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Purinergic responses of calcium-dependent signaling pathways in cultured adult human astrocytes Hashioka, Sadayuki; Wang, Yun F; Little, Jonathan P; Choi, Hyun B; Klegeris, Andis; McGeer, Patrick L; McLarnon, James G Jan 22, 2014

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RESEARCH ARTICLE Open AccessPurinergic responses of calcium-dependentsignaling pathways in cultured adult humanastrocytesSadayuki Hashioka1,4*, Yun Fan Wang2, Jonathan P Little3, Hyun B Choi2, Andis Klegeris3, Patrick L McGeer1and James G McLarnon2*AbstractBackground: The properties of Ca2+ signaling mediated by purinergic receptors are intrinsically linked withfunctional activity of astrocytes. At present little is known concerning Ca2+-dependent purinergic responses in adulthuman astrocytes. This work has examined effects of purinergic stimulation to alter levels of intracellular Ca2+ inadult human astrocytes. Ca2+-sensitive spectrofluorometry was carried out to determine mobilization of intracellularCa2+ following adenosine triphosphate (ATP) or 3′-O-(4-benzoyl)benzoyl-ATP (Bz-ATP) stimulation of adult humanastrocytes. In some experiments pharmacological modulation of Ca2+ pathways was applied to help elucidatemechanisms of Ca2+ signaling. RT-PCR was also performed to confirm human astrocyte expression of specificpurinoceptors which were indicated from imaging studies.Results: The endogenous P2 receptor agonist ATP (at 100 μM or 1 mM) applied in physiological saline solution(PSS) evoked a rapid increase of [Ca2+]i to a peak amplitude with the decay phase of response exhibiting twocomponents. The two phases of decay consisted of an initial rapid component which was followed by a secondaryslower component. In the presence of Ca2+-free solution, the secondary phase of decay was absent indicating thisprolonged component was due to influx of Ca2+. This prolonged phase of decay was also attenuated with thestore-operated channel (SOC) inhibitor gadolinium (at 2 μM) added to standard PSS, suggesting this componentwas mediated by SOC activation. These results are consistent with ATP activation of P2Y receptor (P2YR) in adulthuman astrocytes leading to respective rapid [Ca2+]i mobilization from intracellular stores followed by Ca2+ entrythrough SOC. An agonist for P2X7 receptor (P2X7R), BzATP induced a very different response compared with ATPwhereby BzATP (at 300 μM) elicited a slowly rising increase in [Ca2+]i to a plateau level which was sustained induration. The BzATP-induced increase in [Ca2+]i was not enhanced with lipopolysaccharide pre-treatment of cells aspreviously found for P2X7R mediated response in human microglia. RT-PCR analysis showed that adult humanastrocytes in vitro constitutively express mRNA for P2Y1R, P2Y2R and P2X7R.Conclusion: These results suggest that activation of metabotropic P2YR (P2Y1R and/or P2Y2R) and ionotropicP2X7R could mediate purinergic responses in adult human astrocytes.Keywords: Adult human astrocytes, P2 receptors, Intracellular calcium signaling, ATP, 3′-O-(4-benzoyl)benzoyl-ATP* Correspondence: hashioka@f2.dion.ne.jp; mclarnon@mail.ubc.ca1Kinsmen Laboratory of Neurological Research, Department of Psychiatry,The University of British Columbia, Vancouver, BC, Canada2Department of Anesthesiology, Pharmacology and Therapeutics, TheUniversity of British Columbia, 2176 Health Sciences Mall, Vancouver, BC V6T1Z3, CanadaFull list of author information is available at the end of the article© 2014 Hashioka et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Hashioka et al. BMC Neuroscience 2014, 15:18http://www.biomedcentral.com/1471-2202/15/18BackgroundAstrocytes respond to a variety of physiological and patho-logical stimuli with an increase in intracellular Ca2+ con-centration ([Ca2+]i), often referred to as “Ca2+ signaling”or “Ca2+ excitability” [1,2]. Astrocyte functional processesare intricately linked to, and shaped by, activation ofparticular purinergic receptors. Adenosine triphosphate(ATP) is one of the primary extracellular signaling mole-cules for astrocytes under both physiological and pa-thological conditions and evokes an astrocytic [Ca2+]ielevation through activation of P2 purinoceptors [1]. P2purinoceptors are subdivided into two families consistingof metabotropic P2Y receptor (P2YR) and ionotropic P2Xreceptor (P2XR). In the former case subtypes of P2YR,such as P2Y1R and P2Y2R, are G-protein coupled andlinked to inositol triphosphate-mediated release of Ca2+from intracellular endoplasmic reticulum (ER) stores [3-5].Activation of purinergic receptors alters Ca2+-dependentpathways and intracellular levels of Ca2+ which in turndetermine cellular functional responses to endogenousligand, ATP. For example, ATP stimulation of P2YR notonly mobilizes [Ca2+]i from stores but also leads to influxof Ca2+ through store-operated channels (SOC) subse-quent to store depletion. An alternative pathway for entryof Ca2+ from extracellular medium is provided by acti-vation of family members of P2XR ionotropic channels.Overall, a diversity of astrocyte functional responses suchas cellular growth and proliferation, cytokine productionand regulation of cerebral blood flow can depend on thecharacteristics of Ca2+ signaling in cells [2,6,7].At present, few studies have addressed the expressionand properties of Ca2+ signaling in adult human astro-cytes compared with work on rodent astrocytes. Further-more, the majority of studies on human astrocytes haveinvolved use of fetal cells. Specific properties and activityof astrocytes could differ depending on their species aswell as ages. For example, human astrocytes are substan-tially larger, more complex and propagate Ca2+ signalssignificantly faster than their rodent counterparts [8]. Inhumans, adult astrocytes have been reported to proli-ferate at much lower rate than fetal cells and not to re-capitulate the in vitro differentiation [9]. The manner ofCa2+ signaling mediated by purinoceptor activation inadult human astrocytes may have significance in deter-mining astrocyte characteristics, including expression ofneurotransmitter receptors, ion channels, transportersand gap junction proteins.The main purpose of this study was to characterize Ca2+signaling pathways in adult human astrocytes followingactivation of purinergic receptors. Calcium-sensitive fluo-rescence spectroscopy has been used to determine P2YRand P2XR contributions to [Ca2+]i mobilization in stimu-lated cells. In addition, reverse transcription polymerasechain reaction (RT-PCR) has indicated the expression ofP2Y1R, P2Y2R and P2X7R in the adult human cells. Toour knowledge, this work is the first report describingchanges in intracellular Ca2+ mobilization associated withactivation of purinergic receptors in primary culture ofadult human astrocytes.MethodsChemicals and reagentsATP, 3′-O-(4-benzoyl)benzoyl-ATP (BzATP), lipopoly-saccharide (LPS), gadolinium and dimethyl sulfoxide(DMSO) were obtained from Sigma-Aldrich (St. Louis,MO). ATP and BzATP were dissolved in PBS solu-tion. Fura-2/AM (F-1221) was purchased from InvitrogenCanada (Burlington, ON) and dissolved in DMSO.Cell cultureAdult human astrocytes were obtained from epilepticpatients undergoing temporal lobe surgery with consentsof all patients. Normal brain tissues overlying the epi-leptic foci were obtained from a standard elective surgi-cal procedure where, in order to remove an epilepticfocus, the surgeon first removed normal brain tissuewhich lies superficial to the previously defined epilepticfocus. The epileptic patients were a 27 year-old male,31 year-old female, 36 year-old female and 41 year-oldmale. Every brain sample arrived at our laboratorywithin 24 h after surgery and was immediately used forastrocyte isolation. The use of human brain materialswas approved by the Clinical Research Ethics Board forHuman Subjects of the University of British Columbia.Astrocytes were isolated as described previously [10,11].They were grown in Dulbecco’s modified Eagle medium-nutrient mixture F12 Ham (DMEM-F12) supplementedwith 10% fetal bovine serum and penicillin (200 U/ml)/streptomycin (200 μg/ml) (all from Invitrogen Canada).Astrocytes were cultured for 3-4 weeks before perfor-ming assays. Purity of astrocyte culture was estimated byfluorescent immunocytochemistry with the astrocyticmarker glial fibrillar acidic protein (GFAP) (Z334, Dako,Denmark) and counterstaining nuclei with Hoechst 33258(Hoechst, Frankfurt, Germany). Visualization was achievedusing the Alexa Fluor 546 (Invitrogen Canada)-con-jugated secondary antibody and a fluorescence mi-croscope (Olympus, BX-51, Tokyo, Japan). Under ourculture conditions, more than 99% cells were positivefor GFAP in astrocytic culture.Calcium spectrofluorometryA previous procedure established for measurement ofintracellular Ca2+ [12-15] was modified and followed. Inbrief, 2-5 × 105 of astrocytes plated on 22-mm coverslips(Deckglaser, Sondheim, Germany) were incubated withthe fluorescent Ca2+ indicator Fura-2/AM (at 1 μM) pluspluronic acid (at 1 μM) in normal physiological salineHashioka et al. BMC Neuroscience 2014, 15:18 Page 2 of 8http://www.biomedcentral.com/1471-2202/15/18solution (PSS) for 20 min at 37°C. PSS contained (in mM):NaCl (126), KCl (5), MgCl2 (1.2), HEPES (10), D-glucose(10) and CaCl2 (1); pH of 7.4. In some experiments,Ca2+-free PSS was used; this solution had the same com-position as PSS except that 1 mM of EGTA was addedinstead of CaCl2. All reagents used in this assay wereobtained from Sigma-Aldrich (St. Louis, MO). After a20-minute wash in dye-free PSS at 37°C, coverslips wereplaced on the stage of an inverted microscope equip-ped with a 40× objective (Axiovert, Zeiss, Oberkochen,Germany). Cells were exposed to alternating wavelengthsof 340 nm and 380 nm for excitation at 6-second intervals.Emission light was passed through a 510-nm filter. An im-aging system (Empix Imaging, Mississauga, ON, Canada)was used to record fluorescence ratios using a CCDcamera (DVC-1310, DVC Company Inc., Austin, TX).The bath chamber was designed to maintain a constantbath volume and standard saline PSS was used to rinsethe bath immediately prior to experiments. The bath solu-tion was static with the exception of changes in solution,applied within 60 s after PSS rinse, and associated withthe addition or removal of agonists and antagonists.Responses to purinergic application are presented as fluo-rescence intensity ratios at excitation wavelengths of 340to 380 nm (F340/380) versus time with all experimentsperformed at room temperature. Amplitudes of all re-sponses in this study are described as ratiometric valuesderived from the ratio of excitation wavelengths.ATP-induced responses exhibited fast and slow com-ponents of decay. The time course of the rapid initialdecay was measured at a point at half-amplitude of peakresponse. The time course of the secondary slower phaseof decay was measured at half-amplitude of this compo-nent. The height of the prolonged phase was determinedas the point of intersection of the component with timeat peak response. ATP response in Ca2+-free PSS or instandard Ca2+ solution containing Gd3+ showed singlephase decays from a peak value with time courses deter-mined at half-amplitude values of peak. BzATP-inducedresponse consisted of a single phase of a slowly develop-ing increase to a peak level with amplitude of fluorescentratio used as a measure of response.RT-PCRPrimary human astrocytes were seeded onto 6-well plates(4 × 105 cells per well in 2 ml total volume) in DMEM/F12 containing 10% fetal bovine serum. After 48 h, totalRNA was isolated using a commercially available kitaccording to the manufacturer’s instructions for adherentcells (Aurum™ Total RNA Mini Kit, Bio-Rad Laboratories,Inc., Hercules, CA). RNA concentration was measuredusing a spectrophotometer and purity ensured by 260/280 nm ratio of >1.95 for all samples. cDNA was reverse-transcribed using the qScript™ cDNA Synthesis Kit fromQuanta Biosciences (Gaithersburg, MD). PCR amplifica-tion of cDNA was performed as described previously [16]using GoTaq Green Master Mix (Promega, Madison, WI)on a Bio-Rad C1000 Thermal Cycler. Previously publishedprimer sequences were used: P2Y1 [17]; Forward: 5′ -GAC TTC TTG TAC GTG CTG ACT CT - 3′; Reverse:5′ - GAC CTC TTG TCA CCT GAT ACG TG - 3′; pro-duct size: 647 bp, P2Y2 [18]; Forward: 5′ - CTC TACTTT GTC ACC ACC AG - 3′; Reverse: 5′ - TTC TGCTCC TAC AGC CGA AT - 3′; product size: 638 bp, andP2X7 [19]; Forward: 5′ – TCC GAG AAA CAG GCGATA A- 3′; Reverse: 5′ – ACT CGC ACT TCT TCCTGT A - 3′; product size: 465 bp. PCR conditions con-sisted of an initial denaturation step at 95°C for 2 min,followed by 40 cycles of 30 s denaturation step at 95°C,30 s annealing step at 53.5°C (P2Y1 and P2X7) or 56.5°C(P2Y2), and 1 min extension step at 72°C. A final exten-sion step of 5 min at 72°C was also performed. PCRproducts were separated by electrophoresis on a 1%agarose gel and visualized with SYBR safe DNA gel stain(Invitrogen, Eugene, OR). Digital photographs of the gelswere taken with the Fluorchem FC2 imaging and imageanalysis system from Alpha Innotech (Santa Clara, CA).All PCR results were derived with cycle number pro-ducing a signal in the linear portion of the amplificationcurve.StatisticsData are presented as means ± standard error of mean(SEM). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test. P < 0.05 wasconsidered statistically significant.ResultsATP-induced changes in [Ca2+]iWe first confirmed that in excess of 99% cells in astro-cyte culture were positive for GFAP under our cultureconditions (see Methods). A representative image of cul-tured cells is presented in Figure 1.Calcium-dependent spectrofluorescence was used toexamine effects of ATP on [Ca2+]i in adult human astro-cytes. The experiments generally employed 1 mM ofATP (application time of 200 s); this level of ATP isinsufficient to activate the P2X7 subtype ionotropic re-ceptor in human microglia [4]. We initially measuredthe effect of ATP on intracellular calcium mobilizationin standard PSS with the change in [Ca2+]i exhibiting abiphasic time course (Figure 2A). Overall (N = 4 experi-ments, total of 76 cells), respective time courses for ATPapplied in PSS were 19.1 ± 0.8 s and 55.9 ± 3.6 s for thefast and slow phases of [Ca2+]i. We also examined, in asingle experiment, for effects of a 10-fold lower concen-tration of ATP (at 100 μM). As shown in Figure 2B, theHashioka et al. BMC Neuroscience 2014, 15:18 Page 3 of 8http://www.biomedcentral.com/1471-2202/15/18Figure 1 Immunocytochemistry of cultured adult human astrocytes. Representative image of purified culture of adult human astrocytesstained with GFAP (red). Cellular nuclei were counterstained with Hoechst 33258 (blue).Figure 2 Intracellular calcium responses to ATP in adult human astrocytes. (A) Representative change in intracellular Ca2+ ([Ca2+]i)(response collated from 20 cells) to ATP (applied at 1 mM) in Ca2+-containing physiological saline solution (PSS). The change in [Ca2+]i exhibited abiphasic time course with time components of 18.5 s and 49 s for the fast and slow phases of decay, respectively. The arrow indicates theinflection point between the rapid and prolonged components of the decay phase of response. (B) Typical mobilization of [Ca2+]i (responsecollated from 9 cells) induced by a lower level of ATP (applied at 100 μM) in standard PSS. A biphasic change in [Ca2+]i was observed (arrowindicated inflection point) with respective rapid and slow component decay times of 17.8 s and 58.7 s. (C) Representative [Ca2+]i mobilization(response collated from 22 cells) to 1 mM ATP applied in Ca2+-free PSS. A single component of decay of response was observed with a timecourse of 23.2 s. (D) Representative change in [Ca2+]i (response collated from 20 cells) to 1 mM ATP in the presence of gadolinium (Gd3+ at 2 μMpretreatment for 200 s) in standard PSS. A single monophasic time course of decay for [Ca2+]i was observed with a time course of 25.6 s. All cellsshown in this figure were obtained and cultured from a single human surgical case.Hashioka et al. BMC Neuroscience 2014, 15:18 Page 4 of 8http://www.biomedcentral.com/1471-2202/15/18response to 100 μM ATP showed a similar biphasic timecourse as found with the higher ATP concentration(Figure 2A). The results from control experiments areconsistent with the possibility that Ca2+ responses,induced by different concentrations of ATP in standardPSS, are mediated by a rapid release of intracellular Ca2+followed by a secondary component of influx.Physiological and pharmacological protocols were usedto examine modulation of Ca2+ entry into adult human as-trocytes using application of 1 mM ATP. In one set of ex-periments, extracellular Ca2+ was replaced with Ca2+-freePSS to prevent influx of Ca2+ as a contributing mechan-ism for changes in [Ca2+]i. A typical astrocytic responseinduced by ATP in Ca2+-free PSS is presented inFigure 2C. It showed a single declining phase with no pro-longed component of decay. The absence of the delayedphase of [Ca2+]i in Ca2+-free solution is consistent withinflux of extracellular Ca2+ mediating this component ofresponse. In three additional experiments, the secondaryslow phase of response was absent in Ca2+-free solution.Overall results (N = 4 experiments, total of 85 cells)yielded a single time course of decay in Ca2+-free PSS was28.9 ± 1.8 s. This decay time course was not significantlydifferent (p > 0.05) from the rapid time course of the con-trol response evoked by 1 mM ATP (Figure 2A).The prolonged phase of [Ca2+]i elicited by ATP instandard PSS (Figure 2A, B) and its absence in Ca2+-freePSS (Figure 2C) could reflect entry of Ca2+ throughSOC following the initial release of the divalent ion frominternal stores. To investigate this possibility, ATP-induced responses were studied with 2 μM of gado-linium (Gd3+) added to standard PSS. Inhibition of SOCwith Gd3+ has previously been demonstrated in a varietyof cell types, including smooth muscle and glioma cells[14,20]. A representative response is shown in Figure 2Dfor the [Ca2+]i change induced by 1 mM ATP in humanastrocytes exposed to Gd3+. A single monophasic timecourse of decay for [Ca2+]i was observed, indicating thataddition of Gd3+ to standard PSS inhibits the prolongedcomponent of the ATP response. Overall (N = 4 expe-riments, total of 79 cells), ATP induced a single timecourse of decay with mean value of 29.3 ± 5.2 s when Gd3+was added to PSS. This time course of response was notsignificantly different (p > 0.05) from the rapid phase ofdecay in control induced by 1 mM ATP (Figure 2A).BzATP-induced changes in [Ca2+]iFigure 3A represents a typical intracellular Ca2+ responseevoked by BzATP (at 300 μM). The response was consi-derably different from that induced by ATP (Figure 2A, B)and was characterized by a slow progressive increase in[Ca2+]i to a peak level; experiments were terminated at10 min after BzATP application. Similar results werefound in 3 additional experiments whereby responseswere characterized by a slow increase of [Ca2+]i over a10 min application of BzATP. Overall (N = 4 experiments,total of 57 cells), the mean amplitude of [Ca2+]i was0.21 ± 0.02 in control.Previous work has demonstrated LPS priming of BzATPresponses, measured as amplitudes of fluorescent ratio, inmicroglia which was attributed to inflammatory enhance-ment in numbers of P2X7R [13]. This finding promptedus to examine LPS as a modulatory agent for purinergicresponse in adult human astrocytes. LPS pretreatment(100 ng/ml for 16 h) was used as an inflammatory stimu-lus for adult human astrocytes. Figure 3B shows a repre-sentative change in [Ca2+]i induced by BzATP for cellsadministered LPS treatment. Overall (N = 4 experiments,total of 49 cells), the amplitude of the BzATP-induced res-ponse was 0.24 ± 0.03 with LPS treatment compared withFigure 3 Intracellular calcium responses to BzATP in adulthuman astrocytes. (A) Representative change in intracellular Ca2+([Ca2+]i) (response collated from 21 cells) to BzATP (applied at 300 μM).The amplitude of response, expressed as a ratio of fluorescent intensity340/380, was 0.23. (B) Representative change in [Ca2+]i (responsecollated from 25 cells) to BzATP in the presence of lipopolysaccharide(LPS at 100 ng/ml, pretreatment for 16 h). The amplitude of response(F340/380) was 0.26 from baseline to plateau level. Data shown in thisfigure was recorded from cells obtained and cultured from onesurgical case.Hashioka et al. BMC Neuroscience 2014, 15:18 Page 5 of 8http://www.biomedcentral.com/1471-2202/15/18an amplitude of 0.21 ± 0.02 in the absence of LPS treat-ment. This difference was not significant (p > 0.05) indi-cating LPS was ineffective as a modulatory stimulus toenhance purinergic responses to BzATP in adult humanastrocytes.Expression of P2Y1R, P2Y2R and P2X7R in adult humanastrocytesThe results from imaging experiments for changes in[Ca2+]i suggest functional expression of metabotropicand ionotropic P2R subtypes in cultured adult humanastrocytes. We therefore carried out RT-PCR to examineexpression for particular P2R, including P2Y1R, P2Y2Rand P2X7R, which have previously been reported to me-diate Ca2+ response [4,21]. Figure 4 shows the astrocyticexpression of mRNA encoding P2Y1R, P2Y2R andP2X7R. The mRNA expression of all these subtypes wasdetected in 3 different individuals.DiscussionTo our knowledge, this is the first study that demonstratesintracellular Ca2+ mobilization following activation ofpurinergic receptors in cultures of primary adult humanastrocytes. We report ATP induction of intracellular Ca2+mobilization mediated by depletion of intracellular storesconsistent with activation of metabotropic P2YR in adulthuman astrocytes. This component of [Ca2+]i change isfollowed by a subsequent influx of Ca2+ through SOC.RT-PCR analysis demonstrated the expression of specificsubtype metabotropic P2Y1R and P2Y2R in addition toionotropic P2X7R. Interestingly, this expression pattern ofP2 purinoceptor in adult human astrocytes is consistentwith observations made in fetal human [17] and newbornrat [22] astrocytes.ATP stimulation of adult human astrocytes mobilizedintracellular Ca2+ with a response characterized by twocomponents of decay. The initial rapid transient compo-nent following peak response is consistent with activationof metabotropic P2YR and mediated by Ca2+ release fromER stores independent of extracellular Ca2+ [4,14]. Thesubsequent prolonged component was considerably atten-uated with ATP application in Ca2+-free PSS, indicatingthis phase of response was due to Ca2+ influx throughplasmalemmal membrane [4]. This secondary componentof response likely represents Ca2+ entry through SOCsince the component was inhibited in the presence of theSOC antagonist, Gd3+ [14,20]. The single time courses of[Ca2+]i elicited by ATP in Ca2+-free and in Gd3+ withstandard Ca2+ PSS (200 s treatment) were similar in mag-nitude (near 29 s) and somewhat longer than the rapidphase evoked by ATP in standard Ca2+ solution. Thisresult suggests that only a partial inhibition of SOC wasattained with astrocytes exposed to 2 μM Gd3+ for aduration 200 s. A possible explanation for the longer timecourse of decay in Ca2+-free PSS, relative to the rapidphase of control ATP response in PSS, is that residualCa2+ could remain in nominally Ca2+-free solution. Inorder to minimize effects of non-physiological Ca2+-freePSS on cell viability, we employed relatively short treat-ment times of 60 s with this solution prior to ATP stimu-lation. We did not test Gd3+ at concentrations higher than2 μM nor increase incubation time with Ca2+-free PSS todetect astrocytic responses in a robust and healthy con-dition. The overall results from calcium imaging ex-periments suggest that purinergic response to endogenousligand in adult human astrocytes is mediated by ATPbinding to metabotropic P2YR with subsequent mobi-lization of [Ca2+]i due to intracellular release and influxthrough SOC.Ca2+ spectrofluorometry showed that application ofBzATP elicited a gradual and sustained increase in [Ca2+]iin adult human astrocytes. This finding suggests influx of700 bp600 bp700 bp600 bp500 bp400 bpABCP2Y1 647 bpP2Y2 638 bpP2X7 465 bpFigure 4 Expression of P2X and P2Y purinoceptors in adulthuman astrocytes. RT-PCR assay with 40 amplification cycles detectedbands of expected size for P2Y1R (A, 647 bp), P2Y2R (B, 638 bp) andP2X7R (C, 465 bp) in total RNA extracted from culture of adult humanastrocytes. Photos are representative of results obtained using cells from3 independent surgical cases.Hashioka et al. BMC Neuroscience 2014, 15:18 Page 6 of 8http://www.biomedcentral.com/1471-2202/15/18Ca2+ through the nonselective cationic channel coupled toactivation of P2X7R [1,4] and is consistent with previouswork demonstrating a modest and prolonged [Ca2+]i riseelicited by BzATP in fetal human astrocytes [21].Purinergic agonists and antagonists are notorious fornon-specific activity [23,24]. Although BzATP has beenreported as an activator of P2X7R in numerous studies[12,13,15,25], considerable non-specificity of the ligandhas also been documented. Examples include actions ofBzATP mediated by ionotropic P2X1 and P2X3 [26] andmetabotropic P2Y2 [27] receptors. Recent work on rodentcerebellar astrocytes has demonstrated calcium responsesmediated by P2Y13 receptors in addition to P2X7R acti-vation [28]. In addition, BzATP responses have been at-tributed to activation of adenosine receptors, an effectinvolving dephosphorylation activity of ecto-nucleotidases[29]. It should also be noted that interpretation of BzATP-induced responses is further complicated by the variabilityin actions of P2X7R antagonists with Brilliant blue G [30]exhibiting a greater selectivity for P2X7R inhibitory acti-vity compared with oxidized ATP [31]. Overall, a multipli-city of purinergic receptors could contribute to BzATPresponses in addition to the activation of P2X7R.We found that LPS priming of human astrocytes(100 ng/ml for 16 h) had no significant effect to alteramplitude of BzATP-induced responses compared withcontrols (no LPS pretreatment). Interestingly, this resultis in contrast to previous findings on fetal human micro-glia which demonstrated that exposure of cells to LPS(100 ng/ml for 2 h) significantly enhanced the amplitudeof BzATP-evoked [Ca2+]i [13]. One possibility for thedifferences of LPS treatment on Ca2+ mobilization inastrocytes and microglia may be related to differentialcellular expression of receptors for LPS. In particularCD14, a putative LPS receptor, is not expressed in hu-man astrocytes [32] whereas this receptor is expressedin human microglia, the resident immune respondingcells in brain [33].ConclusionsOur study has presented novel findings concerning ex-pression and activation of specific purinergic Ca2+ sig-naling pathways in cultured adult human astrocytes.Metabotropic P2YR and ionotropic P2XR are putativemediators of purinergic responses in the cells. Futurestudies using adult human astrocytes are warranted tocharacterize the specific roles of the purinergic receptorsin mediating cellular responses. Such work will enableclarification of downstream Ca2+-dependent and in-dependent signaling pathways. P2X7R expression andfunction should be confirmed in these cells followed byexamination of roles of the receptor in mediating astro-cytic responses in pathological microenvironments inhuman brain.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsSH, YFW and JGM participated in the design of the study. SH and YFW carriedout all experiments, collected the data and performed the statistical analysis.JPL and AK performed the RT-PCR analysis. HCB participated in calciumspectrofluorometry. SH, YFW and JGM interpreted the data. SH and JGMwrote the manuscript. JGM, HCB, AK and PLM revised the manuscript. Allauthors read and approved the final manuscript.AcknowledgmentsThis research was supported by the Pacific Alzheimer Research Foundation(SH, PLM and JGM), Grant-in-Aid for Scientific Research #24591721 (SH) andthe Jack Brown and Family Alzheimer’s Disease Research Foundation(JPL and AK).Author details1Kinsmen Laboratory of Neurological Research, Department of Psychiatry,The University of British Columbia, Vancouver, BC, Canada. 2Department ofAnesthesiology, Pharmacology and Therapeutics, The University of BritishColumbia, 2176 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.3Department of Biology, I.K. Barber School of Arts and Sciences, TheUniversity of British Columbia Okanagan Campus, Kelowna, BC, Canada.4Present affiliation; Department of Psychiatry, Faculty of Medicine, ShimaneUniversity, 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan.Received: 5 July 2013 Accepted: 15 January 2014Published: 22 January 2014References1. 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BMCNeuroscience 2014 15:18.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitHashioka et al. BMC Neuroscience 2014, 15:18 Page 8 of 8http://www.biomedcentral.com/1471-2202/15/18


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