The human SH‐SY5Y neuroblastoma cell‐line expresses a functional P2X 7 purinoceptor that modulates voltage‐dependent Ca 2+ channel function

Fura‐2 imaging of purinergic stimulation of non‐differentiated neuronal human SH‐SY5Y cells resulted in a rapid elevation in intracellular Ca 2+ ([Ca 2+ ] i ) that was dependent on extracellular Ca 2+ . The rank order of agonists (200 µ m ) was as follows: 2′,3′‐ O ‐(4‐benzoyl‐benzoyl)‐ATP (BzATP) > ATP 4–  > ATP; whereas 2‐(methylthio)‐ATP, ADP, UTP and α,β‐methylene‐ATP and β,γ‐methylene‐ATP were ineffective. The response to BzATP was inhibited by pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic‐acid (PPADS, 1 µ m ), 1‐( N , O ‐bis[5‐isoquinolinesulfonyl]‐ N ‐methyl‐ l ‐tyrosyl)‐4‐phenylpiperazine (KN‐62, 100 n m ) and 8‐(3‐benzamido‐4–4‐methylbenzamido)‐naphthalene‐1,3,5‐trisulfonic‐acid (suramin, 200 µ m ). The presence of a P2X 7 receptor was confirmed by western blot studies using anti‐P2X 7 . EC 50 for BzATP was 212 ± 6 µ m . BzATP > 30 µ m induced an initial, transient increase in [Ca 2+ ] i before a plateau level was reached. BzATP < 30 µ m only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 µ m ) and to a smaller degree by ω‐conotoxin GVIA (1 µ m ) despite an almost equal presence of L and N‐type Ca 2+ ‐channels. In whole‐cell voltage‐clamp studies at − 90 mV, BzATP (300 µ 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 ATP 4– . BzATP also triggered transmitter release. Thus, the human neuronal SH‐SY5Y cell line expresses a functional P2X 7 receptor coupled to activation of Ca 2+ ‐channels.