A novel non-opioid binding site for endomorphin-1.

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors, such as rodent cerebellum, or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.

Cannabinoid CB1 receptor in the modulation of stress coping behavior in mice: the role of serotonin and different forebrain neuronal subpopulations.

The endocannabinoid system (ECS) may either enhance or inhibit responses to aversive stimuli, possibly caused by its modulatory activity on diverse neurotransmitters. The aim of this work was to investigate the involvement of serotonin (5-HT) and catecholamines, as well as the role of glutamatergic and GABAergic cannabinoid type 1 (CB(1)) receptor, in responses to the antidepressant-like doses of the CB(1) receptor agonist Δ(9)-tetrahydrocannabinol (THC) and the antagonist rimonabant in the forced swim test (FST). Mice received acute injections of low doses of THC (0.1 or 0.5 mg/kg) or high dose of rimonabant (3 or 10 mg/kg) after treatment with the 5-HT synthesis inhibitor pCPA (100 mg/kg, 4 days), the 5-HT(1A) receptor antagonist WAY100635 (1 mg/kg, acute) or the non-selective blocker of catecholamine synthesis, AMPT (20 mg/kg, acute). THC and rimonabant were also tested in mutant mice lacking CB(1) receptor in specific forebrain neuronal subpopulations. Both THC and rimonabant induced antidepressant-like effects, quantified as immobility in the FST. However, only THC effects were reversed by pCPA or WAY100635. In contrast, only AMPT could attenuate the rimonabant effect. We also found decreased immobility in mice lacking the CB(1) receptor in glutamatergic cortical neurons, but not in forebrain GABAergic neurons, as compared with wild-type controls. The effect of THC persisted in mutant mice with CB(1) receptor inactivation in GABAergic neurons, whereas rimonabant effects were alleviated in these mutants. Thus, employing both pharmacological and genetic tools, we could show that the ECS regulates stress responses by influencing GABAergic, glutamatergic and monoaminergic transmission. The antidepressant-like action of THC depends on serotonergic neurotransmission, whereas rimonabant effects are mediated by CB(1) receptor on GABAergic neurons and by catecholamine signaling.

Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: role of glutamatergic transmission.

To investigate the impact of averseness, controllability and familiarity of a test situation on the involvement of the endocannabinoid system in the regulation of exploratory behaviour, we tested conventional and conditional cannabinoid receptor type 1 (CB1)-deficient mice in behavioural paradigms with different emotional load, which depended on the strength of illumination and the ability of the animals to avoid the light stimulus. Complete CB1 null-mutant mice (Total-CB1-KO) showed an anxiogenic-like phenotype under circumstances where they were able to avoid the bright light such as the elevated plus-maze and the light/dark avoidance task. Conditional mutant mice lacking CB1 expression specifically in cortical glutamatergic neurons (Glu-CB1-KO), in contrast, failed to show a similar phenotype under the same experimental conditions. However, both mutant lines showed increased avoidance of open arm exploration during a second exposure to the elevated plus-maze. If tested in situations where the fear eliciting light could not be avoided, Total-CB1-KO mice showed increased thigmotaxis in an open field, decreased social investigation and decreased novel object exploration under aversive light conditions, but not under non-aversive low light. This time, Glu-CB1-KO also showed decreased exploratory behaviour towards objects and conspecific juveniles and increased thigmotaxis in the open field. Taking into consideration that the behavioural performance of wild-type mice was only marginally affected by changes in light intensities, these data indicate that the endocannabinoid system renders exploratory behaviour largely independent of the test averseness. This process differentially involves endocannabinoid-controlled glutamatergic transmission, depending on the controllability of the test situation.

Impaired cannabinoid receptor type 1 signaling interferes with stress-coping behavior in mice.

Dysregulation of the endocannabinoid system is known to interfere with emotional processing of stressful events. Here, we studied the role of cannabinoid receptor type 1 (CB1) signaling in stress-coping behaviors using the forced swim test (FST) with repeated exposures. We compared effects of genetic inactivation with pharmacological blockade of CB1 receptors both in male and female mice. In addition, we investigated potential interactions of the endocannabinoid system with monoaminergic and neurotrophin systems of the brain. Naive CB1 receptor-deficient mice (CB1-/-) showed increased passive stress-coping behaviors as compared to wild-type littermates (CB1+/+) in the FST, independent of sex. These findings were partially reproduced in C57BL/6N animals and fully reproduced in female CB1+/+ mice by pharmacological blockade of CB1 receptors with the CB1 receptor antagonist SR141716. The specificity of SR141716 was confirmed in female CB1-/- mice, where it failed to affect behavioral performance. Sensitivity to the antidepressants desipramine and paroxetine was preserved, but slightly altered in female CB1-/- mice. There were no genotype differences between CB1+/+ and CB1-/- mice in monoamine oxidase A and B activities under basal conditions, nor in monoamine content of hippocampal tissue after FST exposure. mRNA expression of vesicular glutamate transporter type 1 was unaffected in CB1-/- mice, but mRNA expression of brain-derived neurotrophic factor (BDNF) was reduced in the hippocampus. Our results suggest that impaired CB1 receptor function promotes passive stress-coping behavior, which, at least in part, might relate to alterations in BDNF function.

Identification of the cannabinoid receptor type 1 in serotonergic cells of raphe nuclei in mice.

The endocannabinoid system (ECS) possesses neuromodulatory functions by influencing the release of various neurotransmitters, including GABA, noradrenaline, dopamine, glutamate and acetylcholine. Even though there are studies indicating similar interactions between the ECS and the serotonergic system, there are no results showing clear evidence for type 1 cannabinoid receptor (CB1) location on serotonergic neurons. In this study, we show by in situ hybridization that a low but significant fraction of serotonergic neurons in the raphe nuclei of mice contains CB1 mRNA as illustrated by the coexpression with the serotonergic marker gene tryptophane hydroxylase 2, the rate limiting enzyme for the serotonin synthesis. Furthermore, by double immunohistochemistry and confocal microscopy, we were able to detect CB1 protein on serotonergic fibers and synapses expressing the serotonin uptake transporter in the hippocampus and the amygdala. Our findings indicate that the CB1-mediated regulation of serotonin release can depend in part on a direct cross-talk between the two systems at single cell level, which might lead to functional implications in the modulation of emotional states.

Endocannabinoids and the gastrointestinal tract.

In the past centuries, different preparations of marijuana have been used for the treatment of gastrointestinal (GI) disorders, such as GI pain, gastroenteritis and diarrhea. Delta9-tetrahydrocannabinol (THC; the active component of marijuana), as well as endogenous and synthetic cannabinoids, exert their biological functions on the gastrointestinal tract by activating two types of cannabinoid receptors, cannabinoid type 1 receptor (CB1 receptor) and cannabinoid type 2 receptor (CB2 receptor). While CB1 receptors are located in the enteric nervous system and in sensory terminals of vagal and spinal neurons and regulate neurotransmitter release, CB2 receptors are mostly distributed in the immune system, with a role presently still difficult to establish. Under pathophysiological conditions, the endocannabinoid system conveys protection to the GI tract, eg from inflammation and abnormally high gastric and enteric secretion. For such protective activities, the endocannabinoid system may represent a new promising therapeutic target against different GI disorders, including frankly inflammatory bowel diseases (eg, Crohn's disease), functional bowel diseases (eg, irritable bowel syndrome), and secretion- and motility-related disorders.

Effects of cannabinoids in Krox-24 targeted mice.

Krox-24 is an immediate early gene encoding a zinc-finger transcription factor implicated in several adaptive responses, and its induction by cannabinoids has been reported. We used mice targeted in the Krox-24 gene to specifically dissect the role of this protein in the acute and chronic central actions of cannabinoids. We report here on the ability of cannabinoids to activate G-proteins and to inhibit adenylyl cyclase, and to elicit behavioral responses in wild-type and mutant mice. The behavioral parameters and the biochemical correlates of abstinence after delta9-THC withdrawal were evaluated. We show that Krox-24 is not involved in the acute analgesic effects of delta9-THC and in the SR precipitated delta9-THC withdrawal syndrome.

Radioligand binding properties of VV-hemorphin 7, an atypical opioid peptide.

Receptor binding properties of the hemoglobin-derived nonapeptide VV-hemorphin 7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH) were studied using both the unlabelled form and tritium-labelled derivative of the peptide. In binding studies using selective opioid radioligands, VV-hemorphin 7 exhibited a rank order of potency of mu > kappa >> delta. VV-hemorphin 7 was tritiated resulting in a compound with 1.03 TBq/mmol (27.8 Ci/mmol) specific radioactivity. The maximal number of binding sites was found to be 66.5 pmol/mg protein with an affinity of 82.1 nM in rat brain membranes. In competition studies, marked similarity was observed to the binding profile of the naturally occurring opioid heptapeptide Met-enkephalin-Arg-Phe (MERF) and its analogues to their naloxone-insensitive binding site. The common -Arg-Phe sequence at the carboxyl terminal end, which is similar to those of other endogenous peptides (-Arg-Phe-NH(2) in neuropeptide FF and FMRF-NH(2)) brings attention to the C-terminal end of the molecule and points to the possible existence of a common nonopioid binding site in mammals.

Characterization of N,N(Me)2-Dmt-Tic-OH, a delta selective opioid dipeptide antagonist.

N,N(Me)2-Dimethyl-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-OH (N,N(Me)2-Dmt-Tic-OH) is a very selective delta opioid dipeptide with elevated antagonist activity. We have radiolabelled this compound by catalytic tritiation of the N,N(Me)2-Dmt(3',5'-I2)-Tic-OH precursor. The ligand labelled rat brain membranes with a Kd value of 0.42 nM and a Bmax of 63.12 fmol/mg protein. The new tritiated ligand showed high affinity for the delta opioid receptor whereas its binding at mu and kappa opioid receptors was weak. N,N(Me)2-Dmt-Tic-OH was able to inhibit the agonist-stimulated binding of the non-hydrolysable GTP analogue ¿35SGTPgammaS, thus attenuating the activation of G proteins via opioid receptors. This simple opioid dipeptide in both normal and labelled form may serve as a useful tool to study delta opioid receptors in vitro and in vivo.

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2.

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.

Deltorphin II analogues with 6-hydroxy-2-aminotetralin-2-carboxylic acid in position 1.

Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties. Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead of Tyr(1) in the message domain. In the radioreceptor-binding studies, in which type-specific tritiated opioid ligands were used, (R)- and (S)-Hat-deltorphins exhibited similar K(i) values, revealing high delta selectivity. The peptides displayed agonist properties in the in vitro bioassay, with IC(50) values in the subnanomolar range in the mouse vas deferens assay and in the micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity toward delta receptors. The agonist property of the new ligands was confirmed by means of [(35)S]GTPgammaS-binding experiments in membranes of the rat frontal cortex.

Mu-opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPgammaS binding assays.

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K(d) 3. 8+/-0.18 nM) to membranes of rat brain affording a B(max) of 87. 1+/-4.83 fmol/mg. Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for delta- ([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa- (5alpha,7alpha, 8beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPgammaS binding. The EC(50) value of morphine increased about 500-fold in the presence of 10 microM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization.

Nociceptin binding sites in frog (Rana esculenta) brain membranes.

The recently discovered natural heptadecapeptide nociceptin (orphanin FQ) shares some homology with the opioid peptides but it binds to a distinct receptor type, termed nociceptin receptor. This study demonstrates the presence of specific nociceptin recognition sites in brain membrane fractions of an amphibian, Rana esculenta. Para-iodo-Phe(1)-nociceptin-amide was radiolabelled by catalytic dehalotritiation, resulting in p[(3)H]Phe(1)-nociceptin-amide of 25 Ci/mmol specific radioactivity. Specific binding of [(3)H]nociceptin-amide to frog brain membranes was found to be saturable and of high affinity with equilibrium K(d) values in the low nanomolar range. A single set of binding sites with about 180 fmol/mg protein maximal binding capacity was obtained in saturation and competition experiments. [(3)H]Nociceptin-amide binding could easily be inhibited by synthetic nociceptin compounds but not by opioid ligands. Both sodium ions and 5'-guanylylimidodiphosphate decreased the binding of the radioligand by transferring the receptor to a lower affinity state. Nociceptin dose-dependently stimulated the binding of the nonhydrolysable, radiolabeled GTP-analogue guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) to G-proteins in frog brain membranes. Addition of 1 microM naloxone caused no significant change in the curves, indicating that nociceptin-mediated activation of G-proteins occurred through nonopioid mechanism.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans.

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.

In vitro binding and signaling profile of the novel mu opioid receptor agonist endomorphin 2 in rat brain membranes.

The recently discovered endogenous mu receptor selective endomorphin 2 was prepared in tritiated form by a catalytic dehalogenation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), and used for in vitro labelling of rat brain membranes. The binding was saturable, stereospecific and of high affinity (Kd: 0.97 and 1.12 nM based on kinetic and equilibrium binding studies, respectively). The maximal number of binding sites (Bmax) was found to be 114.8 fmol/mg protein. [3H]Endomorphin 2 was displaced by mu-receptor selective specific peptides and heterocyclic compounds with high affinity, whereas kappa and delta receptor specific ligands were much less potent. The Ki values of endomorphin 1 and 2 in inhibiting [3H]naloxone binding increased by 15-fold in the presence of 100 mM NaCl which indicates the agonist property of these peptides. Endomorphins stimulated [35S]GTPgammaS binding and inhibited adenylyl cyclase activity which also provides evidence for the agonist character of endomorphins.