Pool Of Receptors Research Articles

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties

BackgroundChemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. MethodsWe investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. ResultsTwo commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. ConclusionsCCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. General significanceWe provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

The Epilepsy-Related Protein PCDH19 Regulates Tonic Inhibition, GABAAR Kinetics, and the Intrinsic Excitability of Hippocampal Neurons

PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.

On the dynamical aspects of local translation at the activated synapse

BackgroundThe key role in the dynamic regulation of synaptic protein turnover belongs to the Fragile X Mental Retardation Protein, which regulates the efficiency of dendritic mRNA translation in response to stimulation of metabotropic glutamate receptors at excitatory synapses of the hippocampal pyramidal cells. Its activity is regulated via positive and negative regulatory loops that function in different time ranges, which is an absolute factor for the formation of chaotic regimes that lead to disrupted proteome stability. The indicated condition may cause a number of neuropsychiatric diseases, including autism and epilepsy. The present study is devoted to a theoretical analysis of the local translation system dynamic properties and identification of parameters affecting the chaotic potential of the system.ResultsA mathematical model that describes the maintenance of a specific pool of active receptors on the postsynaptic membrane via two mechanisms – de novo synthesis of receptor proteins and restoration of protein function during the recycling process – has been developed. Analysis of the model revealed that an increase in the values of the parameters describing the impact of protein recycling on the maintenance of a pool of active receptors in the membrane, duration of the signal transduction via the mammalian target of rapamycin pathway, influence of receptors on the translation activation, as well as reduction of the rate of synthesis and integration of de novo synthesized proteins into the postsynaptic membrane – contribute to the reduced complexity of the local translation system dynamic state. Formation of these patterns significantly depends on the complexity and non-linearity of the mechanisms of exposure of de novo synthesized receptors to the postsynaptic membrane, the correct evaluation of which is currently problematic.ConclusionsThe model predicts that an increase of “receptor recycling” and reduction of the rate of synthesis and integration of de novo synthesized proteins into the postsynaptic membrane contribute to the reduced complexity of the local translation system dynamic state. Herewith, stable stationary states occur much less frequently than cyclic states. It is possible that cyclical nature of functioning of the local translation system is its “normal” dynamic state.

Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

Interleukin-1 receptor inhibition reduces stroke size in a murine model of sickle cell disease.

Sickle cell disease (SCD) is associated with chronic hemolytic anemia and a heightened inflammatory state. The causal role of inflammatory pathways in stroke associated with SCD is unclear. Therefore, the hypothesis that deletion of the non-hematopoietic interleukin-1 receptor (IL-1R) pool may be beneficial in SCD was pursued. Since potential deleterious effects of IL-1R signaling in SCD could be mediated via downstream production of interleukin-6 (IL-6), the role of the nonhematopoietic IL-6 pool was also addressed. Bone marrow transplantation (BMT) from SCD to wild-type (WT) recipient mice was used to generate SCD mice (Wt,SCDbmt). In order to generate mice with nonhematopoietic deficiency of IL-1R or IL-6, SCD marrow was transplanted into IL-1R deficient (IL1R-/-,SCDbmt) or IL-6 deficient recipients (IL6-/-, SCDbmt). Blood counts, reticulocytes, soluble E-selectin (sEsel), and IL-6 levels were analyzed 14-15 weeks post-BMT. Ischemic stroke was induced by middle cerebral artery (MCA) photothrombosis at 16 weeks post-BMT. A separate group of Wt,SCDbmt mice was given the IL-1R inhibitor, anakinra, following stroke induction. Seventy-two hours after MCA occlusion, stroke volume was assessed by staining brain sections with 2,3,5-triphenyltetrazolium chloride. Formalin-fixed brain sections were also stained for macrophages with MAC3, for endothelial activation with ICAM-1, and for loss of blood brain barrier integrity with fibrin (ogen) staining. All SCD mice generated by BMT were anemic and the severity of anemia was not different between Wt,SCDbmt, IL1R-/-,SCDbmt, and IL-6-/-,SCDbmt mice. Three days following MCA occlusion, stroke volume was significantly reduced in IL1R-/-,SCDbmt mice compared to Wt,SCDbmt mice and IL6-/-,SCDbmt mice. Plasma levels of sEsel were lower in IL1R-/-,SCDbmt compared to Wt,SCDbmt and IL-6-/-,SCDbmt mice. Post-stroke treatment of Wt,SCDbmt mice with anakinra decreased stroke size, leukocyte infiltration, ICAM-1 expression, and fibrin(ogen) accumulation compared to vehicle-treated mice. Deficiency of non-hematopoietic IL-1R or treatment with an IL-1R antagonist is sufficient to confer protection against the increased stroke size associated with SCD. These effects of IL1R deficiency are associated with reduced endothelial activation, leukocyte infiltration, and blood brain barrier disruption, and are independent of non-hematopoietic IL-6 signaling.

Heterologous expression of concatenated nicotinic ACh receptors: Pros and cons of subunit concatenation and recommendations for construct designs.

Concatenation of Cys-loop receptor subunits is a commonly used technique to ensure experimental control of receptor assembly. However, we recently demonstrated that widely used constructs did not lead to the expression of uniform pools of ternary and more complex receptors. The aim was therefore to identify viable strategies for designing concatenated constructs that would allow strict control of resultant receptor pools. Concatenated dimeric, tetrameric, and pentameric α4β2-containing nicotinic ACh (nACh) receptor constructs were designed with successively shorter linker lengths and expressed in Xenopus laevis oocytes. Resulting receptor stoichiometries were investigated by functional analysis in two-electrode voltage-clamp experiments. Molecular dynamics simulations were performed to investigate potential effects of linkers on the 3D structure of concatemers. Dimeric constructs were found to be unreliable and should be avoided for expression of ternary receptors. By introducing two short linkers, we obtained efficient expression of uniform receptor pools with tetrameric and pentameric constructs. However, linkers should not be excessively short as that introduces strain on the 3D structure of concatemers. The data demonstrate that design of concatenated Cys-loop receptors requires a compromise between the desire for control of assembly and avoiding introduction of strain on the resulting protein. The overall best strategy was found to be pentameric constructs with carefully optimised linker lengths. Our findings will advance studies of ternary or more complex Cys-loop receptors as well as enabling detailed analysis of how pharmacological agents interact with stoichiometry-specific binding sites.

A novel method using ambient glutamate for the electrophysiological quantification of extrasynaptic NMDA receptor function in acute brain slices.

We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ∼77nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death. Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35-42 postnatal days, we estimated ambient glutamate to be 72-83nM resulting in a block of more than 82% of ES-NMDARs during a 5min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling.

Remodeling Promotes Proarrhythmic Disruption of Calcium Homeostasis in Failing Atrial Myocytes

It is well known that heart failure (HF) typically coexists with atrial fibrillation (AF). However, until now, no clear mechanism has been established that relates HF to AF. In this study, we apply a multiscale computational framework to establish a mechanistic link between atrial myocyte structural remodeling in HF and AF. Using a spatially distributed model of calcium (Ca) signaling, we show that disruption of the spatial relationship between L-type Ca channels (LCCs) and ryanodine receptors results in markedly increased Ca content of the sarcoplasmic reticulum (SR). This increase in SR load is due to changes in the balance between Ca entry via LCCs and Ca extrusion due to the sodium-calcium exchanger after an altered spatial relationship between these signaling proteins. Next, we show that the increased SR load in atrial myocytes predisposes these cells to subcellular Ca waves that occur during the action potential (AP) and are triggered by LCC openings. These waves are common in atrial cells because of the absence of a well-developed t-tubule system in most of these cells. This distinct spatial architecture allows for the presence of a large pool of orphaned ryanodine receptors, which can fire and sustain Ca waves during the AP. Finally, we incorporate our atrial cell model in two-dimensional tissue simulations and demonstrate that triggered wave generation in cells leads to electrical waves in tissue that tend to fractionate to form wavelets of excitation. This fractionation is driven by the underlying stochasticity of subcellular Ca waves, which perturbs AP repolarization and consequently induces localized conduction block in tissue. We outline the mechanism for this effect and argue that it may explain the propensity for atrial arrhythmias in HF.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Upregulation of α2-adrenoceptor synthesis in SHR cardiomyocytes: Recompense without sense – Increased amounts, impaired commands

Aimsto investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. Methodsexperiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2–2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. Resultspercentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. Conclusiondata indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.

Properties of Individual Hippocampal Synapses Influencing NMDA-Receptor Activation by Spontaneous Neurotransmission.

NMDA receptor (NMDAR) activation is critical for maintenance and modification of synapse strength. Specifically, NMDAR activation by spontaneous glutamate release has been shown to mediate some forms of synaptic plasticity as well as synaptic development. Interestingly, there is evidence that within individual synapses each release mode may be segregated such that postsynaptically there are distinct pools of responsive receptors. To examine potential regulators of NMDAR activation because of spontaneous glutamate release in cultured hippocampal neurons, we used GCaMP6f imaging at single synapses in concert with confocal and super-resolution imaging. Using these single-spine approaches, we found that Ca2+ entry activated by spontaneous release tends to be carried by GluN2B-NMDARs. Additionally, the amount of NMDAR activation varies greatly both between synapses and within synapses, and is unrelated to spine and synapse size, but does correlate loosely with synapse distance from the soma. Despite the critical role of spontaneous activation of NMDARs in maintaining synaptic function, their activation seems to be controlled factors other than synapse size or synapse distance from the soma. It is most likely that NMDAR activation by spontaneous release influenced variability in subsynaptic receptor position, release site position, vesicle content, and channel properties. Therefore, spontaneous activation of NMDARs appears to be regulated distinctly from other receptor types, notably AMPARs, within individual synapses.

The autophagic degradation of cytosolic pools of peroxisomal proteins by a new selective pathway

ABSTRACTDamaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for Pichia pastoris and Atg36 for Saccharomyces cerevisiae, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In P. pastoris, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products.Abbreviations: ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation

Effect of TGF-beta1 on long-term synaptic plasticity and distribution of AMPA receptors in the CA1 field of the hippocampus

Using the methods of electrophysiology and immunohistochemistry, the effect of the transforming factor beta-1(TGF-β1), an anti-inflammatory cytokine, on the long-term post-tetanic potentiation (LTP) in CA1 field hippocampal slices and the distribution of the GluR1 subunit of the AMPA receptor has been studied.It was shown that TGF-β1 at a concentration of 10 ng/ml did not significantly affect the initial stage of LTP and substantially changed the distribution of synaptic AMPA receptors in response to tetanic stimulation. Twenty five minutes after the tetanization, the main pool of AMPA receptors (90%) was due to the postsynaptic density (PSD). By contrast, LTP in the presence of TGF-β1 was accompanied by less pronounced changes in the distribution of AMPA receptors. Their localization in both pre- and postsynaptic regions remained nearly the same as that in the control. It may be suggested that the normal distribution of AMPA receptors in spinous synapses promotes the stabilization of potentiated synapses, thereby retaining LTP for longer terms.

The physiological hypertrophic agonist, norepinephrine, is able to induce PLC‐mediated PI4P hydrolysis in cardiac myocytes via a pool of internal β‐adrenergic receptors

Previously, we have shown that PLC∑‐mediated phosphatidylinositol‐4‐phosphate (PI4P) can be activated by raising cAMP in cardiac myocytes, however, activation by Isoproterenol was unable to induce PI4P hydrolysis. In the accompanying abstract by Wei et al, we have demonstrated that the cell permeant β‐agonist, Dobutamine is able to induce PI4P hydrolysis. Here, we seek to determine if the physiological agonist, norepinephrine is able to induce PI4P hydrolysis, despite being membrane impermeant. In contrast to Iso, norepinephrine induced sustained PI4P hydrolysis that was blocked by the cell permeant β‐adrenergic antagonist, metoprolol (100μM). The cell impermeant βAR antagonist, sotalol (5mM) could not block norepinephrine effects, suggesting that a pool of internal β‐adrenergic receptors is responsible for NE‐mediated PI4P hydrolysis. To directly determine if internal β1‐ARs in NRVMs can be activated by norepinephrine, we monitored translocation of a YFP‐tagged mini Gs protein in β1AR transfected NRVMs2. Norepinephrine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. Golgi localization could be reversed by Metoprolol but not Sotalol. Isoproterenol, on the other hand, induced a strong recruitment to the plasma membrane but not to the Golgi. NE‐mediated PI4P hydrolysis is also abrogated by an inhibitor of a cation transporter Oct3, Corticosterone (100μM). Oct3 has been recently shown to be required for epinephrine‐mediated β1‐AR stimulation at the Golgi envelope in HELA cells3. Taken together, these data suggest that norepinephrine can activate β‐adrenergic receptors in internal membranes, potentially at the Golgi apparatus, stimulating PI4P hydrolysis. These internal GPCRs may be responsible for cardiac hypertrophy and heart failure and targeting them may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fundamental studies of the response patterns of the human immune system in the conditions of the North

The paper presents the facts and patterns that indicate the differentiation of immune responses to adaptive, regulatory and prenosological influences in response to the adverse effects of climatic and geographical factors. The activation of migration reactions of peripheral blood cells in response to an increase in the concentration of antigens or autoantigens causes hemodynamic reactions with the release of hemopoietic cells to the periphery of the depot, urgent catecholamine reactions and prolongation of them, if necessary, with glucocorticoids. Prenosological signs of the risk of disrupting regulatory mechanisms are neutropenia of lymphopenia, lack of phagocytic protection, an increase in the concentration of pro-inflammatory cytokines and the extracellular pool of receptors due to an increase in their shedding.

A High-content Assay for Monitoring AMPA Receptor Trafficking.

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

The Correlation between the Pool of Free Adhesion Molecule Receptors and the Activity of the Immune System in the Murmansk Oblast Residents

The molecules of intercellular adhesion are the surface cellular glycoproteins responsible for interaction between cells of different types with one another and with the components of intercellular matrix. They play the primary role in the formation of tissue structure and participate in a number of physiological processes, such as morphogenesis, embryogenesis, organogenesis, wound healing, and inflammation. Together with the other free receptors and their ligands, free intercellular adhesion molecules are formed mainly through shedding, resulting from the proteolytic cleavage of the transmembrane region under physiological conditions or cell destruction. There are almost no literature data on the influence of free intercellular adhesion molecules on the functional activity of T and B lymphocytes and the contents of immunoglobulins, cytokines, and circulating immune complexes. Therefore, the purpose of this study was to identify the interrelationship between the content of free and membrane-bound intercellular adhesion molecules and the level of immunocompetent cell activity in the residents of the Arctic settlement of Revda (Murmansk oblast), who form one of special populations of residents living in the Arctic region of the Russian Federation. It has been found that the contents of sCD54, sCD62L, and sCD324 free intercellular adhesion molecules vary widely, which results from an increase in the concentration of cells with the corresponding membrane forms. An increase in the levels of sCD54 and sCD62 is accompanied by the activation of migration processes with increasing concentrations of circulating neutrophils and total lymphocyte count. The formation of a pool of membrane-bound and free adhesion molecules occurs together with an increase in the content of activated T cells of the HLADRII and CD8 + phenotypes. An increase in the serum levels of sCD54 and L-selectin (sCD62L) adhesion molecules is accompanied by a decrease in the level of immunocompetent cell activity and a twofold increase in the serum level of a natural immunosuppressant IL-10. Low activity of the sCD54 and sCD62L is associated with an increase in the IgE concentration and in immune responses caused by the basophil and eosinophil activation.

Source relationships between streambank soils and streambed sediments in a mercury-contaminated stream

PurposeIn contaminated streams, understanding the role of streambank and streambed source contributions is essential to developing robust remedial solutions. However, identifying relationships can be difficult because of the lack of identifying signatures in source and receptor pools. East Fork Poplar Creek (EFPC) in Oak Ridge, TN, USA received historical industrial releases of mercury that contaminated streambank soils and sediments. Here, we determined relationships between the contaminated streambank soils and sand-sized streambed sediments.Materials and methodsField surveys revealed the spatial trends of the concentrations of inorganic total mercury (Hg) and methyl mercury (MeHg), Hg lability as inferred by sequential extraction, particle size distribution, and total organic carbon. Statistical tests were applied to determine relationships between streambank soil and streambed sediment properties.Results and discussionConcentrations of Hg in streambank soils in the upper reaches averaged 206 mg kg−1 (all as dry weight) (n = 457), and 13 mg kg−1 in lower reaches (n = 321), while sand-sized streambed sediments were approximately 16 mg kg−1 (n = 57). Two areas of much higher Hg and MeHg concentrations in streambank soils were identified and related to localized higher Hg concentrations in the streambed sediments; however, most of the streambank soils have similar Hg concentrations to the streambed sediments. The molar ratio of Hg to organic carbon, correlation between MeHg and Hg, and particle size distributions suggested similarity between the streambank soils and the fine sand-sized fraction (125–250 μm) collected from the streambed sediments. Mercury in the fine sand-sized streambed sediments, however, was more labile than Hg in the streambank soils, suggesting an in-stream environment that altered the geochemistry of sediment-bound Hg.ConclusionsThis study revealed major source areas of Hg in streambank soils, identified possible depositional locations in streambed sediments, and highlighted potential differences in the stability of Hg bound to streambank soils and sediments. This work will guide future remedial decision making in EFPC and will aid other researchers in identifying source–sink linkages in contaminated fluvial systems.

High-fructose corn syrup consumption in adolescent rats causes bipolar-like behavioural phenotype with hyperexcitability in hippocampal CA3-CA1 synapses.

Children and adolescents are the top consumers of high-fructose corn syrup (HFCS) sweetened beverages. Even though the cardiometabolic consequences of HFCS consumption in adolescents are well known, the neuropsychiatric consequences have yet to be determined. Adolescent rats were fed for a month with 11% weight/volume carbohydrate containing HFCS solution, which is similar to the sugar-sweetened beverages of human consumption. The metabolic, behavioural and electrophysiological characteristics of HFCS-fed rats were determined. Furthermore, the effects of TDZD-8, a highly specific GSK-3B inhibitor, on the HFCS-induced alterations were further explored. HFCS-fed adolescent rats displayed bipolar-like behavioural phenotype with hyperexcitability in hippocampal CA3-CA1 synapses. This hyperexcitability was associated with increased presynaptic release probability and increased readily available pool of AMPA receptors to be incorporated into the postsynaptic membrane, due to decreased expression of the neuron-specific α3-subunit of Na+ /K+ -ATPase and an increased ser845 -phosphorylation of GluA1 subunits (AMPA receptor subunit) respectively. TDZD-8 treatment was found to restore behavioural and electrophysiological disturbances associated with HFCS consumption by inhibition of GSK-3B, the most probable mechanism of action of lithium for its mood-stabilizing effects. This study shows that HFCS consumption in adolescent rats led to a bipolar-like behavioural phenotype with neuronal hyperexcitability, which is known to be one of the earliest endophenotypic manifestations of bipolar disorder. Inhibition of GSK-3B with TDZD-8 attenuated hyperexcitability and restored HFCS-induced behavioural alterations.