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.

Identification of a vesicular nucleotide transporter

“…Finally, we investigated whether SLC17A9 is responsible for vesicular storage and subsequent exocytosis of ATP in purinergic cells. PC12 cells are known to store and secrete ATP through secretory granule-mediated exocytosis (24). We found that mSLC17A9 protein was associated with secretory granules (Fig.…”

ATP release from non-excitable cells

“…In a similar fashion, clusters of PC12 cells display such 'autaptic' behaviour, as detected by spontaneous transient inward currents (STICs) through P2X 2 receptors. There is firm evidence that this phenomenon reflects quantal exocytotic release of ATP from PC12 cells [2]. In addition, the discovery of pannexin 1 as an ATP-releasing channel in taste bud epithelia convincingly used the P2X2/3 biosensor approach [10].…”

“…It is thus established that, e.g. chromaffin cells and the closely related PC12 cells store large amounts of ATP (>100 mM) together with catecholamines [2,102], that insulin secretion from B cells occurs together with ATP [17] and that α-dense granules in thrombocytes contain very high concentrations of ADP, a known key factor in thrombus formation [14]. There is strong evidence that astrocytic release of nucleotides under physiological conditions involves an exocytotic mechanism [45,103,104], in contrast to the above-mentioned controversial issue of connexon hemichannel-mediated ATP release from astrocytes under non-physiological situations [36].…”

“…Recent breakthroughs in this field include the role of adenosine triphosphate (ATP) as neurotransmitter and/or modulator in sensory transduction [1][2][3][4][5][6][7][8][9][10], the role of released ATP as a precursor signalling molecule in renal tubulo-glomerular feedback [11,12], the role of released nucleotides for migrating neutrophils [13] and the crucial function of nucleotides in the control of thrombocyte aggregation and haemostasis [14]. The essential features of the purinergic signalling system are well characterised.…”

“…Apparently, the source of extracellular ATP is the large pool of cytosolic ATP. In classically secreting cells like neurons and neuro-endocrine cells, ample evidence indicates that ATP release occurs via exocytosis [2,[15][16][17]. Cells of non-neuronal origin like epithelia, endothelial cells or astrocytes can also be stimulated to release nucleotides.…”