High Agonist Concentrations Research Articles

Enteroendocrine cell-derived peptide YY signalling is stimulated by pinolenic acid or Intralipid and involves coactivation of fatty acid receptors FFA1, FFA4 and GPR119

Long chain fatty acids are sensed by enteroendocrine L cells that express free-fatty acid receptors, including FFA1, FFA4 and the acylethanolamine receptor GPR119. Here we investigated the acute effects of single or multiple agonism at these G protein-coupled receptors in intestinal mucosae where L cell-derived peptide YY (PYY) is anti-secretory and acts via epithelial Y1 receptors. Mouse ileal or colonic mucosae were mounted in Ussing chambers, voltage-clamped and the resultant short-circuit current (Isc) recorded continuously. The agonists used were; FFA1, TAK-875 or AM-1638; for FFA4, Merck A; or for GPR119, AR231453, PSN632408 or AR440006. Their responses were compared with those of pinolenic acid (PA, a presumed dual FFA1/FFA4 agonist) and the lipid emulsion, Intralipid. The FFA1 agonist AM-1638 (EC50 = 38.2 nM) was more potent than TAK-875 (EC50 = 203.1 nM) but exhibited similar efficacy. GPR119 agonism (AR231453) pretreatment enhanced subsequent FFA1 (AM-1638 or TAK-875) and FFA4 (Merck A) signalling. PA (EC50 = 298.2 nM) co-activated epithelial FFA1 and FFA4 and involved endogenous PYY Y1/Y2-receptor mechanisms but desensitisation was observed between PA and high GPR119 agonist concentrations. Apical Intralipid co-activated FFA1, FFA4 and GPR119 with a residual component not being attributable to PYY, or this trio of fatty acid receptors.

OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids

Abstract Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, encoding the Glucocorticoid Receptor (GR). We identified a novel heterozygous point mutation causing fluctuating resistance to glucocorticoid depending on ligand concentrations. Initially, the propositus, a 45-year-old man, was diagnosed with a right adrenal incidentaloma and an ACTH-independent biological hypercortisolism. Shortly after adrenal surgery, plasma cortisol increased again (612 nmol/L) associated with inadequate ACTH levels (122 pg/mL) without any evidence of pituitary adenoma or ectopic ACTH-secreting tumor, suggesting a glucocorticoid resistance syndrome. Methods: We sequenced NR3C1 gene and performed in vitro analysis to assess contribution of this variant to the patient’s phenotype and how it affects biological activity of GR. Results: Exon sequencing of the NR3C1 gene identified a novel missense variant located on exon 5 (NM_000176.3, c.1706 G>A) resulting in substitution of arginine to glutamine at amino acid 569 (GR R569Q) in the Ligand-Binding Domain (LBD) of GR. We characterized transcriptional activity of GR R569Q in HEK 293T cells transiently co-transfected with either GR WT or GR R569Q, together with a luciferase reporter gene construct placed under control of a promoter containing two glucocorticoid responsive elements. We observed a decreased transcriptional activity of GR R569Q (EC50 4.55 nM [3.8;5.3]) compared to GR WT (EC50 0.19 nM [0.15; 0.24]), in response to dexamethasone. Interestingly, GR R569Q exerted a dominant negative effect on GR WT, with a decrease in transcriptional activity of 80% when stimulated with 1 nM dexamethasone (2-way ANOVA test, P<0.0001), which was blunted when stimulated with a 100-fold higher concentration of dexamethasone. As the variant is located within the second nuclear localization signal, we studied nuclear translocation in response to 1 nM cortisol for 1 h. As expected, nuclear-cytoplasmic ratio was lower in patients’ fibroblasts (2.3 ± 0.14 vs 3.0 ± 0.98, ANOVA 2-Way, P < 0.0001) compared with control fibroblasts, as quantified by automated high-throughput microscopy. Three-dimensional modeling revealed that arginine substitution leads to loss of a hydrogen bond stabilizing the LBD structure. This may result in a lower ligand affinity that can be bypassed by high concentrations of agonist. We are currently testing receptor affinity and reversibility of phenotype after hydrogen bond restoration by directed mutagenesis (c.1705 C>A, p.Q569K). Conclusion: Overall, we identified a novel variant of the NR3C1 gene associated with fluctuating GR sensitivity depending on ligand concentration. This finding may provide new insights into mechanisms involved in GR sensitivity. Presentation: Friday, June 16, 2023

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Opioid-Induced Pronociceptive Signaling in the Gastrointestinal Tract Is Mediated by Delta-Opioid Receptor Signaling.

Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic in vivo administration of morphine by intraperitoneal injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when intraperitoneally injected in previous morphine administration. Patch-clamp studies of DRG neurons following overnight incubation with high concentrations of morphine, the µ-opioid receptors (MOPr) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or the DOPr agonist [D-Ala2, D-Leu5]-Enkephalin evoked hyperexcitability. The pronociceptive actions of these opioids were blocked by the DOPr antagonist SDM25N but not the MOPr antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 The hyperexcitability induced by DAMGO was reversed after a 1 h washout, but reapplication of low concentrations of DAMGO or [D-Ala2, D-Leu5]-Enkephalin restored the hyperexcitability, an effect mediated by protein kinase C. DOPr-dependent DRG neuron hyperexcitability was blocked by the endocytosis inhibitor Pitstop 2, and the weakly internalizing DOPr agonist ARM390 did not cause hyperexcitability. Bioluminescence resonance energy transfer studies in HEK cells showed no evidence of switching of G-protein signaling from Gi to a Gs pathway in response to either high concentrations or overnight incubation of opioids. Thus, chronic high-dose opioid exposure leads to opioid tolerance and features of OIH in the colon. This action is mediated by DOPr signaling and is dependent on receptor endocytosis and downstream protein kinase C signaling.SIGNIFICANCE STATEMENT Opioids are effective in the treatment of abdominal pain, but escalating doses can lead to opioid tolerance and potentially opioid-induced hyperalgesia. We found that δ-opioid receptor (DOPr) plays a central role in the development of opioid tolerance and opioid-induced hyperalgesia in colonic afferent nociceptors following prolonged exposure to high concentrations of MOPr or DOPr agonists. Furthermore, the role of DOPr was dependent on OPr internalization and activation of a protein kinase C signaling pathway. Thus, targeting DOPr or key components of the downstream signaling pathway could mitigate adverse side effects by opioids.

Defining innate immune training potential as a predictor of Bacillus Calmette-Guérin immunotherapy response in nonmuscle-invasive bladder cancer.

522 Background: Intravesical instillation of the live attenuated mycobacterium Bacillus Calmette-Guerin (BCG) remains the most effective therapy for the treatment of non-muscle invasive bladder cancer. The precise mechanism of BCG is thought to involve phagocytosis, induction of inflammation, activation of the innate immune apparatus, and an effective cell-mediated T cell response. An increasing body of evidence suggests that the innate immune system has characteristics that involve a heterologous memory of past insults through a process thought to involve epigenetic reprogramming, termed trained immunity. We seek to investigate the potential for innate immune training as a predictor of the anti-tumor effects of BCG. Methods: A prospective biospecimen collection was performed on venous blood from non-muscle invasive bladder cancer patients prior to receiving intravesical BCG therapy. To investigate the role of trained immunity in primary monocytes, cells were isolated from peripheral blood mononuclear cells (PBMC) by plate adhesion. 2-5 x 105 cells/well were assayed in 96-well format, cultured in RPMI 1640 supplemented with 10% Fetal Bovine Serum and 2mM L-glutamine and stimulated with 10:1 MOI BCG for 24 hours. Cells were then washed in media and allowed to rest 6 days. Media was changed on day 3 of rest. Subsequently, cells were stimulated with 25 ng/ml lipopolysaccharide (a TLR4 agonist) for 24 hours. Cell free supernatant was measured for TNF-alpha production by enzyme-linked immunosorbent assay. Results: In vitro training of primary monocytes induced an increase in proinflammatory cytokine production upon re-stimulation. This phenotype was dose dependent on stimulation with LPS, however, the relative change between trained and untrained cells was diminished at higher concentrations of agonist. In our in vitro training experiments, there was demonstrable interpatient variability seen pre-BCG exposure in a cohort of NMIBC patients. These results will be correlated with future long term BCG response in this prospective cohort. Conclusions: We present data on a trained innate immune phenotype on primary monocytes collected from patient samples. These data serve as proof of principle for the ability to induce a trained phenotype in vitro. These findings will be correlated with prospective clinical data on BCG responsiveness and recurrence free survival to allow us to garner further insight into the role of trained immunity in BCG immunotherapy. Such insight will have potential clinical impact on the development of biomarkers and clinical assays that could predict immune responsiveness to BCG a priori, as well as identify possible immunological adjuvants that could enhance the effect of intravesical BCG.

Flow cytometry identifies an early stage of platelet apoptosis produced by agonists of the P2X1 and P2X7 receptors

Platelets express P2X1 receptors and our data also show the expression of P2X7 receptors. We studied the role of both receptors in platelet apoptosis by incubation of PRP with P2X agonists, then centrifuged to remove viable platelets, and analyzed the supernatant by flow cytometry to identify a sparse platelet-derived population that stained with MitoTracker dyes and CD41. BzATP, a potent agonist of P2X receptors, and ABT737, an activator of intrinsic apoptosis, produced altered platelets that stained moderately for annexin V and corresponded to an early stage apoptotic platelet (ESAP). Over a range of BzATP concentrations, we observed a dose-dependent formation of ESAPs between 5 and 500 uM BzATP, together with a variable formation of ESAPs at nanomolar ATP or BzATP (50–200 nM). Production of ESAPs occurred with αβ-meATP, while responses with either BzATP or αβ-meATP showed desensitization at a higher agonist concentration. Formation of ESAPs by either 100 nM or 0.5 mM BzATP was inhibited by preincubation of platelets with latrunculin A, an inhibitor of the actin cytoskeleton that prevents apoptosis. ESAP production was totally inhibited by preincubation of platelets with methyl-beta-cyclodextrin, which removes cholesterol from lipid rafts. Our data show that both P2X1 and P2X7 receptors are localized in platelet lipid rafts where P2X-agonists act to produce early stage apoptotic platelets.

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.

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.

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.

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.

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.

Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of β-arrestins. Indeed, recently described GLP-1R agonists with reduced β-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both β-arrestin isoforms the duration of G protein–dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, “biased” GLP-1, GCG, and GIP analogs with selective reductions in β-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced β-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

Thyroid hormones reduce nicotinic receptor mediated currents in SH-SY5Y neuroblastoma cells.

Thyroid hormones (THs) are crucial for maturation and functioning of mammalian CNS. THs "classical" signaling involves nuclear receptors binding but alsotheir non genomic actions, as rapid modulators of cell activity, are widely recognized. Since THs imbalance affects cognition and the cholinergic system is deeply involved in learning and memory processes we have studied THs effects at the level of the nicotinic acetylcholine receptors (nAchR). We used the patch-clamp technique to analyze T3 and T4 modulation of nicotine (NIC)-mediated current in SH-SY5Y neuroblastoma cells. Both hormones decreased NIC-evoked current in a dose dependent fashion. The antagonism was reversible, not competitive and not blocked by Tetrac, an integrin αVβ3 receptor antagonist. A similar effect was detected with the endogenous agonist Acetylcholine. THs potencies were higher at 100μM NIC (IC50 = 4.6 ± 2μM for T3 and 4.8 ± 2μM for T4) compared to those measured at 10μM NIC (IC50 = 10 ± 4μM for T3 and 8 ± 4μM for T4). Furthermore, the efficacy of THs reached almost 90% at 100μM NIC while was about 30 % at 10μM NIC. THs inhibited nAchR-mediated currents by enhancing receptor desensitization and this effect was more pronounced at high agonist concentrations. Our results make light on a new non genomic activity of THs at the level of nAchR. This mechanism of action of THs can provide a new explanation for the cognitive deficitsassociated with tyroid dysfunction.

Reduced Activation of the Synaptic-Type GABAA Receptor Following Prolonged Exposure to Low Concentrations of Agonists: Relationship between Tonic Activity and Desensitization.

Synaptic GABAA receptors are alternately exposed to short pulses of a high, millimolar concentration of GABA and prolonged periods of low, micromolar concentration of the transmitter. Prior work has indicated that exposure to micromolar concentrations of GABA can both activate the postsynaptic receptors generating sustained low-amplitude current and desensitize the receptors, thereby reducing the peak amplitude of subsequent synaptic response. However, the precise relationship between tonic activation and reduction of peak response is not known. Here, we have measured the effect of prolonged exposure to GABA or the combination of GABA and the neurosteroid allopregnanolone, which was intended to desensitize a fraction of receptors, on a subsequent response to a high concentration of agonist in human α1β3γ2L receptors expressed in Xenopus oocytes. We show that the reduction in the peak amplitude of the post-exposure test response correlates with the open probability of the preceding desensitizing response. Curve fitting of the inhibitory relationship yielded an IC50 of 12.5 µM and a Hill coefficient of -1.61. The activation and desensitization data were mechanistically analyzed in the framework of a three-state Resting-Active-Desensitized model. Using the estimated affinity, efficacy, and desensitization parameters, we calculated the amount of desensitization that would accumulate during a long (2-minute) application of GABA or GABA plus allopregnanolone. The results indicate that accumulation of desensitization depends on the level of activity rather than agonist or potentiator concentration per se. We estimate that in the presence of 1 µM GABA, approximately 5% of α1β3γ2L receptors are functionally eliminated because of desensitization. SIGNIFICANCE STATEMENT: We present an analytical approach to quantify and predict the loss of activatable GABAA receptors due to desensitization in the presence of transmitter and the steroid allopregnanolone. The findings indicate that the peak amplitude of the synaptic response is influenced by ambient GABA and that changes in ambient concentrations of the transmitter and other GABAergic agents can modify tonically and phasically activated synaptic receptors in opposite directions.

PAK Membrane Translocation and Phosphorylation Regulate Platelet Aggregation Downstream of Gi and G12/13 Pathways.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.

Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline

Light transmission aggregometry (LTA) is considered the gold standard method for evaluation of platelet function. However, there are a lot of variation in protocols (pre-analytical procedures and agonist concentrations) and results. The aim of our study was to establish a national LTA protocol, to investigate the effect of standardization and to define national reference values for LTA. The SSC guideline was used as base for a national procedure. Almost all recommendations of the SSC were followed e.g. no adjustment of PRP, citrate concentration of 109 mM, 21 needle gauge, fasting, resting time for whole blood and PRP, centrifugation time, speed and agonists concentrations. LTA of healthy volunteers was measured in a total of 16 hospitals with 5 hospitals before and after standardization. Results of more than 120 healthy volunteers (maximum aggregation %) were collected, with participating laboratories using 4 different analyzers with different reagents. Use of low agonist concentrations showed high variation before and after standardization, with the exception of collagen. For most high agonist concentrations (ADP, collagen, ristocetin, epinephrine and arachidonic acid) variability in healthy subjects decreased after standardization. We can conclude that a standardized Dutch protocol for LTA, based on the SSC guideline, does not result in smaller variability in healthy volunteers for all agonist concentrations.

A Noncompetitive and Selective Prostaglandin EP2 Antagonist with Anti‐inflammatory Property

The prostaglandin E2 (PGE2) receptor subtype EP2 promotes chronic inflammation, modulates tumorigenesis, and regulates immune function. Inhibition of EP2 receptors reduces inflammation in rodent models of epilepsy, endometriosis, and arthritis, and decreases tumor cell growth. Therefore, inhibiting EP2 receptors could be a promising method for treatment of inflammatory diseases. To date all reported EP2 antagonists have a purely competitive mode of action, which can be overcome by high concentrations of agonist. Herein, we report the characterization of a novel compound belongs to substituted pyrroloquinoxaline scaffold as the first EP2 antagonist that features a noncompetitive mode of action. Cell‐based time‐resolved fluorescence resonance energy transfer (TR‐FRET) assays were developed in 384‐well format to measure the accumulation of cyclic adenosine monophosphate (cAMP) induced by PGE2, on the basis of the fact that activation of the Gas‐coupled EP2 receptor induces cAMP formation. At a low concentration (3 μM) of the pyrroloquinoxaline antagonist, a saturating concentration of PGE2 (3 μM) could reverse the inhibition of EP2 receptor activation in human EP2‐overexpressing C6 glioma (C6G‐EP2) cells. At higher antagonist concentrations (10–30 μM), the inhibition of EP2 receptors could not be fully overcome by the agonist. Meanwhile, the maximal agonist response inhibition by the antagonist increased with increasing concentration. The maximum effect (cAMP induction) by PGE2 was decreased to 81.9 ±1.44%, unlike 100% for a typical competitive antagonist. The antagonist itself had no effect on the TR‐FRET signal since there was no change in the TR‐FRET signal in the absence of C6G‐EP2 cells. The antagonist was reversible as demonstrated by washing out the antagonist three times each at 80% efficiency. After washing out the antagonist, the maximal response of C6G‐EP2 cells to PGE2 increased to 97.6 ± 0.69% from 86.4 ± 0.85% (p < 0.0001, one‐way ANOVA with Bonferroni). Furthermore, the antagonist exhibited little to no effect on the EC50 or maximal response of other Gas‐coupled prostanoid receptors (EP4, IP, and DP1). The agonist EC50s for EP2, EP4, IP and DP1 receptors were shifted 13.2, 2.2, 1.1 and 0.95‐fold to the right, respectively, in 30 μM antagonist. To determine whether the noncompetitive antagonist exerts an anti‐inflammatory action we studied the expression of IL‐6 and IL‐1β in the murine BV2 microglial cell line stably expressing human EP2 receptors (BV2‐hEP2). Treatment of these cells with a >EC90 concentration (30 nM) of the EP2 selective agonist, ONO‐AE1‐259, caused elevated mRNA levels of the two cytokines (measured by qRT‐PCR) that was suppressed by the antagonist (20 μM). Thus, the pyrroloquinoxaline is the first noncompetitive EP2 receptor antagonist. This compound may provide a basis for further development of novel noncompetitive EP2 antagonists.Support or Funding InformationThis work was supported by the National Institutes of Health [Grant Numbers 1R01NS097776 (RD), 1 U01 AG052460 (TG)]. C.J. is a member of the joint training program between Emory University School of Medicine and Xiangya School of Medicine, Central South University, and is supported by the China Scholarship Council. We gratefully acknowledge the gift of ONO‐AE1‐259 by Ono Pharmaceuticals.

Inhibition of GABAergic Transmission as a Model of Hyperactivation of Purkinje Cells in the Rat Cerebellum

Abstract—Pathological changes in the cerebellum are often associated with dysfunction of Purkinje cells, which is manifested in excessive spike activity. In our experiments, the increase in the frequency of spike activity of Purkinje cells was caused by the suppression of inhibitory transmission by the GABA receptor antagonist gabazin (hydrobromide 6-imino-3-(4-methoxyphenyl)-1(6H)-pyrazinobutanic acid) contrary to the classical model of neurodegeneration, in which hyperactivation of neurons is caused by the long-term action of high concentrations of excitatory agonists (glutamate or N-methyl-D-aspartate). In adult and young animals, gabazine caused a significant increase in the frequency of Purkinje cell discharges after 25 and 45 min of action, respectively. Thus, this model worked more effectively in adult animals than in young ones, which is due to the ontogenetic features of the formation of the cerebellar cortex. Moreover, the use of NS 309 (6,7-dichloro-1H-indole-2,3-diketone-3-oxime), a modulator of Ca2+-activated K+ channels of small conductance, in this model made it possible to compensate for the increase in the frequency of simple spikes induced by the gabazine in the discharge of Purkinje cells to the level of the control values in young and adult rats.

Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta.

An increase in intracellular Ca2+ concentration ([Ca2+]i) plays a key role in controlling endothelial functions; however, it is still unclear whether endothelial Ca2+ handling is altered by type 2 diabetes mellitus, which results in severe endothelial dysfunction. Herein, we analyzed for the first time the Ca2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca2+-sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The “Ca2+ add-back” protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca2+ pool, while store-operated Ca2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit.