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1 Department of Pharmacology, University of Vermont College of Medicine, Burlington, VT 05405-0068, USA
The translation of nerve transmission to Ca2+ signals in urinary bladder smooth muscle (UBSM) is incompletely understood. Thus, we sought to characterize Ca2+ signals in strips of UBSM loaded with the Ca2+-sensitive fluorescent dye, fluo-4, using laser scanning confocal microscopy. Two types of Ca2+ signals occurred spontaneously and could be evoked with field stimulation: large, rapid, global Ca2+ transients termed ‘global Ca2+ flashes’, and much smaller, localized Ca2+ transients. Global Ca2+ flashes were inhibited by the L-type voltage-dependent Ca2+ channel (VDCC) inhibitor, diltiazem and with P2X receptor blockade. Simultaneous intracellular recordings and Ca2+ measurements indicated that these events are caused by Ca2+ influx through VDCCs during action potentials. Small, local Ca2+ transients occurred spontaneously, and their frequency could be elevated with field stimulation. Atropine, an inhibitor of muscarinic receptors, did not affect these local Ca2+ transients. However, the desensitizing P2X receptor agonist 
,ß-methylene ATP, and the purinergic antagonist, suramin, effectively inhibited the local Ca2+ transients. The frequency of these ‘purinergic Ca2+ transients’ was increased about 7-fold by a 10 s stimulus train (1 Hz). The amplitude, duration at one-half amplitude and the spatial spread of the evoked purinergic Ca2+ transients were F/Fo
= 2.4 ± 0.13, 111.7 ± 9.3 ms and 14.0 ± 1.0 µm2, respectively. Tetrodotoxin inhibited evoked purinergic Ca2+ transients, indicating that they were dependent on nerve fibre activation. Purinergic Ca2+ transients were not dependent on VDCC activity. Neither 2-APB, an inhibitor of inositol 1,4,5-triphosphate (Ins(1,4,5)P3) (IP3)-induced Ca2+ release, nor ryanodine inhibited the purinergic Ca2+ transients. We have identified two novel Ca2+ signals in rat UBSM. Large, rapid, global Ca2+ flashes that represent Ca2+ influx through VDCCs during action potentials, and local, purinergic Ca2+ transients that represent Ca2+ entry through P2X receptors. Our results indicate that purinergic Ca2+ transients evoked by release of ATP from nerve varicosities are elementary signals in the process of nerve-smooth muscle communication.
(Received 20 October 2004;
accepted after revision 23 December 2004;
first published online 6 January 2005)
Corresponding author T. Heppner: Department of Pharmacology, University of Vermont, Given Building, Rm C-315, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Email: thomas.heppner{at}uvm.edu
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