Dopamine D receptor (DR) activation triggers both G protein- and β-arrestin-dependent signaling. ... more Dopamine D receptor (DR) activation triggers both G protein- and β-arrestin-dependent signaling. Biased DR ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of DR to heteromerize with adenosine A receptor (AR) has been associated to DR agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the AR dependence of DR/β-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between β-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of DR/AR homomers and heteromers, we demonstrated that the DR agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when AR/DR heteromers were expressed. Subsequently, the role of AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and AR mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in AR mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that DR/AR heteromerization facilitates DR β-arrestin recruitment, and furthermore, reveal a pivotal role for AR in the antipsychotic-like activity of the β-arrestin-biased DR ligand, UNC9994.
SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 ... more SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 receptor (D2R), however, the binding mode and allosteric mechanism of action of this ligand remain incompletely understood. SB269,652 comprises an orthosteric, primary pharmacophore and a secondary (or allosteric) pharmacophore joined by a hydrophilic cyclohexyl linker and is known to form corresponding interactions with the orthosteric binding site (OBS) and the secondary binding pocket (SBP) in the D2R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D2R-mediated activation of G protein-coupled inward-rectifier potassium channels (GIRK) and decided to perform a more detailed investigation of the interaction between dopamine and SB269,652. The results indicated that the SB269,652 inhibitory potency is increased 6.6-fold upon ligand pre-incubation, compared to the simultaneous co-application with dopamine. Mutagenesis experiments implicated both S193 ...
G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotrans... more G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotransmitter, and sensory functions. These receptors are important drug targets; in fact, about 27 % of prescribed drugs are GPCR ligands. The dopamine D2 receptor is prominently expressed within the CNS as two distinct isoforms; D2L (long isoform) and D2S (short isoform). The former is mainly expressed postsynaptically, whereas the latter functions primarily as an inhibitory autoand heteroreceptor. The D2 receptor is of considerable pharmacological interest, as it constitutes the main target for most antiparkinsonian and antipsychotic drugs in clinical use. While many ion channels have long been known to be voltage sensitive, this property has not been attributed to GPCRs until quite recently. As a notable example, the muscarinic M2 receptor was shown to display depolarization-induced decreases in agonist binding and functional potency. M2 receptor voltage sensitivity has been implicated in t...
The forward (kon) and reverse (koff) rate constants of drug–target interactions have important im... more The forward (kon) and reverse (koff) rate constants of drug–target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and re...
Background: Clinically, corticosteroids are used mainly for their immune-modulatory properties bu... more Background: Clinically, corticosteroids are used mainly for their immune-modulatory properties but are also known to influence mood. Despite evidence of a role in regulating tryptophan hydroxylases (TPH), key enzymes in serotonin biosynthesis, a direct action of corticosteroids on these enzymes has not been systematically investigated. Methodology & results: Corticosteroid effects on TPHs were tested using an in vitro assay. The compound with the strongest modulatory effect, beclomethasone dipropionate, activated TPH1 and TPH2 with low micromolar potency. Thermostability assays suggested a stabilizing mechanism, and computational docking indicated that beclomethasone dipropionate interacts with the TPH active site. Conclusion: Beclomethasone dipropionate is a stabilizer of TPHs, acting as a pharmacological chaperone. Our findings may inspire further development of steroid scaffolds as putative antidepressant drugs.
Neurotransmitter contacts within the receptor binding site differentially contribute to the overa... more Neurotransmitter contacts within the receptor binding site differentially contribute to the overall functional response: transmembrane helix (TM) 5 contacts promote G protein coupling whereas concerted TM5–TM6 contacts enhance β-arrestin recruitment.
Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wi... more Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TP...
RATIONALE:
The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by ... more RATIONALE: The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo.
OBJECTIVES: The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses.
METHODS: fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride. RESULTS: Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes.
CONCLUSIONS: The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.
G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotrans... more G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotransmitter, and sensory functions. These receptors are important drug targets; in fact, about 27% of prescribed drugs are GPCR ligands. The dopamine D2 receptor is ...
Newer, "atypical" antipsychotics carry a lower ... more Newer, "atypical" antipsychotics carry a lower risk of motor side-effects than older, "typical" compounds. It has been proposed that a ~100-fold faster dissociation from the dopamine D2 receptor (D2R) distinguishes atypical from typical antipsychotics. Furthermore, differing antipsychotic D2R affinities have been suggested to reflect differences in dissociation rate constants (koff), while association rate constants (kon) were assumed to be similar. However, it was recently demonstrated that lipophilic accumulation of ligand in the cell interior and/or membrane can cause underestimation of koff, and as high-affinity D2R antagonists are frequently lipophilic, this may have been a confounding factor in previous studies. In the present work, a functional electrophysiology assay was used to measure the recovery of dopamine-mediated D2R responsivity from antipsychotic antagonism, using elevated concentrations of dopamine to prevent the potential bias of re-binding of lipophilic ligands. The variability of antipsychotic kon was also reexamined, capitalizing on the temporal resolution of the assay. kon was estimated from the experimental recordings using a simple mathematical model assumed to describe the binding process. The time course of recovery from haloperidol (typical antipsychotic) was only 6.4- to 2.5-fold slower than that of the atypical antipsychotics, amisulpride, clozapine, and quetiapine, while antipsychotic kons were found to vary more widely than previously suggested. Finally, affinities calculated using our kon and koff estimates correlated well with functional potency and with affinities reported from radioligand binding studies. In light of these findings, it appears unlikely that typical and atypical antipsychotics are primarily distinguished by their D2R binding kinetics.
Facilitated anion transport potentially represents a powerful tool to modulate various cellular f... more Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores are still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.
Rationale
Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory ac... more Rationale Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding. Objectives The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats. Methods Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer 11C-SA4503 (sigma-1R) or 11C-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with 11C-raclopride. Cerebral 11C-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V T) calculated by Logan graphical analysis. 11C-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model. Results Cunningham-Lassen plots indicated sigma-1R occupancies of 57 ± 2 and 85 ± 2 % after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44–66 %) reduction of 11C-raclopride binding was only observed at 60 mg/kg pridopidine. Conclusions At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D2Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity.
The sigma-2 receptor is a steroid-binding membrane-associated receptor which has been implicated ... more The sigma-2 receptor is a steroid-binding membrane-associated receptor which has been implicated in cell survival. Sigma-2 has recently been shown to bind amyloid-β (Aβ) oligomers in Alzheimer's disease (AD) brain. Furthermore, blocking this interaction was shown to prevent or reverse the effects of Aβ to cause cognitive impairment in mouse models and synaptic loss in neuronal cultures. In the present work, the density of sigma-2 receptors was measured in a double transgenic mouse model of amyloid-β deposition (APP/PS1). Comparisons were made between males and females and between transgenic and wt animals. Sigma-2 receptor density was assessed by quantitative autoradiography performed on coronal brain slices using [3H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methyl-benzamide ([3H]RHM-1), which has a 300-fold selectivity for the sigma-2 receptor over the sigma-1 receptor. The translocator protein of 18 kDa (TSPO) is expressed on activated microglia and is a marker for neuroinflammation. TSPO has been found to be upregulated in neurodegenerative disorders, including AD. Therefore, in parallel with the sigma-2 autoradiography experiments, we measured TSPO expression using the selective radioligand, [3H]PBR28. We also quantified Aβ plaque burden in the same animals using a monoclonal antibody raised against aggregated Aβ. Sigma-2 receptor density was significantly decreased in piriform and motor cortices as well as striata of 16-month old female, but not male, APP/PS1 mice as compared to their wt counterparts. [3H]PBR28 binding and immunostaining for Aβ plaques were significantly increased in piriform and motor cortices of both male and female transgenic mice. In striatum however, significant increases were observed only in females.
It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. cl... more It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. clozapine and amisulpride) are related to their ∼100-fold faster dissociation from dopamine D2 receptors (D2R) compared with typical antipsychotics (e.g. haloperidol and chlorpromazine). Fast dissociation would entail rapidly reversible antagonism; however, this has not been thoroughly studied using functional assays. We compared the reversibilities of D2R antagonism by 17 compounds using an electrophysiological method to measure dopamine-evoked potassium channel activation via D2R. Varying rates and amplitudes of D2R response recovery were observed following antagonist removal. Whereas recovery rates differed 15-fold between atypical drugs, recovery from clozapine and amisulpride antagonism was, unexpectedly, less than twofold faster than from chlorpromazine. The recovery amplitude correlated with calculated water solubility and lipid/water distribution coefficients, suggesting variable drug partitioning into cell membranes. Our data do not support the notion that the rate of reversibility of D2R antagonism is what distinguishes atypical from typical antipsychotics.
Dopamine D receptor (DR) activation triggers both G protein- and β-arrestin-dependent signaling. ... more Dopamine D receptor (DR) activation triggers both G protein- and β-arrestin-dependent signaling. Biased DR ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of DR to heteromerize with adenosine A receptor (AR) has been associated to DR agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the AR dependence of DR/β-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between β-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of DR/AR homomers and heteromers, we demonstrated that the DR agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when AR/DR heteromers were expressed. Subsequently, the role of AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and AR mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in AR mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that DR/AR heteromerization facilitates DR β-arrestin recruitment, and furthermore, reveal a pivotal role for AR in the antipsychotic-like activity of the β-arrestin-biased DR ligand, UNC9994.
SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 ... more SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 receptor (D2R), however, the binding mode and allosteric mechanism of action of this ligand remain incompletely understood. SB269,652 comprises an orthosteric, primary pharmacophore and a secondary (or allosteric) pharmacophore joined by a hydrophilic cyclohexyl linker and is known to form corresponding interactions with the orthosteric binding site (OBS) and the secondary binding pocket (SBP) in the D2R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D2R-mediated activation of G protein-coupled inward-rectifier potassium channels (GIRK) and decided to perform a more detailed investigation of the interaction between dopamine and SB269,652. The results indicated that the SB269,652 inhibitory potency is increased 6.6-fold upon ligand pre-incubation, compared to the simultaneous co-application with dopamine. Mutagenesis experiments implicated both S193 ...
G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotrans... more G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotransmitter, and sensory functions. These receptors are important drug targets; in fact, about 27 % of prescribed drugs are GPCR ligands. The dopamine D2 receptor is prominently expressed within the CNS as two distinct isoforms; D2L (long isoform) and D2S (short isoform). The former is mainly expressed postsynaptically, whereas the latter functions primarily as an inhibitory autoand heteroreceptor. The D2 receptor is of considerable pharmacological interest, as it constitutes the main target for most antiparkinsonian and antipsychotic drugs in clinical use. While many ion channels have long been known to be voltage sensitive, this property has not been attributed to GPCRs until quite recently. As a notable example, the muscarinic M2 receptor was shown to display depolarization-induced decreases in agonist binding and functional potency. M2 receptor voltage sensitivity has been implicated in t...
The forward (kon) and reverse (koff) rate constants of drug–target interactions have important im... more The forward (kon) and reverse (koff) rate constants of drug–target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and re...
Background: Clinically, corticosteroids are used mainly for their immune-modulatory properties bu... more Background: Clinically, corticosteroids are used mainly for their immune-modulatory properties but are also known to influence mood. Despite evidence of a role in regulating tryptophan hydroxylases (TPH), key enzymes in serotonin biosynthesis, a direct action of corticosteroids on these enzymes has not been systematically investigated. Methodology & results: Corticosteroid effects on TPHs were tested using an in vitro assay. The compound with the strongest modulatory effect, beclomethasone dipropionate, activated TPH1 and TPH2 with low micromolar potency. Thermostability assays suggested a stabilizing mechanism, and computational docking indicated that beclomethasone dipropionate interacts with the TPH active site. Conclusion: Beclomethasone dipropionate is a stabilizer of TPHs, acting as a pharmacological chaperone. Our findings may inspire further development of steroid scaffolds as putative antidepressant drugs.
Neurotransmitter contacts within the receptor binding site differentially contribute to the overa... more Neurotransmitter contacts within the receptor binding site differentially contribute to the overall functional response: transmembrane helix (TM) 5 contacts promote G protein coupling whereas concerted TM5–TM6 contacts enhance β-arrestin recruitment.
Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wi... more Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TP...
RATIONALE:
The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by ... more RATIONALE: The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo.
OBJECTIVES: The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses.
METHODS: fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride. RESULTS: Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes.
CONCLUSIONS: The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.
G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotrans... more G protein coupled receptors (GPCRs) mediate a multitude of responses serving hormonal, neurotransmitter, and sensory functions. These receptors are important drug targets; in fact, about 27% of prescribed drugs are GPCR ligands. The dopamine D2 receptor is ...
Newer, "atypical" antipsychotics carry a lower ... more Newer, "atypical" antipsychotics carry a lower risk of motor side-effects than older, "typical" compounds. It has been proposed that a ~100-fold faster dissociation from the dopamine D2 receptor (D2R) distinguishes atypical from typical antipsychotics. Furthermore, differing antipsychotic D2R affinities have been suggested to reflect differences in dissociation rate constants (koff), while association rate constants (kon) were assumed to be similar. However, it was recently demonstrated that lipophilic accumulation of ligand in the cell interior and/or membrane can cause underestimation of koff, and as high-affinity D2R antagonists are frequently lipophilic, this may have been a confounding factor in previous studies. In the present work, a functional electrophysiology assay was used to measure the recovery of dopamine-mediated D2R responsivity from antipsychotic antagonism, using elevated concentrations of dopamine to prevent the potential bias of re-binding of lipophilic ligands. The variability of antipsychotic kon was also reexamined, capitalizing on the temporal resolution of the assay. kon was estimated from the experimental recordings using a simple mathematical model assumed to describe the binding process. The time course of recovery from haloperidol (typical antipsychotic) was only 6.4- to 2.5-fold slower than that of the atypical antipsychotics, amisulpride, clozapine, and quetiapine, while antipsychotic kons were found to vary more widely than previously suggested. Finally, affinities calculated using our kon and koff estimates correlated well with functional potency and with affinities reported from radioligand binding studies. In light of these findings, it appears unlikely that typical and atypical antipsychotics are primarily distinguished by their D2R binding kinetics.
Facilitated anion transport potentially represents a powerful tool to modulate various cellular f... more Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores are still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.
Rationale
Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory ac... more Rationale Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding. Objectives The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats. Methods Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer 11C-SA4503 (sigma-1R) or 11C-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with 11C-raclopride. Cerebral 11C-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V T) calculated by Logan graphical analysis. 11C-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model. Results Cunningham-Lassen plots indicated sigma-1R occupancies of 57 ± 2 and 85 ± 2 % after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44–66 %) reduction of 11C-raclopride binding was only observed at 60 mg/kg pridopidine. Conclusions At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D2Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity.
The sigma-2 receptor is a steroid-binding membrane-associated receptor which has been implicated ... more The sigma-2 receptor is a steroid-binding membrane-associated receptor which has been implicated in cell survival. Sigma-2 has recently been shown to bind amyloid-β (Aβ) oligomers in Alzheimer's disease (AD) brain. Furthermore, blocking this interaction was shown to prevent or reverse the effects of Aβ to cause cognitive impairment in mouse models and synaptic loss in neuronal cultures. In the present work, the density of sigma-2 receptors was measured in a double transgenic mouse model of amyloid-β deposition (APP/PS1). Comparisons were made between males and females and between transgenic and wt animals. Sigma-2 receptor density was assessed by quantitative autoradiography performed on coronal brain slices using [3H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methyl-benzamide ([3H]RHM-1), which has a 300-fold selectivity for the sigma-2 receptor over the sigma-1 receptor. The translocator protein of 18 kDa (TSPO) is expressed on activated microglia and is a marker for neuroinflammation. TSPO has been found to be upregulated in neurodegenerative disorders, including AD. Therefore, in parallel with the sigma-2 autoradiography experiments, we measured TSPO expression using the selective radioligand, [3H]PBR28. We also quantified Aβ plaque burden in the same animals using a monoclonal antibody raised against aggregated Aβ. Sigma-2 receptor density was significantly decreased in piriform and motor cortices as well as striata of 16-month old female, but not male, APP/PS1 mice as compared to their wt counterparts. [3H]PBR28 binding and immunostaining for Aβ plaques were significantly increased in piriform and motor cortices of both male and female transgenic mice. In striatum however, significant increases were observed only in females.
It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. cl... more It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. clozapine and amisulpride) are related to their ∼100-fold faster dissociation from dopamine D2 receptors (D2R) compared with typical antipsychotics (e.g. haloperidol and chlorpromazine). Fast dissociation would entail rapidly reversible antagonism; however, this has not been thoroughly studied using functional assays. We compared the reversibilities of D2R antagonism by 17 compounds using an electrophysiological method to measure dopamine-evoked potassium channel activation via D2R. Varying rates and amplitudes of D2R response recovery were observed following antagonist removal. Whereas recovery rates differed 15-fold between atypical drugs, recovery from clozapine and amisulpride antagonism was, unexpectedly, less than twofold faster than from chlorpromazine. The recovery amplitude correlated with calculated water solubility and lipid/water distribution coefficients, suggesting variable drug partitioning into cell membranes. Our data do not support the notion that the rate of reversibility of D2R antagonism is what distinguishes atypical from typical antipsychotics.
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Papers by Kristoffer Sahlholm
The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo.
OBJECTIVES:
The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses.
METHODS:
fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride.
RESULTS:
Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes.
CONCLUSIONS:
The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.
Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding.
Objectives
The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats.
Methods
Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer 11C-SA4503 (sigma-1R) or 11C-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with 11C-raclopride. Cerebral 11C-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V T) calculated by Logan graphical analysis. 11C-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model.
Results
Cunningham-Lassen plots indicated sigma-1R occupancies of 57 ± 2 and 85 ± 2 % after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44–66 %) reduction of 11C-raclopride binding was only observed at 60 mg/kg pridopidine.
Conclusions
At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D2Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity.
The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo.
OBJECTIVES:
The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses.
METHODS:
fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride.
RESULTS:
Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes.
CONCLUSIONS:
The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.
Dopamine stabilizers have stimulatory actions under low dopamine tone and inhibitory actions under high dopamine tone without eliciting catalepsy. These compounds are dopamine D2 receptor (D2R) antagonists or weak partial agonists and may have pro-mnemonic and neuroprotective effects. The mechanism underlying their stimulatory and neuroprotective actions is unknown but could involve sigma-1R binding.
Objectives
The present study examined sigma-1R and D2R occupancy by the dopamine stabilizer pridopidine (ACR16) at behaviorally relevant doses in living rats.
Methods
Rats were administered 3 or 15 mg/kg pridopidine, or saline, before injection of the radiotracer 11C-SA4503 (sigma-1R) or 11C-raclopride (D2R). Some animals received 60 mg/kg pridopidine and were only scanned with 11C-raclopride. Cerebral 11C-SA4503 binding was quantified using metabolite-corrected plasma input data and distribution volume (V T) calculated by Logan graphical analysis. 11C-raclopride binding was quantified using striatum-to-cerebellum ratios and binding potentials calculated with a simplified reference tissue model.
Results
Cunningham-Lassen plots indicated sigma-1R occupancies of 57 ± 2 and 85 ± 2 % after pretreatment of animals with 3 and 15 mg/kg pridopidine. A significant (44–66 %) reduction of 11C-raclopride binding was only observed at 60 mg/kg pridopidine.
Conclusions
At doses shown to elicit neurochemical and behavioral effects, pridopidine occupied a large fraction of sigma-1Rs and a negligible fraction of D2Rs. Significant D2R occupancy was only observed at a dose 20-fold higher than was required for sigma-1R occupancy. The characteristics of dopamine stabilizers may result from the combination of high sigma-1R and low D2R affinity.