Abstract
Neuroblastoma SH-SY5Y (SH) cells endogenously express A2A adenosine receptors and can be differentiated into a sympathetic neuronal phenotype, capable of depolarisation-dependent noradrenaline release. Using differentiated SH culture, we here explored the link between A2A-receptor signalling and neurotransmitter release. In response to the receptor agonist CGS21680, the cells produced cyclic AMP (cAMP), and when depolarised, they released increased amounts of noradrenaline. An A2A-receptor antagonist, XAC, as well as an inhibitor of cAMP-dependent protein kinase A (PKA), H89, depressed agonist-dependent release. In the presence of XAC or H89, noradrenaline release was found to be below basal values. This suggested that release facilitation also owes to constitutive receptor activity. We demonstrate that even in the absence of an agonist, the native A2A-receptor stimulated cAMP production, leading to the activation of PKA and enhanced noradrenaline release. Ancillary, non-cAMP-dependent effects of the receptor (i.e. phosphorylation of CREB, of Rabphilin3A) were refractory to constitutive activation. PKA-dependent facilitation of noradrenaline release was recapitulated with membrane-permeable 8-Br-cAMP; in addition to facilitation, 8-Br-cAMP caused marked inhibition of release, an effect not observed upon receptor activation. Inhibition by receptor-independent cAMP was likely due to suppression of voltagedependent calcium current (VDCC) and increased activity of Src-family kinases. Receptor-mediated release facilitation was reproduced in the presence of tetrodotoxin (blocking action potentials); hence, the signalling occurred at the active zone comprising release sites. Our findings thus support (1) presynaptic localisation of the A2A-receptor and (2) suggest that compartmentalised pathways transmit cAMP signalling in order to facilitate depolarisation-dependent neurotransmitter release.
Similar content being viewed by others
References
Haas HL, Selbach O (2000) Functions of neuronal adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol 362:375–381
Cunha RA (2001) Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors. Neurochem Int 38:107–125
Ferré S, Quiroz C, Woods AS, Cunha R, Popoli P, Ciruela F, Lluis C, Franco R, Azdad K, Schiffmann SN (2008) An update on adenosine A2A-dopamine D2 receptor interactions: implications for the function of G protein-coupled receptors. Curr Pharm Des 14:1468–1474
Azdad K, Gall D, Woods AS, Ledent C, Ferré S, Schiffmann SN (2009) Dopamine D2 and adenosine A2A receptors regulate NMDA-mediated excitation in accumbens neurons through A2A–D2 receptor heteromerization. Neuropsychopharmacol 34:972–986
Sebastião AM, Ribeiro JA (1996) Adenosine A2 receptor-mediated excitatory actions on the nervous system. Prog Neurobiol 48:167–189
Popoli P, Betto P, Reggio R, Ricciarello G (1995) Adenosine A2A receptor stimulation enhances striatal extracellular glutamate levels in rats. Eur J Pharmacol 287:215–217
Kurokawa M, Koga K, Kase H, Nakamura J, Kuwana Y (1996) Adenosine A2a-receptor-mediated modulation of striatal acetylcholine release in vivo. J Neurochem 66:1882–1888
Wirkner K, Gerevich Z, Krause T, Günther A, Köles L, Schneider D, Nörenberg W, Illes P (2004) Adenosine A2A receptor-induced inhibition of NMDA and GABAA receptor-mediated synaptic currents in a subpopulation of rat striatal neurons. Neuropharmacol 46:994–1007
Gomes CA, Vaz SH, Ribeiro JA, Sebastião AM (2006) Glial cell line-derived neurotrophic factor (GDNF) enhances dopamine release from striatal nerve endings in an adenosine A2A receptor-dependent manner. Brain Res 1113:129–136
Mayfield RD, Suzuki F, Zahniser NR (1993) Adenosine A2a receptor modulation of electrically evoked endogenous GABA release from slices of rat globus pallidus. J Neurochem 60:2334–2337
Kirk IP, Richardson PJ (1994) Adenosine A2a receptor-mediated modulation of striatal [3H]GABA and [3H]acetylcholine release. J Neurochem 62:960–966
Mori A, Shindou T, Ichimura M, Nonaka H, Kase H (1996) The role of adenosine A2a receptors in regulating GABAergic synaptic transmission in striatal medium spiny neurons. J Neurosci 16:605–611
Chergui K, Bouron A, Normand E, Mulle C (2000) Functional GluR6 kainate receptors in the striatum: indirect downregulation of synaptic transmission. J Neurosci 20:2175–2182
Shindou T, Nonaka H, Richardson PJ, Mori A, Kase H, Ichimura M (2002) Presynaptic adenosine A2A receptors enhance GABAergic synaptic transmission via a cyclic AMP dependent mechanism in the rat globus pallidus. Br J Pharmacol 136:296–302
Shindou T, Richardson PJ, Mori A, Kase H, Ichimura M (2003) Adenosine modulates the striatal GABAergic inputs to the globus pallidus via adenosine A2A receptors in rats. Neurosci Lett 352:167–170
Seino S, Shibasaki T (2005) PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis. Physiol Rev 85:1303–1342
Szaszák M, Christian F, Rosenthal W, Klussmann E (2008) Compartmentalized cAMP signalling in regulated exocytic processes in non-neuronal cells. Cell Signal 20:590–601
Zhong N, Beaumont V, Zucker RS (2004) Calcium influx through HCN channels does not contribute to cAMP-enhanced transmission. J Neurophysiol 92:644–647
Neitz A, Mergia E, Eysel UT, Koesling D, Mittmann T (2011) Presynaptic nitric oxide/cGMP facilitates glutamate release via hyperpolarization-activated cyclic nucleotide-gated channels in the hippocampus. Eur J Neurosci 33:1611–1621
Klinger M, Freissmuth M, Nanoff C (2002) Adenosine receptors: G protein-mediated signalling and the role of accessory proteins. Cell Signal 14:99–108
Cunha RA, Ribeiro JA (2000) Adenosine A2A receptor facilitation of synaptic transmission in the CA1 area of the rat hippocampus requires protein kinase C but not protein kinase A activation. Neurosci Lett 289:127–130
Gardner AM, Olah ME (2003) Distinct protein kinase C isoforms mediate regulation of vascular endothelial growth factor expression by A2A adenosine receptor activation and phorbol esters in pheochromocytoma PC12 cells. J Biol Chem 278:15421–15428
Kubista H, Boehm S (2006) Molecular mechanisms underlying the modulation of exocytotic noradrenalinee release via presynaptic receptors. Pharmacol Ther 112:213–242
Sebastião AM, Ribeiro JA (2009) Tuning and fine-tuning of synapses with adenosine. Curr Neuropharmacol 7:180–194
Cristóvão-Ferreira S, Vaz SH, Ribeiro JA, Sebastião AM (2009) Adenosine A2A-receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1. J Neurochem 109:336–347
Smith FD, Langeberg LK, Scott JD (2006) The where’s and when’s of kinase anchoring. Trends Biochem Sci 31:316–323
Appert-Collin A, Baisamy L, Diviani D (2006) Regulation of g protein-coupled receptor signaling by a-kinase anchoring proteins. J Recept Signal Transduct Res 26:631–646
Johansson S (1994) Graded action potentials generated by differentiated human neuroblastoma cells. Acta Physiol Scand 151:331–341
Brown NA, Kemp JA, Seabrook GR (1994) Block of human voltage-sensitive Na+ currents in differentiated SH-SY5Y cells by lifarizine. Br J Pharmacol 113:600–606
Klinger M, Kuhn M, Just H, Stefan E, Palmer T, Freissmuth M, Nanoff C (2002) Removal of the carboxy terminus of the A2A-adenosine receptor blunts constitutive activity: differential effect on cAMP accumulation and MAP kinase stimulation. Naunyn Schmiedebergs Arch Pharmacol 366:287–298
Lissandron V, Terrin A, Collini M, D’Alfonso L, Chirico G, Pantano S, Zaccolo M (2005) Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction. J Mol Biol 354:546–555
Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Ceña V, Gallego C, Comella JX (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem 75:991–1003
Boehm S (1999) ATP stimulates sympathetic transmitter release via presynaptic P2X purinoceptors. J Neurosci 19:737–746
Charalambous C, Gsandtner I, Keuerleber S, Milan-Lobo L, Kudlacek O, Freissmuth M, Zezula J (2008) Restricted collision coupling of the A2A receptor revisited: evidence for physical separation of two signaling cascades. J Biol Chem 283:9276–9288
Feige JN, Sage D, Wahli W, Desvergne B, Gelman L (2005) PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs. Microsc Res Tech 68:51–58
Lechner SG, Hussl S, Schicker KW, Drobny H, Boehm S (2005) Presynaptic inhibition via a phospholipase C- and phosphatidylinositol bisphosphate-dependent regulation of neuronal Ca2+ channels. Mol Pharmacol 68:1387–1396
Pankevych H, Korkhov V, Freissmuth M, Nanoff C (2003) Truncation of the A1 adenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling. J Biol Chem 278:30283–30293
Vaughan PF, Peers C, Walker JH (1995) The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release. Gen Pharmacol 26:1191–1201
Goodall AR, Danks K, Walker JH, Ball SG, Vaughan PF (1997) Occurrence of two types of secretory vesicles in the human neuroblastoma SH-SY5Y. J Neurochem 68:1542–1552
Sarkanen JR, Nykky J, Siikanen J, Selinummi J, Ylikomi T, Jalonen TO (2007) Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells. J Neurochem 102:1941–1952
Pousinha PA, Diogenes MJ, Ribeiro JA, Sebastião AM (2006) Triggering of BDNF facilitatory action on neuromuscular transmission by adenosine A2A receptors. Neurosci Lett 404:143–147
Boehm S, Huck S (1996) Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release. Eur J Neurosci 8:1924–1931
Fykse EM, Li C, Südhof TC (1995) Phosphorylation of rabphilin-3A by Ca2+/calmodulin- and cAMP-dependent protein kinases in vitro. J Neurosci 15:2385–2395
Ohnishi H, Yamamori S, Ono K, Aoyagi K, Kondo S, Takahashi M (2001) A src family tyrosine kinase inhibits neurotransmitter release from neuronal cells. Proc Natl Acad Sci U S A 98:10930–10935
Baldwin ML, Cammarota M, Sim ATR, Rostas JAP (2006) Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals. Neurochem Int 49:80–86
Vela J, Pérez-Millán MI, Becu-Villalobos D, Díaz-Torga G (2007) Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells. Am J Physiol Cell Physiol 293:C951–C959
Walker F, deBlaquiere J, Burgess AW (1993) Translocation of pp 60c-src from the plasma membrane to the cytosol after stimulation by platelet-derived growth factor. J Biol Chem 268:19552–19558
Murray D, Hermida-Matsumoto L, Buser CA, Tsang J, Sigal CT, Ben-Tal N, Honig B, Resh MD, McLaughlin S (1998) Electrostatics and the membrane association of Src: theory and experiment. Biochemistry 37:2145–2159
Yu VC, Hochhaus G, Chang FH, Richards ML, Bourne HR, Sadée W (1988) Differentiation of human neuroblastoma cells: marked potentiation of prostaglandin E-stimulated accumulation of cyclic AMP by retinoic acid. J Neurochem 51:1892–1899
Brooke RE, Deuchars J, Deuchars SA (2004) Input-specific modulation of neurotransmitter release in the lateral horn of the spinal cord via adenosine receptors. J Neurosci 24:127–137
Shindou T, Arbuthnott GW, Wickens JR (2008) Actions of adenosine A 2A receptors on synaptic connections of spiny projection neurons in the neostriatal inhibitory network. J Neurophysiol 99:1884–1889
Nanoff C, Jacobson KA, Stiles GL (1991) The A2 adenosine receptor: guanine nucleotide modulation of agonist binding is enhanced by proteolysis. Mol Pharmacol 39:130–135
Nanoff C, Stiles GL (1993) Solubilization and characterization of the A2-adenosine receptor. J Recept Res 13:961–973
Roka F, Brydon L, Waldhoer M, Strosberg AD, Freissmuth M, Jockers R, Nanoff C (1999) Tight association of the human Mel(1a)-melatonin receptor and G(i): precoupling and constitutive activity. Mol Pharmacol 56:1014–1024
Hervé D (2011) Identification of a specific assembly of the g protein golf as a critical and regulated module of dopamine and adenosine-activated cAMP pathways in the striatum. Front Neuroanat 5:1–9
Huang Z, Lujan R, Kadurin I, Uebele VN, Renger JJ, Dolphin AC, Shah MM (2011) Presynaptic HCN1 channels regulate Cav3.2 activity and neurotransmission at select cortical synapses. Nat Neurosci 14:478–486
Weiss JL, Burgoyne RD (2001) Voltage-independent inhibition of P/Q-type Ca2+ channels in adrenal chromaffin cells via a neuronal Ca2+ sensor-1-dependent pathway involves Src family tyrosine kinase. J Biol Chem 276:44804–44811
Zong X, Eckert C, Yuan H et al (2005) A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase. J Biol Chem 280:34224–34232
Bjelfman C, Meyerson G, Cartwright CA, Mellström K, Hammerling U, Påhlman S (1990) Early activation of endogenous pp 60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells. Mol Cell Biol 10:361–370
Beene DL, Scott JD (2007) A-kinase anchoring proteins take shape. Curr Opin Cell Biol 19:192–198
Burgueño J, Blake DJ, Benson MA et al (2003) The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin. J Biol Chem 278:37545–37552
Kirk IP, Richardson PJ (1995) Inhibition of striatal GABA release by the adenosine A2a receptor is not mediated by increases in cyclic AMP. J Neurochem 64:2801–2809
Acknowledgements
The authors wish to thank their colleague Dr Sonja Sucic for assistance in the preparation of the manuscript. Work was supported by funding from FWF, the Austrian Science Fund (P 18150).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ibrisimovic, E., Drobny, H., Yang, Q. et al. Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release. Purinergic Signalling 8, 677–692 (2012). https://doi.org/10.1007/s11302-012-9298-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11302-012-9298-3