Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons

Activation of P2X receptors by a Ca2+- and soluble N-ethylmaleimidesensitive factor attachment protein receptor ( SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca2+ ionophore ionomycin (1.5 and 3 mu M). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca2+, depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba2+ for Ca2+ augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca2+] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca2+ with Mg2+, demonstrating a Ca2+ requirement. ASTICs were Na+ dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) (10 mu M) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 mu M and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 mu M). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.