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New molecular insights into the mechanism of lithium action at the dopaminergic D2 receptor
P.1.g. Basic and clinical neuroscience − Neuropharmacology [3] Conrad CD., 2008. Chronic Stress-Induced Hippocampal Vulnerability, The Glucocorticoid Vulnerability Hypothesis, Rev Neurosci. 19(6), 395–411.
P.1.g.044 New molecular insights into the mechanism of lithium action at the dopaminergic D2 receptor Caballero1 ° ,
Pastor2 ,
Fabritiis2 ,
Selent2
J. Le´on M. G. De J. 1 Insitut de neuropsiquiatria i addiccions, Psiquiatria, Barcelona, Spain; 2 Pompeu Fabra University UPF - Hospital del Mar Medical Research Institute IMIM, Research Programme on Biomedical Informatics GRIB, Barcelona, Spain Introduction: Despite the therapeutic relevance of lithium in the treatment of psychiatric disorders, its mechanism of action remains unknown. Several of the proposed mechanisms of action of lithium are related to the downstream signaling of G proteincoupled receptors (GPCRs) (e.g. inositol depletion, interference with cAMP mediated processes or disruption of the Akt/bArr2/PP2A complex) [1]. The pharmacological response of GPCRs is typically triggered by ligand binding to the orthosteric site, but can be fine-tuned by allosteric modulation (monovalent cations are an example of the agents that can exert such allosteric modulation) [2]. The dopamine D2 receptor (D2R) is a GPCR, and our group previously simulated a cation-binding site for this receptor. There a Na+ ion is trapped in a hydrogen-bonding network (HBN), stabilizing the receptor in a distinct conformation [3]. The ability of other monovalent cations that may interact with the D2R cationbinding site has not been explored yet. Aim of the study: The similarity of Na+ with the Li+ ion suggests that the mechanism of Li+ pharmacological activity could involve competing for binding with Na+ deep inside receptor the D2R and, by doing so, destabilizing the sodium-induced GPCR conformation. Methods: A realistic three-dimensional structure of the D2R was modelled using a previously reported modeling protocol [3]. Three all-atom models were generated using the AMBER99SB force-field and simulated using ACEMD software. The three simulated all-atom systems consisted of the dopaminergic D2 receptor embedded in a hydrated membrane bilayer environment ionized up to a physiological ionic strength of 150 mM K+, Na+ or Li+ ions. Results: All three ions are able to spontaneously penetrate the D2R. The propensity of ions to enter the D2 receptor is higher for larger-sized Na+ and K+ ions. In addition, there is a preference of smaller Li+ and Na+ ions to bind at the allosteric site over the orthosteric site. The most interesting observation of our simulation is an ion size dependent binding pattern at the extracellular loop (ECL) region that produces changes on the conformation of the receptor entrance. The binding of lithium at ECL produces a structural rearrangement of the ECL region. Interestingly, this closed loop conformation seems to be specific for the small Li+ ion. Conclusions: The most important finding is the formation of an unprecedented ionic salt bridge at the extracellular receptor region (at the ECL region) induced by lithium that can have major implications for D2R functionality. This results in an obstruction of the receptor entrance. Since the ECL is known to serve as a “lid” of the receptor entrance, the observed Li+-induced partial receptor closure will most likely affect the entrance/exit of small molecules into/out of the binding pocket. The tendency of Li+
S239
ions to partially close the D2 receptor entrance suggests an ioninduced impact on the kinetics of ligand binding. There are several cases reported of severe neurological toxicity in patients treated in combination with lithium and haloperidol [4], and our study could partially explain the molecular mechanisms underlying this effect. References [1] Beaulieu, J.M., Caron, M.G., 2008. Looking at lithium: molecular moods and complex behaviour. Mol Interv 8, 230−41. [2] Schetz, J.A., 2005. Allosteric modulation of dopamine receptors. Mini Rev Med Chem 5, 555−61. [3] Selent, J., Sanz, F., Pastor, M., De Fabritiis, G., 2010. Induced effects of sodium ions on dopaminergic G-protein coupled receptors. PLoS Comput Biol 6, pii, e1000884. [4] Spring, G., Frankel, M., 1981. New data on lithium and haloperidol incompatibility. Am J Psychiatry 138, 818−21.
P.1.g.045 Effect of individual Valeriana officinalis and Melissa officinalis extracts and a combination on learning, memory and locomotor activity of rats I. Kandilarov1 ° , M. Katsarova2 , H. Zlatanova1 , I. Kostadinova1 , S. Dimitrova2 , L. Lukanov2 , F. Sadakov3 1 Medical university Plovdiv, Pharmacology and clinical pharmacology, Plovdiv, Bulgaria; 2 Medical university Plovdiv, Chemistry and biochemistry, Plovdiv, Bulgaria; 3 Avicena herb-Ltd., Avicena herb-Ltd., Plovdiv, Bulgaria Background: Adaptogens are plant products which normalize bodily functions and strengthen organs and systems damaged by stress [1]. Studies show that they affect learning and memory and improve locomotor activity of rats [2,3]. Melissa officinalis contains rosmarinic acid which inhibits GABA-transaminase − the enzyme that breaks down gamma aminobutyric acid (GABA) thus enhances its depressive action on the central nervous system [4]. Valeriana officinalis contains essential oils (mainly Bornyl acetate), valerenic acid and valepotriates that regulate the functions of the autonomic nervous system [5]. The aim of the current study was to investigate the effect of individual powdered extracts obtained from Valeriana officinalis and Melissa officinalis and a combination (Melissa officinalis, Hypericum perforatum and Serratula coronata 4.5:4.5:1) on learning, memory and locomotor activity of rats. Methods: The quantification of the bioactive compounds was made by using HPLC system Varian ProStar with PDA detector. Each extract was quantified based on the amount of its standard substance: Valeriana officinalis − valerenic acid, Melissa officinalis − rosmarinic acid, Hypericum perforatum − hypericin, Serratula coronata − 20-hydroxyecdysone. 32 adult male Wistar rats (weight 150–180 g) were divided in 4 groups (8 per group). They were treated for 4 weeks (once daily, p.os) 1st group − saline 10 ml/kg b.w.; 2nd group − Melissa officinalis 500 mg/kg b.w.; 3rd group − Valeriana officinalis 500 mg/kg b.w.; 4th group − combination 500 mg/kg b.w. Passive avoidance test for learning and memory was performed 60 minutes after the last treatment, using Step-through apparatus. A learning session was performed for 2 days and a short memory retention test was made on the 3rd day. Criterion for learning and memory retention was latency of reaction 180s during which subjects were required to stay in the lighted chamber. Locomotor activity was evaluated by Activity cage which measured horizontal and vertical movements of the animals. Statistical assessment of the results was made with
P.1.g.044 New molecular insights into the mechanism of lithium action at the dopaminergic D2 receptor Caballero1 ° ,
Pastor2 ,
Fabritiis2 ,
Selent2
J. Le´on M. G. De J. 1 Insitut de neuropsiquiatria i addiccions, Psiquiatria, Barcelona, Spain; 2 Pompeu Fabra University UPF - Hospital del Mar Medical Research Institute IMIM, Research Programme on Biomedical Informatics GRIB, Barcelona, Spain Introduction: Despite the therapeutic relevance of lithium in the treatment of psychiatric disorders, its mechanism of action remains unknown. Several of the proposed mechanisms of action of lithium are related to the downstream signaling of G proteincoupled receptors (GPCRs) (e.g. inositol depletion, interference with cAMP mediated processes or disruption of the Akt/bArr2/PP2A complex) [1]. The pharmacological response of GPCRs is typically triggered by ligand binding to the orthosteric site, but can be fine-tuned by allosteric modulation (monovalent cations are an example of the agents that can exert such allosteric modulation) [2]. The dopamine D2 receptor (D2R) is a GPCR, and our group previously simulated a cation-binding site for this receptor. There a Na+ ion is trapped in a hydrogen-bonding network (HBN), stabilizing the receptor in a distinct conformation [3]. The ability of other monovalent cations that may interact with the D2R cationbinding site has not been explored yet. Aim of the study: The similarity of Na+ with the Li+ ion suggests that the mechanism of Li+ pharmacological activity could involve competing for binding with Na+ deep inside receptor the D2R and, by doing so, destabilizing the sodium-induced GPCR conformation. Methods: A realistic three-dimensional structure of the D2R was modelled using a previously reported modeling protocol [3]. Three all-atom models were generated using the AMBER99SB force-field and simulated using ACEMD software. The three simulated all-atom systems consisted of the dopaminergic D2 receptor embedded in a hydrated membrane bilayer environment ionized up to a physiological ionic strength of 150 mM K+, Na+ or Li+ ions. Results: All three ions are able to spontaneously penetrate the D2R. The propensity of ions to enter the D2 receptor is higher for larger-sized Na+ and K+ ions. In addition, there is a preference of smaller Li+ and Na+ ions to bind at the allosteric site over the orthosteric site. The most interesting observation of our simulation is an ion size dependent binding pattern at the extracellular loop (ECL) region that produces changes on the conformation of the receptor entrance. The binding of lithium at ECL produces a structural rearrangement of the ECL region. Interestingly, this closed loop conformation seems to be specific for the small Li+ ion. Conclusions: The most important finding is the formation of an unprecedented ionic salt bridge at the extracellular receptor region (at the ECL region) induced by lithium that can have major implications for D2R functionality. This results in an obstruction of the receptor entrance. Since the ECL is known to serve as a “lid” of the receptor entrance, the observed Li+-induced partial receptor closure will most likely affect the entrance/exit of small molecules into/out of the binding pocket. The tendency of Li+
S239
ions to partially close the D2 receptor entrance suggests an ioninduced impact on the kinetics of ligand binding. There are several cases reported of severe neurological toxicity in patients treated in combination with lithium and haloperidol [4], and our study could partially explain the molecular mechanisms underlying this effect. References [1] Beaulieu, J.M., Caron, M.G., 2008. Looking at lithium: molecular moods and complex behaviour. Mol Interv 8, 230−41. [2] Schetz, J.A., 2005. Allosteric modulation of dopamine receptors. Mini Rev Med Chem 5, 555−61. [3] Selent, J., Sanz, F., Pastor, M., De Fabritiis, G., 2010. Induced effects of sodium ions on dopaminergic G-protein coupled receptors. PLoS Comput Biol 6, pii, e1000884. [4] Spring, G., Frankel, M., 1981. New data on lithium and haloperidol incompatibility. Am J Psychiatry 138, 818−21.
P.1.g.045 Effect of individual Valeriana officinalis and Melissa officinalis extracts and a combination on learning, memory and locomotor activity of rats I. Kandilarov1 ° , M. Katsarova2 , H. Zlatanova1 , I. Kostadinova1 , S. Dimitrova2 , L. Lukanov2 , F. Sadakov3 1 Medical university Plovdiv, Pharmacology and clinical pharmacology, Plovdiv, Bulgaria; 2 Medical university Plovdiv, Chemistry and biochemistry, Plovdiv, Bulgaria; 3 Avicena herb-Ltd., Avicena herb-Ltd., Plovdiv, Bulgaria Background: Adaptogens are plant products which normalize bodily functions and strengthen organs and systems damaged by stress [1]. Studies show that they affect learning and memory and improve locomotor activity of rats [2,3]. Melissa officinalis contains rosmarinic acid which inhibits GABA-transaminase − the enzyme that breaks down gamma aminobutyric acid (GABA) thus enhances its depressive action on the central nervous system [4]. Valeriana officinalis contains essential oils (mainly Bornyl acetate), valerenic acid and valepotriates that regulate the functions of the autonomic nervous system [5]. The aim of the current study was to investigate the effect of individual powdered extracts obtained from Valeriana officinalis and Melissa officinalis and a combination (Melissa officinalis, Hypericum perforatum and Serratula coronata 4.5:4.5:1) on learning, memory and locomotor activity of rats. Methods: The quantification of the bioactive compounds was made by using HPLC system Varian ProStar with PDA detector. Each extract was quantified based on the amount of its standard substance: Valeriana officinalis − valerenic acid, Melissa officinalis − rosmarinic acid, Hypericum perforatum − hypericin, Serratula coronata − 20-hydroxyecdysone. 32 adult male Wistar rats (weight 150–180 g) were divided in 4 groups (8 per group). They were treated for 4 weeks (once daily, p.os) 1st group − saline 10 ml/kg b.w.; 2nd group − Melissa officinalis 500 mg/kg b.w.; 3rd group − Valeriana officinalis 500 mg/kg b.w.; 4th group − combination 500 mg/kg b.w. Passive avoidance test for learning and memory was performed 60 minutes after the last treatment, using Step-through apparatus. A learning session was performed for 2 days and a short memory retention test was made on the 3rd day. Criterion for learning and memory retention was latency of reaction 180s during which subjects were required to stay in the lighted chamber. Locomotor activity was evaluated by Activity cage which measured horizontal and vertical movements of the animals. Statistical assessment of the results was made with