Heterodimerization With 5-HT2BR Is Indispensable for ß2AR-Mediated Cardioprotection.

RATIONALE: The ß2-adrenoceptor (ß2-AR), a prototypical GPCR (G protein-coupled receptor), couples to both Gs and Gi proteins. Stimulation of the ß2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-PI3K-Akt cell survival pathway. Cardiac ß2-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. OBJECTIVE: Here, we aim to investigate the potential cardioprotective effect of ß2-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of ß2-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT2BRs). METHODS AND RESULTS: Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the ß2-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H2O2) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H2O2 or hypoxia/reoxygenation. Mechanistically, we found that MNF or another ß2-agonist zinterol markedly promoted heterodimerization of ß2-ARs with 5-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and ß2-ARs enhanced ß2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT2BR attenuated ß2-AR-stimulated Gi signaling and cardioprotection. CONCLUSIONS: These data demonstrate that the ß2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of ß2-ARs and 5-HT2BRs.

NMDAR inhibition-independent antidepressant actions of ketamine metabolites.

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

Ketamine in the Past, Present, and Future: Mechanisms, Metabolites, and Toxicity.

PURPOSE OF REVIEW: While ketamine's analgesia has mostly been attributed to antagonism of N-methyl-D-aspartate receptors, evidence suggests multiple other pathways are involved in its antidepressant and possibly analgesic activity. These mechanisms and ketamine's role in the nociplastic pain paradigm are discussed. Animal studies demonstrating ketamine's neurotoxicity have unclear human translatability and findings from key rodent and human studies are presented. RECENT FINDINGS: Ketamine's metabolites, and (2R,6R)-hydroxynorketamine in particular, may play a greater role in its clinical activity than previously believed. The activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and the mammalian target of rapamycin by ketamine are mechanisms that are still being elucidated. Ketamine might work best in nociplastic pain, which involves altered pain processing. While much is known about ketamine, new studies will continue to define its role in clinical medicine. Evidence supporting ketamine's neurotoxicity in humans is lacking and should not impede future ketamine clinical trials.

Discovery of ß-arrestin-biased ß2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives.

ß-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). ß-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the ß-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of ß2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their ß-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or the cardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter ß-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and ß-arrestin recruitment were applied to the Black-Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of ß-arrestin-biased ß2-adrenoceptor agonists, whereas salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

d-Serine is a potential biomarker for clinical response in treatment of post-traumatic stress disorder using (R,S)-ketamine infusion and TIMBER psychotherapy: A pilot study.

Post-traumatic stress disorder (PTSD) is a chronic and debilitating condition that is often refractory to standard frontline antidepressant therapy. A promising new approach to PTSD therapy is administration of a single sub-anesthetic dose of (R,S)-ketamine (Ket). The treatment produces rapid and significant therapeutic response, which lasts for only 4-7 days. In one of our studies, the mean duration of response was increased to 33 days when Ket administration was combined with a mindfulness-based cognitive therapy, Trauma Interventions using Mindfulness Based Extinction and Reconsolidation (TIMBER). We now report the results from a 20-patient study, which examined the duration of sustained response with combined TIMBER-Ket therapy, TIMBER-K arm, relative to the response observed in a placebo-controlled arm, TIMBER-P. A significant difference in the duration of response was observed between TIMBER-K and TIMBER-P arms: 34.44 ±â€¯19.12 days and 16.50 ±â€¯11.39 days, respectively (p = 0.022). Previous studies identified a negative correlation between antidepressant response to Ket and basal plasma concentrations of d-serine (DSR). In this study, the basal DSR levels positively correlated with the pre-treatment severity of PTSD symptoms (Pearson's r = 0.42, p = 0.07) and patients with basal DSR level ≥ 3.5 µM displayed not only higher PTSD severity but also shorter duration of response. The data indicate that basal DSR levels may serve as a biomarker of the severity of PTSD symptoms and as a predictor of clinical response. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

Stereochemical and structural effects of (2R,6R)-hydroxynorketamine on the mitochondrial metabolome in PC-12 cells.

BACKGROUND: Impairment in mitochondrial biogenesis and function plays a key role in depression and anxiety, both of which being associated with changes in fatty acid and phospholipid metabolism. The antidepressant effects of (R,S)-ketamine have been linked to its conversion into (2S,6S;2R,6R)-hydroxynorketamine (HNK); however, the connection between structure and stereochemistry of ketamine and HNK in the mitochondrial homeostatic response has not yet been fully elucidated at a metabolic level. METHODS: We used a multi-platform, non-targeted metabolomics approach to study the change in mitochondrial metabolome of PC-12 cells treated with ketamine and HNK enantiomers. The identified metabolites were grouped into pathways in order to assess global responses. RESULTS: Treatment with (2R,6R)-HNK elicited the significant change in 49 metabolites and associated pathways implicated in fundamental mitochondrial functions such as TCA cycle, branched-chain amino acid biosynthetic pathway, glycoxylate metabolic pathway, and fatty acid ß-oxidation. The affected metabolites included glycerate, citrate, leucine, N,N-dimethylglycine, 3-hexenedioic acid, and carnitine and attenuated signals associated with 9 fatty acids and elaidic acid. Important metabolites involved in the purine and pyrimidine pathways were also affected by (2R-6R)-HNK. This global metabolic profile was not as strongly impacted by treatment with (2S,6S)-HNK, (R)- and (S)-ketamine and in some instances opposite effects were observed. CONCLUSIONS: The present data provide an overall view of the metabolic changes in mitochondrial function produced by (2R,6R)-HNK and related ketamine compounds and offer an insight into the source of the observed variance in antidepressant response elicited by the compounds.

GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts.

The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to l-lactate and is associated with upregulated expression of HIF-1α and activation of the EGFR-MEK-ERK, Wnt-ß-catenin, and PI3K-AKT signaling pathways. (R,R')-4'-methoxy-1-naphthylfenoterol ((R,R')-MNF) significantly reduces proliferation, survival, and motility of PANC-1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R')-MNF's effect on glycolysis in PANC-1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R')-MNF-treated cells. LC/MS analysis was used to quantify intracellular concentrations of ß-hydroxybutyrate, carnitine, and l-lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC-1 tumor xenografts after administration of (R,R')-MNF. Metabolomics data indicate that (R,R')-MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of ß-hydroxybutyrate and carnitine while reducing l-lactate content. The cellular content of phosphoinositide-dependent kinase-1 and hexokinase 2 was reduced consistent with diminished PI3K-AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R')-MNF. Mice treated with (R,R')-MNF had significant accumulation of l-lactate in tumor tissue relative to vehicle-treated mice, together with reduced levels of the selective l-lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, ß-catenin, hexokinase 2, and p-glycoprotein were also observed. The data suggest that (R,R')-MNF reduces glycolysis in PANC-1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway.

Effects of Ketamine and Ketamine Metabolites on Evoked Striatal Dopamine Release, Dopamine Receptors, and Monoamine Transporters.

Following administration at subanesthetic doses, (R,S)-ketamine (ketamine) induces rapid and robust relief from symptoms of depression in treatment-refractory depressed patients. Previous studies suggest that ketamine's antidepressant properties involve enhancement of dopamine (DA) neurotransmission. Ketamine is rapidly metabolized to (2S,6S)- and (2R,6R)-hydroxynorketamine (HNK), which have antidepressant actions independent of N-methyl-d-aspartate glutamate receptor inhibition. These antidepressant actions of (2S,6S;2R,6R)-HNK, or other metabolites, as well as ketamine's side effects, including abuse potential, may be related to direct effects on components of the dopaminergic (DAergic) system. Here, brain and blood distribution/clearance and pharmacodynamic analyses at DA receptors (D1-D5) and the DA, norepinephrine, and serotonin transporters were assessed for ketamine and its major metabolites (norketamine, dehydronorketamine, and HNKs). Additionally, we measured electrically evoked mesolimbic DA release and decay using fast-scan cyclic voltammetry following acute administration of subanesthetic doses of ketamine (2, 10, and 50 mg/kg, i.p.). Following ketamine injection, ketamine, norketamine, and multiple hydroxynorketamines were detected in the plasma and brain of mice. Dehydronorketamine was detectable in plasma, but concentrations were below detectable limits in the brain. Ketamine did not alter the magnitude or kinetics of evoked DA release in the nucleus accumbens in anesthetized mice. Neither ketamine's enantiomers nor its metabolites had affinity for DA receptors or the DA, noradrenaline, and serotonin transporters (up to 10 µM). These results suggest that neither the side effects nor antidepressant actions of ketamine or ketamine metabolites are associated with direct effects on mesolimbic DAergic neurotransmission. Previously observed in vivo changes in DAergic neurotransmission following ketamine administration are likely indirect.

Selective GPR55 antagonism reduces chemoresistance in cancer cells.

G protein-coupled receptor 55 (GPR55) possesses pro-oncogenic activity and its function can be competitively inhibited with (R,R')-4'-methoxy-1-naphthylfenoterol (MNF) through poorly defined signaling pathways. Here, the anti-tumorigenic effect of MNF was investigated in the human pancreatic cancer cell line, PANC-1, by focusing on the expression of known cancer biomarkers and the expression and function of multidrug resistance (MDR) exporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Incubation of PANC1 cells with MNF (1µM) for 24h significantly decreased EGF receptor, pyruvate kinase M2 (PKM2), and ß-catenin protein levels and was accompanied by significant reduction in nuclear accumulation of HIF-1α and the phospho-active forms of PKM2 and ß-catenin. Inhibition of GPR55 with either MNF or the GPR55 antagonist CID 16020046 lowered the amount of MDR proteins in total cellular extracts while diminishing the nuclear expression of Pgp and BCRP. There was significant nuclear accumulation of doxorubicin in PANC-1 cells treated with MNF and the pre-incubation with MNF increased the cytotoxicity of doxorubicin and gemcitabine in these cells. Potentiation of doxorubicin cytotoxicity by MNF was also observed in MDA-MB-231 breast cancer cells and U87MG glioblastoma cells, which express high levels of GPR55. The data suggest that inhibition of GPR55 activity produces antitumor effects via attenuation of the MEK/ERK and PI3K-AKT pathways leading to a reduction in the expression and function of MDR proteins.

Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ß2-adrenoceptor.

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, ß2-adrenoceptor (ß2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of ß2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of ß2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most ß2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates ß2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on ß2-AR essential for Gs-biased signaling. Following stimulation with a ß2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type ß2-AR. Interestingly, Y308F substitution on ß2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering ß2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of ß2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of ß2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Recruitment of ß-arrestin 1 and 2 to the ß2-adrenoceptor: analysis of 65 ligands.

UNLABELLED: Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at ß2-adrenoceptors (ß2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or ß-arrestins (ßarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for ßarr recruitment experiments based on a split-luciferase assay in a cellular system expressing ß2AR. Many agonists showed only (weak) partial agonism regarding ßarr recruitment. Potencies and/or efficacies increased depending on the number of chirality centers in (R) configuration; no (S)-configured distomer was more effective at inducing ßarr recruitment other than the eutomer. ßarr2 was recruited more effectively than ßarr1. The analysis of antagonists revealed no significant effects on ßarr recruitment. Several agonists showed preference for activation of Gαs GTPase relative to ßarr recruitment, and no ßarr-biased ligand was identified. IN CONCLUSION: 1) agonists show strong bias for Gαs activation relative to ßarr recruitment; 2) agonists recruit ßarr1 and ßarr2 with subtle differences; and 3) there is no evidence for ßarr recruitment by antagonists.

(R,S)-Ketamine metabolites (R,S)-norketamine and (2S,6S)-hydroxynorketamine increase the mammalian target of rapamycin function.

BACKGROUND: Subanesthetic doses of (R,S)-ketamine are used in the treatment of neuropathic pain and depression. In the rat, the antidepressant effects of (R,S)-ketamine are associated with increased activity and function of mammalian target of rapamycin (mTOR); however, (R,S)-ketamine is extensively metabolized and the contribution of its metabolites to increased mTOR signaling is unknown. METHODS: Rats (n = 3 per time point) were given (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine and their effect on the mTOR pathway determined after 20, 30, and 60 min. PC-12 pheochromocytoma cells (n = 3 per experiment) were treated with escalating concentrations of each compound and the impact on the mTOR pathway was determined. RESULTS: The phosphorylation of mTOR and its downstream targets was significantly increased in rat prefrontal cortex tissue by more than ~2.5-, ~25-, and ~2-fold, respectively, in response to a 60-min postadministration of (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine (P < 0.05, ANOVA analysis). In PC-12 pheochromocytoma cells, the test compounds activated the mTOR pathway in a concentration-dependent manner, which resulted in a significantly higher expression of serine racemase with ~2-fold increases at 0.05 nM (2S,6S)-hydroxynorketamine, 10 nM (R,S)-norketamine, and 1,000 nM (R,S)-ketamine. The potency of the effect reflected antagonistic activity of the test compounds at the α7-nicotinic acetylcholine receptor. CONCLUSIONS: The data demonstrate that (R,S)-norketamine and (2S,6S)-hydroxynorketamine have potent pharmacological activity both in vitro and in vivo and contribute to the molecular effects produced by subanesthetic doses of (R,S)-ketamine. The results suggest that the determination of the mechanisms underlying the antidepressant and analgesic effects of (R,S)-ketamine requires a full study of the parent compound and its metabolites.

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2-AR) agonist [(3)H]-(R,R')-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series of 4'-methoxyfenoterol analogs in which the length of the alkyl substituent at α' position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [(3)H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC50 values, were determined in HEK293 cells expressing the ß2-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α' position. The results also indicate that the Ki values obtained using [(3)H]-(R,R')-methoxyfenoterol as the marker ligand modeled the EC50 values obtained from cAMP stimulation better than the data obtained using [(3)H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the ß2-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized ß2-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the ß2-AR is governed to a greater extend by steric effects than binding to the [(3)H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.

Synthesis, in vitro and in vivo studies, and molecular modeling of N-alkylated dextromethorphan derivatives as non-competitive inhibitors of α3ß4 nicotinic acetylcholine receptor.

9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3ß4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3ß4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3ß4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3ß4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction.

Observational study of the effect of ketamine infusions on sedation depth, inflammation, and clinical outcomes in mechanically ventilated patients with SARS-CoV-2.

Severely ill patients with COVID-19 are challenging to sedate and often require high-dose sedation and analgesic regimens. Ketamine can be an effective adjunct to facilitate sedation of critically ill patients but its effects on sedation level and inflammation in COVID-19 patients have not been studied. This retrospective, observational cohort study evaluated the effect of ketamine infusions on inflammatory biomarkers and clinical outcomes in mechanically ventilated patients with SARS-CoV-2 infection. A total of 186 patients were identified (47 received ketamine, 139 did not). Patients who received ketamine were significantly younger than those who did not (mean (standard deviation) 59.2 (14.2) years versus 66.3 (14.4) years; P = 0.004), but there was no statistically significant difference in body mass index (P = 0.25) or sex distribution (P = 0.91) between groups. Mechanically ventilated patients who received ketamine infusions had a statistically significant reduction in Richmond Agitation-Sedation Scale score (-3.0 versus -2.0, P < 0.001). Regarding inflammatory biomarkers, ketamine was associated with a reduction in ferritin (P = 0.02) and lactate (P = 0.01), but no such association was observed for C-reactive protein (P = 0.27), lactate dehydrogenase (P = 0.64) or interleukin-6 (P = 0.87). No significant association was observed between ketamine administration and mortality (odds ratio 0.971; 95% confidence interval 0.501 to 1.882; P = 0.93). Ketamine infusion was associated with improved sedation depth in mechanically ventilated COVID-19 patients and provided a modest anti-inflammatory benefit but did not confer benefit with respect to mortality or intensive care unit length of stay.

Thermodynamics and docking of agonists to the ß(2)-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand.

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the ß(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total ß(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The ß(2)-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K(i) values determined for fenoterol analogs model much better the cAMP activity of the ß(2)-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [(3)H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel ß(2)-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the ß(2)-adrenoceptor.

Cannabinoid receptor activation correlates with the proapoptotic action of the ß2-adrenergic agonist (R,R')-4-methoxy-1-naphthylfenoterol in HepG2 hepatocarcinoma cells.

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ß(2)-adrenergic receptor (ß(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ß(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of ß(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ß(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ß(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ß(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ß(2)-AR-CBR ligand.

Capillary electrophoresis-laser-induced fluorescence (CE-LIF) assay for measurement of intracellular D-serine and serine racemase activity.

An enantioselective capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for the analysis of D-serine (D-Ser) in cellular matrices has been developed. The assay involves derivatization with FITC followed by CE-LIF using 0.5 mM hydroxyl propyl-ß-cyclodextrin in borate buffer [80 mM, pH 9.3]. The method was able to resolve D-Ser and L-Ser with an enantioselectivity (α) of 1.03 and a resolution (R(s)) of 1.37. Linearity was established from 0.25 to 100.00 µM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intracellular D-Ser concentrations in PC-12, C6, 1312N1, and HepG2 cell lines. This method was used to determine the concentration-dependent increases in D-Ser and associated EC50 values produced by L-Ser and the concentration-dependent decreases in d-Ser and associated IC50 values produced by glycine, a competitive inhibitor of serine racemase (SR). Western blot analysis determined that the PC-12 and C6 cell lines contained monomeric and dimeric forms of SR while the 1321N1 and HepG2 cells contained only the monomeric form. Although the SR dimer has been identified as the active form of the enzyme, all four of the tested cell lines expressed enzymatically active SR.

Simultaneous population pharmacokinetic modelling of ketamine and three major metabolites in patients with treatment-resistant bipolar depression.

AIM: To construct a population pharmacokinetic (popPK) model for ketamine (Ket), norketamine (norKet), dehydronorketamine (DHNK), hydroxynorketamine (2S,6S;2R,6R)-HNK) and hydroxyketamine (HK) in patients with treatment-resistant bipolar depression. METHOD: Plasma samples were collected at 40, 80, 110, 230 min on day 1, 2 and 3 in nine patients following a 40 min infusion of (R,S)-Ket (0.5 mg kg⁻¹) and analyzed for Ket, norKet and DHNK enantiomers and (2S,6S;2R,6R)-HNK, (2S,6S;2R,6R)-HK and (2S,6R;2R,6S)-HK. A compartmental popPK model was constructed that included all quantified analytes, and unknown parameters were estimated with an iterative two-stage algorithm in ADAPT5. RESULTS: Ket, norKet, DHNK and (2S,6S;2R,6R)-HNK were present during the first 230 min post infusion and significant concentrations (>5 ng ml⁻¹) were observed on day 1. Plasma concentrations of (2S,6S;2R,6R)-HK and (2S,6R;2R,6S)-HK were below the limit of quantification. The average (S) : (R) plasma concentrations for Ket and DHNK were <1.0 while no significant enantioselectivity was observed for norKet. There were large inter-patient variations in terminal half-lives and relative metabolite concentrations; at 230 min (R,S)-DHNK was the major metabolite in four out of nine patients, (R,S)-norKet in three out of nine patients and (2S,6S;2R,6R)-HNK in two out of nine patients. The final PK model included three compartments for (R,S)-Ket, two compartments for (R,S)-norKet and single compartments for DHNK and HNK. All PK profiles were well described, and parameters for (R,S)-Ket and (R,S)-norKet were in agreement with prior estimates. CONCLUSION: This represents the first PK analysis of (2S,6S;2R,6R)-HNK and (R,S)-DHNK. The results demonstrate that while norKet is the initial metabolite, it is not the main metabolite suggesting that future Ket studies should include the analysis of the major metabolites.