Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle

The translation of nerve transmission to Ca 2 + signals in urinary bladder smooth muscle (UBSM) is incompletely understood. Thus, we sought to characterize Ca 2 + signals in strips of UBSM loaded with the Ca 2 + -sensitive fluorescent dye, fluo-4, using laser scanning confocal microscopy. Two types of Ca 2 + signals occurred spontaneously and could be evoked with field stimulation: large, rapid, global Ca 2 + transients termed ‘global Ca 2 + flashes’, and much smaller, localized Ca 2 + transients. Global Ca 2 + flashes were inhibited by the L-type voltage-dependent Ca 2 + channel (VDCC) inhibitor, diltiazem and with P2X receptor blockade. Simultaneous intracellular recordings and Ca 2 + measurements indicated that these events are caused by Ca 2 + influx through VDCCs during action potentials. Small, local Ca 2 + transients occurred spontaneously, and their frequency could be elevated with field stimulation. Atropine, an inhibitor of muscarinic receptors, did not affect these local Ca 2 + transients. However, the desensitizing P2X receptor agonist α,β-methylene ATP, and the purinergic antagonist, suramin, effectively inhibited the local Ca 2 + transients. The frequency of these ‘purinergic Ca 2 + 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 Ca 2 + transients were F / F o = 2.4 ± 0.13, 111.7 ± 9.3 ms and 14.0 ± 1.0 μm 2 , respectively. Tetrodotoxin inhibited evoked purinergic Ca 2 + transients, indicating that they were dependent on nerve fibre activation. Purinergic Ca 2 + transients were not dependent on VDCC activity. Neither 2-APB, an inhibitor of inositol 1,4,5-triphosphate (Ins(1,4,5)P 3 ) (IP 3 )-induced Ca 2 + release, nor ryanodine inhibited the purinergic Ca 2 + transients. We have identified two novel Ca 2 + signals in rat UBSM. Large, rapid, global Ca 2 + flashes that represent Ca 2 + influx through VDCCs during action potentials, and local, purinergic Ca 2 + transients that represent Ca 2 + entry through P2X receptors. Our results indicate that purinergic Ca 2 + transients evoked by release of ATP from nerve varicosities are elementary signals in the process of nerve-smooth muscle communication.