Purinergic responses of calcium-dependent signaling pathways in cultured adult human astrocytes

BMC Neuroscience

James G McLarnon¹  Patrick L McGeer²  Andis Klegeris⁴  Hyun B Choi¹  Jonathan P Little⁴  Yun Fan Wang¹  Sadayuki Hashioka³ 

[1] Department of Anesthesiology, Pharmacology and Therapeutics, The University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada;Kinsmen Laboratory of Neurological Research, Department of Psychiatry, The University of British Columbia, Vancouver, BC Canada;Present affiliation; Department of Psychiatry, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan;Department of Biology, I.K. Barber School of Arts and Sciences, The University of British Columbia Okanagan Campus, Kelowna, BC Canada

关键词: 3′-O-(4-benzoyl)benzoyl-ATP;    ATP;    Intracellular calcium signaling;    P2 receptors;    Adult human astrocytes;   

Others  :  1122562
DOI  :  10.1186/1471-2202-15-18

 received in 2013-07-05, accepted in 2014-01-15,  发布年份 2014

【 摘 要 】

Background

The properties of Ca²⁺ signaling mediated by purinergic receptors are intrinsically linked with functional activity of astrocytes. At present little is known concerning Ca²⁺-dependent purinergic responses in adult human astrocytes. This work has examined effects of purinergic stimulation to alter levels of intracellular Ca²⁺ in adult human astrocytes. Ca²⁺-sensitive spectrofluorometry was carried out to determine mobilization of intracellular Ca²⁺ following adenosine triphosphate (ATP) or 3′-O-(4-benzoyl)benzoyl-ATP (Bz-ATP) stimulation of adult human astrocytes. In some experiments pharmacological modulation of Ca²⁺ pathways was applied to help elucidate mechanisms of Ca²⁺ signaling. RT-PCR was also performed to confirm human astrocyte expression of specific purinoceptors 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 two components. The two phases of decay consisted of an initial rapid component which was followed by a secondary slower component. In the presence of Ca²⁺-free solution, the secondary phase of decay was absent indicating this prolonged component was due to influx of Ca²⁺. This prolonged phase of decay was also attenuated with the store-operated channel (SOC) inhibitor gadolinium (at 2 μM) added to standard PSS, suggesting this component was mediated by SOC activation. These results are consistent with ATP activation of P2Y receptor (P2YR) in adult human astrocytes leading to respective rapid [Ca²⁺]_i mobilization from intracellular stores followed by Ca²⁺ entry through SOC. An agonist for P2X7 receptor (P2X7R), BzATP induced a very different response compared with ATP whereby BzATP (at 300 μM) elicited a slowly rising increase in [Ca²⁺]_i to a plateau level which was sustained in duration. The BzATP-induced increase in [Ca²⁺]_i was not enhanced with lipopolysaccharide pre-treatment of cells as previously found for P2X7R mediated response in human microglia. RT-PCR analysis showed that adult human astrocytes in vitro constitutively express mRNA for P2Y1R, P2Y2R and P2X7R.

Conclusion

These results suggest that activation of metabotropic P2YR (P2Y1R and/or P2Y2R) and ionotropic P2X7R could mediate purinergic responses in adult human astrocytes.

【 授权许可】

   
2014 Hashioka et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Butt AM: ATP: a ubiquitous gliotransmitter integrating neuron-glial networks. Semin Cell Dev Biol 2011, 22:205-213.
• [2]James G, Butt AM: P2Y and P2X purinoceptor mediated Ca2+ signalling in glial cell pathology in the central nervous system. Eur J Pharmacol 2002, 447:247-260.
• [3]Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, Weisman GA: International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 2006, 58:281-341.
• [4]McLarnon JG: Purinergic mediated changes in Ca2+ mobilization and functional responses in microglia: effects of low levels of ATP. J Neurosci Res 2005, 81:349-356.
• [5]Neary JT, Zimmermann H: Trophic functions of nucleotides in the central nervous system. Trends Neurosci 2009, 32:189-198.
• [6]Mulligan SJ, MacVicar BA: Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature 2004, 431:195-199.
• [7]Straub SV, Nelson MT: Astrocytic calcium signaling: the information currency coupling neuronal activity to the cerebral microcirculation. Trends Cardiovasc Med 2007, 17:183-190.
• [8]Oberheim NA, Goldman SA, Nedergaard M: Heterogeneity of astrocytic form and function. Methods Mol Biol 2012, 814:23-45.
• [9]Sharif A, Prevot V: Isolation and culture of human astrocytes. Methods Mol Biol 2012, 814:137-151.
• [10]Hashioka S, Klegeris A, Schwab C, McGeer PL: Interferon-gamma-dependent cytotoxic activation of human astrocytes and astrocytoma cells. Neurobiol Aging 2009, 30:1924-1935.
• [11]Hashioka S, Klegeris A, Qing H, McGeer PL: STAT3 inhibitors attenuate interferon-gamma-induced neurotoxicity and inflammatory molecule production by human astrocytes. Neurobiol Dis 2011, 41:299-307.
• [12]Choi HB, Hong SH, Ryu JK, Kim SU, McLarnon JG: Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia. Glia 2003, 43:95-103.
• [13]Choi HB, Ryu JK, Kim SU, McLarnon JG: Modulation of the purinergic P2X7 receptor attenuates lipopolysaccharide-mediated microglial activation and neuronal damage in inflamed brain. J Neurosci 2007, 27:4957-4968.
• [14]Jantaratnotai N, Choi HB, McLarnon JG: ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells. BMC Cancer 2009, 9:442. BioMed Central Full Text
• [15]McLarnon JG, Ryu JK, Walker DG, Choi HB: Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus. J Neuropathol Exp Neurol 2006, 65:1090-1097.
• [16]Villanueva EB, Little JP, Lambeau G, Klegeris A: Secreted phospholipase A(2) group IIA is a neurotoxin released by stimulated human glial cells. Mol Cell Neurosci 2012, 49:430-438.
• [17]John GR, Simpson JE, Woodroofe MN, Lee SC, Brosnan CF: Extracellular nucleotides differentially regulate interleukin-1beta signaling in primary human astrocytes: implications for inflammatory gene expression. J Neurosci 2001, 21:4134-4142.
• [18]Adrian K, Bernhard MK, Breitinger HG, Ogilvie A: Expression of purinergic receptors (ionotropic P2X1-7 and metabotropic P2Y1-11) during myeloid differentiation of HL60 cells. Biochim Biophys Acta 2000, 1492:127-138.
• [19]Budagian V, Bulanova E, Brovko L, Orinska Z, Fayad R, Paus R, Bulfone-Paus S: Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B. J Biol Chem 2003, 278:1549-1560.
• [20]Flemming R, Xu SZ, Beech DJ: Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells. Br J Pharmacol 2003, 139:955-965.
• [21]Narcisse L, Scemes E, Zhao Y, Lee SC, Brosnan CF: The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes. Glia 2005, 49:245-258.
• [22]Jimenez AI, Castro E, Communi D, Boeynaems JM, Delicado EG, Miras-Portugal MT: Coexpression of several types of metabotropic nucleotide receptors in single cerebellar astrocytes. J Neurochem 2000, 75:2071-2079.
• [23]Anderson CM, Nedergaard M: Emerging challenges of assigning P2X7 receptor function and immunoreactivity in neurons. Trends Neurosci 2006, 29:257-262.
• [24]North RA: Molecular physiology of P2X receptors. Physiol Rev 2002, 82:1013-1067.
• [25]Ryu JK, Jantaratnotai N, Serrano-Perez MC, McGeer PL, McLarnon JG: Block of purinergic P2X7R inhibits tumor growth in a C6 glioma brain tumor animal model. J Neuropathol Exp Neurol 2011, 70:13-22.
• [26]Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, Alexander KM, Park HS, Yu H, Metzger R, Kowaluk E, et al.: Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 1999, 376:127-138.
• [27]Fedorov IV, Rogachevskaja OA, Kolesnikov SS: Modeling P2Y receptor-Ca2+ response coupling in taste cells. Biochim Biophys Acta 2007, 1768:1727-1740.
• [28]Carrasquero LM, Delicado EG, Bustillo D, Gutierrez-Martin Y, Artalejo AR, Miras-Portugal MT: P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes. J Neurochem 2009, 110:879-889.
• [29]Kukley M, Stausberg P, Adelmann G, Chessell IP, Dietrich D: Ecto-nucleotidases and nucleoside transporters mediate activation of adenosine receptors on hippocampal mossy fibers by P2X7 receptor agonist 2′-3′-O-(4-benzoylbenzoyl)-ATP. J Neurosci 2004, 24:7128-7139.
• [30]Jiang LH, Mackenzie AB, North RA, Surprenant A: Brilliant blue G selectively blocks ATP-gated rat P2X(7) receptors. Mol Pharmacol 2000, 58:82-88.
• [31]Evans RJ, Lewis C, Buell G, Valera S, North RA, Surprenant A: Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol 1995, 48:178-183.
• [32]Farina C, Aloisi F, Meinl E: Astrocytes are active players in cerebral innate immunity. Trends Immunol 2007, 28:138-145.
• [33]Lambert C, Desbarats J, Arbour N, Hall JA, Olivier A, Bar-Or A, Antel JP: Dendritic cell differentiation signals induce anti-inflammatory properties in human adult microglia. J Immunol 2008, 181:8288-8297.