Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons.
Activation of P2X receptors by a Ca(2+)- and soluble N-ethylmaleimide-sensitive 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 Ca(2+) ionophore ionomycin (1.5 and 3 microM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca(2+), depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba(2+) for Ca(2+) 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 [Ca(2+)] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca(2+) with Mg(2+), demonstrating a Ca(2+) 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 microM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 microM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 microM). 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.
Tompkins
John D
JD
University of Vermont, College of Medicine, Dept. of Anatomy and Neurobiology, Burlington, VT 05405, USA. john.tompkins@uvm.edu
Parsons
Rodney L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001464:Barium / Q000378:metabolism; D001905:Botulinum Toxins / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D005089:Exocytosis; D006168:Guinea Pigs; D018738:Hexamethonium / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D012964:Sodium / Q000378:metabolism; D013233:Stellate Ganglion / Q000166:cytology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002712:Chlorides; D007473:Ion Channels; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D001464:Barium; D018738:Hexamethonium; D015759:Ionomycin; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D001905:Botulinum Toxins; D013755:Tetradecanoylphorbol Acetate; C096326:botulinum toxin type E