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

Abstract

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.

Somatic ATP release from guinea pig sympathetic neurons does not require calcium-induced calcium release from internal stores

“…2A). These transient inward currents were similar in time course to the ATP-generated ASTICs described previously (26). Caffeine could elicit both SICs and ASTICs in cells that were bathed in a Ca 2ϩ -deficient solution in which Mg 2ϩ was substituted for Ca 2ϩ (Fig.…”

“…All efforts were made to minimize the number of animals used and their suffering. The methods for cell dissociation were slightly modified from those described previously (12,26). In brief, the right and left stellate ganglia were exposed rostral to the first rib, removed, and placed in Krebs solution containing (in mM) 120.9 NaCl, 5.9 KCl, 25 NaHCO 3, 1.2 NaH2PO4, 2.5 CaCl 2, 1.2 MgCl2, and 11 glucose (bubbled with 95% O2-5% CO2).…”

“…All experiments were conducted at 33-34°C, with the temperature maintained by an in-line heater (Warner Instruments, Hamden, CT). Whole cell currents were recorded under voltage-clamp conditions using the perforated patch configuration of the patch-clamp recording technique (8), as described in our laboratory's prior studies (14,26). Cells were superfused with a HEPES-buffered salt solution (extracellular solution) composed of (in mM) 121 NaCl, 26 Na-HEPES, 5.9 KCl, 1.2 MgCl 2, and 2.5 CaCl2 (pH 7.36).…”

“…nonclassical transmitter release; autonomic neurons; asynchronous transient inward currents; slow inward currents CLASSICALLY, IT WAS PROPOSED that calcium (Ca 2ϩ )-dependent neurotransmitter release primarily occurs at highly organized active zones in nerve terminals (11,19,24). However, neurotransmitter release is also now known to occur from somatodendritic areas of neurons (9,10,16,17,26,27,30,31). Although nonclassical transmitter release is Ca 2ϩ dependent, it can occur from areas lacking highly organized release sites.…”

“…Previously, our laboratory demonstrated the regulated release of ATP quanta from guinea pig sympathetic neurons dissociated from the stellate ganglion (26). The released ATP activates P2X receptors on the same cell to generate asynchronous transient inward currents (ASTICs) in cells voltageclamped to negative potentials.…”