The Impact of Gabapentin Administration on Brain GABA and Glutamate Concentrations: A 7T 1H-MRS Study

Gamma-aminobutyric acid (GABA) and glutamate are implicated in numerous neuropsychiatric and substance abuse conditions, but their spectral overlap with other resonances makes them a challenge to quantify in humans. Gabapentin, marketed for the treatment of seizures and neuropathic pain, has been shown to increase in vivo GABA concentration in the brain of both rodents and humans. Gabapentin effects on glutamate are not known. We conducted a gabapentin (900 mg) challenge in healthy human subjects to confirm and explore its effects on GABA and glutamate concentrations, respectively, and to test the ability of single voxel localized proton magnetic resonance spectroscopy (¹H-MRS) to reliably measure GABA and glutamate in the visual cortex at the ultra-high magnetic field of 7 Tesla. Reproducibility of GABA and glutamate measurements was determined in a comparison group without drug twice within day and 2 weeks apart. Although GABA concentration changes were small both within day (average 5.6%) and between day (average 4.8%), gabapentin administration was associated with an average increase in GABA concentration of 55.7% (6.9–91.0%). Importantly, drug-induced change in GABA levels was inversely correlated to the individual''s baseline GABA level (R²=0.72). Mean glutamate concentrations did not change significantly with or without drug administration. In conclusion, localized ¹H-MRS at 7 Tesla can be successfully applied to the measurement of GABA concentration and is sensitive to acute drug-induced changes in cortical GABA. Whether baseline GABA concentrations predict clinical efficacy of gabapentin is an area worthy of exploration.     

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

1. The γ-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA_A receptors are favoured by β₂- or β₃- versus β₁-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β_{2 N290}, β_{3 N289}, β_{1 S290}). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster.
2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at −60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC₅₀ for GABA of 20±0.4 μM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (Rdl_M314N) or a serine (Rdl_M314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC₅₀ of 4.8±0.2 μM.
3. Utilizing the appropriate GABA EC₁₀, etomidate (300 μM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at Rdl_M314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC₅₀ of 64±3 μM and an E_max of 68±2% (of the maximum response to GABA).
4. The actions of etomidate at Rdl_M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μM R-(+)-etomidate and S-(−)-etomidate enhancing the current induced by GABA (EC₁₀) to 52±6% and 12±1% of the GABA maximum respectively.
5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and Rdl_M314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one (Rdl=25±4% of the GABA maximum) was not altered by this mutation (Rdl_M314N=18±3% of the GABA maximum).
6. Etomidate acting at β₁ (S290)-containing mammalian GABA_A receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at Rdl_M314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to Rdl_M314N receptors.
7. Etomidate acting at human α₆β₃γ_2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA_A receptor complex. Mutation of the human β₃ subunit asparagine to methionine (β_{3 N289M} found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10–300 μM) acting at α₆β_{3 N289M}γ_2L receptors.
8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

     

NLRP3 Inflammasome Mediates Chronic Mild Stress-Induced Depression in Mice via Neuroinflammation

Background:

Evidence from both clinical and experimental research indicates that the immune-brain interaction plays a pivotal role in the pathophysiology of depression. A multi-protein complex of the innate immune system, the NLRP3 inflammasome regulates cleavage and secretion of proinflammatory cytokine interleukin-1β. The inflammasome detects various pathogen-associated molecule patterns and damage-associated molecule patterns, which then leads to a series of immune-inflammatory reactions.

Methods:

To explore the role of inflammasome activation in the underlying biological mechanisms of depression, we established a mouse model of depression with unpredictable chronic mild stress.

Results:

Mice subjected to chronic mild stress for 4 weeks had significantly higher serum corticosterone levels, serum interleukin-1β levels, and hippocampal active interleukin-1β protein levels. They also displayed depressive-like symptoms, including decreased sucrose preference and increased immobility time. Moreover, the hippocampi of chronic mild stress-exposed mice had significantly higher activity of caspase-1, which accompanied by higher protein levels of NLRP3 and the apoptotic speck-containing protein with a card. Pretreatment with the NLRP3 inflammasome inhibitor VX-765 decreased serum and hippocampal levels of interleukin-1β protein and significantly moderated the depressive-like behaviors induced by chronic mild stress.

Conclusions:

These data suggest the NLRP3 inflammasome mediates stress-induced depression via immune activation. Future procedures targeting the NLRP3 inflammasome may have promising effects in the prevention and treatment of depression.     

Adaptive changes in the pharmacodynamics of midazolam in different experimental models of epilepsy: kindling,cortical stimulation and genetic absence epilepsy

1. The objective of this investigation was to determine quantitatively whether experimental epilepsy is associated with a change in the pharmacodynamics of benzodiazepines in vivo. For that purpose the pharmacodynamics of midazolam were quantified by an integrated pharmacokinetic-pharmacodynamic approach in three different models of experimental epilepsy: amygdala kindling, cortical stimulation and genetic absence epilepsy.
2. The time course of the EEG effect was determined in conjunction with the decline of drug concentrations after intravenous administration of 10 mg kg⁻¹ midazolam. The pharmacokinetics of midazolam were most adequately described by a bi-exponential equation. No influence of epilepsy on the pharmacokinetics of midazolam was observed.
3. The increase in β activity (11.5–30 Hz) of the EEG as derived by Fast Fourier Transformation analysis was used as pharmacodynamic endpoint. For each individual rat the increase in β activity was directly related to the concentration in blood on the basis of the sigmoidal E_max pharmacodynamic model. In all three models a significant reduction in the maximal effect was observed, in amygdala kindling 28%, in the cortical stimulation model 49% and in genetic absence epilepsy 37%. No differences in the other pharmacodynamic parameters, E₀, EC_50,u and Hill factor, were observed.
4. It is inferred that in three different models of epilepsy there is a similar change in GABAergic functioning which is associated with a significant reduction in the intrinsic activity of midazolam in vivo. These models provide therefore a useful basis for further studies on the mechanism of epilepsy-induced changes in pharmacodynamics of anti-epileptic drugs.

     

Treatment with resveratrol attenuates sublesional bone loss in spinal cord-injured rats

BACKGROUND AND PURPOSE

Sublesional osteoporosis predisposes individuals with spinal cord injury (SCI) to an increased risk of low-trauma fracture. The aim of the present work was to investigate the effect of treatment with resveratrol (RES) on sublesional bone loss in spinal cord-injured rats.

EXPERIMENTAL APPROACH

Complete SCI was generated by surgical transaction of the cord at the T_10–12 level. Treatment with RES (400 mg·kg⁻¹ body mass per day⁻¹, intragastrically) was initiated 12 h after the surgery for 10 days. Then, blood was collected and femurs and tibiae were removed for evaluation of the effects of RES on bone tissue after SCI.

KEY RESULTS

Treatment of SCI rats with RES prevented the reduction of bone mass including bone mineral content and bone mineral density in tibiae, preserved bone structure including trabecular bone volume fraction, trabecular number, and trabecular thickness in tibiae, and preserved mechanical strength including ultimate load, stiffness, and energy in femurs. Treatment of SCI rats with RES enhanced femoral total sulfhydryl content, reduced femoral malondialdehyde and IL-6 mRNA levels. Treatment of SCI rats with RES suppressed the up-regulation of mRNA levels of PPARγ, adipose-specific fatty-acid-binding protein and lipoprotein lipase, and restored mRNA levels of Wnt1, low-density lipoprotein-related protein 5, Axin2, ctnnb1, insulin-like growth factor 1 (IGF-1) and receptor for IGF-1 in femurs and tibiae.

CONCLUSIONS AND IMPLICATIONS

Treatment with RES attenuated sublesional bone loss in spinal-cord-injured rats, associated with abating oxidative stress, attenuating inflammation, depressing PPARγ signalling, and restoring Wnt/β-catenin and IGF-1 signalling.     

The Discovery of MK-4256, a Potent SSTR3 Antagonist as a Potential Treatment of Type 2 Diabetes

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The Phosphodiesterase-4 Inhibitor Roflumilast,a Potential Treatment for the Comorbidity of Memory Loss and Depression in Alzheimer’s Disease: A Preclinical Study in APP/PS1 Transgenic Mice

BackgroundDepression is highly related to Alzheimer’s disease (AD), yet no effective treatment is available. Phosphodiesterase-4 (PDE4) has been considered a promising target for treatment of AD and depression. Roflumilast, the first PDE4 inhibitor approved for clinical use, improves cognition at doses that do not cause side effects such as emesis.MethodsHere we examined the effects of roflumilast on behavioral dysfunction and the related mechanisms in APPswe/PS1dE9 transgenic mice, a widely used model of AD. Mice at 10 months of age were examined for memory in the novel object recognition and Morris water-maze tests and depression-like behavior in the tail-suspension test and forced swimming test before killing for neurochemical assays.ResultsIn the novel object recognition and Morris water-maze, APPswe/PS1dE9 mice showed significant cognitive declines, which were reversed by roflumilast at 5 and 10 mg/kg orally once per day. In the tail-suspension test and forced swimming test, the AD mice showed prolonged immobility time, which was also reversed by roflumilast. In addition, the staining of hematoxylin–eosin and Nissl showed that roflumilast relieved the neuronal cell injuries, while terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling analysis indicated that roflumilast ameliorated cell apoptosis in AD mice. Further, roflumilast reversed the decreased ratio of B-cell lymphoma-2/Bcl-2-associated X protein and the increased expression of PDE4B and PDE4D in the cerebral cortex and hippocampus of AD mice. Finally, roflumilast reversed the decreased levels of cyclic AMP (cAMP) and expression of phosphorylated cAMP response element-binding protein and brain derived neurotrophic factor in AD mice.ConclusionsTogether, these results suggest that roflumilast not only improves learning and memory but also attenuates depression-like behavior in AD mice, likely via PDE4B/PDE4D-mediated cAMP/cAMP response element-binding protein/brain derived neurotrophic factor signaling. Roflumilast can be a therapeutic agent for AD, in particular the comorbidity of memory loss and depression.     

Induction of mucosal immunity by pulmonary administration of a cell-targeting nanoparticle

We previously found that a nanoparticle constructed with an antigen, benzalkonium chloride (BK) and γ-polyglutamic acid (γ-PGA) showed high Th1 and Th2-type immune induction after subcutaneous administration. For prophylaxis of respiratory infections, however, mucosal immunity should be induced. In this study, we investigated the effect of pulmonary administration of a nanoparticle comprising ovalbumin (OVA) as a model antigen, BK, and γ-PGA on induction of mucosal immunity in the lungs and serum. The complex was strongly taken up by RAW264.7 and DC2.4cells. After pulmonary administration, lung retention was longer for the OVA/BK/γ-PGA complex than for OVA alone. OVA-specific serum immunoglobulin (Ig)G was highly induced by the complex. High IgG and IgA levels were also induced in the bronchoalveolar lavage fluid, and in vivo toxicities were not observed. In conclusion, we effectively and safely induced mucosal immunity by pulmonary administration of an OVA/BK/γ-PGA complex.     

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

1. The electrophysiological actions of several agonists which may differentiate between P2X₁- and P2X₃-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.
2. β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P¹,P⁵-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.
3. The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.
4. The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.
5. This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X₃-subtype to produce these effects.

     

Herbal Insomnia Medications that Target GABAergic Systems: A Review of the Psychopharmacological Evidence

Insomnia is a common sleep disorder which is prevalent in women and the elderly. Current insomnia drugs mainly target the γ-aminobutyric acid (GABA) receptor, melatonin receptor, histamine receptor, orexin, and serotonin receptor. GABA_A receptor modulators are ordinarily used to manage insomnia, but they are known to affect sleep maintenance, including residual effects, tolerance, and dependence. In an effort to discover new drugs that relieve insomnia symptoms while avoiding side effects, numerous studies focusing on the neurotransmitter GABA and herbal medicines have been conducted. Traditional herbal medicines, such as Piper methysticum and the seed of Zizyphus jujuba Mill var. spinosa, have been widely reported to improve sleep and other mental disorders. These herbal medicines have been applied for many years in folk medicine, and extracts of these medicines have been used to study their pharmacological actions and mechanisms. Although effective and relatively safe, natural plant products have some side effects, such as hepatotoxicity and skin reactions effects of Piper methysticum. In addition, there are insufficient evidences to certify the safety of most traditional herbal medicine. In this review, we provide an overview of the current state of knowledge regarding a variety of natural plant products that are commonly used to treat insomnia to facilitate future studies.     

Evidence for direct and indirect mechanisms in the potent modulatory action of interleukin-2 on the release of acetylcholine in rat hippocampal slices

1. The biphasic nature of the potent modulatory action of interleukin-2 (IL-2) on hippocampal acetylcholine (ACh) release was investigated by use of brain slice superfusion.
2. Both the potentiating (10⁻¹³ M) and inhibitory (10⁻⁹ M) effects of IL-2 on hippocampal ACh release were stimulation-dependent and were blocked by a neutralizing IL-2 receptor antibody, suggesting the activation of typical IL-2 receptors in both cases.
3. Tetrodotoxin (TTX; 10 μM) failed to block the potentiation of ACh release induced by a very low concentration of IL-2 (10⁻¹³M) suggesting a direct effect on cholinergic nerve terminals.
4. In contrast, the inhibitory effect seen at a higher concentration (10⁻⁹ M) was TTX-sensitive, and hence indicative of an indirect action.
5. To establish the nature of this intermediate mediator, blockers of nitric oxide synthesis, and of opioid and γ-aminobutyric acid (GABA) receptors were used. Only GABA_A and GABA_B receptor antagonists altered the inhibitory action of IL-2, suggesting the participation of GABA as mediator.
6. Taken together, these results provide further evidence for the potent role of IL-2 in the modulation of cholinergic function in the rat hippocampus.

     

Comparison of anaesthetic and non-anaesthetic effects on depolarization-evoked glutamate and GABA release from mouse cerebrocortical slices

1. Investigation with substances that are similar in structure, but different in anaesthetic properties, may lead to further understanding of the mechanisms of general anaesthesia.
2. We have studied the effects of two cyclobutane derivatives, the anaesthetic, 1-chloro-1,2,2-trifluorocyclobutane (F3), and the non-anaesthetic, 1,2-dichlorohexafluorocyclobutane (F6), on K⁺-evoked glutamate and γ-aminobutyric acid (GABA) release from isolated, superfused, cerebrocortical slices from mice, by use of h.p.l.c. with fluorescence detection for quantitative analysis.
3. At clinically relevant concentrations, the anaesthetic, F3, inhibited 40 mM K⁺-evoked glutamate and GABA release by 72% and 47%, respectively, whereas the structurally similar non-anaesthetic, F6, suppressed evoked glutamate release by 70% but had no significant effects on evoked GABA release. A second exposure to 40 mM KCl after a ∼30 min washout of F3 or F6 showed recovery of K⁺-evoked release, suggesting that F3 and F6 did not cause any non-specific or irreversible changes in the brain slices.
4. Our findings suggest that suppression of excitatory neurotransmitter release may not be directly relevant to the primary action of general anaesthetics. A mechanism involving inhibitory postsynaptic action is implicated, in which a moderate suppression of depolarization-evoked GABA release by the anaesthetic may be consistent with the enhancement of postsynaptic GABAergic activities.

     

SB-205384: a GABAA receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA_A receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods.
2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA_A currents in oocytes expressing human GABA_A subunits. This temporal effect was observed for the α3β2γ2 subunit combination with little effect in subunit combinations containing either α1 or α2.
3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 μM drug was seen on the α3β2γ2 subunit combination.
4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA_A currents in oocytes expressing the α3β2γ2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the γ2 subunit compared to those containing the γ1.
5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the α3β2γ2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.

     

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors

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TGF-B3 Dependent Modification of Radiosensitivity in Reporter Cells Exposed to Serum From Whole-Body Low Dose-Rate Irradiated Mice

Prior findings in vitro of a TGF-β3 dependent mechanism induced by low dose-rate irradiation and resulting in increased radioresistance and removal of low dose hyper-radiosensitivity (HRS) was tested in an in vivo model. DBA/2 mice were given whole-body irradiation for 1 h at low dose-rates (LDR) of 0.3 or 0.03 Gy/h. Serum was harvested and added to RPMI (4% mouse serum and 6% bovine serum).This medium was transferred to reporter cells (T-47D breast cancer cells or T98G glioblastoma cells). The response to subsequent challenge irradiation of the reporter cells was measured by the colony assay. While serum from unirradiated control mice had no effect on the radiosensitivity in the reporter cells, serum from mice given 0.3 Gy/h or 0.03 Gy/h for 1 h removed HRS and also increased survival in response to doses up to 5 Gy. The effect lasted for at least 15 months after irradiation. TGF-β3 neutralizer added to the medium containing mouse serum inhibited the effect. Serum from mice given irradiation of 0.3 Gy/h for 1 h and subsequently treated with iNOS inhibitor 1400W did not affect radiosensitivity in reporter cells; neither did serum from the unirradiated progeny of mice given 1h LDR whole-body irradiation.     

Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells

1. In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon γ (IFNγ) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time- and concentration-dependent manner.
2. Incubation with the direct activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) alone, did not result in detectable iNOS expression in either cell type.
3. Chronic PMA pretreatment resulted in significant down-regulation of α, β and ε isoforms of PKC in RAW 264.7 macrophages and corresponded to a 20–30% reduction in LPS-induced iNOS expression. In contrast, IFNγ alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively.
4. Pre-incubation with PKC inhibitors (calphostin C and H-7) showed similar effects upon stimulated induction of iNOS.
5. In RASM cells chronic PMA pretreatment resulted in down-regulation of α and ε PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin.
6. Co-incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin.
7. Differential sensitivity to PKC inhibitors (calphostin C and H-7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction.
8. In addition the PKC inhibitor compound Ro-31-8220 abolished stimulated induction in both cell types in response to all treatments.
9. These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.

     

Novel Cyclic Phosphinic Acids as GABAC ρ Receptor Antagonists: Design,Synthesis, and Pharmacology

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Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

Physicochemical,Pharmacokinetic and Cytotoxicity of the Compounds Isolated from an Endophyte Fusarium oxysporum: In Vitro and In Silico Approaches

The present study was intended to characterize the secondary metabolites of the endophyte Fusarium oxysporum isolated from the plant Aglaonema hookerianum Schott. And to investigate the cytotoxic and other pharmacological properties of the isolated compounds as part of the drug discovery and development process. Different chromatographic techniques were adopted to isolate the bioactive compounds that were identified by spectroscopic techniques. The cytotoxic properties of the compounds were assessed in the Vero cell line via the trypan blue method. Moreover, physicochemical, pharmacokinetic, bioactivity and toxicity profiles of the compounds were also investigated through in silico approaches. After careful spectral analysis, the isolated compounds were identified as 3β,5α-dihydroxy-ergosta-7,22-dien-6-one (1), 3β,5α,9α-trihydroxy-ergosta-7,22-dien-6-one (2), p-hydroxybenzaldehyde (3), 3-(R)-7-butyl-6,8-dihydroxy-3-pent-11-enylisochroman-1-one (4) and beauvericin (5). An in vitro study in the Vero cell line revealed that the presence of the compounds reduced the number of cells, as well as the percentage of viable cells, in most cases. An in silico cytotoxic analysis revealed that compounds 1, 2 and 5 might be explored as cytotoxic agents. Moreover, compounds 3 and 4 were found to be highly mutagenic. The present study suggested that further thorough investigations are necessary to use these molecules as leads for the cytotoxic drug development process.