Selective block of adenosine A2A receptors prevents ischaemic-like effects induced by oxygen and glucose deprivation in rat medium spiny neurons

BACKGROUND AND PURPOSE: Ischemia is known to cause massive neuronal depolarization, termed anoxic depolarization (AD), due to energy failure and loss of membrane ion gradients. The neuromodulator adenosine accumulates extracellularly during ischemia and activates four metabotropic receptors: A1 , A2A , A2B and A3 . Striatal medium spiny neurons (MSNs) express high levels of A2A receptors (A2A Rs) and are particularly vulnerable to ischemic insults. It is known that A2A R blockade reduces acute striatal post-ischemic damage but the cellular mechanisms involved are still unknown. EXPERIMENTAL APPROACH: We performed patch-clamp recordings from MSNs in rat striatal slices subjected to oxygen and glucose deprivation (OGD) in order to investigate the effects of A2A R ligands or ion channel blockers on AD and OGD-induced ionic imbalance, measured as a positive shift in Erev of ramp currents. KEY RESULTS: Our data indicate that the A2A R antagonist SCH58261 (10μM) significantly attenuated ionic imbalance and AD appearance in MSNs exposed to OGD. The K+ channel blocker Ba2+ (2mM) or the Na+ channel blocker tetrodotoxin (1μM) exacerbated and attenuated, respectively, OGD-induced changes. Spontaneous excitatory post-synaptic current (sEPSC) analysis in MSNs revealed that the A2A R agonist CGS21680 (1μM) prevented OGD-induced decrease of sEPSCs within the first 5 min of the insult, an effect shared by the K+ channel blocker Ba2+ , thus indicating facilitated glutamate release. CONCLUSION AND IMPLICATIONS: We conclude that adenosine, released during striatal OGD, activates A2A Rs that may exacerbate OGD-induced damage through K+ channel inhibition. Our results could help to develop A2A R-selective therapeutic tools for the treatment of brain ischemia

The Nuclear Envelope as a Regulator of Immune Cell Function

The traditional view of the nuclear envelope (NE) was that it represented a relatively inert physical barrier within the cell, whose main purpose was to separate the nucleoplasm from the cytoplasm. However, recent research suggests that this is far from the case, with new and important cellular functions being attributed to this organelle. In this review we describe research suggesting an important contribution of the NE and its constituents in regulating the functions of cells of the innate and adaptive immune system. One of the standout properties of immune cells is their ability to migrate around the body, allowing them to carry out their physiological/pathophysiology cellular role at the appropriate location. This together with the physiological role of the tissue, changes in tissue matrix composition due to disease and aging, and the activation status of the immune cell, all result in immune cells being subjected to different mechanical forces. We report research which suggests that the NE may be an important sensor/transducer of these mechanical signals and propose that the NE is an integrator of both mechanical and chemical signals, allowing the cells of the innate immune system to precisely regulate gene transcription and functionality. By presenting this overview we hope to stimulate the interests of researchers into this often-overlooked organelle and propose it should join the ranks of mitochondria and phagosome, which are important organelles contributing to immune cell function

Adenosine A2A receptor blockade attenuates excitotoxicity in rat striatal medium spiny neurons during an ischemic-like insult

During brain ischemia, excitotoxicity and peri-infarct depolarization injuries occur and cause cerebral tissue damage. Indeed, anoxic depolarization, consisting of massive neuronal depolarization due to the loss of membrane ion gradients, occurs in vivo or in vitro during an energy failure. The neuromodulator adenosine is released in huge amounts during cerebral ischemia and exerts its effects by activating specific metabotropic receptors, namely: A1, A2A, A2B, and A3. The A2A receptor subtype is highly expressed in striatal medium spiny neurons, which are particularly susceptible to ischemic damage. Evidence indicates that the A2A receptors are upregulated in the rat striatum after stroke and the selective antagonist SCH58261 protects from exaggerated glutamate release within the first 4 hours from the insult and alleviates neurological impairment and histological injury in the following 24 hours. We recently added new knowledge to the mechanisms by which the adenosine A2A receptor subtype participates in ischemia-induced neuronal death by performing patch-clamp recordings from medium spiny neurons in rat striatal brain slices exposed to oxygen and glucose deprivation. We demonstrated that the selective block of A2A receptors by SCH58261 significantly reduced ionic imbalance and delayed the anoxic depolarization in medium spiny neurons during oxygen and glucose deprivation and that the mechanism involves voltage-gated K+ channel modulation and a presynaptic inhibition of glutamate release by the A2A receptor antagonist. The present review summarizes the latest findings in the literature about the possibility of developing selective ligands of A2A receptors as advantageous therapeutic tools that may contribute to counteracting neurodegeneration after brain ischemia

The contribution of ion channels to shaping macrophage behaviour

The expanding roles of macrophages in physiological and pathophysiological mechanisms now include normal tissue homeostasis, tissue repair and regeneration, including neuronal tissue; initiation, progression, and resolution of the inflammatory response and a diverse array of anti-microbial activities. Two hallmarks of macrophage activity which appear to be fundamental to their diverse cellular functionalities are cellular plasticity and phenotypic heterogeneity. Macrophage plasticity allows these cells to take on a broad spectrum of differing cellular phenotypes in response to local and possibly previous encountered environmental signals. Cellular plasticity also contributes to tissue- and stimulus-dependent macrophage heterogeneity, which manifests itself as different macrophage phenotypes being found at different tissue locations and/or after different cell stimuli. Together, plasticity and heterogeneity align macrophage phenotypes to their required local cellular functions and prevent inappropriate activation of the cell, which could lead to pathology. To execute the appropriate function, which must be regulated at the qualitative, quantitative, spatial and temporal levels, macrophages constantly monitor intracellular and extracellular parameters to initiate and control the appropriate cell signaling cascades. The sensors and signaling mechanisms which control macrophages are the focus of a considerable amount of research. Ion channels regulate the flow of ions between cellular membranes and are critical to cell signaling mechanisms in a variety of cellular functions. It is therefore surprising that the role of ion channels in the macrophage biology has been relatively overlooked. In this review we provide a summary of ion channel research in macrophages. We begin by giving a narrative-based explanation of the membrane potential and its importance in cell biology. We then report on research implicating different ion channel families in macrophage functions. Finally, we highlight some areas of ion channel research in macrophages which need to be addressed, future possible developments in this field and therapeutic potential

Activity-dependent regulation of NMDA receptors in substantia nigra dopaminergic neurones.

N-Methyl-d-aspartate receptors (NMDARs) are Ca(2+)-permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca(2+)-dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca(2+)-independent mechanisms. We have investigated the activity-dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole-cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run-down) that was use dependent and not readily reversible. Run-down was reduced by increasing intracellular Ca(2+) buffering or by reducing Ca(2+) influx but did not appear to be mediated by the same regulatory proteins that cause Ca(2+)-dependent run-down in hippocampal neurones. The NMDAR current run-down may be mediated in part by a Ca(2+)-independent mechanism, because intracellular dialysis with a dynamin-inhibitory peptide reduced run-down, suggesting a role for clathrin-mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run-down during repeated low-frequency synaptic stimulation in a Ca(2+)-dependent but dynamin-independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca(2+) at both synaptic and extrasynaptic sites and provide evidence for activity-dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca(2+) homeostasis in dopaminergic neurones by limiting Ca(2+) influx through the NMDAR

Absence of Whisker-Related Pattern Formation in Mice with NMDA Receptors Lacking Coincidence Detection Properties and Calcium Signaling

Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement

First Pharmacokinetic Data of Tenofovir Alafenamide Fumarate and Tenofovir With Dolutegravir or Boosted Protease Inhibitors in African Children: A Substudy of the CHAPAS-4 Trial

BACKGROUND: We evaluated the pharmacokinetics of tenofovir alafenamide fumarate (TAF) and tenofovir in a subset of African children enrolled in the CHAPAS-4 trial. METHODS: Children aged 3-15 years with human immunodeficiency virus infection failing first-line antiretroviral therapy were randomized to emtricitabine/TAF versus standard-of-care nucleoside reverse transcriptase inhibitor combination, plus dolutegravir, atazanavir/ritonavir, darunavir/ritonavir, or lopinavir/ritonavir. Daily emtricitabine/TAF was dosed according to World Health Organization (WHO)-recommended weight bands: 120/15 mg in children weighing 14 to <25 kg and 200/25 mg in those weighing ≥25 kg. At steady state, 8-9 blood samples were taken to construct pharmacokinetic curves. Geometric mean (GM) area under the concentration-time curve (AUC) and the maximum concentration (Cmax) were calculated for TAF and tenofovir and compared to reference exposures in adults. RESULTS: Pharmacokinetic results from 104 children taking TAF were analyzed. GM (coefficient of variation [CV%]) TAF AUClast when combined with dolutegravir (n = 18), darunavir/ritonavir (n = 34), or lopinavir/ritonavir (n = 20) were 284.5 (79), 232.0 (61), and 210.2 (98) ng*hour/mL, respectively, and were comparable to adult reference values. When combined with atazanavir/ritonavir (n = 32), TAF AUClast increased to 511.4 (68) ng*hour/mL. For each combination, tenofovir GM (CV%) AUCtau and Cmax remained below reference values in adults taking 25 mg TAF with a boosted protease inhibitors. CONCLUSIONS: In children, TAF combined with boosted PIs or dolutegravir and dosed according to WHO-recommended weight bands provides TAF and tenofovir concentrations previously demonstrated to be well tolerated and effective in adults. These data provide the first evidence for use of these combinations in African children. CLINICAL TRIALS REGISTRATION: ISRCTN22964075