ABSTRACT
Background Posttranslational protein modification with O-linked N-acetylglucosamine (O-GlcNAc) is linked to high glucose levels in type 2 diabetes mellitus (T2DM) and may alter cellular function. We sought to elucidate the involvement of O-GlcNAc modification in endothelial dysfunction in patients with T2DM. Methods and Results Freshly isolated endothelial cells obtained by J-wire biopsy from a forearm vein of patients with T2DM (n=18) was compared with controls (n=10). Endothelial O-GlcNAc levels were 1.8-ford higher in T2DM patients than in nondiabetic controls (P=0.003). Higher endothelial O-GlcNAc levels correlated with serum fasting blood glucose level (r=0.433, P=0.024) and hemoglobin A1c (r=0.418, P=0.042). In endothelial cells from patients with T2DM, normal glucose conditions (24 hours at 5 mmol/L) lowered O-GlcNAc levels and restored insulin-mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O-GlcNAc levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O-GlcNAcase inhibitor, increased O-GlcNAc levels and blunted the improvement of insulin-mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O-GlcNAc modification in the dynamic, glucose-induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2DM. O-GlcNAc protein modification may be a treatment target for vascular dysfunction in T2DM.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Endothelial Cells/drug effects , Forearm/blood supply , Glucose/pharmacology , Glucose/toxicity , Protein Processing, Post-Translational/drug effects , Adult , Case-Control Studies , Cells, Cultured , Diabetes Mellitus, Type 2/diagnosis , Endothelial Cells/metabolism , Female , Glycosylation , Humans , Insulin/pharmacology , Male , Middle Aged , Nitric Oxide Synthase Type III/metabolism , Phenotype , Phosphorylation , beta-N-Acetylhexosaminidases/metabolismABSTRACT
Background Prior studies have shown that nutrient excess induces endoplasmic reticulum ( ER ) stress in nonvascular tissues from patients with diabetes mellitus ( DM ). ER stress and the subsequent unfolded protein response may be protective, but sustained activation may drive vascular injury. Whether ER stress contributes to endothelial dysfunction in patients with DM remains unknown. Methods and Results To characterize vascular ER stress, we isolated endothelial cells from 42 patients with DM and 37 subjects without DM. Endothelial cells from patients with DM displayed higher levels of ER stress markers compared with controls without DM. Both the early adaptive response, evidenced by higher phosphorylated protein kinase-like ER eukaryotic initiation factor-2a kinase and inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.02, P=0.007, respectively), and the chronic ER stress response evidenced by higher C/ EBP α-homologous protein ( P=0.02), were activated in patients with DM . Higher inositol-requiring ER-to-nucleus signaling protein 1 activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction ( r=0.53, P=0.02). Acute treatment with liraglutide, a glucagon-like peptide 1 receptor agonist, reduced p-inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.01), and the activation of its downstream target c-jun N-terminal kinase ( P=0.025) in endothelial cells from patients with DM . Furthermore, liraglutide restored insulin-stimulated endothelial nitric oxide synthase activation in patients with DM ( P=0.019). Conclusions In summary, our data suggest that ER stress contributes to vascular insulin resistance and endothelial dysfunction in patients with DM . Further, we have demonstrated that liraglutide ameliorates ER stress, decreases c-jun N-terminal kinase activation and restores insulin-mediated endothelial nitric oxide synthase activation in endothelial cells from patients with DM .
Subject(s)
Diabetes Mellitus/drug therapy , Endoplasmic Reticulum Stress/drug effects , Endothelium, Vascular/physiopathology , Insulin Resistance/physiology , Insulin/blood , Liraglutide/pharmacology , Vasodilation/physiology , Brachial Artery/diagnostic imaging , Brachial Artery/physiopathology , Cells, Cultured , Diabetes Mellitus/metabolism , Diabetes Mellitus/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Humans , Hypoglycemic Agents/pharmacology , Male , Middle Aged , Ultrasonography, DopplerABSTRACT
OBJECTIVE: Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function. APPROACH AND RESULTS: Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1-100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction. CONCLUSIONS: Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity.
Subject(s)
Electronic Nicotine Delivery Systems , Endothelial Cells/drug effects , Flavoring Agents/toxicity , Tobacco Products/toxicity , Adult , Case-Control Studies , Cell Death/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Humans , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Male , Middle Aged , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Risk Assessment , Smoking/adverse effects , Vaping/adverse effectsABSTRACT
BACKGROUND: Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. METHODS AND RESULTS: We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. CONCLUSION: Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease.
Subject(s)
Autophagy/drug effects , Diabetes Mellitus, Type 2/pathology , Diabetic Angiopathies/pathology , Endothelial Cells/drug effects , Nitric Oxide/metabolism , Signal Transduction/drug effects , Spermidine/pharmacology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adult , Aged , Biomarkers/metabolism , Case-Control Studies , Cell Separation/methods , Cells, Cultured , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/physiopathology , Diabetic Angiopathies/blood , Diabetic Angiopathies/physiopathology , Diabetic Angiopathies/prevention & control , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Humans , Macrolides/pharmacology , Male , Middle Aged , Nitric Oxide Synthase Type III/metabolismABSTRACT
OBJECTIVE: Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. APPROACH AND RESULTS: We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. CONCLUSIONS: Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus.
Subject(s)
Brachial Artery/enzymology , Diabetes Mellitus, Type 2/enzymology , Endothelial Cells/enzymology , Endothelium, Vascular/enzymology , JNK Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Vasodilation , Wnt Proteins/metabolism , Wnt Signaling Pathway , Adult , Aged , Brachial Artery/drug effects , Brachial Artery/physiopathology , Case-Control Studies , Cells, Cultured , Diabetes Mellitus, Type 2/physiopathology , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiopathology , Enzyme Activation , Female , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Male , Middle Aged , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidative Stress , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/pharmacology , Vasodilation/drug effects , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/pharmacology , Wnt Signaling Pathway/drug effects , Wnt-5a ProteinABSTRACT
Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ~12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH.
Subject(s)
Dual Specificity Phosphatase 6/genetics , Fibroblast Growth Factors/genetics , Genetic Predisposition to Disease/genetics , Hypogonadism/genetics , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Receptors, Interleukin/genetics , Algorithms , Animals , Base Sequence , Computational Biology , Female , Genetic Association Studies , Humans , Immunohistochemistry , Inheritance Patterns/genetics , Male , Membrane Glycoproteins , Mice , Molecular Sequence Data , Mutation/genetics , Sequence Analysis, DNA , Sequence Homology , Surface Plasmon ResonanceABSTRACT
CONTEXT: Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. OBJECTIVE: The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. DESIGN AND PARTICIPANTS: A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. RESULTS: Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). CONCLUSIONS: Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.
Subject(s)
Fibroblast Growth Factor 8/genetics , Hypopituitarism/genetics , Kallmann Syndrome/genetics , Mutation , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Septo-Optic Dysplasia/genetics , Animals , Female , Fibroblast Growth Factor 8/metabolism , Genetic Association Studies , Heterozygote , Humans , Hypopituitarism/metabolism , Kallmann Syndrome/metabolism , Male , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Neurons/pathology , Pituitary Gland, Posterior/metabolism , Pituitary Gland, Posterior/pathology , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Recombinant Fusion Proteins/metabolism , Septo-Optic Dysplasia/metabolism , Signal Transduction , United Kingdom , United StatesABSTRACT
CaMKII plays a critical role in long-term potentiation (LTP). The kinase is a major component of the postsynaptic density (PSD); however, it is also contained in the spine cytoplasm. CaMKII can now be monitored optically in living neurons, and it is therefore important to understand the contribution of the PSD and cytoplasmic pools to optical signals. Here, we estimate the size of these pools under basal conditions. From EM immunolabeling data, we calculate that the PSD/cytoplasmic ratio is ~5%. A second independent estimate is derived from measurements indicating that the average mushroom spine PSD contains 90 to 240 holoenzymes. A cytoplasmic concentration of 16 µM (~2590 holoenzymes) in the spine can be estimated from the total measured CaMKII content of hippocampal tissue, the relative volume of different compartments, and the spine-dendrite ratio of CaMKII (2:1). These numbers yield a second estimate (6%) of the PSD/spine ratio in good agreement with the first. The CaMKII bound to the NMDAR is important because preventing the formation of this complex blocks LTP induction. We estimate that the percentage of spine CaMKII held active by binding to the NMDAR is ~0.2%. Implications of the high spine concentration of CaMKII (> 100 µM alpha subunits) and the small fraction within the PSD are discussed. Of particular note, the finding that the CaMKII signal in spines shows only transient activation (open state) after LTP induction is subject to the qualification that it does not reflect the small but important pool bound to the NMDAR.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cytoplasm/enzymology , Dendritic Spines/enzymology , Hippocampus/enzymology , Post-Synaptic Density/enzymology , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Cytoplasm/ultrastructure , Dendritic Spines/ultrastructure , Hippocampus/ultrastructure , Mice , Post-Synaptic Density/ultrastructure , Primary Cell Culture , Rats , Receptors, N-Methyl-D-Aspartate/ultrastructureABSTRACT
N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (-) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 muM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor-mediated CA3-CA1 synaptic response in rat hippocampal slices. UBP125, UBP128, UBP141, and UBP145 may be useful tools for determining the function of NMDA receptor subtypes.
Subject(s)
Dicarboxylic Acids/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Hippocampus/metabolism , Piperazines/pharmacology , Receptors, N-Methyl-D-Aspartate/drug effects , Synapses/drug effects , Animals , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Drug Design , Electrophysiology , Hippocampus/drug effects , In Vitro Techniques , Kainic Acid/metabolism , Oocytes , Rats , Rats, Wistar , Receptors, AMPA/drug effects , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/chemistry , Structure-Activity Relationship , Substrate Specificity , Xenopus laevisABSTRACT
Exposure to endothelin-1 (ET-1, 50 nM) of sensory neurons, acutely isolated from rat dorsal root ganglia (DRG), results in an increase in the number of action potentials elicited by a linear ramp of stimulating current. The changes are complete in 5 min after ET-1 treatment and do not reverse in 5-10 min after ET-1's removal. Neither the resting potential, nor the threshold potential for the first or second action potentials, nor their rate-of-rise or decay, are changed by ET-1 exposure, but the slow depolarizations which occur before the first and second action potentials during the current ramp are increased by ca. 50% by ET-1. The delayed rectifier type of K(+) currents (I(K)), measured under whole-cell voltage clamp, are depressed by approximately 30% during such exposure to ET-1. The voltage-dependent gating of steady-state I(K) and the current kinetics are unchanged by ET-1, leaving the sole effect as a drop in the number of available channels. I(K) is affected by ET-1 only in Isolectin B(4)-positive cells, suggesting that there may be a selective action in enhancing impulse activity on this class of nociceptive neuron. This decrease in I(K) will potentiate the excitability-inducing actions of the previously reported negative shift in tetrodotoxin-resistant Na(+) channel gating in such neurons.
Subject(s)
Delayed Rectifier Potassium Channels/metabolism , Endothelin-1/metabolism , Potassium/metabolism , Sensory Receptor Cells/physiology , Action Potentials/physiology , Analysis of Variance , Animals , Cell Size , Cells, Cultured , Endothelin-1/pharmacology , Ganglia, Spinal/cytology , Ganglia, Spinal/physiology , Glycoproteins/metabolism , Lectins/metabolism , Male , Membrane Potentials/physiology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Sensory Receptor Cells/cytology , Time Factors , VersicansABSTRACT
In the present study, we examined the interaction of (+/-)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-d-aspartate (NMDA) and sigma(1) receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the l-glutamate or glycine binding sites. The low potency of donepezil (IC(50) = 0.7-3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the sigma(1) receptor with high affinity (K(i) = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5-8, 1999). Thus, we sought to determine whether an interaction with the sigma(1) receptor may occur in vivo under physiologically relevant conditions by evaluating the sigma(1) receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the sigma(1) receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the sigma(1) receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective sigma(1) receptor agonist on these behavioral responses, and an interaction of the drug with the sigma(1) receptor must be considered in its pharmacological actions.
Subject(s)
Behavior, Animal/drug effects , Cholinesterase Inhibitors/pharmacology , Indans/pharmacology , Piperidines/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, sigma/antagonists & inhibitors , Animals , Avoidance Learning/drug effects , Binding Sites , Cloning, Molecular , Donepezil , Ethylenediamines/pharmacology , Female , Ligands , Male , Maze Learning/drug effects , Mice , Oligodeoxyribonucleotides, Antisense/pharmacology , Oocytes/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, sigma/metabolism , Swimming , Xenopus , Sigma-1 ReceptorABSTRACT
The binding site for competitive NMDA receptor antagonists is on the NR2 subunit, of which there are four types (NR2A-D). Typical antagonists such as (R)-AP5 have a subunit selectivity of NR2A > NR2B > NR2C > NR2D. The competitive NMDA receptor antagonist (2R,3S)-(1-biphenylyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD, 16b) displays an unusual selectivity with improved relative affinity for NR2C and NR2D vs NR2A and NR2B. Analogues of 16b bearing aroyl or aryl substituents attached to the N(1) position of piperazine-2,3-dicarboxylic acid have been synthesized to probe the structural requirements for NR2C/NR2D selectivity. A phenanthrenyl-2-carbonyl analogue, 16e, had >60-fold higher affinity for NR2C and NR2D and showed 3-5-fold selectivity for NR2C/NR2D vs NR2A/NR2B. The phenanthrenyl-3-carbonyl analogue (16f) was less potent but more selective, having 5- and 7-fold selectivity for NR2D vs NR2A and NR2B, respectively. Thus, antagonists bearing bulky hydrophobic residues have a different NR2 subunit selectivity than that of typical antagonists.
Subject(s)
Piperazines/chemical synthesis , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Animals , Animals, Newborn , Female , In Vitro Techniques , Male , Molecular Structure , Motor Neurons/drug effects , Motor Neurons/physiology , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Piperazines/chemistry , Piperazines/pharmacology , Protein Subunits/antagonists & inhibitors , Protein Subunits/physiology , Rats , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/physiology , Recombinant Proteins/antagonists & inhibitors , Spinal Cord/cytology , Stereoisomerism , Xenopus laevisABSTRACT
The resolved X-ray crystal structures of the glutamate-binding domain (S1/S2 domains) of the GluR2 and NR1 glutamate receptor subunits were used to model the homologous regions of the N-methyl-D-aspartate (NMDA) receptor's NR2 subunits. To test the predictive value of these models, all four stereoisomers of the antagonist 1-(phenanthren-2-carbonyl) piperazine-2,3-dicarboxylic acid (PPDA) were docked into the NR2B glutamate-binding site model. This analysis suggested an affinity order for the PPDA isomers of d-cis > L-cis > L-trans = D-trans and predicted that the 2-position carboxylate group of the cis-PPDA isomers, but not of the trans-PPDA isomers, may be interacting with histidine 486 in NR2B. Consistent with these predictions, cis-PPDA displays a 35-fold higher affinity for NR2B-containing NMDA receptors than trans-PPDA. In addition, mutating NR2B's H486 to phenylalanine decreased cis-PPDA affinity 8-fold but had no effect on trans-PPDA affinity. In contrast, the NR2B H486F mutation increased the affinity of the typical antagonists CGS-19755 [(2R*,4S*)-4-phosphonomethyl-2-piperidine carboxylic acid] and 4-(3-phosphonopropyl) piperidine-2-carboxylic acid. In the NR1-based NR2 models, there were only four subunit-specific amino acid residues exposed to the ligand-binding pocket (and six in the GluR2-based models). These residues are located at the edge of the binding pocket, suggesting that large antagonists may be necessary for subtype specificity. Of these residues, mutational analysis and modeling suggest that A414, R712, and G713 (NR2B numbering) may be especially useful for developing NR2C- and NR2D-selective NMDA receptor antagonists and that residues A414 and T428 may determine subunit variations in agonist affinity.
Subject(s)
Glutamic Acid/metabolism , Receptors, N-Methyl-D-Aspartate/chemistry , Amino Acid Sequence , Animals , Binding Sites , Female , Humans , Models, Molecular , Molecular Sequence Data , Point Mutation , Protein Subunits , Rats , Receptors, N-Methyl-D-Aspartate/metabolism , Structure-Activity Relationship , Xenopus laevisABSTRACT
The widely-used N-methyl-D-aspartate (NMDA) receptor antagonists (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid ((R)-CPP) and (R)-2-amino-7-phosphonoheptanoate ((R)-AP7) are frequently used as general NMDA receptor antagonists and assumed not to display significant selectivity among NMDA receptor NR2 subunits. However, electrophysiological studies have suggested that certain longer chain N-methyl-D-aspartate (NMDA) receptor competitive antagonists, such as (R)-CPP are ineffective at subpopulations of NMDA receptors in the red nucleus, superior colliculus, and hippocampus. Using recombinant receptors expressed in Xenopus oocytes, we have examined the effect of antagonist chain length on NR2 subunit selectivity. All antagonists displayed the potency order (high to low affinity) of NR2A > NR2B > NR2C > NR2D, however the longer chain antagonists (having 7 instead of 5 bond lengths between acidic groups) displayed much greater subunit selectivity than their short-chain homologues. For example (R)-CPP displayed a 50-fold difference in affinity between NR2A-containing and NR2D-containing NMDA receptors, while the shorter chain homologue 4-(phosphonomethyl) piperazine-2-carboxylic acid (PMPA) displayed only a 5-fold variation in affinity. These results can account for the earlier physiological findings and suggest that longer chain antagonists such as (R)-CPP and (R)-AP7 should not be used as general NMDA receptor antagonists.
Subject(s)
Excitatory Amino Acid Antagonists/chemistry , Excitatory Amino Acid Antagonists/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Female , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , Xenopus laevisABSTRACT
In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA receptor-mediated EPSCs (EPSC(NMDA)). Such a time course has been attributed to the cooperation of synapses through glutamate spillover. Imitating a natural pattern of activity, we have applied short bursts (2-7 stimuli) of high-frequency stimulation and observed a spike-to-spike slow-down of the EPSC(NMDA) kinetics, which accompanied synaptic facilitation. It was found that the early component of the EPSC(NMDA) and the burst-induced late component of the EPSC(NMDA) have distinct pharmacological properties. The competitive NMDA antagonist R-(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP), which has higher affinity to NR2A than to NR2B subunits and lowest affinity at NR2D subunits, significantly slowed down the decay rate of the afterburst EPSC while leaving the kinetics of the control current unaffected. In contrast, ifenprodil, a highly selective NR2B antagonist, and [+/-]-cis-1-[phenanthren-2yl-carbonyl]piperazine-2,3-dicarboxylic acid (PPDA), a competitive antagonist that is moderately selective for NR2D subunits, more strongly inhibited the late component of the afterburst EPSC(NMDA). The receptors formed by NR2B and (especially) NR2D subunits are known to have higher agonist sensitivity and much slower deactivation kinetics than NR2A-containing receptors. Furthermore, NR2B is preferentially and NR2D is exclusively located on extrasynaptic membranes. As the density of active synapses increases, the confluence of released glutamate makes EPSC decay much longer by activating more extrasynaptic NR2B- and NR2D-subunit-containing receptors. Long-term potentiation (LTP) induced by successive rounds of burst stimulation is accompanied by a long-term increase in the contribution of extrasynaptic receptors in the afterburst EPSC(NMDA.)
Subject(s)
Excitatory Postsynaptic Potentials/physiology , Hippocampus/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Animals , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Oocytes/physiology , Piperidines/pharmacology , Rats , Rats, Wistar , Synapses/physiology , XenopusABSTRACT
(2S*,3R*)-1-(biphenyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD) is a moderate affinity, competitive N-methyl-d-aspartate (NMDA) receptor antagonist with an atypical pattern of selectivity among NMDA receptor 2 subunit (NR2) subunits. We now describe the activity of several derivatives of PBPD tested at both rat brain NMDA receptors using l-[3H]-glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. Substituting various branched ring structures for the biphenyl group of PBPD reduced NMDA receptor activity. However, substituting linearly arranged ring structures - fluorenone or phenanthrene groups - retained or enhanced activity. Relative to PBPD, the phenanthrene derivative (2S*, 3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) displayed a 30- to 78-fold increase in affinity for native NMDA receptors. At recombinant receptors, PPDA displayed a 16-fold (NR2B) to 94-fold (NR2C) increase in affinity over PBPD. Replacement of the biphenyl group of PBPD with a 9-oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 2'-Bromo substitution on the biphenyl group of PBPD reduced antagonist affinity 3- to 5-fold at NR2A-, NR2B- and NR2D-containing receptors, but had little effect on NR2C-containing receptors. In contrast, 4'-fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. The aromatic rings of PBPD and PPDA increase antagonist affinity and appear to interact with a region of the NMDA receptor displaying subunit heterogeneity. PPDA is the most potent and selective NR2C/NR2D-preferring antagonist yet reported and thus may be useful in defining NR2C/NR2D function and developing related antagonists with improved NMDA receptor subtype selectivity. British Journal of Pharmacology (2004) 141, 508-516. doi:10.1038/sj.bjp.0705644