Visualization of ATP release in pancreatic acini in response to cholinergic stimulus - Use of fluorescent probes and confocal microscopy

The energy providing substrate ATP can be released from various cells and act extracellularly to regulate the same cells or neighboring cells. However, the pathway for ATP release and the eliciting physiological stimulus are unclear. Recently, we showed that ATP activates P2X and P2Y purinergic receptors on pancreatic ducts. Thus, it was relevant to ask whether the upstream acini could be the source of releasable ATP and what the stimulus might be. We used freshly prepared rat pancreatic acini and applied conventional luminescence measurements of luciferin/luciferase reaction. As a new application of this reaction in confocal microscopy, we monitored luciferin fluorescence as a sign of ATP release by single acini. In addition we used quinacrine to mark ATP stores, which were similar to those marked with fluorescent ATP, 2 '-(or-3 ')-O-(N-methylanthraniloyl) adenosine 5 ' -triphosphate, but only partially overlapping with those marked by aeridine orange and LysoTracker Red. In functional studies we show that native pancreatic acini release ATP in response to various stimuli but most importantly to cholinergic stimulation, a very likely physiological stimulus in this epithelium. In a close vicinity of acini we detect about 9 mum ATP after cholinergic stimulation. Thus, ATP is poised as the paracrine mediator between pancreatic acini and ducts.