Agonist Concentrations Research Articles

Continuum modeling of thrombus formation and growth under different shear rates

Obstruction of blood flow due to thrombosis is a major cause of ischemic stroke, myocardial infarction, and in severe cases, mortality. In particular, in blood wetting medical devices, thrombosis is a common reason for failure. The prediction of thrombosis by understanding signaling pathways using computational models, lead to identify the risk of thrombus formation in blood-contacting devices and design improvements. In this study, a mathematical model of thrombus formation and growth is presented. A biochemical model of platelet activation and aggregation is developed to predict thrombus size and shape at the site of vascular injury. Computational fluid dynamics using the finite volume method is employed to compute the velocity and pressure fields which are influenced by the growing thrombi. The passive transport of platelets, agonists, the platelet activation kinetics, their adhesion to the growing thrombi and embolization of platelets are solved by a fully coupled set of convection–diffusion–reaction equations. The thrombogenic surface representing blood-contacting material or injured blood vessel was incorporated into the model as a surface flux boundary condition to initiate thrombus formation. The blood is considered as a Newtonian fluid, while the thrombus is treated as a porous medium. The results are compared with in vitro experiments of a microfluidic chamber at an initial inlet venous shear rate of 200s−1 using a pressure–inlet boundary condition. The thrombus development due to agonist concentrations and change in the shear rate as well as thromboembolism for this benchmark problem is successfully computed. Furthermore, to extend the current model to a physiologically relevant configuration, thrombus formation in a blood tube is simulated. Two different heterogeneous reaction rates for platelet aggregation are used to simulate thrombus growth under a constant inlet flow rate. The findings show that the thrombus shape can be substantially altered by the hemodynamic conditions, increase in the shear rate and due to the combined effects of shear induced platelet activation and the heterogeneous reaction rates. It is also concluded that the model is able to predict thrombus formation in different physiological and pathological hemodynamics.

History-dependent dopamine release increases cAMP levels in most basal amygdala glutamatergic neurons to control learning.

Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. This learning depends on intracellular plasticity signals such as cyclic adenosine monophosphate (cAMP), which is regulated by activation of dopamine receptors. We examine the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We perform real-time tracking and manipulation of cAMP in BA neurons invitro and invivo. Optogenetically evoked release of dopamine drives proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. This cAMP response decreases across trials with short intertrial intervals owing to depression of dopamine release. No such depression is evident when photostimulating cAMP production directly. cAMP and protein kinase A responses to repeated appetitive or aversive stimuli also exhibit pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells.

Simple SummaryBreast cancer shows a high heterogeneity which accounts for cancer subtype aggressiveness. Store-operated calcium entry (SOCE) is significantly remodeled in breast cancer cells and supports a variety of cancer hallmarks. Here we show that breast cancer cells from different subtypes exhibit a variable Orai1:Orai2 expression ratio. In cells with a high Orai2 expression, Orai2 modulates the magnitude SOCE and support Ca2+ oscillations in response to agonists, thus modulating the activation of the transcription factors NFAT1 and NFAT4. Furthermore, Orai2 plays a relevant role in cell cycle progression and apoptosis resistance.Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.

Ghrelin and Cannabinoid Functional Interactions Mediated by Ghrelin/CB1 Receptor Heteromers That Are Upregulated in the Striatum From Offspring of Mice Under a High-Fat Diet.

There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits. The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors. In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes. Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling. On the one hand, just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling. On the other hand, cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist, arachidonyl-2’-chloroethylamide (ACEA). The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays. Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet. Surprisingly, the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM). These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain.

Screening for Activity Against AMPA Receptors Among Anticonvulsants-Focus on Phenytoin.

The interest in AMPA receptors as a target for epilepsy treatment increased substantially after the approval of perampanel, a negative AMPA receptor allosteric antagonist, for the treatment of partial-onset seizures and generalized tonic-clonic seizures. Here we performed a screening for activity against native calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) among different anticonvulsants using the whole-cell patch-clamp method on isolated Wistar rat brain neurons. Lamotrigine, topiramate, levetiracetam, felbamate, carbamazepine, tiagabin, vigabatrin, zonisamide, and gabapentin in 100-µM concentration were practically inactive against both major subtypes of AMPARs, while phenytoin reversibly inhibited them with IC50 of 30 ± 4 μM and 250 ± 60 µM for CI-AMPARs and CP-AMPARs, respectively. The action of phenytoin on CI-AMPARs was attenuated in experiments with high agonist concentrations, in the presence of cyclothiazide and at pH 9.0. Features of phenytoin action matched those of the CI-AMPARs pore blocker pentobarbital, being different from classical competitive inhibitors, negative allosteric inhibitors, and CP-AMPARs selective channel blockers. Close 3D similarity between phenytoin and pentobarbital also suggests a common binding site in the pore and mechanism of inhibition. The main target for phenytoin in the brain, which is believed to underlie its anticonvulsant properties, are voltage-gated sodium channels. Here we have shown for the first time that phenytoin inhibits CI-AMPARs with similar potency. Thus, AMPAR inhibition by phenytoin may contribute to its anticonvulsant properties as well as its side effects.

Effect of Clonidine Hydrochloride on Isolated Newborn Rat Heart.

The concentration dependenies of the chronotropic response and changes in blood supply to the isolated heart of 7-day-old newborn rats induced by application of α2-adrenergic receptor agonist clonidine hydrochloride in concentrations of 10-9-10-6 M were revealed. The minimum concentration of α2-adrenergic receptor agonist caused tachycardia, while higher concentrations led to bradycardia. The maximum effect manifesting in a decrease in coronary flow was recorded at the minimum concentration of the agonist, while the highest concentration had no effect on the coronary flow. When comparing these results with those obtained in control adult rats, we found that the most pronounced differences in the chronotropic effects were observed after addition of the minimum concentration of the α2-adrenergic receptor agonist: bradycardia in adult rats and tachycardia in newborns. The maximum differences in coronary flow parameters were observed after addition of α2-adrenergic receptor agonist in the maximum concentration that induced a two-phase response in adult rats and had no effect on the blood supply in newborns.

Cell-Surface Loss of Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptors

The present study aimed to investigate the mechanism of cell surface receptor loss by two constitutively activating mutants (designated L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the luteinizing hormone receptor (LHR) in the Japanese eel Anguilla japonica, known to naturally occur in human LHR transmembrane domains. We investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in HEK 293 cells. The expression level of wild-type eel LHR was considered to be 100%, and the expression levels of L469R and D417N were 97% and 101%, respectively, whereas the expression levels of D590Y and Y558F slightly increased to approximately 110% and 106%, respectively. The constitutively activating mutants L469R and D590Y exhibited a decrease in cell surface loss in a manner similar to that of wild-type eel LHR. The rates of loss of cell surface agonist-receptor complexes were observed to be very rapid (2.6-6.2 min) in both the wild-type eel LHR and activating mutants. However, cell surface receptor loss in the cells expressing inactivating mutants D417N and Y558F was slightly observed in the cells expressing inactivating mutants D417N and Y558F, despite treatment with a high concentration of agonist. These results provide important information on LHR function in fish and the regulation of mutations of highly conserved amino acids in glycoprotein hormone receptors.

Effects of mechanosensitive ion channel Piezo1 on proliferation and osteogenic differentiation of human dental follicle cells

BackgroundTo explore the role of the mechanosensitive ion channel Piezo1 in the proliferation and osteogenic differentiation of human dental follicle cells (hDFCs), and its mechanism, so as to provide the basis for the use of hDFCs to achieve bone regeneration. MethodshDFCs were obtained from fresh dental follicle tissues by enzymatic digestion, and cell phenotype and multipotential differentiation were identified. Identification of the expression of mechanosensitive ion channel Piezo1 was performed by immunofluorescence and immunohistochemistry. CCK-8 was used to determine the optimal concentration of the Piezo1 agonist, Yoda1. Then, according to the obtained results, Alizarin red staining, RT-PCR quantitative analysis and Western blot were used to further observe the osteogenic differentiation of hDFCs and its probable mechanism via Wnt/β-catenin signalling. The data were analysed by SPSS 22.0 software. ResultsThe results of the concentration gradient experiments indicated that 0.5 µM Piezo1 agonist (Yoda1) enhanced the proliferation of hDFCs. Compared with the control group, a considerable number of calcium nodules showed that activating Piezo1 could promote the osteogenic differentiation of hDFCs. The relative mRNA and protein expression of Piezo1, ALP, RUNX2, OCN and BMP2 in the Piezo1 agonist group were higher than that of the control group. Furthermore, the expression of Wnt3a and β-catenin related to the classical osteogenic pathway were significantly up-regulated in the Piezo1 agonist group. ConclusionActivating mechanosensitive ion channel Piezo1 with an appropriate concentration of Yoda1 has a positive effect on the proliferation and osteogenic differentiation of hDFCs. This mechanism of promoting osteogenic differentiation may be mediated by the Wnt/β-catenin pathway.

Time and metabolic state-dependent effects of GLP-1R agonists on NPY/AgRP and POMC neuronal activity in vivo

ObjectiveLong-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs), like liraglutide and semaglutide, are viable treatments for diabetes and obesity. Liraglutide directly activates hypothalamic proopiomelanocortin (POMC) neurons while indirectly inhibiting Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons ex vivo. While temporal control of GLP-1R agonist concentration as well as accessibility to tissues/cells can be achieved with relative ease ex vivo, in vivo this is dependent upon the pharmacokinetics of these agonists and relative penetration into structures of interest. Thus, whether liraglutide or semaglutide modifies the activity of POMC and NPY/AgRP neurons in vivo as well as mechanisms required for any changes in cellular activity remains undefined. MethodsIn order to resolve this issue, we utilized neuron-specific transgenic mouse models to examine changes in the activity of POMC and NPY/AgRP neurons after injection of either liraglutide or semaglutide (intraperitoneal - I.P. and subcutaneous - S·C.). POMC and NPY/AgRP neurons were targeted for patch-clamp electrophysiology as well as in vivo fiber photometry. ResultsWe found that liraglutide and semaglutide directly activate and increase excitatory tone to POMC neurons in a time-dependent manner. This increased activity of POMC neurons required GLP-1Rs in POMC neurons as well as a downstream mixed cation channel comprised of TRPC5 subunits. We also observed an indirect upregulation of excitatory input to POMC neurons originating from glutamatergic cells that also required TRPC5 subunits. Conversely, GLP-1Ra's decreased excitatory input to and indirectly inhibited NPY/AgRP neurons through activation of K-ATP and TRPC5 channels in GABAergic neurons. Notably, the temporal activation of POMC and inhibition of NPY/AgRP neuronal activity after liraglutide or semaglutide was injected [either intraperitoneal (I.P.) or subcutaneous (S·C.)] was dependent upon the nutritional state of the animals (fed vs food-deprived). ConclusionsOur results support a mechanism of liraglutide and semaglutide in vivo to activate POMC while inhibiting NPY/AgRP neurons, which depends upon metabolic state and mirrors the pharmacokinetic profile of these compounds in vivo.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Pharmacological characterization of a novel negative allosteric modulator of NMDA receptors, UBP792

N-methyl-d-aspartate (NMDA) receptors (NMDARs) are a subtype of ionotropic glutamate receptor with important roles in CNS function. Since excessive NMDAR activity can lead to neuronal cell death and epilepsy, there is interest in developing NMDAR negative allosteric modulators (NAMs) as neuroprotective agents. In this study, we characterize the inhibitory properties of a novel NMDAR antagonist, UBP792. This compound displays partial subtype-selectivity by having a varied maximal inhibition of GluN2A-, GluN2B-, GluN2C-, and GluN2D-containing receptors (52%, 70%, 87%, 89%, respectively) with IC50s 4–10 μM. UBP792 inhibited NMDAR responses by reducing l-glutamate and glycine potencies and efficacies. Consistent with non-competitive inhibition, increasing agonist concentrations 30-fold did not reduce UBP792 potency. UBP792 inhibition was also not competitive with the structurally-related positive allosteric modulator (PAM) UBP684. UBP792 activity was voltage-independent, unaffected by GluN1's exon-5, and reduced at low pH (except for GluN1/GluN2A receptors which were more sensitive at acidic pH). UBP792 binding appeared independent of agonist binding and may be entering the plasma membrane to gain access to its binding site. Inhibition by UBP792 is reduced when the ligand-binding domain (LBD) of the GluN2 subunit, but not that of the GluN1 subunit, is cross-linked in the closed-cleft, activated conformation. Thus, UBP792 may be inhibiting by stabilizing an open GluN2-LBD cleft associated with channel inactivation or by stabilizing downstream closed channel conformations allosterically-coupled to the GluN2-LBD. These findings further expand the repertoire displayed by NMDAR NAMs thus expanding the opportunities for developing NMDAR modulators with the most appropriate selectivity and physiological actions for specific therapeutic indications.This article is part of the Neuropharmacology Special Issue on ‘Glutamate Receptors – NMDA receptors’.

Computational modeling of blood component transport related to coronary artery thrombosis in Kawasaki disease.

Coronary artery thrombosis is the major risk associated with Kawasaki disease (KD). Long-term management of KD patients with persistent aneurysms requires a thrombotic risk assessment and clinical decisions regarding the administration of anticoagulation therapy. Computational fluid dynamics has demonstrated that abnormal KD coronary artery hemodynamics can be associated with thrombosis. However, the underlying mechanisms of clot formation are not yet fully understood. Here we present a new model incorporating data from patient-specific simulated velocity fields to track platelet activation and accumulation. We use a system of Reaction-Advection-Diffusion equations solved with a stabilized finite element method to describe the evolution of non-activated platelets and activated platelet concentrations [AP], local concentrations of adenosine diphosphate (ADP) and poly-phosphate (PolyP). The activation of platelets is modeled as a function of shear-rate exposure and local concentration of agonists. We compared the distribution of activated platelets in a healthy coronary case and six cases with coronary artery aneurysms caused by KD, including three with confirmed thrombosis. Results show spatial correlation between regions of higher concentration of activated platelets and the reported location of the clot, suggesting predictive capabilities of this model towards identifying regions at high risk for thrombosis. Also, the concentration levels of ADP and PolyP in cases with confirmed thrombosis are higher than the reported critical values associated with platelet aggregation (ADP) and activation of the intrinsic coagulation pathway (PolyP). These findings suggest the potential initiation of a coagulation pathway even in the absence of an extrinsic factor. Finally, computational simulations show that in regions of flow stagnation, biochemical activation, as a result of local agonist concentration, is dominant. Identifying the leading factors to a pro-coagulant environment in each case—mechanical or biochemical—could help define improved strategies for thrombosis prevention tailored for each patient.

PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α 1 -ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α 1 -BLOCKERS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (PD11)1 Sep 2021PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α1-ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α1-BLOCKERS Ru Huang, Yuhan Liu, Bingsheng Li, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Ru HuangRu Huang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Bingsheng LiBingsheng Li More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001986.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Mirabegron inhibits detrusor smooth muscle contractions and is available for treatment of overactive bladder. Consequently, its effects on lower urinary tract symptoms (LUTS) suggestive of bengin prostatic hyperplasia (BPH) were subject of recent trials. However, available results are not yet conclusive, and effects of mirabegron on human prostate smooth muscle contraction are hardly characterized. Voiding symptoms suggestive of BPH may be driven by increased prostate smooth muscle tone. Here, we examined effects of mirabegron on contractions of human prostate tissues. METHODS: Human prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Comparisons were performed between whole groups (one-way ANOVA), and at single frequencies and agonist concentrations (multivariate analysis). RESULTS: Electric field stimulation (EFS) induced neurogenic, frequence-dependent contractions, while agonists induced concentration-dependent contractions. Mirabegron (10 µM) inhibited EFS-induced contractions (p <0.001 for controls vs. mirabegron between whole groups), which was observed at different frequencies and reflected by reduced Emax values calculated by curve fitting (contractions in controls 84±11% of KCl-induced contractions, but 14±2% after mirabegron, p <0.01). Miragebron increased the EC50 values for noradrenaline (controls 0.55±0.32 µM, mirabegron 5.1±2.4 µM, p <0.004), resulting in rightshifts of concentration response curves for noradrenaline-induced contractions (p <0.001) and in reduced contractions up to 10 µM noradrenaline, but in recovery at higher noradrenaline concentrations and unchanged Emax values. Similarly, EC50 values for the α1-adrenergic agonist phenylephrine were increased by mirabegron (controls 7.4±4.1 µM, mirabegron 29.2±8.9 µM, p=0.05), resulting in rightshifts of concentration response curves for phenylephrine (p <0.001) and in reduced contractions up to 30 µM phenylephrine, but in recovery at higher phenylephrine concentrations and in unchanged Emax values. Mirabegron slightly reduced contractions induced by the thromboxane A2 analog U46619 (p <0.03), without changing EC50 values. Contractions induced by endothelin-1 remained unchanged by mirabegron. CONCLUSIONS: Mirabegron inhibits neurogenic and α1-adrenergic smooth muscle contractions in the human prostate. Surprisingly, the pharmacologic pattern is similar to that of α1-blockers, including rightshifts of α1-adrenergic concentration response curves, reduced Emax values for neurogenic contractions, and lacking or small effects on non-adrenergic contractions. Source of Funding: China Scholarship Council (CSC) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e200-e200 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ru Huang More articles by this author Yuhan Liu More articles by this author Bingsheng Li More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Loading ...

A de novo GRIN1 Variant Associated With Myoclonus and Developmental Delay: From Molecular Mechanism to Rescue Pharmacology.

N-Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology.

The residence of synaptically released dopamine on D2 autoreceptors.

SUMMARYNeuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition.

Brain-Derived Neurotrophic Factor Induces Platelet Aggregation

Background: A large extra-cerebral pool of Brain-Derived Neurotrophic Factor (BDNF) is found in platelets. With concentrations reaching 100-1000 fold those of neurons, platelet-stored BDNF is hypothesized to play an important role in the vasculature.Aims: To investigate the effect of BDNF on platelets, alone and synergistically with traditional agonists.Methods: The presence of BDNF in platelets was confirmed by Western Blotting and confocal microscopy. The ability of platelets to release BDNF upon stimulation was assessed by ELISA on platelet releasates following stimulation by maximal concentrations of arachidonic acid, adenosine diphosphate (ADP), epinephrine, collagen, or thrombin-receptor activating peptide (TRAP). To investigate the effect of BDNF on platelet function, we used a recombinant BDNF protein (rBDNF, Peprotech) added exogenously to a preparation of washed platelets obtained from healthy volunteers. Aggregation was recorded by light transmission aggregometry over 20 minutes. Synergistic assays were used with priming (sub-threshold) concentrations of either collagen or TRAP. The dependence on secondary mediators was assessed by blocking either COX-1-dependent thromboxane formation with aspirin or the ADP-P2Y12 receptor with AR-C66096. Platelet aggregation was blocked with a GPIIbIIIa inhibitor (abciximab) to assess the contribution of outside-in signalling.Results: Western blotting showed a 14kD band for the washed platelet protein preparations and for rBDNF used as a positive control. BDNF appeared stored in granules on confocal microscopy, consistent with previous reports. BDNF in the platelet releasate was increased 5-fold following platelet aggregation induced by traditional agonists, as compared with baseline. Exogenous rBDNF had no effect on platelet aggregation at low doses (1 and 3 µg/ml), but induced complete biphasic platelet aggregation at a high dose (10 µg/ml). The secondary wave of platelet aggregation was abrogated in the presence of aspirin and AR-C66096, but they had no effect on the primary wave. Platelet aggregation was completely abolished in the presence of the GPIIbIIIa inhibitor abciximab. Synergistic studies were conducted with priming concentrations of rBDNF, collagen and TRAP which were insufficient to elicit platelet aggregation on their own. Incubation of platelets with rBDNF (3 µg/ml) potentiated the platelet responses to priming concentrations of both collagen and TRAP. Indeed, in the absence of BDNF, no platelet aggregation was seen at the concentrations used, whereas incubation with BDNF led to complete and irreversible platelet aggregation to priming concentrations of collagen and TRAP.Conclusion: Platelets contain BDNF within their granules, which they release upon platelet activation with traditional agonists. Stimulation of washed platelets with high concentrations of rBDNF induced platelets to aggregate; in the presence of low concentrations of rBDNF, platelet aggregation was potentiated in response to priming concentrations of traditional agonists. Platelet responses to rBDNF were partly dependent on secondary mediators, as inhibition of COX-1 and the ADP-P2Y12 receptor inhibited the secondary wave of aggregation. However, the primary wave of aggregation was unaltered, thereby suggesting that platelets either possess a BDNF receptor or a traditional receptor that can signal in the presence of BDNF. Further studies are needed to uncover which receptor is implicated in platelet responses to BDNF, and the physiological role of BDNF-induced platelet responses. DisclosuresNo relevant conflicts of interest to declare.

Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation.Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized.As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA2's effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA2's lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548.These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. DisclosuresNo relevant conflicts of interest to declare.

Concentration-dependent alpha1-Adrenoceptor Antagonism and Inhibition of Neurogenic Smooth Muscle Contraction by Mirabegron in the Human Prostate

Introduction: Mirabegron is available for treatment of storage symptoms in overactive bladder, which may be improved by β3-adrenoceptor-induced bladder smooth muscle relaxation. In addition to storage symptoms, lower urinary tract symptoms in men include obstructive symptoms attributed to benign prostatic hyperplasia, caused by increased prostate smooth muscle tone and prostate enlargement. In contrast to the bladder and storage symptoms, effects of mirabegron on prostate smooth muscle contraction and obstructive symptoms are poorly understood. Evidence from non-human smooth muscle suggested antagonism of α1-adrenoceptors as an important off-target effect of mirabegron. As α1-adrenergic contraction is crucial in pathophysiology and medical treatment of obstructive symptoms, we here examined effects of mirabegron on contractions of human prostate tissues and on proliferation of prostate stromal cells. Methods: Contractions were induced in an organ bath. Effects of mirabegron on proliferation, viability, and cAMP levels in cultured stromal cells were examined by EdU assays, CCK-8 assays and enzyme-linked immunosorbent assay. Results: Mirabegron in concentrations of 5 and 10 μM, but not 1 µM inhibited electric field stimulation-induced contractions of human prostate tissues. Mirabegron in concentrations of 5 and 10 µM shifted concentration response curves for noradrenaline-, methoxamine- and phenylephrine-induced contractions to the right, including recovery of contractions at high concentrations of α1-adrenergic agonists, increased EC50 values, but unchanged Emax values. Rightshifts of noradrenaline concentration response curves and inhibition of EFS-induced contractions were resistant to L-748,337, l-NAME, and BPIPP. 1 µM mirabegron was without effect on α1-adrenergic contractions. Endothelin-1- and U46619-induced contractions were not affected or only inhibited to neglectable extent. Effects of mirabegron (0.5–10 µM) on proliferation and viability of stromal cells were neglectable or small, reaching maximum decreases of 8% in proliferation assays and 17% in viability assays. Mirabegron did not induce detectable increases of cAMP levels in cultured stromal cells. Conclusion: Mirabegron inhibits neurogenic and α1-adrenergic human prostate smooth muscle contractions. This inhibition may be based on antagonism of α1-adrenoceptors by mirabegron, and does not include activation of β3-adrenoceptors and requires concentrations ranging 50-100fold higher than plasma concentrations reported from normal dosing. Non-adrenergic contractions and proliferation of prostate stromal cells are not inhibited by mirabegron.

β1-Blockers Enhance Inotropy of Endogenous Catecholamines in Chronic Heart Failure.

Although β1-blockers impressively reduce mortality in chronic heart failure (CHF), there are concerns about negative inotropic effects and worsening of hemodynamics in acute decompensated heart failure. May receptor theory dispel these concerns and confirm clinical practice to use β1-blockers? In CHF, concentrations of catecholamines at the β1-adrenoceptors usually exceed their dissociation constants (KDs). The homodimeric β1-adrenoceptors have a receptor reserve and display negative cooperativity. We considered the binomial distribution of occupied receptor dimers with respect to the interaction of an exogenous β1-blocker and elevated endogenous agonist concentrations > [KDs], corresponding to an elevated sympathetic tone. Modeling based on binomial distribution suggests that despite the presence of a low concentration of the antagonist, the activation of the dimer receptors is higher than that in its absence. Obviously, the antagonist improves the ratio of the dimer receptors with only single agonist activation compared with the dimer receptors with double activation. This leads to increased positive inotropic effects of endogenous catecholamines due to a β1-blocker. To understand the positive inotropic sequels of β1-blockers in CHF is clinically relevant. This article may help to eliminate the skepticism of clinicians about the use of β1-blockers because of their supposed negative inotropic effect, since, on the contrary, a positive inotropic effect can be expected for receptor-theoretical reasons.

Randialic acid B and tomentosolic acid block formyl peptide receptor 1 in human neutrophils and attenuate psoriasis-like inflammation in vivo

Psoriasis is a long-lasting inflammatory skin disease lacking proper cure. Dysregulated activation of neutrophils is a major pathogenic factor in psoriasis. Formyl peptide receptor 1 (FPR1) triggers neutrophil activation in response to bacteria- or mitochondria-derived N-formyl peptides, but its significance in neutrophilic psoriasis remains unknown. In this study, we discovered two derivatives of ursolic acid, 3β-hydroxyurs-12,18-dien-28-oic acid (randialic acid B, RAB) and 3β-hydroxyurs-12,19-dien-28-oic acid (tomentosolic acid, TA), as FPR1 inhibitors in human neutrophils with ability to suppress psoriatic symptoms in mice. Both RAB and TA, triterpenoids of traditional medicinal plant Ilex kaushue, selectively inhibited reactive oxygen species production, elastase release, and CD11b expression in human neutrophils activated by FPR1, but not non-FPR1 agonists. Importantly, RAB and TA inhibited the binding of N-formyl peptide to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells, indicating FPR1 antagonism. Moreover, in assays induced by various concentrations of FPR1 agonist, both RAB and TA acted competitively for its binding to the FPR1 receptor. The FPR1-downstream signaling such as Ca2+ mobilisation and activation of Akt and MAPKs was also competitively inhibited. In addition, imiquimod-induced psoriasis-like symptoms, including epidermal hyperplasia, desquamation with scaling, neutrophil skin infiltration, and transepidermal water loss were significantly reduced by both RAB and TA. The results illustrate a possible role of human neutrophils FPR1 receptor in psoriasis-like inflammation. Accordingly, triterpenoids RAB and TA represent novel FPR1 antagonists and exhibit therapeutic potential for treating neutrophilic inflammatory skin diseases.