Clinical Correlates of the NR3C1 Gene Methylation at Various Stages of Psychosis

BackgroundDysregulation of epigenetic processes might account for alterations of the hypothalamic-pituitary-adrenal axis observed in patients with schizophrenia. Therefore, in this study, we aimed to investigate methylation of the glucocorticoid receptor (NR3C1) gene in patients with schizophrenia-spectrum disorders, individuals at familial high risk of schizophrenia (FHR-P), and healthy controls with respect to clinical manifestation and a history of psychosocial stressors.MethodsWe recruited 40 first-episode psychosis patients, 45 acutely relapsed schizophrenia (SCZ-AR) patients, 39 FHR-P individuals, and 56 healthy controls. The level of methylation at 9 CpG sites of the NR3C1 gene was determined using pyrosequencing.ResultsThe level of NR3C1 methylation was significantly lower in first-episode psychosis patients and significantly higher in SCZ-AR patients compared with other subgroups of participants. Individuals with FHR-P and healthy controls had similar levels of NR3C1 methylation. A history of adverse childhood experiences was associated with significantly lower NR3C1 methylation in all subgroups of participants. Higher methylation of the NR3C1 gene was related to worse performance of attention and immediate memory as well as lower level of general functioning in patients with psychosis.ConclusionsPatients with schizophrenia-spectrum disorders show altered levels of NR3C1 methylation that are significantly lower in first-episode psychosis patients and significantly higher in SCZ-AR patients. Higher methylation of the NR3C1 gene might be related to cognitive impairment observed in this clinical population. The association between a history of adverse childhood experiences and lower NR3C1 methylation is not specific to patients with psychosis. Longitudinal studies are needed to establish causal mechanisms underlying these observations.     

Inactivation of prostaglandins in the perfused rat lung

Inactivation in the isolated perfused rat lung of prostaglandins (PG) D₂, E₁, F_2α, I₂ and the metabolites 6-keto PGF_1α (= 6KF_1α) and 13,14-dihydro-15-keto POP_2α (= KH₂F_2α) was studied using 5 min perfusions of 7–10 ng/ml PG in Krebs' solution containing 0.02 μCi/ml tritiated PG and 4.5% bovine serum albumin (BSA). The parameters measured were (a) extent of inactivation (F_2α > E₁ > D₂ > 6KF_1α > I₂; KH₂F_2α unchanged), (b) the accumulation of PG within the lung measured as tissue to medium ratio (F_2α = D₂ > E₁ > 6KF_1α > I₁ = KH₂F_2α), and (c) rate of equi within the lung measured as “wash-in t$\text{1}\text{2}$” (D₂ > F_2α > e₁ > I₂ = 6KF_1α = KH₂F_2α). Removal of sodium ions produced a small decrease in PGD₂ and PGE₁ breakdown but not of PGF_2α whereas breakdown of all PGs was markedly inhibited at 5°. Removal of BSA enhanced PGE₁ and PGI₂ breakdown but not that of PGF_2α. Addition of 10% BSA inhibited PGE₁ breakdown but not that of PGF_2α. Binding of PGs to 4.5% BSA was PGE₁ = KH₂F_2α > D₂ > F_2α, and increased at 10% BSA or after removal of sodium ions. These data support the view that PGs must be taken up into pulmonary cells by a transmembrane carrier process as a prerequisite for enzymatic breakdown. The metabolites are then released back into the pulmonary circulation.     

Low Dopamine D2/D3 Receptor Availability is Associated with Steep Discounting of Delayed Rewards in Methamphetamine Dependence

Background:

Individuals with substance use disorders typically exhibit a predilection toward instant gratification with apparent disregard for the future consequences of their actions. Indirect evidence suggests that low dopamine D₂-type receptor availability in the striatum contributes to the propensity of these individuals to sacrifice long-term goals for short-term gain; however, this possibility has not been tested directly. We investigated whether striatal D₂/D₃ receptor availability is negatively correlated with the preference for smaller, more immediate rewards over larger, delayed alternatives among research participants who met DSM-IV criteria for methamphetamine (MA) dependence.

Methods:

Fifty-four adults (n = 27 each: MA-dependent, non-user controls) completed the Kirby Monetary Choice Questionnaire, and underwent positron emission tomography scanning with [¹⁸F]fallypride.

Results:

MA users displayed steeper temporal discounting (p = 0.030) and lower striatal D₂/D₃ receptor availability (p < 0.0005) than controls. Discount rate was negatively correlated with striatal D₂/D₃ receptor availability, with the relationship reaching statistical significance in the combined sample (r = -0.291, p = 0.016) and among MA users alone (r = -0.342, p = 0.041), but not among controls alone (r = -0.179, p = 0.185); the slopes did not differ significantly between MA users and controls (p = 0.5).

Conclusions:

These results provide the first direct evidence of a link between deficient D₂/D₃ receptor availability and steep temporal discounting. This finding fits with reports that low striatal D₂/D₃ receptor availability is associated with a higher risk of relapse among stimulant users, and may help to explain why some individuals choose to continue using drugs despite knowledge of their eventual negative consequences. Future research directions and therapeutic implications are discussed.     

[3H]sumatriptan labels both 5-HT1D and 5-HT1F receptor binding sites in the guinea pig brain: an autoradiographic study

We have used in vitro autoradiography to visualize [³H]sumatriptan binding sites in sections of guinea-pig and rat brain. In saturation studies, this ligand recognized a single saturable population of high affinity binding sites in all regions examined (pK_D = 8.3–9.3). While 5-HT and the sumatriptan derivative CP-122,288 (5-methyl-aminosulfonylmethyl-3-(N-methylpyrrolidin-2R-yl-methyl)-1H-indole) competed for [³H]sumatriptan binding sites with a high affinity and monophasic profile, displacement experiments with 5-carboxamidotryptamine revealed the existence of 2 classes of binding sites. The high affinity component (pKD = 9.2–9.9) probably corresponded to 5-HT_1B (rat) or 5-HT_1D (guinea-pig) receptors. The intermediate affinity (pK_D = 5.7–7.3) of the other component, taken together with their high affinity for [³H]sumatriptan, was similar to that of the cloned 5-HT_1F receptor. The regional distribution of the 5-HT_1B/1D [³H]sumatriptan binding sites was in agreement with previously published studies (striatonigral system, hypothalamus, central gray, superficial layer of the superior colliculus) and corresponded to the pattern of serotonin-5-O-carboxymethyl-glycyl [¹²⁵I]tyrosinamide labeling in consecutive sections. [³H]sumatriptan binding sites with a low affinity for 5-CT predominated in the intermediate neocortical layers, the claustrum (in the guinea-pig only), the mammillary nuclei, most of the thalamic nuclei and the principal oculomotor nucleus (in the guinea-pig only). This distribution is very similar to that of 5-HT_1F mRNA, indicating further the identity of these sites with 5-HT_1F receptors. Very high densities of 5-HT_1F sites were also found in the rat parafascicular nucleus.Some regions, such as the caudate/nucleus, the lateral geniculate nuclei and the spinal trigeminal nucleus appeared to contain both 5-HT_1B/1D and 5-HT_1F binding sites. Ketanserin had a low affinity for [³H]sumatriptan binding sites in all guinea-pig brain regions, compatible with the presence of the 5-HT_1D\ subtype. An exception was the substantia nigra, where a significant proportion of sites displayed an intermediate affinity for this compound, suggesting the presence of 5-HT_1D receptors. [³H]5-HT labeled 5-HT_1F sites in the claustrum and intermediate cortical layers in the guinea-pig. However these data show that [³H]sumatriptan, in the presence of 10 nM 5-carboxamidotryptamine, is a more suitable radioligand to study the distribution of 5-HT_1F binding sites.     

Activation of 5-hydroxytryptamine1B/1D/1F receptors as a mechanism of action of antimigraine drugs

Introduction: The introduction of the triptans (5-hydroxytryptamine (5-HT)_1B/1D receptor agonists) was a great improvement in the acute treatment of migraine. However, shortcomings of the triptans have prompted research on novel serotonergic targets for the treatment of migraine.

Areas covered: In this review the different types of antimigraine drugs acting at 5-HT receptors, their discovery and development are discussed. The first specific antimigraine drugs were the ergot alkaloids, consisting of ergotamine, dihydroergotamine and methysergide, which are agonists at 5-HT receptors, but can also bind α-adrenoceptors and dopamine receptors. In the 1990s, the triptans became available on the market. They are 5-HT_1B/1D receptor agonists, showing fewer side effects due to their receptor specificity. In the last years, compounds that bind specifically to 5-HT_1D, 5-HT_1F and 5-HT₇ receptors have been explored for their antimigraine potential. Furthermore, the serotonergic system seems to act in tight connection with the glutamatergic as well as the CGRP-ergic systems, which may open novel therapeutic avenues.

Expert opinion: Although the triptans are very effective in treating migraine attacks, their shortcomings have stimulated the search for novel drugs. Currently, the focus is on 5-HT_1F receptor agonists, which seem devoid of vascular side effects. Moreover, novel compounds that affect multiple transmitter and/or neuropeptide systems that are involved in migraine could be of therapeutic relevance.     

The crystal structure of a β-allyl type phenylpropanoid, 2-(4-allyl-2,6-dimethoxyphenoxy)-1-(4-hydroxy-3-methoxyphenyl) propan-1-ol,from the seeds ofMyristica fragrans

The structure of a β-allyl type phenylpropanoid was determined by single crystal X-ray diffraction analysis. The compound was recrystallized from a mixture ofn-hexane and benzene in monoclinic crystal system, witha=24.782 92),b=10.537 (1),c=7.871 (1) å, β=95.74 (1)^o,D _x =1.216,D _m =1.22g/cm³, space groupP2₁/a, andZ=4. The structure was solved by direct method and refined by least-squares procedure to the finalR value of 0.054 for 2824 observed reflections {F≥3σ(F)}. The molecular geometry shows a most stabletrans-form with respect to the bulky phenyls, and this conformation is settled by an intramolecular hydrogen bond. In the crystal, the molecules are arranged along with the screw axis, and stabilized by the O?H…O type intermolecular hydrogen bonds. The other intermolecular contacts appear to be the normalvan der Waals’ interactions. The compound is a dimeric phenylpropanoid, and belongs to the neolignan analogues.     

Pharmacological characterization of recombinant NR1/NR2A NMDA receptors with truncated and deleted carboxy termini expressed in Xenopus laevis oocytes

Background and purpose:

The carboxy terminal domain (CTD) of NR2 N-methyl-d-aspartate receptor (NMDAR) subunits interacts with numerous scaffolding and signal transduction proteins. Mutations of this region affect trafficking and downstream signalling of NMDARs. This study determines to what extent characteristic pharmacological properties of NR2A-containing NMDARs are influenced by this key functional domain.

Experimental approach:

Using recombinant receptor expression in Xenopus laevis oocytes and two electrode voltage clamp recordings we characterized pharmacological properties of rat NR1/NR2A NMDARs with altered CTDs. We assessed the effects of truncating [at residue Iso1098; NR2A(trunC)] and deleting [from residue Phe822; NR2A(delC)] the CTD of NR2A NMDAR subunits on agonist potencies, channel block by Mg²⁺ and memantine and potentiation of NMDAR-mediated responses by chelating contaminating divalent cations.

Key results:

Truncation or deletion of the CTD of NR2A NMDAR subunits did not affect glutamate potency [EC₅₀ = 2.2 µmol·L⁻¹, NR2A(trunC); 2.7 µmol·L⁻¹, NR2A(delC) compared with 3.3 µmol·L⁻¹, NR2A(WT)] but did significantly increase glycine potency [EC₅₀ = 500 nmol·L⁻¹, NR2A(trunC); 900 nmol·L⁻¹, NR2A(delC) compared with 1.3 µmol·L⁻¹, NR2A(WT)]. Voltage-dependent Mg²⁺ block of NR2A(WT)- and NR2A(trunC)-containing NMDARs was similar but low concentrations of Mg²⁺ (1 µmol·L⁻¹) potentiated NR1/NR2A(delC) NMDARs. Memantine block was not affected by changes to the structure of the NR2A CTD. EDTA-induced potentiation was similar at each of the three NMDAR constructs.

Conclusions and implications:

Of the parameters studied only minor influences of the CTD were observed; these are unlikely to compromise interpretation of studies that make use of CTD-mutated recombinant receptors or transgenic mice in investigations of the role of the CTD in NMDAR signalling.     

Metabolism of 26,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 26,27-hexafluoro1α,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafl uoro-1α, 25-dihydroxyvitamin D₃ [F₆-1,25(OH)₂D₃] to 26,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D₃ [F₆-1,23,25(OH)₃D₃ and that of F₆-1,23,25(OH)₃D₃ to 26,27-hexafluoro-23-oxo-1α,25-dihydroxyvitamin D₃ [F₆-23-oxo-1,25(OH)₂D₃] are catalysed by 25-hydroxyvitamin D 24-hydroxylase (CYP24), ROS17}2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24–] and a corresponding blank plasmid [pSLVCYP24R(®)] were used. 2. Incubation of [1β-³H]-F-1,25(OH)₂D₃ for 2 and 5 days with ROS17}2.8 cells transfected with pSVL-CYP24– generated a metabolite that co-migrated with authentic F₆ -1,23,25(OH)₃D₃ in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1β-³H]-F₆ -1,23,25(OH)₃D₃ for 5 days with pSVL-CYP24– - transfected ROS 17}2.8 cells generated a metabolite that co-migrated with authentic F₆ -23-oxo-1,25(OH)₂D₃. In contrast, the metabolites F₆ -1,23,25(OH)₃D₃ or F₆ -23-oxo- 1,25(OH)₂D₃ were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F₆ -1,25(OH)₂D₃ to F₆ -1,23,25(OH)₃D₃ and that of F₆ -1,23,25(OH)₃D₃ to F₆ -23-oxo-1,25(OH)₂D₃.     

Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects

Aim

Interindividual epigenetic variation is likely to be an important mechanism contributing to the interindividual variability in the expression and function of ATP-binding cassette, sub-family B, member 1 (ABCB1). The aim of the present study was to explore the effect of interindividual epigenetic variability in the ABCB1 promoter on ABCB1 expression and function in healthy Chinese subjects.

Methods

Using bisulfite sequencing polymerase chain reaction (PCR) and chromatin immunoprecipitation assays, the DNA methylation and histone acetylation status of the ABCB1 promoter in stool DNA and exfoliated colonic epithelial cells of 157 healthy Chinese male volunteers was analysed. ABCB1 mRNA levels in colonic epithelial cells were detected by real-time PCR. The digoxin pharmacokinetics in subjects with different epigenetic profiles was investigated after a single oral administration of digoxin (0.5 mg).

Results

The methylation levels of ABCB1 promoter in stool DNA showed a significant interindividual variation, from 0.84% to 18.05%. A high methylation level of the ABCB1 promoter was closely related to the low levels of acetylated histone H3 and ABCB1 mRNA expression. In the high methylation group, the area under the concentration–time curves (AUC_(0–4 h) and AUC_(0–10 h)) of digoxin was increased by 19% [95% confidence interval (CI) 10%, 31%; P = 0.024] and 13% (95% CI 8%, 26%; P = 0.026), respectively, and the peak concentration (C_max) of digoxin was increased by 30% (95% CI 12%, 41%; P = 0.021) compared with the low methylation group.

Conclusions

The epigenetic modifications of the ABCB1 promoter show high interindividual variability in healthy Chinese subjects, and are closely related to the interindividual variation in ABCB1 mRNA expression and digoxin 0–4 h plasma concentrations in vivo.     

A two-generation reproductive toxicity study of octamethylcyclotetrasiloxane (D4) in rats exposed by whole-body vapor inhalation

This study evaluated the potential toxicity of whole-body vapor inhalation of octamethylcyclotetrasiloxane (D₄) on reproductive capabilities in exposed F₀ and F₁ parental animals and the potential effects on neonatal survival, growth, and development of the F₁ and F₂ offspring. F₀ male and female Sprague–Dawley rats (30/sex/group) were exposed to D₄ vapor at concentrations of 0, 70, 300, 500 or 700 ppm 6 h per day for at least 70 consecutive days prior to mating and lasted through weaning of the pups on postnatal day (PND) 21. Female exposures were suspended from gestation day (GD) 21 through PND 4 to allow for parturition and permit continuous maternal care for the early neonates. Starting on PND 22, F₁ weanlings were exposed to D₄ as described for the F₀ generation. The F₂ pups were not directly exposed to D₄. F₀ animals were mated once to produce the F₁ generation; F₁ parental animals were mated twice to produce two F₂ litters. In addition, the F₁ males were mated with unexposed females. Prolonged estrous cycles, decreased mating and fertility indices were observed in the F₁ generation exposed to D₄ for the first and second matings. Significant reductions in the mean number of pups born and mean live litter size were observed in the 500 and 700 ppm groups for both the F₀ and F₁ generations. Implantation sites were also reduced at 700 ppm for both F₀ and F₁ generations. No adverse effects were observed at any exposure level on anogenital distance, vaginal patency and preputial separation. No adverse effects were seen on male functional reproductive parameters, spermatogenic endpoints, microscopic evaluation of male reproductive tissue, or when the D₄-exposed F₁ males were mated with the unexposed females, demonstrating that the reproductive toxicity observed was due to D₄ exposure to the females. Based on the lack of effect on reproduction when the D₄-exposed males were mated to näive females, the NOAEL for male reproductive toxicity was considered to be 700 ppm. Based on the statistically significant effects on fertility and litter size, NOAEL for female reproductive toxicity was considered to be 300 ppm. The findings observed in this study are consistent with suppression or delaying of LH surge as well as acceleration of the onset of female reproductive senescence in the rat. While analogous pathways control ovulation in both rats and humans, there are significant differences in the mechanism for timing and release of LH and resulting changes in the control of ovulation and mating behavior between the two species. If D₄ delays rather than causes a prolonged suppression or ablation of the LH surge, the reproductive mode of action of D₄ would not likely be relevant for humans.     

K+ release from rat parotid cells: An α1-adrenergic mediated event

To characterize α-adrenergic receptors in rat parotid gland tissue, 9,10-[9,10-³H(N)]-dihydro-α-ergocryptine ([³h]dhe) and [³H]prazosin binding to membranes and stimulated K⁺ release from parotid cell aggregates were examined. Prazosin (selective α₁-adrenergic antagonist), displacement of [³H]DHE binding from parotid membranes was biphasic, indicating the presence of both α₁- and α₂-adrenergic receptors. The numbers of α₁- and α₂-receptors were about equal. α₁-Adrenergic receptors were further studied by [³H]prazosin binding. [³H]Prazosin binding was a rapid, reversible, saturable and stereospecific process, with high affinity (K_D = 0.38 nM) and low capacity (B_max = 380 fmoles ligand bound/g tissue, 10.1 fmoles/mg protein) as determined by Scatchard analysis. The characteristics of [³H]prazosin binding were in good agreement with those of catecholamine-stimulated K⁺ release, suggesting that K⁺ release from rat parotid gland cells is an α₁-adrenergic mediated effect.     

Suppression of glucocorticoid secretion induces a behaviorally depressive state in rotarod performance in rat

Glucocorticoid hormones are important in the maintenance of many brain functions, and their receptors distribute abundantly throughout the brain. In patients with several neuropsychiatric disorders such as depression, dysregulation of the glucocorticoid negative feedback system is the consistent observations, which is thought to be caused by reduced glucocorticoid response at the several feedback sites including the brain. In the present study, we examined whether reduced glucocorticoid actions via suppression of circulating glucocorticoids by adrenalectomy (ADX) induced a behavioral depressive state using the rotarod test. We found that ADX impaired the rotarod performance while it did not affect the traction performance and locomotor activity. Moreover, this impairment was significantly reversed by corticosterone replacement treatment and was ameliorated by the infusion of D₁ receptor agonist SKF 81297 into the prefrontal cortex (PFC) in a dose-dependent manner. Considering the previous findings that ADX reduces dopaminergic transmission in the PFC, the present results suggest that suppression of circulating glucocorticoids induces a behaviorally depressive state that is caused by a D₁ receptor-mediated hypodopaminergic mechanism in the PFC. This finding would help to understand the involvement of the dysregulated feedback system in the pathogenesis of depression.     

High-affinity binding of [3H]doxepin to histamine H1-receptors in rat brain: Possible identification of a subclass of histamine H1-receptors

The binding of the radioactively labeled tricyclic antidepressant, [³H]doxepin, to rat brain tissie was examined. Scatchard plots of specific [³H]doxepin binding indicated the presence of two distinct binding sites. The equilibrium dissociation constant (K_D) of the high-affinity site was 0.020 nM with a maximal binding capacity (B_max) of 13.7 fmol/mg protein. The corresponding values for the low-affinity site were 3.6 nM and 740 fmol/mg protein, respectively. The high-affinity site was sensitive to competition by pharmacologically relevant concentrations of histamine H₁ antagonists such as pyrilamine (K_D = 1.0 nM), diphenhydramine (K_D = 20 nM), d-chlorpheniramine (K_D = 1.7 nM), and 1-chlorpheniramine (K_D = 97 nM). The B_max for [³H]doxepin binding in the high-affinity H₁-receptor, however, was approximately 10% of the B_max obtained using [³H]pyrilamine to label the H₁-receptor. Various tricyclic antidepressants were very potent inhibitors at the high-affinity [³H]doxepin site. Their potencies, however, did not correlated with their potencies previously reported for the H₁-receptor. The regional distribution of [³H]doxepin high-affinity sites correlated with the known distribution of H₁-receptors in the rat brain. These results suggest that [³H]doxepin is binding to a subclass of histamine H₁-receptors.     

Crystal structures,absolute configurations and molecular docking studies of naftopidil enantiomers as α1D-adrenoceptor antagonists

Chiral drug naftopidil (NAF), a specific α_1D-adrenoceptor (AR) antagonist for the treatment of benign prostatic hyperplasia, was used in racemic form for several decades. Our recent work declared that NAF enantiomers showed the same antagonistic effects on the α_1D-AR, but the binding mechanism of these two stereochemical NAF isomers to the α_1D receptor remained unclear. Herein, we reported the crystallographic structures of optically pure NAF stereoisomers for the first time and unambiguously determined their absolute configurations. The crystal data of R and S enantiomers matched satisfactorily the pharmacophore model for α_1D-selective antagonists. Based on the constructed α_1D homology model, molecular docking studies shed light on the molecular mechanism of NAF enantiomers binding to α_1D-AR. The results indicated that NAF enantiomers exhibited the very similar binding poses and occupied the same binding pocket.     

Evaluation of FAO/WHO pesticide standards in relation to polish and honduran diets

The FAO/WHO Codex Alimentarius Commission publishes a Guide to Codex Maximum Limits for Pesticide Residues (1978) which gives both acceptable daily intake (ADI) values and maximum residue limit (MRL) values for many pesticides in raw agricultural commodities (rac's) from crops on which their use is approved. ADI values are determined from animal toxicology data which give a no-observable-effect level (NOEL) and use of a safety factor. Specific MRL values are calculated from the ADI and a food factor (F), which is the decimal fraction of that rac in the assumed typical diet. One of the difficulties with this procedure is that dietary patterns differ substantially between countries. Whatever dietary pattern is assumed in calculation of the MRLs may be inappropriate for that country. Theoretical maximum residue intakes (TMRIs) were calculated from the Codex ADIs and MRLs using F values calculated from the national dietary patterns published separately in Poland and Honduras. The equation is TMRI = ΣMRL_i × F_i × D, where D is the daily individual diet weight of rac's in kilograms for the particular country. The TMRI should be lower than the maximum permitted intake (MPI) calculated as MPI = ADI × 60, where the 60 is an arbitrary human body weight in kilograms. The TMRI exceeded the MPI for 16 of 33 pesticides used in Poland and for 10 of 20 used in Honduras. At the extreme, the Polish TMRI/MPI ratio for aldrin-dieldrin is 19.5 and 18.1 for fenithion; nine pesticides had ratios of 4.0 or higher. For Honduras, piperonyl butoxide gave the highest ratio, 5.7 and three pesticides had ratios of 4.0 or higher. For Poland, the MRLs for 13 pesticides would cause the MPI to be exceeded by the theoretical residue concentrations of only one crop; in Honduras, seven pesticides have single-crop TMRIs greater than the MPI. Thus, these standards appear inappropriate for Poland and Honduras.     

Computational Study and Modified Design of Selective Dopamine D3 Receptor Agonists

Dopamine D₃ receptor (D₃R) is considered as a potential target for the treatment of nervous system disorders, such as Parkinson's disease. Current research interests primarily focus on the discovery and design of potent D₃ agonists. In this work, we selected 40 D₃R agonists as the research system. Comparative molecular field analysis (CoMFA) of three‐dimensional quantitative structure–activity relationship (3D‐QSAR), structure–selectivity relationship (3D‐QSSR), and molecular docking was performed on D₃ receptor agonists to obtain the details at atomic level. The results indicated that both the CoMFA model (r² = 0.982, q² = 0.503, = 0.893, SEE = 0.057, F = 166.308) for structure–activity and (r² = 0.876, q² = 0.436, = 0.828, F = 52.645) for structure–selectivity have good predictive capabilities. Furthermore, docking studies on three compounds binding to D₃ receptor were performed to analyze the binding modes and interactions. The results elucidate that agonists formed hydrogen bond and hydrophobic interactions with key residues. Finally, we designed six molecules under the guidance of 3D‐QSAR/QSSR models. The activity and selectivity of designed molecules have been improved, and ADMET properties demonstrate they have low probability of hepatotoxicity (<0.5). These results from 3D‐QSAR/QSSR and docking studies have great significance for designing novel dopamine D₃ selective agonists in the future.     

Mitochondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone

BACKGROUND AND PURPOSE

3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F₀F₁-ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme.

EXPERIMENTAL APPROACH

Kinetic analyses were performed on F₀F₁-ATP synthase in sub-mitochondrial particles and soluble F₁-ATPase. Activity assays and immunodetection of the inhibitor protein IF₁ were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis.

KEY RESULTS

T1AM was a non-competitive inhibitor of F₀F₁-ATP synthase whose binding was mutually exclusive with that of the inhibitors IF₁ and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF₁ binding site, causing IF₁ release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F₀F₁-ATP synthase consistent with displacement of endogenous IF_1,, reinforcing the in vitro results.

CONCLUSIONS AND IMPLICATIONS

Effects of T1AM on F₀F₁-ATP synthase were twofold: IF₁ displacement and enzyme inhibition. By targeting F₀F₁-ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF₁ and aurovertin binding sites are described.     

Alterations in serotonin1B (5HT1B) receptor subtypes in the brain of ethanol-treated rats

The effects of acute or chronic ethanol treatment and of withdrawal (24 h) after chronic ethanol treatment on 5HT_1B receptor subtypes in different regions of the rat brain were investigated. Male Sprague-Dawley rats were fed the ethanol (9% v/v)-containing Lieber-DeCarli liquid diet or the control liquid diet for 1 day in the acute study and for 15 days in the chronic study. The ethanol-withdrawn group received the Lieber-DeCarli control liquid diet instead of the ethanol diet on the 15th night. Ethanol-withdrawn rats after 15 days of ethanol treatment were rated for withdrawal symptoms (e.g. hyperactivity, piloerection, squealing, and enhanced startle reflex) and were found to exhibit such symptoms after 24 h of ethanol withdrawal. The rats were decapitated, and cortices, cerebelli, striata, and hippocampi were separated for measurement of 5HT_1B receptors by receptor binding techniques using ¹²⁵I-cyanopindolol (CYP) as the ligand. It was observed that acute ethanol treatment had no significant effect on the maximum number of binding sites (B_max) or the apparent dissociation constant (K_D) of 5HT_1B receptor binding sites in the various brain regions. On the other hand, chronic ethanol treatment produced a significant increase in B_max of ¹²⁵I-CYP binding to 5HT_1B receptors in the rat cortex and hippocampus, which remained increased after 24 h of ethanol withdrawal. In contrast, in the striatum and the cerebellum of chronic ethanol-treated and withdrawn rats, the 5HT_1B binding parameters (B_maxand K_D) were unchanged. These results suggest the possible involvement of cortical and hippocampal 5HT_1B receptors in ethanol dependence.