Dysmagnesemia as a predictor of developing diabetic levels of hemoglobin A1c.

The aim of this study was to assess the possible association between P-Mg and subsequent high levels of HbA1c. The study involves data from primary health care patients and data from patients treated in hospitals located in the capital region of Denmark. P-Mg and HbA1c levels were analyzed from 121,575 patients in the period 2010-2022. Patients were categorized in a diabetic and non-diabetic group. Out of 121,575 patients, 8,532 were categorized as diabetic. A reverse J-shaped association between P-Mg and HbA1c levels ≥ 48 mmol/mol was observed with nadir at P-Mg of 0.90 mmol/L. The unadjusted hazard ratio (HR) for having a first HbA1c measurement ≥ 48 mmol/mol is 1.54 (95% Cl 1.50; 1.57) per 0.1 mmol/L decrease in P-Mg when P-Mg is lower than 0.90 mmol/L. After adjusting for age and gender, the HR remained significant at 1.45 (95% Cl 1.41; 1.48).For P-Mg levels above 0.90 mmol/L, the unadjusted HR per 0.1 mmol/L increase in P-Mg was 1.04 (95% Cl 1.02; 1.06). After adjusting for age and gender the HR remained significant at 1.06 (95% Cl 1.05; 1.08). In conclusion, this study found that patients with dysmagnesemia have a higher risk of developing diabetes even after adjusting for age and gender. Hyper- or hypomagnesemia in patients could be a biomarker for predicting the risk of developing diabetes.

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aß, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy.

Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities.

Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [³H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious 'mixing' experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among five pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [³H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. The dopamine transporter (DAT) can exist as an oligomer but it is unknown whether the protomers function independently. The present results indicate that protomers that are superpotent or deficient in cocaine analog binding can confer enhanced or reduced potency to the oligomer, respectively. In this respect, positive or negative cooperativity is revealed in the DAT oligomer.

Novel C-1 substituted cocaine analogs unlike cocaine or benztropine.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.

SKF-83566, a D1-dopamine receptor antagonist, inhibits the dopamine transporter.

Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ∼ 65% in 5 µM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 µM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 µM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 µM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.

Filamin A mutant lacking actin-binding domain restores mu opioid receptor regulation in melanoma cells.

We have previously reported that the protein filamin A (FLA) binds to the carboxyl tail of the mu opioid receptor (MOPr). Using human melanoma cells, which do not express filamin A, we showed that receptor down-regulation, functional desensitization and trafficking are deficient in the absence of FLA (Onoprishvili et al. Mol Pharmacol 64:1092-1100, 2003). Since FLA has a binding domain for actin and is a member of the family of actin cytoskeleton proteins, it is usually assumed that FLA functions via the actin cytoskeleton. We decided to test this hypothesis by preparing cDNA coding for mutant FLA lacking the actin binding domain (FLA-ABD) and expressing FLA-ABD in the human melanoma cell line M2 (M2-ABD cell line). We report here that this mutant is capable of restoring almost as well as full length FLA the down-regulation of the human MOPr. It is similarly very effective in restoring functional desensitization of MOPr, as assessed by the decrease in G-protein activation after chronic exposure of M2-ABD cells to the mu agonist DAMGO. We also found that A7 cells, expressing wild type FLA, exhibit rapid activation of the MAP kinases, ERK 1 and 2, by DAMGO, as shown by a rise in the level of phospho-ERK 1 and 2. This is followed by rapid dephosphorylation (inactivation), which reaches basal level between 30 and 60 min after DAMGO treatment. M2 cells show normal activation of ERK 1 and 2 in the presence of DAMGO, but very slow inactivation. The rapid rate of MAPK inactivation is partially restored by FLA-ABD. We conclude that some functions of FLA do not act via the actin cytoskeleton. It is likely that other functions, not studied here, may require functional binding of the MOPr-FLA complex to actin.

Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTPγS assays.

BACKGROUND: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [(35)S]GTPγS to G protein cannot be observed in many "non-functional" D3 expressing cell lines. NEW METHOD: The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [(3)H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay). COMPARISON WITH EXISTING METHOD: The current study describes the determination of GTP shifts in [(3)H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope (35)S. CONCLUSION: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [(35)S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.

Characterization of [³H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive.

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [(3)H]nisoxetine. However, traditional [(3)H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [(3)H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [(3)H]CFT binding to whole cells (at physiological [Na(+)] and 25°C) were similar to potencies observed in the [(3)H]NE uptake assay. Inhibition constants (K(i)) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R(2)=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [(3)H]nisoxetine binding to membrane preparations (4°C for 3h, [Na(+)] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [(3)H]CFT binding became greater than that measured in a functional assay of [(3)H]NE uptake at physiological [Na(+)]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [(3)H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [(3)H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.

The novel trisubstituted pyran derivative D-142 has triple monoamine reuptake inhibitory activity and exerts potent antidepressant-like activity in rodents.

Major depression disorder is a significant health problem with 10-20% of all adults suffering from this disease. The underlying causes of depression are still unclear and 15% of depressed patients are resistant to all known therapies. Monoamine therapies have so far been the most successful approach for treating depression. Triple monoamine reuptake inhibitors have recently been implicated in generation of potent antidepressant activity while possibly exhibiting a low side-effect profile in addition to treating anhedonia. The additional, previously under-appreciated involvement of dopaminergic systems in depression prompted our efforts to develop novel asymmetric trisubstituted and disubstituted pyran derivatives as triple reuptake inhibitors. One of the lead compounds, D-142, exhibited uptake inhibition (K(i)) values of 29.3 nM, 14.7 nM and 59.3 ± 13.7 nM for norepinephrine, serotonin and dopamine transporters, respectively. Its affinity for serotonin transporter was comparable to fluoxetine, a well known SSRI. In the rat forced swimming test, compound D-142 exhibited potent antidepressant activity in the dose range tested (2.5, 5 and 10mg/kg) and was far more efficacious than the reference compound imipramine. In the mouse tail suspension test, compound D-142 reduced immobility in a dose (2.5, 5 and 10mg/kg) dependent manner, indicating a potent antidepressant effect. In locomotor activity tests, compound D-142 did not exhibit any stimulation in the same dose ranges. In the extended CNS receptors screening assay this molecule exhibited little or no non-specific interaction in the CNS, indicating high specificity for monoamine transporters. These results advance D-142 as a potential potent antidepressant.

Further structure-activity relationship studies on 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol: identification of compounds with triple uptake inhibitory activity as potential antidepressant agents.

To investigate structural alterations of the lead triple uptake inhibitor molecule, disubstituted 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol, we have carried out structure-activity relationship (SAR) studies to investigate the effect of alteration of aromatic substitutions and introduction of heterocyclic aromatic moieties on this molecular template. The novel compounds were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. SAR results indicate dopamine norepinephrine reuptake inhibitory (DNRI) type activity in thiophene (10g) and pyrrole (10i) derivatives. On the other hand, 3-hydroxyphenyl derivative 10f and 4-methoxyphenyl derivative 10j exhibited a triple reuptake inhibitory (TUI) activity profile, as these molecules exhibited potent uptake inhibition for all the monoamine transporters (K(i) of 31.3, 40, 38.5 and K(i) of 15.9, 12.9, 29.3 for DAT, SERT, and NET for 10f and 10g, respectively). Compound 10f was further evaluated in the rat forced swim test to evaluate its potential antidepressant effect. The results show significant reduction of immobility by TUI 10f at 10 mg/kg dose, indicating potential antidepressant activity.