The human SH-SY5Y neuroblastoma cell-line expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function.
Fura-2 imaging of purinergic stimulation of non-differentiated neuronal human SH-SY5Y cells resulted in a rapid elevation in intracellular Ca2+ ([Ca2+]i) that was dependent on extracellular Ca2+. The rank order of agonists (200 micro m) was as follows: 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) > ATP4- > ATP; whereas 2-(methylthio)-ATP, ADP, UTP and alpha,beta-methylene-ATP and beta,gamma-methylene-ATP were ineffective. The response to BzATP was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic-acid (PPADS, 1 micro m), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN-62, 100 nm) and 8-(3-benzamido-4-4-methylbenzamido)-naphthalene-1,3,5-trisulfonic-acid (suramin, 200 micro m). The presence of a P2X7 receptor was confirmed by western blot studies using anti-P2X7. EC50 for BzATP was 212 +/- 6 micro m. BzATP > 30 micro m induced an initial, transient increase in [Ca2+]i before a plateau level was reached. BzATP < 30 micro m only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 micro m) and to a smaller degree by omega-conotoxin GVIA (1 micro m) despite an almost equal presence of L and N-type Ca2+-channels. In whole-cell voltage-clamp studies at - 90 mV, BzATP (300 micro m) produced a fast activating inward current with a similar pharmacology as observed with Fura-2 imaging. Current clamp studies showed a dose-dependent depolarization to BzATP and ATP4-. BzATP also triggered transmitter release. Thus, the human neuronal SH-SY5Y cell line expresses a functional P2X7 receptor coupled to activation of Ca2+-channels.
Larsson
Kim Per
KP
Department of Medical Physiology, The Panum Institute, Copenhagen, Denmark.
Hansen
Anker Jon
AJ
Dissing
Steen
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism; D018408:Patch-Clamp Techniques; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis*; D058486:Receptors, Purinergic P2X7; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D018377:Neurotransmitter Agents; C546468:P2RX7 protein, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D002118:Calcium; D016257:Fura-2; D009638:Norepinephrine