Long-term systemic administration of kynurenic acid brain region specifically elevates the abundance of functional CB1 receptors in rats.

Kynurenic acid (KYNA) is one of the most significant metabolite of the kynurenine pathway both in terms of functional and potential therapeutic value. It is an N-methyl-D-aspartate (NMDA) receptor antagonist, but it can also activate the G-protein coupled receptor 35 (GPR35), which shares several structural and functional properties with cannabinoid receptors. Previously our group demonstrated that systemic chronic KYNA treatment altered opioid receptor G-protein activity. Opioid receptors also overlap in many features with cannabinoid receptors. Thus, our aim was to examine the direct in vitro and systemic, chronic in vivo effect of KYNA on type 1 cannabinoid receptor (CB1R) binding and G-protein activity. Based on competition and [35S]GTPγS G-protein binding assays in rat brain, KYNA alone did not show significant binding towards the CB1R, nor did it alter CB1R ligand binding and agonist activity in vitro. When rats were chronically treated with KYNA (single daily, i.p., 128 mg/kg for 9 days), the KYNA plasma and cerebrospinal fluid levels significantly increased compared to vehicle treated group. Furthermore, in G-protein binding assays, in the whole brain the amount of G-proteins in basal and in maximum activity coupled to the CB1R also increased due to the treatment. At the same time, the overall stimulatory properties of the receptor remained unaltered in vehicle and KYNA treated samples. Similar observations were made in rat hippocampus, but not in the cortex and brainstem. In saturation binding assays the density of CB1Rs in rat whole brain and hippocampus were also significantly enhanced after the same treatment, without significantly affecting ligand binding affinity. Thus, KYNA indirectly and brain region specifically increases the abundance of functional CB1Rs, without modifying the overall binding and activity of the receptor. Supposedly, this can be a compensatory mechanism on the part of the endocannabinoid system induced by the long-term KYNA exposure.

Kynurenic acid and its analogue can alter the opioid receptor G-protein signaling after acute treatment via NMDA receptor in rat cortex and striatum.

Previously, we have shown that the N-methyl d-aspartate (NMDA)-receptor antagonist kynurenic acid (KYNA) and its analogue KYNA1 do not bind directly to mu, kappa and delta opioid receptors in vitro. On the other hand, chronic administration of KYNA and KYNA1 resulted in region (cortex vs striatum) and opioid receptor-type specific alterations in G-protein activation of mouse brain homogenates. Here we describe for the first time the acute effect of KYNA and KYNA1 on opioid receptor function with the possible involvement of the NMDA receptor. The acute 30minute in vivo KYNA1 and KYNA treatments altered opioid receptor G-protein signaling or ligand potency depending on the opioid receptor type and brain region (rat cortex vs striatum) using [35S]GTPγS binding assays. Pretreatment with the NMDA receptor antagonist MK-801 impaired or reversed the effects of KYNA1 and KYNA. These results suggest an NMDA receptor mediated effect. After acute 30minute treatment HPLC measurements revealed a similar KYNA1 and a higher KYNA plasma concentration compared to cerebrospinal fluid concentrations. Finally, KYNA, KYNA1 and MK-801 showed comparable results in opioid receptor G-protein activity and ligand potency with acute in vivo treatments when they were administered in vitro for 30min on isolated cortex and striatum slices. We previously demonstrated that KYNA1 and KYNA acutely altered opioid receptor function in vivo and in vitro through the NMDA receptor depending on the opioid receptor type and brain region. This study may lead to a new, indirect approach to influence opioid receptor signaling.

Characterization of [3H] oxymorphone binding sites in mouse brain: Quantitative autoradiography in opioid receptor knockout mice.

Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [3H]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7nM and Bmax of 147fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [3H]oxymorphone in mouse brain. The distribution of [3H]oxymorphone binding sites was found to be similar to the selective MOP agonist [3H]DAMGO in the mouse brain. [3H]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [3H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone.

The effects of progesterone on the alpha2-adrenergic receptor subtypes in late-pregnant uterine contractions in vitro.

BACKGROUND: The adrenergic system and progesterone play major roles in the control of the uterine function. Our aims were to clarify the changes in function and expression of the α2-adrenergic receptor (AR) subtypes after progesterone pretreatment in late pregnancy. METHODS: Sprague Dawley rats from pregnancy day 15 were treated with progesterone for 7 days. The myometrial expressions of the α2-AR subtypes were determined by RT-PCR and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C) and spiroxatrine (α2A). The accumulation of myometrial cAMP was also measured. The activated G-protein level was investigated via GTPγS binding assays. RESULTS: Progesterone pretreatment decreased the contractile effect of (-)-noradrenaline through the α2-ARs. The most significant reduction was found through the α2B-ARs. The mRNA of all of the α2-AR subtypes was increased. Progesterone pretreatment increased the myometrial cAMP level in the presence of BRL 44408 (p < 0.001), spiroxatrine (p < 0.001) or the spiroxatrine + BRL 44408 combination (p < 0.05). Progesterone pretreatment increased the G-protein-activating effect of (-)-noradrenaline in the presence of the spiroxatrine + BRL 44408 combination. CONCLUSIONS: The expression of the α2-AR subtypes is progesterone-sensitive. It decreases the contractile response of (-)-noradrenaline through the α2B-AR subtype, blocks the function of α2A-AR subtype and alters the G protein coupling of these receptors, promoting a Gs-dependent pathway. A combination of α2C-AR agonists and α2B-AR antagonists with progesterone could be considered for the treatment or prevention of preterm birth.

The effects of estrogen on the α2-adrenergic receptor subtypes in rat uterine function in late pregnancy in vitro.

AIM: To assess the effect of 17ß-estradiol pretreatment on the function and expression of α2- adrenergic receptors (ARs) subtypes in late pregnancy in rats. METHODS: Sprague-Dawley rats (n=37) were treated with 17ß-estradiol for 4 days starting from the 18th day of pregnancy. The myometrial expression of the α2-AR subtypes was determined by real time polymerase chain reaction and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C), and spiroxatrine (α2A). The cyclic adenosine monophosphate (cAMP) accumulation was also measured. The activated G-protein level was investigated by guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding assay. RESULTS: 17ß-estradiol pretreatment decreased the contractile effect of (-)-noradrenaline via the α2-ARs, and abolished the contractile effect via the α2B-ARs. All the α2-AR subtypes' mRNA was significantly decreased. 17ß-estradiol pretreatment significantly increased the myometrial cAMP level in the presence of BRL 44408 (P=0.001), ARC 239 (P=0.007), and spiroxatrine (P=0.045), but did not modify it in the presence of spiroxatrine + BRL 44408 combination (P=0.073). It also inhibited the G-protein-activating effect of (-)-noradrenaline by 25% in the presence of BRL 44408 + spiroxatrine combination. CONCLUSIONS: The expression of the α2-AR subtypes is sensitive to 17ß-estradiol, which decreases the contractile response of (-)-noradrenaline via the α2B-AR subtype, and might cause changes in G-protein signaling pathway. Estrogen dysregulation may be responsible for preterm labor or uterine inertia via the α2-ARs.

The effects of female sexual hormones on the expression and function of α1A- and α1D-adrenoceptor subtypes in the late-pregnant rat myometrium.

The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pregnant rat uterus in vitro by the administration of subtype-specific antagonists (the α1A-adrenoceptor antagonist WB 4101 and the α1D-adrenoceptor antagonist BMY 7378) after 17ß-estradiol or progesterone pretreatment. In isolated organ bath studies, contractions were elicited with (-)-noradrenaline (10(-8)-10(-5)M) in the presence of propranolol (10(-5)M) and yohimbine (10(-6)M) in order to avoid ß-, and α2-adrenergic action. The myometrial expressions of the α1-adrenoceptor subtypes were determined by means of the real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques. The activated G protein levels were investigated through radiolabelled GTP binding assays. Both 17ß-estradiol and progesterone pretreatment changed the myometrial contracting effect of (-)-noradrenaline. In the presence of WB 4101, progesterone pretreatment decreased the (-)-noradrenaline-induced myometrial contraction. In the presence of BMY 7378, both the 17ß-estradiol and the progesterone pretreatment reduced the effect of (-)-noradrenaline. The mRNA and protein expressions of the α1A-adrenoceptors were decreased after 17ß-estradiol pretreatment. (-)-Noradrenaline increased the [(35)S]GTPγS binding of the α1-adrenoceptors, which was most markedly elevated by progesterone. Pertussis toxin inhibited the [(35)S]GTPγS binding-stimulating effect of (-)-noradrenaline, indicating the role of Gi proteins in the signal mechanisms. 17ß-estradiol pretreatment blocks the expression of the α1A-adrenoceptors, whereas it does not influence the expression of the α1D-adrenoceptors. Progesterone pretreatment does not have any effect on the myometrial mRNA and protein expressions of the α1-adrenoceptors, but it alters the G protein coupling of these receptors, promoting a Gi-dependent pathway.

Further Characterization of Hemopressin Peptide Fragments in the Opioid and Cannabinoid Systems.

BACKGROUND: Hemopressin, so-called because of its hypotensive effect, belongs to the derivatives of the hemoglobin α-chain. It was isolated from rat brain membrane homogenate by the use of catalytically inactive forms of endopeptidase 24.15 and neurolysin. Hemopressin has antihyperalgesic features that cannot be prevented by the opioid receptor antagonist, naloxone. METHODS: In the present study, we investigated whether hemopressin (PVNFKFLSH) and its C-terminally truncated fragment hemopressin 1-7 (PVNFKFL) have any influence on opioid-dependent signaling. Peptides have been analyzed using G-protein-stimulating functional and receptor bindings in this experimental setup. RESULTS: These 2 compounds efficiently activated the G-proteins, and naloxone slightly blocked this stimulation. At the same time, they were able to displace radiolabeled [3H]DAMGO, a selective ligand for µ-opioid system, at micromolar concentrations. Displacement caused by the heptapeptide was more modest compared with hemopressin. Experiments performed on cell lines overexpressing µ-opioid receptors verified the opioid activity of both hemopressins. Moreover, the CB1 cannabinoid receptor antagonist, AM251, significantly decreased their G-protein stimulatory effect. CONCLUSIONS: Here, we further confirm that hemopressins can modulate CB1 receptors and can have a slight modulatory effect on the opioid system.

Design, Synthesis and Biological Evaluation of Two Opioid Agonist and Cav 2.2 Blocker Multitarget Ligands.

N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first ω-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of ω-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity.

Low dosage of rimonabant leads to anxiolytic-like behavior via inhibiting expression levels and G-protein activity of kappa opioid receptors in a cannabinoid receptor independent manner.

WHAT IS KNOWN: There is an increasing number of studies demonstrating the direct effect of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant on the opioid system. The kappa opioid receptors (KORs) are well known to mediate depression- and anxiety-like behavior. Clinical studies on chronic rimonabant administration have revealed that rimonabant leads to a very similar pathophysiology, suggesting a potential impact of rimonabant on KORs. OBJECTIVES: Our objectives were to examine the putative effects of rimonabant on KOR ligand binding, G-protein activity, protein expression and how all these contribute to the development of depression- and anxiety-like behavior. RESULTS: In Chinese hamster ovary (CHO) cell membranes transfected with rat KOR (CHO-rKOR) rimonabant inhibited KOR agonist [3H]U69593 binding in the micromolar range in competition binding experiments and specifically reduced KOR basal activity at lower micromolar concentrations in [35S]GTPγS binding assays. Rimonabant significantly inhibited dynorphin (1-11)-induced [35S]GTPγS binding in micromolar range in CHO-rKOR cells, CB1 knockout (CB1 K.O.) and CB1/CB2 double knockout mouse forebrain membranes. A single dose of i.p. 0.1 mg/kg rimonabant significantly reduced dynorphin (1-11)-induced KOR G-protein activity and KOR protein expression levels 24 h following the administration in both wild type and CB1 K.O. mice forebrain. Furthermore, in elevated plus maze mice showed an anxiolytic-like effect upon rimonabant injection that could be reversed by 1 mg/kg KOR antagonist norbinaltorphimine. The anxiolytic-like effects were further confirmed with the light­dark box test. CONCLUSION: Rimonabant reduced KOR ligand binding, receptor mediated G-protein activity and protein expression level, which overall leads to altered anxiety-like behavior.

Inhibition of opioid receptor mediated G-protein activity after chronic administration of kynurenic acid and its derivative without direct binding to opioid receptors.

There is an increasing number of evidence showing analgesic properties of the kynurenic acid (KYNA), and also some studies demonstrate that kynurenine might interact with the opioid system. Therefore in this study, for the first time we investigated the direct binding affinity of KYNA and its structural analog KYNA-1 towards mu, kappa and delta opioid receptor in competition binding experiments applying opioid receptor specific radioligands. The binding affinity measurements were performed in Chinese hamster ovary cell lines overexpressing the corresponding opioid receptor (mu and kappa opioid receptor were rat, delta opioid receptor were mouse sequence). Additionally we also examined the chronic effect of these compounds on mu, kappa and delta opioid receptor and also nociceptin peptide receptor mediated G-protein activity in [(35)S]GTPγS binding assays performed in mouse cortex and striatum membranes. Our results showed that KYNA and KYNA-1 had no affinity towards any of the three classic opioid receptors. On the other hand the compounds significantly decreased opioid and nociceptin receptor mediated G-protein activity or in some cases enhanced the potency of the activating ligand. Moreover, the alterations were receptor and brain region specific. Accordingly, we conclude that KYNA and KYNA-1 do not interact directly with the opioid receptors, but more likely alter the receptor functions intracellularly.