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J. Biol. Chem., Vol. 276, Issue 35, 32925-32932, August 31, 2001
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From the August Krogh Institute, Universitetsparken 13, University
of Copenhagen, DK-2100 Copenhagen Ø, Denmark
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 acridine 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 µM ATP after cholinergic stimulation.
Thus, ATP is poised as the paracrine mediator between pancreatic acini
and ducts.
Visualization of ATP Release in Pancreatic Acini in
Response to Cholinergic Stimulus
USE OF FLUORESCENT PROBES AND CONFOCAL MICROSCOPY*
*
This work was supported by the Danish Medical and Science
Research Councils.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
45-3532-1645; Fax: 45-3532-1567; E-mail: inovak@aki.ku.dk.
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