Anandamide hydrolysis: a new target for anti-anxiety drugs?

The major psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol, affects emotional states in humans and laboratory animals by activating brain cannabinoid receptors. A primary endogenous ligand of these receptors is anandamide, the amide of arachidonic acid with ethanolamine. Anandamide is released in selected regions of the brain and is deactivated through a two-step process consisting of transport into cells followed by intracellular hydrolysis. Pharmacological blockade of the enzyme fatty acid amide hydrolase (FAAH), which is responsible for intracellular anandamide degradation, produces anxiolytic-like effects in rats without causing the wide spectrum of behavioral responses typical of direct-acting cannabinoid agonists. These findings suggest that anandamide contributes to the regulation of emotion and anxiety, and that FAAH might be the target for a novel class of anxiolytic drugs.     

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.     

Tachykinin receptors and the airways.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.     

The NK-1 Receptor and a Calcium-Phospholipid Pathway: Inositol Trisphosphate Production and Calcium Movements Induced by Selective Agonists of Neurokinin Receptors in Rat Parotid Glands

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.     

Substance P induces migration of capillary endothelial cells: a novel NK-1 selective receptor mediated activity.

Substance P (SP) has been indicated as a main mediator of neurogenic inflammation, leading to vasodilation, increase in vascular permeability and modulation of immune cell function. Certain vascular effects produced by SP are endothelium mediated. We have studied the effect of SP and of selective NK-1, NK-2 and NK-3 receptor agonists on migration of cultured capillary endothelial cells of bovine origin. Our results indicate that SP (10(-14)-10(-6) M) induces a concentration-dependent migration of endothelial cells with maximal activity at 10(-10) M. This effect was mimicked by the selective NK-1 receptor agonist which showed a similar concentration-dependent curve, while selective NK-2 and NK-3 receptor agonists were ineffective. Our conclusions are that endothelial cells possess specific receptors for SP of the NK-1 type which affect mobilization of capillary endothelial cells.     

Progestin receptor-mediated reduction of anxiety-like behavior in male rats

Background

It is well known progesterone can have anxiolytic-like effects in animals in a number of different behavioral testing paradigms. Although progesterone is known to influence physiology and behavior by binding to classical intracellular progestin receptors, progesterone''s anxiety reducing effects have solely been attributed to its rapid non-genomic effects at the GABA_A receptor. This modulation occurs following the bioconversion of progesterone to allopregnanolone. Seemingly paradoxical results from some studies suggested that the function of progesterone to reduce anxiety-like behavior may not be entirely clear; therefore, we hypothesized that progesterone might also act upon progestin receptors to regulate anxiety.

Methodology/Principal Findings

To test this, we examined the anxiolytic-like effects of progesterone in male rats using the elevated plus maze, a validated test of anxiety, and the light/dark chamber in the presence or absence of a progestin receptor antagonist, RU 486. Here we present evidence suggesting that the anxiolytic-like effects of progesterone in male rats can be mediated, in part, by progestin receptors, as these effects are blocked by prior treatment with a progestin receptor antagonist.

Conclusion/Significance

This indicates that progesterone can act upon progestin receptors to regulate anxiety-like behavior in the male rat brain.     

Synthesis and biological activity of analogues of the C-terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues. Structure-activity studies.

Analogues of [Orn6]-SP6-11 have been synthesized in which the methionyl residue is replaced by glutamine gamma-carboxamide substituted derivatives. These analogues where tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by CONHCH3, CON(CH3)2, CONHPh and CONCH3Ph groups results in analogues which are full agonists in NK-1 and NK-2 preparations with the exception of the Glu[N(CH3)2]11 and the Glu(NHCH3)11 analogues, which are partial agonists at NK-1 and NK-2 receptors respectively. The Glu(NHCH3)11 analogue shows selectivity for the NK-1 receptor type and is equipotent to the Glu(NCH3Ph)11 analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK-2 preparation. The Glu(NHPh)11 analogue is a full agonist in the NK-3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine gamma-carboxamide substituted residues. It is concluded that for NK-1 receptor type the lipophilic character of Met11 side chain is not a determining factor for activity but it is an important factor for activity in the NK-2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.     

Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.     

The dehydroepiandrosterone sulfate influence on anxiety and depressive behaviour: participation of mu-opioid receptors

The dehydroepiandrosterone sulfate (DHEAS) influence on anxiety and depressive behaviour was studied in animals after chronic stress exposures. It is shown that DHEAS (30 mg/kg) reduced anxiety in submission (with increased level of stress-induced anxiety resulting from chronic (20-days) defeats in aggressive interactions) of male mice of CBA/Lac strain in the "partition" test and in the plus-maze test, whereas in the control--only in the plus-maze test. DHEAS injection (30 mg/kg) in male Wistar rats decreased depressive behaviour in the Porsolt test in control and multiple (shuttling on the laboratory scrambler for 18 days by 1 hour in day) stressed animals. NaItrexone (0.25 mg/kg in mice and 0.1 mg/kg in rats) blocked anxiolytic and antidepressant-like effects of DHEAS. The findings suggest that these DHEAS effects are mediated by mu-opioid receptors.     

Substance induces migration of capillary endothelial cells: A novel NK-1 selective receptor mediated activity

Substance P (SP) has been indicated as a main mediator of neurogenic inflammation, leading to vasodilation, increase in vascular permeability and modulation of immune cell function. Certain vascular effects produced by SP are endothelium mediated. We have studied the effect of SP and of selective NK-1, NK-2 and NK-3 receptor agonists on migration of cultured capillary endothelial cells of bovine origin. Our results indicate that SP (10⁻¹⁴–10⁻⁶ M) induces a concentration-dependent migration of endothelial cells with maximal activity at 10⁻¹⁰ M. This effect was mimicked by the selective NK-1 receptor agonist which showed a similar concentration-dependent curve, while selective NK-2 and NK-3 receptor agonists were ineffective. Our conclusions are that endothelial cells possess specific receptors for SP of the NK-1 type which affect mobilization of capillary endothelial cells.     

The 5,7-DHT-induced anticonflict effect is dependent on intact adrenocortical function.

We have recently reported that the anxiolytic-like effect observed in rats severely depleted of brain serotonin (5-HT) by means of 5,7-DHT is indirect and probably involves the GABA(A)/benzodiazepine chloride ionophore receptor complex (GABAA/BDZ-RC). One tentative explanation for this effect considered the involvement of corticosteroids. In the present series of experiments we have therefore investigated the effect of adrenalectomy (ADX) on the 5,7-DHT-induced anxiolytic-like effect displayed by rats in Vogel's conflict test. ADX totally abolished the anticonflict effect of the 5,7-DHT lesion. Replacement treatment with corticosterone, but not with dexamethasone, reinstated the anticonflict effect. These results indicate that an intact adrenocortical function, possibly via brain steroid type I receptors, is required for the expression of the 5,7-DHT-induced anxiolytic-like effect. It is postulated that ADX lowers the concentration of endogenous positive modulators at the GABAA/BDZ-RC to a level no longer sufficient to produce anxiolytic-like effects in 5,7-DHT-lesioned animals. The finding that 5,7-DHT-lesioned animals were more sensitive than sham-lesioned controls to the anticonflict effect of the barbiturate-like corticosteroid THDOC provides further support for the contention that an increased endogenous activity at the GABAA/BDZ-RCes is involved in the anxiolytic-like effect observed in rats with a severe depletion of brain 5-HT.     

Design, synthesis and structure-affinity relationships of 4-methylidenepiperidine and 4-aryl-1,2,3,6-tetrahydropyridine derivatives as corticotropin-releasing factor1 receptor antagonists

Recently, various non-peptide corticotropin-releasing factor1 (CRF1) receptor antagonists have been reported. Structure-affinity relationships (SARs) of non-peptide CRF antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Our interest focused on the Up-Area in deriving the novel methylidenepiperidine derivatives 8-10 and 4-aryl-1,2,3,6-tetrahydropyridine derivatives 11-13 as non-peptide CRF1 receptor antagonists. Compounds 8a and 11a had moderate affinity for CRF1 receptor, but compounds 9, 10, 12 and 13 did not exhibit CRF1 receptor affinity. Modification of derivatives 11 afforded compounds 11i (CRA1001) and 11x (CRA1000), which had high affinity and selectivity for CRF1 receptors with potent anxiolytic-like and antidepressant-like properties in some experimental animal models. These findings suggest that the hydrophonic unit (Up-Area) may be useful for design of CRF1 antagonists. We report here the design, synthesis and SARs of the derivatives 8 and 11 and isosteres 9, 10, 12 and 13.     

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.     

Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of substance P. Structure-activity relationships.

Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11 residue is replaced by glutamate gamma-alkylesters. These analogues were tested in three in vitro preparations representative of NK-1, NK-2, and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonists in NK-1 and NK-2 preparations but show little agonist activity in the NK-3 preparation. When the SCH3 group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH3 at NK-1 receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by gamma-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Orn6]-SP6-11. Other gamma-alkyl esters of the glutamic acid reduce its biological activity.     

The synergistic effect of selective sigma receptor agonists and uncompetitive NMDA receptor antagonists in the forced swim test in rats.

Data acquired to date show that some sigma receptor ligands reveal "antidepressant-like" activity in the forced swim test in mice and rats. Moreover, our preliminary results suggested that joint administration of sigma receptor ligands and amantadine (AMA, a glutamatergic/NMDA receptor antagonist) caused a positive interaction in the Porsolt test in rats, as had already been observed in the case of co-treatment with clinically active antidepressants and AMA. The aim of the present study was to examine the effect of combined administration of sigma1 or sigma2 receptor agonists, SA4503 or siramesine, respectively, and AMA or memantine (MEM) (uncompetitive NMDA receptor antagonist). SA4503 or siramesine given jointly with MEM (as well as with AMA) decreased the immobility time in rats. The effect of SA4503 and AMA co-administration was antagonized by progesterone, a sigma1 receptor antagonistic neurosteroid. Combined treatment with siramesine and AMA was modified by neither progesterone nor BD1047 (a novel sigma antagonist with preferential affinity for sigma1 sites); but it was counteracted by sulpiride and prazosin (a dopamine D2- and an alpha1-adrenergic receptor antagonist, respectively). The "antidepressant-like" effect induced by siramesine and MEM was not antagonized by progesterone, but was attenuated by BD1047, sulpiride and prazosin. The obtained results give support to the hypothesis that sigma (particularly sigma1) receptors may be one of the possible mechanisms by which drugs induce antidepressant-like activity in the forced swim test, and that this effect may be enhanced by NMDA receptor antagonists. Combined treatment with sigma ligands and AMA or MEM (applied in the clinic) may be an alternative to the treatment of antidepressant-resistant depressive patients in the future.     

Neurokinin-1 receptor antagonism by SR140333: enhanced in vivo ACh in the hippocampus and promnestic post-trial effects

Substance P (SP) has memory-promoting, reinforcing and anxiolytic-like effects when applied systemically or centrally. Such effects may be mediated by the neurokinin-1 (NK-1) receptor, since SP preferentially binds to this receptor. We measured the effects of a selective non-peptide NK-1 receptor antagonist, SR140333 (1, 3 and 9 mg/kg i.p.) on ACh levels in frontal cortex, amygdala and hippocampus by microdialysis and HPLC. Levels of ACh in the hippocampus increased dose-dependently immediately after treatment. The same doses of SR140333 given post-trial had minor facilitative effects on inhibitory avoidance learning and open-field habituation, but did not have reinforcing effects in a conditioned place preference (CPP) task. The selective action of NK-1 receptor antagonism on hippocampal ACh may be related to its positive influence on learning.     

Selective agonists for receptors of substance P and related neurokinins

Neurokinins and their receptors are a complex system consisting of at least three endogenous agents--substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)--and their corresponding receptor types, respectively, NK-1, NK-2, and NK-3. Investigations on receptors have been made using sensitive and fairly selective pharmacological preparations (the dog carotid artery for the NK-1, the rabbit pulmonary artery devoid of endothelium for the NK-2, and the rat portal vein for the NK-3 receptor), and some natural peptides of mammalian and nonmammalian origin. Because of the nonselectivity of the natural peptides, analogues of the neurokinins have been found that act on one receptor only and show therefore high selectivity. The selective agonists [Sar9,Met(O2)11]SP, [Nle10]NKA (4-10), and [MePhe7]-NKB have been used successfully for (a) characterizing the three neurokinin receptors, (b) identifying isolated organs whose responses to neurokinins depend on the activation of a single (monoreceptor systems) or of more than one (multireceptor systems) receptor, and (c) elucidating some of the physiological function of the three receptor types. It is suggested that NK-1 mediate peripheral vasodilatation and exocrine secretions, NK-2 stimulate bronchial muscles and facilitate the release of catecholamines, and NK-3 promote the release of acetylcholine in peripheral organs.     

The neurokinin-1 and neurokinin-2 receptor binding sites of MDL103,392 differ

Several small molecule non-peptide antagonists of the NK-1 and NK-2 receptors have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these non-peptide antagonists lies within the bundle created by transmembrane domains IV–VII of the receptor and differs from the binding sites of peptide agonists and antagonists. The current investigation uses site-directed mutagenesis of the NK-1 and NK-2 receptors to elucidate the amino acids that are important for binding and functional activity of the first potent dual NK-1/NK-2 antagonist MDL103,392. The amino acids found to be important for MDL103,392 binding to the NK-1 receptor are Gln-165, His-197, Leu-203, Ile-204, Phe-264, His-265 and Tyr-272. The amino acids found to be important for MDL103,392 binding to the NK-2 receptor are Gln-166, His-198, Tyr-266 and Tyr-289. While residues in transmembrane (TM) domains IV and V are important in both receptors (Gln-165/166 and His-197/198), residues in TM V and VI are more important for the NK-1 receptor and residues in TM VII play a more important role in the NK-2 receptor. These data are the first report of the analysis of the binding site of a dual tachykinin receptor antagonist and indicate that a single compound (MDL103,392) binds to each receptor in a different manner despite there being a high degree of homology in the transmembrane bundles. In addition, this is the first report in which a model for the binding of a non-peptide antagonist to the NK-2 receptor is proposed.     

Antidepressant- and Anxiolytic-Like Effects of New Dual 5-HT1A and 5-HT7 Antagonists in Animal Models

The aim of this study was to further characterize pharmacological properties of two phenylpiperazine derivatives: 1-{2-[2-(2,6-dimethlphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazynine hydrochloride (HBK-14) and 2-[2-(2-chloro-6-methylphenoxy)ethoxy]ethyl-4-(2- methoxyphenyl)piperazynine dihydrochloride (HBK-15) in radioligand binding and functional in vitro assays as well as in vivo models. Antidepressant-like properties were investigated in the forced swim test (FST) in mice and rats. Anxiolytic-like activity was evaluated in the four-plate test in mice and elevated plus maze test (EPM) in rats. Imipramine and escitalopram were used as reference drugs in the FST, and diazepam was used as a standard anxiolytic drug in animal models of anxiety. Our results indicate that HBK-14 and HBK-15 possess high or moderate affinity for serotonergic 5-HT₂, adrenergic α₁, and dopaminergic D₂ receptors as well as being full 5-HT_1A and 5-HT₇ receptor antagonists. We also present their potent antidepressant-like activity (HBK-14—FST mice: 2.5 and 5 mg/kg; FST rats: 5 mg/kg) and (HBK-15—FST mice: 1.25, 2.5 and 5 mg/kg; FST rats: 1.25 and 2.5 mg/kg). We show that HBK-14 (four-plate test: 2.5 and 5 mg/kg; EPM: 2.5 mg/kg) and HBK-15 (four-plate test: 2.5 and 5 mg/kg; EPM: 5 mg/kg) possess anxiolytic-like properties. Among the two, HBK-15 has stronger antidepressant-like properties, and HBK-14 displays greater anxiolytic-like activity. Lastly, we demonstrate the involvement of serotonergic system, particularly 5-HT_1A receptor, in the antidepressant- and anxiolytic-like actions of investigated compounds.     

Altered anxiety-related behavior in nociceptin/orphanin FQ receptor gene knockout mice

Studies showed that nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) agonists produce anxiolytic-like actions, while little is known about the effects of blockade of NOP receptor signaling in anxiety. To this aim, we investigated the behavioral phenotype of NOP receptor gene knockout mice (NOP(-/-)) in different assays. In the elevated plus-maze and light-dark box, NOP(-/-) mice displayed increased anxiety-related behavior. In the novelty-suppressed feeding behavior and elevated T-maze, NOP(-/-) mice showed anxiolytic-like phenotype, while no differences were found in the open-field, hole-board, marble-burying, and stress-induced hyperthermia. Altogether, these findings suggest that the N/OFQ-NOP receptor system modulates anxiety-related behavior in a complex manner.