Design, Synthesis, and Pharmacological Evaluation of Haloperidol Derivatives as Novel Potent Calcium Channel Blockers with Vasodilator Activity

Several haloperidol derivatives with a piperidine scaffold that was decorated at the nitrogen atom with different alkyl, benzyl, or substituted benzyl moieties were synthesized at our laboratory to establish a library of compounds with vasodilator activity. Compounds were screened for vasodilatory activity on isolated thoracic aorta rings from rats, and their quantitative structure–activity relationships (QSAR) were examined. Based on the result of QSAR, N-4-tert-butyl benzyl haloperidol chloride (16c) was synthesized and showed the most potent vasodilatory activity of all designed compounds. 16c dose-dependently inhibited the contraction caused by the influx of extracellular Ca2+ in isolated thoracic aorta rings from rats. It concentration-dependently attenuated the calcium channel current and extracellular Ca2+ influx, without affecting the intracellular Ca2+ mobilization, in vascular smooth muscle cells from rats. 16c, possessing the N-4-tert-butyl benzyl piperidine structure, as a novel calcium antagonist, may be effective as a calcium channel blocker in cardiovascular disease

Mechanisms Of Cannabinoid Cb 2 Receptor-Mediated Reduction Of Dopamine Neuronal Excitability In Mouse Ventral Tegmental Area

Background: We have recently reported that activation of cannabinoid type 2 receptors (CB 2 Rs)reduces dopamine (DA)neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms. Methods: Patch-clamp recordings were performed in mouse VTA slices and dissociated single VTA DA neurons. Findings: Using cell-attached recording in VTA slices, bath-application of CB 2 R agonists (JWH133 or five other CB 2 R agonists)significantly reduced VTA DA neuron action potential (AP)firing rate. Under the patch-clamp whole-cell recording model, JWH133 (10 μM)mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs)but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 (1 μM)enhanced M-type K + currents and this effect was absent in CB 2−/− mice and abolished by co-administration of a selective CB 2 R antagonist (10 μM, AM630). CB 2 R-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener (10 μM retigabine)and blocked by M-current blocker (30 μM XE991). In addition, enhancement of neuronal cAMP by forskolin (10 μM)reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX (10 μM), D-APV (50 μM)and picrotoxin (100 μM)in VTA slices failed to prevent CB 2 R-mediated inhibition, while intracellular infusion of guanosine 5\u27-O-2-thiodiphosphate (600 μM, GDP-β-S)through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing. Interpretation: Our results suggest that CB 2 Rs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CB 2 R-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K + currents. Fund: This research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, and CNSF (81771437)

Gap Junctions Contribute To Ictal/Interictal Genesis In Human Hypothalamic Hamartomas

Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patient\u27s HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cβ2β3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 μM. Interestingly, in the presence of 30 μM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine’s EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cβ2β3-nAChRs and α6M211L/α3ICβ2β3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cβ2β3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence

Identification of Signature Genes in the PD-1 Relative Gastric Cancer Using a Combined Analysis of Gene Expression and Methylation Data

Background. The morbidity and mortality rates for gastric cancer (GC) rank second among all cancers, indicating the serious threat it poses to human health, as well as human life. This study aims to identify the pathways and genes as well as investigate the molecular mechanisms of tumor-related genes in gastric cancer (GC). Method. We compared differentially expressed genes (DEGs) and differentially methylated genes (DMGs) in gastric cancer and normal tissue samples using The Cancer Genome Atlas (TCGA) data. The Kyoto Encyclopedia of Gene and Genome (KEGG) and the Gene Ontology (GO) enrichment analysis’ pathway annotations were conducted on DMGs and DEGs using a clusterProfiler R package to identify the important functions, as well as the biological processes and pathways involved. The intersection of the two was chosen and defined as differentially methylated and expressed genes (DMEGs). For DMEGs, we used the principal component analysis (PCA) to differentiate gastric cancer from adjacent samples. The linear discriminant analysis method was applied to categorize the samples using DMEGs methylation data and DMEGs expression profiles data and was validated using the leave-one-out cross-validation (LOOCV) method. We plotted the ROC curve for the classification and calculated the AUC (area under the ROC curve) value for a more intuitive view of the classification effect. We also used the NetworkAnalyst 3.0 tool to analyze DMEGs, using DrugBank to acquire information on protein-drug interactions and generate a network map of gene-drug interactions. Results. We identified a total of 971 DMGs in 188 PD-1 negative and 187 PD-1 positive gastric cancer samples obtained from TCGA. The KEGG and GO enrichment analysis showed the involvement of the regulation of ion transmembrane transport, collagen-containing extracellular matrix, cell-cell junction, and peptidase regulator activity. We simultaneously obtained 1,189 DEGs, out of which 986 were downregulated, while 203 were upregulated in tumors. The enriched analysis of the GO’s and KEGG’s pathways indicated that the most significant pathways included an intestinal immune network for IgA production, Staphylococcus aureus infection, cytokine-cytokine receptor interaction, and viral protein interaction with cytokine and cytokine receptor, which have previously been linked with gastric cancer. The compound DB01830 can bind well to the active site of the LCK protein and shows good stability, thus making it a potential inhibitor of the LCK protein. To observe the relationship between DMEGs’ expression and prognosis, we observed 10 genes, among which were TRIM29, TSPAN8, EOMES, PPP1R16B, SELL, PCED1B, IYD, JPH1, CEACAM5, and RP11-44K6.2. Their high expressions were related to high risks. Besides, those genes were validated in different internal and external validation sets. Conclusion. These results may provide potential molecular biological therapy for PD-1 negative gastric cancer

Suramin Is A Novel Competitive Antagonist Selective To α1β2γ2 Gaba A Over Ï1 Gaba C Receptors

GABA A and GABA C receptors are both GABA-gated chloride channels with distinct pharmacological properties, mainly in their sensitivity to bicuculline and gabazine. In this study, we found that suramin, a purinergic receptor antagonist, is a novel competitive antagonist selective to GABA A over GABA C receptors. Specifically, suramin antagonized the GABA-induced current and the spontaneous opening current of the wild type α1β2γ2 GABA A receptor with high-level expression in Xenopus oocytes. The antagonism was concentration dependent with an IC 50 that varied depending on the concentration of GABA, and with the lowest IC 50 of 0.43 μM when antagonizing the spontaneous current. Thus, its potency is slightly higher than bicuculline on the same GABA A receptor. Suramin also antagonized the mouse native brain GABA receptors micro-transplanted into the Xenopus oocytes with its potency depending on the GABA concentration. In addition, in the presence of two fixed concentrations of suramin, the GABA concentration response of the receptor was shifted to the right without reduction of the maximum current. Thus, our results are consistent with that suramin is a competitive antagonist for the α1β2γ2 GABA A receptor. Interestingly, the rank order of maximum allosteric inhibition (efficacy) of spontaneous current of the GABA A receptor by three competitive antagonists was suramin \u3e bicuculline \u3e gabazine, similar to the rank order of their molecular weight. In contrast, similar to bicuculline, suramin has much lower potency in antagonizing the GABA-induced current of the Ï1 GABA C receptor. In conclusion, we have identified a novel GABA A receptor competitive antagonist, which is selective to the α1β2γ2 over Ï1 GABA receptors

Hypoxia Suppresses TGF-B1-Induced Cardiac Myocyte Myofibroblast Transformation by Inhibiting Smad2/3 and Rhoa Signaling Pathways

Background/Aims: Hypoxia modulation of transforming growth factor (TGF)- β-induced signaling during myofibroblast transformation is dependent on the specific cell type. The purpose of this study was to explore the effects of hypoxia on myofibroblast transformation of TGF-β1-induced cardiomyocyte H9c2 cells. Methods: H9c2 cells were cultured for intermittent hypoxia treatment and TGF-β1 treatment. α-Smooth muscle actin (α-SMA) expression was examined by western blotting and immunofluorescence after treatment. To further explore the possible mechanism for this effect, the effects of hypoxia on three early TGF-β-dependent signaling pathways, i.e. the Smad2/3, RhoA and mitogen-activated protein kinase (MAPK) pathways, were screened by western blotting. Results: Intermittent hypoxia induced TGF-β1 expression, but had no effect on α-SMA expression. Exogenous TGF-β1 alone upregulated α-SMA expression in H9c2 cells in a concentration- and time-dependent manner. α-SMA expression declined with the duration of hypoxia after intermittent hypoxia and exogenous TGF-β1 co-treatment. Phospho-JNK and phospho-p38 levels were not significantly altered after TGF-β1 and hypoxia treatment. However, levels of phospho-ERK increased after TGF-β1 treatment and continued to increase after hypoxia co-treatment. The activation of phospho-Smad2/3 and phospho-RhoA induced by TGFβ1 was significantly reduced after hypoxia co-treatment. Conclusion: Hypoxia can inhibit TGF-β1-induced H9c2 myofibroblast transformation, based on inhibition of α-SMA expression by suppressing signaling downstream of TGF-β1, Smad2/3 and RhoA. It suggested that TGF-β-mediated cardiomyocyte transformation is not involved in hypoxia-mediated fibrosis

Buckling Analysis on Resin Base Laminated Plate Reinforced with Uniform and Functional Gradient Distribution of Carbon Fiber in Thermal Environment

The present paper aims to investigate the buckling load of functionally graded carbon-fiber-reinforced polymer (FG-CFRP) composite laminated plates under in-plane loads in a thermal environment. The effective material properties of the CFRP composite are calculated by the Mori–Tanaka homogenization method. The theoretical formulations are based on classical laminate plate theory (CLPT) and the von Kármán equations for large deflections. The governing equations are derived based on the principle of virtual work and then solved through the Navier solution. Results are obtained for the critical buckling load and temperature effect of a simply supported plate subjected to in-plane loading. A detailed numerical study is conducted to provide important insights into the effects of the functionally graded carbon fiber (CF) distribution pattern and volume fraction, total number of layers, temperature, geometrical dimension and lamination angle on the buckling load of functionally carbon-fiber-reinforced composite plates. Finally, the validation is compared with the Reddy and finite element analyses, which show consistency with each other