Quantal release of ATP from clusters of PC12 cells

Abstract

Although ATP is important for intercellular communication, little is known about the mechanism of endogenous ATP release due to a dearth of suitable models. Using PC12 cells known to express the P2X2 subtype of ATP receptors and to store ATP with catecholamines inside dense-core vesicles, we found that clusters of PC12 cells cultured for 3-7 days generated small transient inward currents (STICs) after an inward current elicited by exogenous ATP. The amplitude of STICs in individual cells correlated with the peak amplitude of ATP-induced currents. STICs appeared as asynchronous responses (approximately 20 pA average amplitude) for 1-20 s and were investigated with a combination of patch clamping, Ca2+ imaging, biochemistry and electron microscopy. Comparable STICs were produced by focal KCl pulses and were dependent on extracellular Ca2+. STICs were abolished by the P2X antagonist PPADS and potentiated by Zn2+, suggesting they were mediated by P2X2 receptor activation. The highest probability of observing STICs was after the peak of intracellular Ca2+ increase caused by KCl. Biochemical measurements indicated that KCl application induced a significant release of ATP from PC12 cells. Electron microscopy studies showed narrow clefts without 'synaptic-like' densities between clustered cells. Our data suggest that STICs were caused by quantal release of endogenous ATP by depolarized PC12 cells in close juxtaposition to the recorded cell. Thus, STICs may be a new experimental model to characterize the physiology of vesicular release of ATP and to study the kinetics and pharmacology of P2X2 receptor-mediated quantal currents.

Figure 1. Correlation between growth and functional properties of PC12 cells in culture

A–D, time profile (days) for increase in cell number in control or in the presence of PPADS (A), amplitude of intracellular Ca²⁺ transients induced by K⁺ pulse; (B, number of cells ranged from 7 to 78); size of membrane currents elicited by 1 mm ATP (C, number of recorded cells ranged from 17 to 86) and per cent of cells generating small transient inward currents (STICs) to 1 mm ATP application (D). E and F, examples of morphology of PC12 cells that, despite cell clustering, maintained spherical shape without processes. Cells were visualized on the basis of their Ca²⁺ signals before (E) and after (F) K⁺ pulse (day 4 after plating). Calibration bar = 12 mm

Figure 2. Small transient inward currents (STICs) evoked by ATP and their sensitivity to Zn²⁺

Aa, application of ATP (1 mm; 2 s) evoked large membrane current followed by STICs after current decay during washout (black trace). In the same cell washout of ATP with 30 μm Zn²⁺ increased the number, amplitude, rise time and duration of STICs (grey trace). Ab, superimposed records to show the effect of Zn²⁺ on average STIC. Note monoexponential decay both in control (decay time constant, τ = 24 ms; n = 64 events) and in the presence of Zn²⁺ (τ = 104 ms; n = 48 events). Examples of cumulative distribution of amplitude (B) and decay (C) of STICs before (open circles) and after addition of 30 μm Zn²⁺ (grey circles). D, distribution of STIC latency (from the end of ATP application) in control (open bars) and in the presence of Zn²⁺ (grey bars). E, the amplitude, rise time and decay time constant of STICs in Zn²⁺ solution (30 μm) normalized to control (dashed line). Data from 9 cells. *P < 0.05, **P < 0.001.

Figure 3. High K⁺ increased intracellular Ca²⁺ and generated STICs in clustered cells

A, application of 100 mm KCl (2 s) generated single STIC (*) during KCl-induced inward current and several STICs (*) during washout. Ba, distribution of amplitude of KCl-induced STICs. Bb, distribution of decay of STICs. Note that both distributions could be fitted by a single Gaussian curve, suggesting a mainly monoquantal release process. C, representative records of the time course of intracellular Ca²⁺ transients ([Ca²⁺]_i) induced by KCl (100 mm; 2 s) in a cell cluster (schematized in inset) loaded with Fluo-3AM. Signals were scaled to the Ca²⁺ transient from cell no. 1 (to which calibration applies) to compare their time course. Note almost synchronous generation of Ca²⁺ transients in all three cells of this cluster.

Figure 4. STICs were generated in a Ca²⁺-dependent manner

A, test showing that removal of Ca²⁺ from physiological solution reversibly abolished KCl-induced [Ca²⁺]_i transients. KCl application was 2 s long. B, STICs (*) generated by KCl pulse were reversibly abolished after removal of Ca²⁺. C, average data for generation of STICs by KCl in control solution, Ca²⁺-free media and upon return to normal saline (n = 5; *P < 0.05). D, average data for generation of STICs by 1 mm ATP in control solution, Ca²⁺-free media and upon return to normal saline (n = 8; *P < 0.05).

Figure 5. STICs were abolished by PPADS, an antagonist of P2X receptors

A, membrane currents induced by 1 mm ATP (2 s) in control (a) and after 3 min application of 10 μm PPADS (b). Note that PPADS reduced ATP-induced current and abolished STICs (compare inset to 5Aa with inset to 5Ab). B, STICs generated by 10 s pulse of KCl (a) were abolished in 10 μm PPADS solution (b).

Figure 6. Correlation between amplitude of ATP-induced membrane current and of STICs

Aa, recording from cell in which 1 mm ATP evoked large membrane current (−1480 pA, left trace) followed by average STICs with relatively large amplitude (−29 pA, 42 events, right trace). Ab, recording from another cell with smaller amplitude of ATP current (−315 pA) followed by STICs with average amplitude of −11 pA (55 events). Ba, the amplitude of currents evoked by 1 mm ATP was plotted against the amplitude of subsequent STICs (n = 25). Note strong correlation between these two parameters (r = 0.86; P < 0.001). Bb, similar plot for the amplitude of membrane current and STICs decay. Note lack of correlation in this case (r = 0.06; P = 0.8).

Figure 7. Zn²⁺ strongly augmented the amplitude and prolonged the decay of currents induced by brief puffer application of ATP

A, fast ATP current elicited by 10 ms pulse of pressure-applied 1 mm ATP in control (a) and in the presence of 30 μm Zn²⁺ (b). B, histograms showing potentiating action of 30 μm Zn²⁺ on the amplitude or decay of ATP-induced currents (n = 4). *P < 0.05.

Figure 8. Cell contacts, intracellular vesicles and amount of released ATP

A, EM micrograph with example of cross section of clustered PC12 cells showing narrow intercellular gap and group of dense core vesicles (dark organelles) in perimembrane regions. Note lack of any ‘synapse-like’ densities between apposed cells. Scale bar, 200 nm. B, quantification of ATP release into the extracellular medium using luciferin–luciferase assay at rest, after stimulation of release with 100 mm KCl, and KCl plus 30 μm Zn²⁺. n = 3 dishes. *P < 0.05.

Figure 9. Idealized scheme to account for P2X₂ receptor-mediated STICs

Application of exogenous ATP or KCl (boxes) to intact (not voltage-clamped) cell (top) is supposed to generate depolarization with Ca²⁺ influx via activated P2X₂ receptors and voltage-activated Ca²⁺ channels (VACC). Raised [Ca²⁺]_i promotes vesicular release of catecholamines (not shown) and endogenous ATP (black circles). When this process occurs within the close gap between two apposed PC12 cells, extracellular ATP can bind P2X₂ receptors to generate STICs in patched cell under voltage clamp. For sake of simplicity and because of its rare occurrence, the possibility that the patched cell can release ATP to self-generate STICs is not included.