Functional distribution of Ca2+-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells

Background Adrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca2+ concentration ([Ca2+]i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells. Results The [Ca2+]i and the [Na+]i were recorded from immunolabeled bovine chromaffin cells by single-cell fluorescence imaging. Among the ATP-sensitive cells ~40% did not yield [Ca2+]i responses to ATP in the absence of extracellular Ca2+ (Ca2+o), indicating that they expressed P2X receptors and did not express Ca2+- mobilizing P2Y receptors; the remainder expressed Ca2+-mobilizing P2Y receptors. Relative to AD-cells approximately twice as many NA-cells expressed P2X receptors while not expressing Ca2+- mobilizing P2Y receptors, as indicated by the proportion of cells lacking [Ca2+]i responses and exhibiting [Na+]i responses to ATP in the absence and presence of Ca2+o, respectively. The density of P2X receptors in NA-cells appeared to be 30–50% larger, as suggested by comparing the average size of the [Na+]i and [Ca2+]i responses to ATP. Conversely, approximately twice as many AD-cells expressed Ca2+-mobilizing P2Y receptors, and they appeared to exhibit a higher (~20%) receptor density. UTP raised the [Ca2+]i in a fraction of the cells and did not raise the [Na+]i in any of the cells tested, confirming its specificity as a P2Y agonist. The cell density of UTP-sensitive P2Y receptors did not appear to vary among AD- and NA-cells. Conclusion Although neither of the major purinoceptor types can be ascribed to a particular cell phenotype, P2X and Ca2+-mobilizing P2Y receptors are preferentially located to noradrenergic and adrenergic chromaffin cells, respectively. ATP might, in addition to an UTP-sensitive P2Y receptor, activate an UTP-insensitive P2Y receptor subtype. A model for a short-loop feedback interaction is presented whereby locally released ATP acts upon P2Y receptors in adrenergic cells, inhibiting Ca2+ influx and contributing to terminate evoked epinephrine secretio

Naloxone inhibits nicotine-induced receptor current and catecholamine secretion in bovine chromaffin cells

Nicotine-induced catecholamine (CA) secretion and inward ionic currents were inhibited by the opioid antagonist naloxone in cultured bovine chromaffin cells. Naloxone inhibited nicotine-induced CA secretion, as detected by an on-line real-time electrochemical technique, in a dose-dependent manner (IC50=29 [mu]M). In voltage-clamped chromaffin cells, nicotine (10 [mu]M) evoked an average peak inward current of -146 pA that was inhibited by low concentrations of naloxone (42% at 0.1 [mu]M). The antagonist also inhibited total charge influx associated with nicotinic receptor activation (53% at 0.1 [mu]M). This provides strong evidence that naloxone modulation of nicotine-induced CA secretion does not involve opioid receptors but results from the direct interaction with the nicotinic receptor itself, which might also be the case for other related opioid compounds.http://www.sciencedirect.com/science/article/B6SYR-43MC9YT-C/1/1994796a9d2862757ab52186bcacc5f

The effects of different concentrations of the α2-Adrenoceptor Agonist Medetomidine on basal excitatory synaptic transmission and synaptic plasticity in hippocampal slices of adult mice

α2-Adrenoceptor agonists are used frequently in human and veterinary anesthesia as sedative/analgesic drugs. However, they can impair cognition. Little is known about the concentration-dependent effects of α2-adrenoceptor agonists on synaptic plasticity, the neurophysiological basis of learning and memory. Therefore, we investigated the effects of different concentrations of medetomidine, an α2-adrenoceptor agonist, on basal excitatory synaptic transmission and on 2 forms of synaptic plasticity: paired-pulse facilitation (PPF) and long-term potentiation (LTP).Funding: This work was supported by FCT (Lisbon, Portugal) and cofunded by COMPETE: 01-0124-FEDER-009497 (Lisbon, Portugal), through the project grants PTDC/CVT/099022/2008 and PTDC/SAU-NSC/122254/2010 and through a personal PhD grant (SFRH /BD/48883/2008) to Patrícia do Céu Oliveira Ribeiro and by QREN (09-68-ESR-FP-010).info:eu-repo/semantics/publishedVersio

Neuromodulation and neuroprotective effects of chlorogenic acids in excitatory synapses of mouse hippocampal slices

The increased healthspan afforded by coffee intake provides novel opportunities to identify new therapeutic strategies. Caffeine has been proposed to afford benefits through adenosine A2A receptors, which can control synaptic dysfunction underlying some brain disease. However, decaffeinated coffee and other main components of coffee such as chlorogenic acids, also attenuate brain dysfunction, although it is unknown if they control synaptic function. We now used electrophysiological recordings in mouse hippocampal slices to test if realistic concentrations of chlorogenic acids directly affect synaptic transmission and plasticity. 3-(3,4-dihydroxycinnamoyl)quinic acid (CA, 1–10 μM) and 5-O-(trans-3,4-dihydroxycinnamoyl)-D-quinic acid (NCA, 1–10 μM) were devoid of effect on synaptic transmission, paired-pulse facilitation or long-term potentiation (LTP) and long-term depression (LTD) in Schaffer collaterals-CA1 pyramidal synapses. However, CA and NCA increased the recovery of synaptic transmission upon re-oxygenation following 7 min of oxygen/glucose deprivation, an in vitro ischemia model. Also, CA and NCA attenuated the shift of LTD into LTP observed in hippocampal slices from animals with hippocampal-dependent memory deterioration after exposure to β-amyloid 1–42 (2 nmol, icv), in the context of Alzheimer’s disease. These findings show that chlorogenic acids do not directly affect synaptic transmission and plasticity but can indirectly affect other cellular targets to correct synaptic dysfunction. Unraveling the molecular mechanisms of action of chlorogenic acids will allow the design of hitherto unrecognized novel neuroprotective strategies

Selective stimulation of catecholamine release from bovine adrenal chromaffin cells by an ionotropic purinergic receptor sensitive to 2-methylthio ATP

Background 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), formerly regarded as a specific P2Y (metabotropic) purinergic receptor agonist, stimulates Ca2+ influx and evokes catecholamine release from adrenal chromaffin cells. These cells express P2Y and P2X (ionotropic) purinoceptors, with the latter providing an important Ca2+ influx pathway. Using single cell calcium imaging techniques, we have determined whether 2-MeSATP might be a specific P2X receptor agonist in bovine chromaffin cells and assessed the relative role of P2X and P2Y receptors on catecholamine secretion from these cells. Results ATP raised the [Ca2+]i in ~50% of the cells. Removing extracellular Ca2+ suppressed the [Ca2+]i-raising ability of 2-MeSATP, observed in ~40% of the ATP-sensitive cells. This indicates that 2-MeSATP behaves as a specific ionotropic purinoceptor agonist in bovine chromaffin cells. The 2-MeSATP-induced [Ca2+]i-rises were suppressed by PPADS. UTP raised the [Ca2+]i in ~40% of the ATP-sensitive cells, indicating that these expressed Ca2+-mobilizing P2Y receptors. UTP-sensitive receptors may not be the only P2Y receptors present, as suggested by the observation that ~20% of the ATP-sensitive pool did not respond to either 2-MeSATP or UTP. The average sizes of the ATP- and 2-MeSATP-evoked [Ca2+]i responses were identical in UTP-insensitive cells. 2-MeSATP stimulated Ca2+ influx and evoked catecholamine release, whereas UTP elicited Ca2+ release from intracellular stores but did not evoke secretion. 2-MeSATP-induced secretion was strongly inhibited by Cd2+ and suppressed by extracellular Ca2+ or Na+ removal. TTX inhibited 2-MeSATP-evoked secretion by ~20%. Conclusion 2-MeSATP is a specific P2X purinoceptor agonist and a potent secretagogue in bovine chromaffin cells. Activation of 2-MeSATP-sensitive receptors stimulates Ca2+ influx mainly via voltage-sensitive Ca2+ channels. For the most part, these are activated by the depolarization brought about by Na+ influx across P2X receptor pore

ATP as a multi-target danger signal in the brain

ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, in astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R) as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR), which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection

Electrophysiology extracellular in caffeine-treated corticostriatal slices

Caffeine causes a shift in neuronal plasticity in corticostriatal dorsolateral slice

Physical basis for the resolution of intra- and extracellular cesium-133 NMR resonances in cesium(+) loaded human erythrocyte suspensions in the presence and absence of shift reagents

For human red blood cells (RBCs) loaded with Cs' and suspended in a shift reagent (SR) free medium, the extracellular 13'Cs+ NMR resonance was shifted upfield from the intracellular resonance. However, in the presence of the SRs Dy(PPP);-, Dy- (TTHA))-, and Tm(DOTP)S- [where Dy3+ and Tm3' denote dysprosium and thulium ions and PPPs-, TTHA6, and DOTPsrepresent the triphosphate, triethylenetetraminehexaacetate, and 1,4,7,10-tetraazacyclododecane-N,N:N""'-tetra~s(methylenephosphonate) ligands, respectively], the extracellular 133C~NrM R resonance was shifted downfield from the intracellular resonance. The magnitudes of the '33Cs+ shifts observed with Tm(DOTP)S- were much larger than those for Dy(TTHA)'- and Dy(PPP)J- at the same concentration. The direction of the IS3Cst shift induced by Dy(PPP)J- was the opposite of that previously reported for 'Lit, 23Nat, and 39K+N MR resonances. The negative sign of the pseudocontact 133Csh ift induced by Dy(PPP)?- is related to the large size of the Cs' cation and its location in the equatorial region formed by the cone around the effective magnetic axis of the triphosphate SR. At physiologically relevant RBC concentrations, 2,3-diphosphoglycerate (DE), of all intracellular phosphates tested, caused the largest '33Cs+ shift. The 13'Cs' resonance in carbonmonoxygenated RBC lysate shifted downfield by approximately 2.0 ppm with increasing hemoglobin concentration, whereas an increase in the diamagnetic susceptibility of the sample induced by hemoglobin is expected to induce an upfield shift of 0.1 ppm. The 13'Cs+ resonance was shifted downfield with increasing concentrations of two unrelated proteins, carbonmonoxyhemoglobin and lysozyme. We conclude that, in the absence of SRs, the physical basis for the resolution of intra- and extracellular NMR resonances in Cs+-loaded human RBC suspensions arises from Cs+ binding to intracellular phosphates, in particular DPG, and from the nonideality of intracellular water induced by hemoglobin

Functional distribution of Ca-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells-3

<p><b>Copyright information:</b></p><p>Taken from "Functional distribution of Ca-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells"</p><p>http://www.biomedcentral.com/1471-2202/8/39</p><p>BMC Neuroscience 2007;8():39-39.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC1906789.</p><p></p>acellular calcium (B) and during stimulation in the virtual absence of extracellular calcium (C, "UTP+EGTA"). At the end of each experiment cells were stimulated with 10 μM nicotine. Cells were allowed to rest for 10 min between consecutive stimulations. The fura-2 fluorescence ratio F/Fwas determined for each cell in a field on a pixel-by-pixel basis. Images were coded in pseudocolor to show differences in the F/Fratio. The images corresponding to UTP stimulation (B and C) were captured ~5 s after the UTP challenges (i.e. at the response peak in presence of extracellular calcium); . Pseudocolor image of PNMT/TH immunofluorescence ratio (labeling index) and corresponding scale. Intensely colored red and green cells are adrenergic and noradrenergic cells, respectively. Scale bar = 50 μm; . Time courses of changes in F/Ffluorescence ratio for two AD-cells (cells 1 and 2, also depicted in A-D) and two NA-cells (cells 3 and 4). The lines denote superfusions with UTP or nicotine in the presence or virtual absence (middle traces) of extracellular calcium

Selective stimulation of catecholamine release from bovine adrenal chromaffin cells by an ionotropic purinergic receptor sensitive to 2-methylthio ATP-2

<p><b>Copyright information:</b></p><p>Taken from "Selective stimulation of catecholamine release from bovine adrenal chromaffin cells by an ionotropic purinergic receptor sensitive to 2-methylthio ATP"</p><p>http://www.biomedcentral.com/1471-2202/8/41</p><p>BMC Neuroscience 2007;8():41-41.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1906790.</p><p></p>owed to rest for ~10 min between successive applications. Nominal agonist concentrations (in μM) are indicated beneath each trace. Nominal concentrations of test agents are used throughout this legend. Actual concentrations at the cell bed differ from nominal concentrations by a factor of 1.64 (see Methods). Concentrations along the X axis in Fig. 3B are actual concentrations at the cell bed; . Dose-response curves of catecholamine release evoked by 2-MeSATP and ATP (circles and squares, respectively). Data from the experiment depicted in A and two similar experiments. Also depicted are data for UTP (100 μM; filled square) as well as for 2-MeSATP stimulations in presence of 50 μM PPADS (diamond), in the virtual absence of extracellular Ca(EGTA-containing solution, inverted triangle) and in absence of extracellular Na(Nareplacement for NMG, triangle) (n = 3 experiments for each condition)