on the immobility in the forced swimming test was studied in mouse lines selectively bred for divergent magnitudes of stress-induced analgesia. The high analgesia (HA) line is known to display enhanced, and the low analgesia (LA) line displays reduced activity of the opioid system. Aminosenktide at doses of 125 microg/kg or 250 microg/kg intraperitoneally (IP) reduced, in naltrexone-reversible manner, the immobility more of opioid receptor-dense HA than of unselected mice, but was ineffective in the opioid receptor-deficient LA line. The effect of aminosenktide was quite similar to the antiimmobility action of desipramine (10 mg/kg IP), a prototypic antidepressant agent. None of the compounds increased animals' locomotion as found with an open field test; therefore their antiimmobility effect cannot be attributed to a change in general motility. The results claim that aminosenktide causes an antidepressant effect, and endogenous opioids are involved in this process.