ABSTRACT
Reduced haloperidol (1) was previously reported to act as a potent sigma-1 receptor (S1R) ligand with substantially lower affinity to the dopamine D2 receptor (D2R) compared to haloperidol. It was also found to facilitate brain-derived neurotrophic factor (BDNF) secretion from astrocytic glial cell lines in a sigma-1 receptor (S1R)-dependent manner. Although an increase in BDNF secretion may have beneficial effects in some neurological conditions, the therapeutic utility of reduced haloperidol (1) is limited because it can be oxidized back to haloperidol in the body, a potent dopamine D2 receptor antagonist associated with well-documented adverse effects. A difluorinated analogue of reduced haloperidol, (±)-4-(4-chlorophenyl)-1-(3,3-difluoro-4-(4-fluorophenyl)-4-hydroxybutyl)piperidin-4-ol (2), was synthesized in an attempt to minimize the oxidation. Compound (±)-2 was found to exhibit high affinity to S1R and facilitate BDNF release from mouse brain astrocytes. It was also confirmed that compound 2 cannot be oxidized back to the corresponding haloperidol analogue in liver microsomes. Furthermore, compound 2 was distributed to the brain following intraperitoneal administration in mice and reversed the learning deficits in active avoidance tasks. These findings suggest that compound 2 could serve as a promising S1R ligand with therapeutic potential for the treatment of cognitive impairments.
Subject(s)
Haloperidol , Receptors, sigma , Mice , Animals , Haloperidol/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Ligands , Sigma-1 ReceptorABSTRACT
Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)-induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.
Subject(s)
ORAI1 Protein , Podocytes , Calcium/metabolism , Calcium Channels/metabolism , Calcium Signaling/physiology , Calpain/genetics , Calpain/metabolism , Glucose/metabolism , Glucose/pharmacology , Humans , Neoplasm Proteins/metabolism , ORAI1 Protein/genetics , ORAI1 Protein/metabolism , Podocytes/metabolism , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolismABSTRACT
To determine whether (+)-catharanthine induces sedative- or anxiolytic/anxiogenic-like activity in male mice, proper animal paradigms were used. The results showed that (+)-catharanthine induces sedative-like activity in the 63-72 mg/Kg dose range in a flumazenil-insensitive manner, but neither this effect nor anxiolytic/anxiogenic-like activity was observed at lower doses. To determine the underlying molecular mechanism of the sedative-like activity, electrophysiological and radioligand binding experiments were performed with (+)-catharanthine and (±)-18-methoxycoronaridine [(±)-18-MC] on GABAA (GABAARs) and glycine receptors (GlyRs). Coronaridine congeners both activated and potentiated a variety of human (h) GABAARs, except hρ1. (+)-Catharanthine-induced potentiation followed this receptor selectivity (EC50's in µM): hα1ß2 (4.6 ± 0.8) > hα2ß2γ2 (12.6 ± 3.8) ~ hα1ß2γ2 (14.4 ± 4.6) indicating that both α1 and α2 are equally important, whereas γ2 is not necessary. (+)-Catharanthine was >2-fold more potent and efficient than (±)-18-MC at hα1ß2γ2. (+)-Catharanthine also potentiated, whereas (±)-18-MC inhibited, hα1 GlyRs with very low potency. Additional [3H]-flunitrazepam competition binding experiments using rat cerebellum membranes clearly demonstrated that these ligands do not bind to the benzodiazepine site. This is supported by the observed activity at hα1ß2 (lacking the BDZ site) and similar effects between α1- and α2-containing GABAARs. Our study shows, for the first time, that (+)-catharanthine induced sedative-like effects in mice, and coronaridine congeners potentiated human α1ß2γ2, α1ß2, and hα2ß2γ2, but not ρ1, GABAARs, both in a benzodiazepine-insensitive fashion, whereas only (+)-catharanthine slightly potentiated GlyRs.
Subject(s)
Benzodiazepines/metabolism , Hypnotics and Sedatives/metabolism , Ibogaine/analogs & derivatives , Ibogaine/metabolism , Receptors, GABA-A/metabolism , Animals , Benzodiazepines/pharmacology , Dose-Response Relationship, Drug , GABA-A Receptor Agonists/metabolism , GABA-A Receptor Agonists/pharmacology , HEK293 Cells , Humans , Hypnotics and Sedatives/pharmacology , Ibogaine/pharmacology , Locomotion/drug effects , Locomotion/physiology , Male , Maze Learning/physiology , MiceABSTRACT
Stroke leads to devastating outcomes including impairments of sensorimotor and cognitive function that may be long lasting. New intervention strategies are needed to overcome the long-lasting effects of ischemic injury. Previous studies determined that treatment with 5-methoxyindole-2-carboxylic acid (MICA) conferred chemical preconditioning and neuroprotection against stroke. The purpose of the current study was to determine whether the preconditioning can lead to functional improvements after stroke (done by transient middle cerebral artery occlusion). After 4 weeks of MICA feeding, half the rats underwent ischemic injury, while the other half remained intact. After one week recovery, all the rats were tested for motor and cognitive function (rotorod and water maze). At the time of euthanasia, measurements of long-term potentiation (LTP) were performed. While stroke injury led to motor and cognitive dysfunction, MICA supplementation did not reverse these impairments. However, MICA supplementation did improve stroke-related impairments in hippocampal LTP. The dichotomy of the outcomes suggest that more studies are needed to determine optimum duration and dosage for MICA to lead to substantial motor and cognitive improvements, along with LTP change and neuroprotection.
Subject(s)
Hippocampus/drug effects , Indoles/pharmacology , Ischemic Stroke/drug therapy , Ischemic Stroke/prevention & control , Ischemic Stroke/physiopathology , Long-Term Potentiation/drug effects , Maze Learning/drug effects , Neuroprotective Agents/pharmacology , Psychomotor Performance/drug effects , Recovery of Function/drug effects , Animals , Behavior, Animal/drug effects , Dietary Supplements , Dihydrolipoamide Dehydrogenase/drug effects , Disease Models, Animal , Indoles/administration & dosage , Infarction, Middle Cerebral Artery/complications , Ischemic Stroke/etiology , Male , Neuroprotective Agents/administration & dosage , Rats , Rats, Sprague-DawleyABSTRACT
Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca2+) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca2+ results in channel opening, with the site's location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.
Subject(s)
Acid Sensing Ion Channels/chemistry , Binding Sites/physiology , Calcium/metabolism , Ion Channel Gating/physiology , Acid Sensing Ion Channels/genetics , Acid Sensing Ion Channels/metabolism , Animals , CHO Cells , Cations, Divalent/metabolism , Cricetulus , Glutamic Acid/genetics , Glutamic Acid/metabolism , Glycine/genetics , Glycine/metabolism , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Domains/physiology , Structure-Activity RelationshipABSTRACT
This study aimed to address the mechanisms and reinforcing effects of three synthetic cathinone analogs of MDMA commonly reported in "Ecstasy" formulations: methylone, butylone, and pentylone. Whole-cell patch clamp techniques were used to assess the mechanism of each compound at the dopamine and serotonin transporters. Separate groups of rats were trained to discriminate methamphetamine, DOM, or MDMA from vehicle. Substitution studies were performed in each group and antagonism studies with SCH23390 were performed against each compound that produced substitution. Self-administration of each compound was evaluated under a progressive ratio schedule of reinforcement. Each compound produced an inward current at the serotonin transporter, but little or no current at the dopamine transporter. Each of the test compounds substituted fully for the discriminative stimulus effects of methamphetamine, methylone and butylone substituted partially for DOM and fully for MDMA, whereas pentylone failed to substitute for DOM and substituted only partially for MDMA. SCH23390 fully and dose-dependently attenuated methamphetamine-appropriate responding produced by each test compound, but was least potent against pentylone. MDMA-appropriate responding was minimally affected by SCH23390. Each test compound was robustly self-administered with pentylone producing the greatest self-administration at the doses tested. Given the prevalence of synthetic cathinones in "Ecstasy" formulations, these data indicate that adulterated "Ecstasy" formulations may drive more compulsive drug use than those containing only MDMA.
Subject(s)
Hallucinogens/pharmacology , N-Methyl-3,4-methylenedioxyamphetamine/pharmacology , Reinforcement, Psychology , Substance-Related Disorders/physiopathology , Animals , Benzazepines/pharmacology , Central Nervous System Stimulants/pharmacology , Conditioning, Operant/drug effects , Disease Models, Animal , Dopamine Antagonists/pharmacology , Dopamine Plasma Membrane Transport Proteins/genetics , Dopamine Plasma Membrane Transport Proteins/metabolism , Drug Combinations , HEK293 Cells , Humans , Male , Methamphetamine/analogs & derivatives , Methamphetamine/pharmacology , N-Methyl-3,4-methylenedioxyamphetamine/analogs & derivatives , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Self Administration , Serotonin/pharmacology , Substance-Related Disorders/etiologyABSTRACT
It is well established that NADH/NAD+ redox balance is heavily perturbed in diabetes, and the NADH/NAD+ redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD+ regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD+ redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and ß cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.
ABSTRACT
Efavirenz is a widely prescribed medicine used to treat type 1 human immunodeficiency virus (HIV-1), the most prevalent pathogenic strain of the virus responsible for the acquired immune deficiency syndrome (AIDS) pandemic. Under prescribed dosing conditions, either alone or in combination therapy, efavirenz-induced CNS disturbances are frequently reported. Efavirenz was recently reported to interact in a similar concentration range with a number of receptors, transporters and ion channels including recombinant rat α1ß2γ2 GABAA receptors whose actions were potentiated (Gatch et al., 2013; Dalwadi et al., 2016). Now we report on the molecular mechanism of efavirenz on GABAA receptors as a function of concentration and subunit composition via whole-cell recordings of GABA-activated currents from HEK293 cells expressing varying subunit configurations of GABAA receptors. Efavirenz elicited dual effects on the GABA response; it allosterically potentiated currents at low concentrations, whereas it inhibited currents at higher concentrations. The allosteric potentiating action on GABAA receptors was pronounced in the α1ß2γ2, α2ß2γ2 and α4ß2γ2 configurations, greatly diminished in the α6ß2γ2 configuration, and completely absent in the α3ß2γ2 or α5ß2γ2 configuration. In stark contrast, the inhibitory modulation of efavirenz at higher concentrations was evident in all subunit configurations examined. Moreover, efavirenz-induced modulatory effects were dependent on GABA concentration ([GABA]), with a pronounced impact on currents activated by low [GABA] but little effect at saturating [GABA]. Mutation of a highly-conserved threonine to phenylalanine in transmembrane domain 2 of the α1 subunit abolished the inhibitory effect of efavirenz in α1ß2 receptors. Finally, mutations of any of the three conserved extracellular residues in α1/2/4 subunits to the conserved residues at the corresponding positions in α3/5 subunits (i.e., R84P, M89L or I120L) completely eliminated the potentiating effect of efavirenz in α1ß2γ2 configuration. These findings demonstrate that efavirenz's positive allosteric modulation of the GABAA receptor is mediated via a novel allosteric site associated with the extracellular domain of the receptor.
Subject(s)
Benzoxazines/pharmacology , Receptors, GABA-A/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Alkynes , Allosteric Regulation , Animals , Cyclopropanes , Diazepam/pharmacology , Dose-Response Relationship, Drug , Flumazenil/pharmacology , GABA Modulators/pharmacology , HEK293 Cells , Humans , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mutagenesis/genetics , Patch-Clamp Techniques , Protein Domains/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Receptors, GABA-A/genetics , Transfection , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 µM, and it had an IC50 of 31 µM in human α1ß2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the ß2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1ß2 receptors to MB was similar to that observed in α1ß2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and ß2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.
Subject(s)
Binding Sites/drug effects , Enzyme Inhibitors/pharmacology , Methylene Blue/pharmacology , Neurons/drug effects , Receptors, GABA-A/metabolism , Animals , Animals, Newborn , Binding Sites/genetics , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , HEK293 Cells , Hippocampus/cytology , Humans , In Vitro Techniques , Inhibitory Postsynaptic Potentials/drug effects , Inhibitory Postsynaptic Potentials/genetics , Male , Mice , Mice, Inbred C57BL , Neurons/metabolism , Protein Binding/drug effects , Rats , Receptors, GABA-A/genetics , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Efavirenz is highly effective at suppressing HIV-1, and the WHO guidelines list it as a component of the first-line antiretroviral (ARV) therapies for treatment-naïve patients. Though the pharmacological basis is unclear, efavirenz is commonly associated with a risk for neuropsychiatric adverse events (NPAEs) when taken at the prescribed dose. In many patients these NPAEs appear to subside after several weeks of treatment, though long-term studies show that in some patients the NPAEs persist. In a recent study focusing on the abuse potential of efavirenz, its receptor psychopharmacology was reported to include interactions with a number of established molecular targets for known drugs of abuse, and it displayed a prevailing behavioral profile in rodents resembling an LSD-like activity. In this report, we discovered interactions with additional serotonergic targets that may be associated with efavirenz-induced NPAEs. The most robust interactions were with 5-HT3A and 5-HT6 receptors, with more modest interactions noted for the 5-HT2B receptor and monoamine oxidase A. From a molecular mechanistic perspective, efavirenz acts as a 5-HT6 receptor inverse agonist of Gs-signaling, 5-HT2A and 5-HT2C antagonist of Gq-signaling, and a blocker of the 5-HT3A receptor currents. Efavirenz also completely or partially blocks agonist stimulation of the M1 and M3 muscarinic receptors, respectively. Schild analysis suggests that efavirenz competes for the same site on the 5-HT2A receptor as two known hallucinogenic partial agonists (±)-DOI and LSD. Prolonged exposure to efavirenz reduces 5-HT2A receptor density and responsiveness to 5-HT. Other ARVs such as zidovudine, nevirapine and emtricitabine did not share the same complex pharmacological profile as efavirenz, though some of them weakly interact with the 5-HT6 receptor or modestly block GABAA currents.
Subject(s)
Anti-HIV Agents/toxicity , Benzoxazines/toxicity , Brain/drug effects , HIV Infections/drug therapy , HIV-1/drug effects , Receptors, Serotonin/drug effects , Reverse Transcriptase Inhibitors/toxicity , Serotonin Antagonists/toxicity , Alkynes , Animals , Anti-HIV Agents/metabolism , Benzoxazines/metabolism , Binding, Competitive , Brain/metabolism , CHO Cells , Calcium Signaling/drug effects , Cricetulus , Cyclopropanes , Dose-Response Relationship, Drug , Drug Partial Agonism , Guinea Pigs , HEK293 Cells , HIV Infections/diagnosis , HIV Infections/virology , HIV-1/pathogenicity , HeLa Cells , Humans , Membrane Potentials , Monoamine Oxidase Inhibitors/toxicity , Protein Binding , Radioligand Assay , Receptors, Muscarinic/drug effects , Receptors, Muscarinic/genetics , Receptors, Muscarinic/metabolism , Receptors, Serotonin/genetics , Receptors, Serotonin/metabolism , Reverse Transcriptase Inhibitors/metabolism , Time Factors , TransfectionABSTRACT
Progesterone (P4) exerts robust cytoprotection in brain slice cultures (containing both neurons and glia), yet such protection is not as evident in neuron-enriched cultures, suggesting that glia may play an indispensable role in P4's neuroprotection. We previously reported that a membrane-associated P4 receptor, P4 receptor membrane component 1, mediates P4-induced brain-derived neurotrophic factor (BDNF) release from glia. Here, we sought to determine whether glia are required for P4's neuroprotection and whether glia's roles are mediated, at least partially, via releasing soluble factors to act on neighboring neurons. Our data demonstrate that P4 increased the level of mature BDNF (neuroprotective) while decreasing pro-BDNF (potentially neurotoxic) in the conditioned media (CMs) of cultured C6 astrocytes. We examined the effects of CMs derived from P4-treated astrocytes (P4-CMs) on 2 neuronal models: 1) all-trans retinoid acid-differentiated SH-SY5Y cells and 2) mouse primary hippocampal neurons. P4-CM increased synaptic marker expression and promoted neuronal survival against H2O2. These effects were attenuated by Y1036 (an inhibitor of neurotrophin receptor [tropomysin-related kinase] signaling), as well as tropomysin-related kinase B-IgG (a more specific inhibitor to block BDNF signaling), which pointed to BDNF as the key protective component within P4-CM. These findings suggest that P4 may exert its maximal protection by triggering a glia-neuron cross talk, in which P4 promotes mature BDNF release from glia to enhance synaptogenesis as well as survival of neurons. This recognition of the importance of glia in mediating P4's neuroprotection may also inform the design of effective therapeutic methods for treating diseases wherein neuronal death and/or synaptic deficits are noted.
Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Membrane Proteins/metabolism , Neuroglia/metabolism , Neurons/metabolism , Progesterone/pharmacology , Receptors, Progesterone/metabolism , Signal Transduction/drug effects , Animals , Apoptosis/drug effects , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Mice , Neuroglia/cytology , Neuroglia/drug effects , Neurons/cytology , Neurons/drug effects , Propionates/pharmacology , Thiazolidines/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: Cognitive deficits in patients with Alzheimer's disease, Parkinson's disease, traumatic brain injury and stroke often involve alterations in cholinergic signalling. Currently available therapeutic drugs provide only symptomatic relief. Therefore, novel therapeutic strategies are needed to retard and/or arrest the progressive loss of memory. EXPERIMENTAL APPROACH: Scopolamine-induced memory impairment provides a rapid and reversible phenotypic screening paradigm for cognition enhancement drug discovery. Male C57BL/6J mice given scopolamine (1 mg·kg(-1) ) were used to evaluate the ability of LS-1-137, a novel sigma (σ1) receptor-selective agonist, to improve the cognitive deficits associated with muscarinic antagonist administration. KEY RESULTS: LS-1-137 is a high-affinity (Ki = 3.2 nM) σ1 receptor agonist that is 80-fold selective for σ1, compared with σ2 receptors. LS-1-137 binds with low affinity at D2-like (D2, D3 and D4) dopamine and muscarinic receptors. LS-1-137 was found to partially reverse the learning deficits associated with scopolamine administration using a water maze test and an active avoidance task. LS-1-137 treatment was also found to trigger the release of brain-derived neurotrophic factor from rat astrocytes. CONCLUSIONS AND IMPLICATIONS: The σ1 receptor-selective compound LS-1-137 may represent a novel candidate cognitive enhancer for the treatment of muscarinic receptor-dependent cognitive deficits.
Subject(s)
Acetanilides/pharmacology , Acetanilides/therapeutic use , Cognition Disorders/chemically induced , Cognition Disorders/drug therapy , Muscarinic Antagonists/pharmacology , Piperidines/pharmacology , Piperidines/therapeutic use , Receptors, sigma/agonists , Receptors, sigma/metabolism , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Avoidance Learning/drug effects , Brain-Derived Neurotrophic Factor/metabolism , Ligands , Male , Maze Learning/drug effects , Mice , Rats , Scopolamine/pharmacology , Sigma-1 ReceptorABSTRACT
Vascular dementia ranks as the second leading cause of dementia in the United States. However, its underlying pathophysiological mechanism is not fully understood and no effective treatment is available. The purpose of the current study was to evaluate long-term cognitive deficits induced by transient middle cerebral artery occlusion (tMCAO) in rats and to investigate the underlying mechanism. Sprague-Dawley rats were subjected to tMCAO or sham surgery. Behavior tests for locomotor activity and cognitive function were conducted at 7 or 30days after stroke. Hippocampal long term potentiation (LTP) and involvement of GABAergic neurotransmission were evaluated at 30days after sham surgery or stroke. Immunohistochemistry and Western blot analyses were conducted to determine the effect of tMCAO on cell signaling in the hippocampus. Transient MCAO induced a progressive deficiency in spatial performance. At 30days after stroke, no neuron loss or synaptic marker change in the hippocampus were observed. LTP in both hippocampi was reduced at 30days after stroke. This LTP impairment was prevented by blocking GABAA receptors. In addition, ERK activity was significantly reduced in both hippocampi. In summary, we identified a progressive decline in spatial learning and memory after ischemic stroke that correlates with suppression of hippocampal LTP, elevation of GABAergic neurotransmission, and inhibition of ERK activation. Our results indicate that the attenuation of GABAergic activity or enhancement of ERK/MAPK activation in the hippocampus might be potential therapeutic approaches to prevent or attenuate cognitive impairment after ischemic stroke.
Subject(s)
Cognition Disorders/etiology , Gene Expression Regulation/physiology , Infarction, Middle Cerebral Artery/complications , Signal Transduction/physiology , Animals , Central Nervous System Stimulants/pharmacology , Cognition Disorders/pathology , Disease Models, Animal , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Functional Laterality , Hippocampus/physiopathology , In Vitro Techniques , Male , Maze Learning/physiology , Membrane Proteins/metabolism , Motor Activity/physiology , Nerve Tissue Proteins/metabolism , Picrotoxin/pharmacology , Rats , Rats, Sprague-Dawley , Time Factors , Visual Perception/physiologyABSTRACT
AIM: To explore the effect of alloxan time administerDrug on establishing diabetic rabbit model. METHODS: Thirty-six healthy rabbits, weighed 2-2.5kg, were randomly divided into one time administerDrug group (Group A, n=12), two times administerDrug group (Group B, n=12) and three times administerDrug group(Group C, n=12). Every rabbit was injected with alloxan of 150mg/kg. The three groups were measured for fasting blood-glucose. The success rate and death rate of each group were also calculated. RESULTS: The success rate of diabetic rabbit model in Group B was higher than that in Group A (P<0.01) but its death rate was lower than that of Group A (P<0.01); the success rate of diabetic rabbit model in Group C was highest but the death rate was the lowest in the three groups. CONCLUSION: Separate administration of alloxan can improve success rate in establishing diabetic rabbit model, decrease the death rate and keep the stability of model.
ABSTRACT
Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.
Subject(s)
Endocytosis/physiology , Receptors, Glycine/physiology , Cell Line , Dynamins/physiology , Humans , Kinetics , Microscopy, Confocal , Patch-Clamp Techniques , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
We previously identified H109 of the glycine alpha1 subunit as a putative proton binding site. In the present studies, we explored additional proton binding site(s) as well as the mechanism underlying modulation of glycine receptors by protons. Whole-cell glycine currents were recorded from HEK 293 cells transiently expressing wild type or mutant glycine receptors. Individual mutation of 3 of 4 remaining extracellular histidine residue into alanine (i.e., alpha1 H107A, H215A or H419A), reduced the receptor sensitivity to protons to a varying extent. In contrast, mutation of alpha1 H201A did not affect proton sensitivity. Double, triple or quadruple histidine mutation of these residues caused a further reduction of proton sensitivity, suggesting multiple binding sites for proton action on glycine receptors. Furthermore, the substitution T133A, which mediates Zn(2+) inhibition, virtually abolished the proton effect on peak amplitude and current kinetics of glycine response. Replacement of T with S on position 133 partially restored receptor sensitivity to protons, suggesting the hydroxyl group of residue T133 is essential for proton-mediated modulation. In heteromeric alpha1beta receptors, mutations beta H132A and S156A, which correspond to H109 and T133 of the alpha1 subunit, respectively, also affected proton inhibition. In conclusion, multiple extracellular histidine residues (H107, H109, H215 and H419) and threonine residues of the alpha1 and beta Zn(2+) coordination sites are critical for modulation of the glycine receptor by protons.
Subject(s)
Protons , Receptors, Glycine/chemistry , Cell Line, Transformed , Cloning, Molecular , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Glycine/pharmacology , Histidine/metabolism , Humans , Hydrogen-Ion Concentration , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Mutagenesis/physiology , Patch-Clamp Techniques , Receptors, Glycine/genetics , Transfection , Zinc/pharmacologyABSTRACT
Endogenous divalent cations Cu2+ and Zn2+ suppress the activity of glycine receptors (glyRs). Whereas residues critical for the effects of Zn2+ on glyRs have been identified, little is known about the determinants of Cu2+-mediated inhibition. In the present studies, we have assessed the potential commonality of Zn2+ and Cu2+-mediated inhibition of glyRs. Cu2+ potently inhibited recombinant human glycine alpha1 receptors, with an IC50 of 4.1+/-0.7 microM. Systematic mutation of extracellular histidine residues revealed that mutation H215A greatly reduced the inhibitory modulation by Cu2+. Substitution of H215 with C produced receptors with Cu2+ sensitivity similar to the wild type. Furthermore, modification of H215C with a thio-specific reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), reduced Cu2+ sensitivity of H215C receptors. However, mutation of other extracellular histidine residues including H107 and H109, which are known inhibitory Zn2+coordination sites, failed to influence inhibition of glycine currents by Cu2+. Moreover, mutation to alanine of two threonine residues (T112, T133) critical for Zn2+ inhibition had no effect (T133A) or only partial inhibitory effects (T112A) on Cu2+-induced inhibition. The double mutation, T112A/H215A, caused greater effects on Cu2+-mediated inhibition than either mutation alone. In addition, the glycine currents recorded from T112A/H215A mutant receptors were significantly potentiated by low concentrations of Cu2+. Our results have identified critical determinants of Cu2+-mediated inhibition of glyRs. Moreover, we demonstrate for the first time a clear difference in residues responsible for Cu2+-mediated compared to Zn2+-mediated inhibition of glyRs.
Subject(s)
Copper/metabolism , Receptors, Glycine/chemistry , Receptors, Glycine/physiology , Cell Line, Transformed , Cloning, Molecular/methods , Copper/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Glycine/pharmacology , Histidine/genetics , Histidine/metabolism , Humans , Inhibitory Concentration 50 , Membrane Potentials/drug effects , Mesylates/pharmacology , Mutagenesis/physiology , Patch-Clamp Techniques/methods , Sulfhydryl Reagents/pharmacology , Transfection/methods , Zinc/pharmacologyABSTRACT
We have previously shown that extracellular protons inhibit recombinant and native GABA(A) receptors. In this report, we studied the site(s) and mechanism by which protons modulate the GABA(A) receptor. Whole cell GABA-activated currents were recorded from human embryonic kidney (HEK) 293 cells expressing recombinant alpha1beta2gamma2 GABA(A) receptors. Protons competitively inhibited the response to GABA and bicuculline. In contrast, change in pH did not influence direct gating of the channel by pentobarbital, and it did not influence spontaneous channel openings in alpha1(L264T)beta2gamma2 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABA(A) receptors at the ligand binding site, we systemically mutated N-terminal residues known to be involved in GABA binding and assessed effects of pH on these mutant receptors. Site-specific mutation of beta2 Y205 to F or alpha1 F64 to A, both of which are known to influence GABA binding, significantly reduced pH sensitivity of the GABA response. These mutations did not affect Zn(2+) sensitivity, suggesting that H(+) and Zn(2+) do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethylpyrocarbonate (DEPC) reduced Zn(2+)-mediated inhibition of GABA(A) receptors but had no effect on proton-induced inhibition of GABA currents. In addition, mutation of residues known to be involved in Zn(2+) modulation had no effect on pH modulation of GABA(A) receptors. Our results support the hypothesis that protons inhibit GABA(A) receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H(+) and Zn(2+) in GABA(A) receptors are distinct.
Subject(s)
Mutation , Protons , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Bicuculline/pharmacology , Binding Sites/genetics , Cell Line , Diethyl Pyrocarbonate/pharmacology , Electrophysiology , GABA Antagonists/pharmacology , GABA Modulators/pharmacology , Humans , Hydrogen-Ion Concentration , Ion Channel Gating/drug effects , Patch-Clamp Techniques , Pentobarbital/pharmacology , Receptors, GABA-A/drug effects , Recombinant Proteins/metabolism , Zinc/pharmacology , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life. Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders. The Pro-Pro-hGHRH(1-44) peptide has better activity. The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro-pro-hGHRH(1-44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp-Pro. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH(1-44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography. The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body. The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy. Its purity was determined by SDS-PAGE. In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH. The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 microg/ml used, respectively, released the GH values of 0.1+/-0.1, 12.5+/-7.3, 16.6+/-5.8, 49.8+/-7.6, and 79.5+/-5.7 ng/ml whereas their blank controls, respectively, were 0.5+/-0.8, 4.1+/-2.6, 3.1+/-3.1, 4.7+/-1.8, and 1.2+/-0.3 ng/ml. The activity results of all dose groups except 0.01 microg/ml Pro-Pro-hGHRH(1-44) group and hGHRH(1-40) group showed that there were significant differences between GH released by the peptide and that by its blank control. With the increase of dosage, the differences were more significant. hGHRH(1-40) showed no measured GH release when the dose was up to 2 microg/ml. The activity results show that the Pro-Pro-hGHRH(1-44) peptide is a potential GH releasing analog.
Subject(s)
Asparaginase/metabolism , Growth Hormone-Releasing Hormone/metabolism , Growth Hormone/metabolism , Pituitary Gland/metabolism , Animals , Chromatography , Dipeptides/chemistry , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Growth Hormone-Releasing Hormone/genetics , Growth Hormone-Releasing Hormone/isolation & purification , Humans , Inclusion Bodies/genetics , Mass Spectrometry , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.