Nerve-released acetylcholine contracts urinary bladder smooth muscle by inducing action potentials independently of IP3-mediated calcium release

Abstract

Nerve-released ACh is the main stimulus for contraction of urinary bladder smooth muscle (UBSM). Here, the mechanisms by which ACh contracts UBSM are explored by determining Ca2+ and electrical signals induced by nerve-released ACh. Photolysis of caged inositol 1,4,5-trisphosphate (IP3) evoked Ca2+ release from the sarcoplasmic reticulum. Electrical field stimulation (20 Hz) induced Ca2+ waves within the smooth muscle that were present only during stimulus application. Ca2+ waves were blocked by inhibition of muscarinic ACh receptors (mAChRs) with atropine and depletion of sarcoplasmic reticulum Ca2+ stores with cyclopiazonic acid (CPA), and therefore likely reflect activation of IP3 receptors (IP3Rs). Electrical field stimulation also increased excitability to induce action potentials (APs) that were accompanied by Ca2+ flashes, reflecting Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) during the action potential. The evoked Ca2+ flashes and APs occurred as a burst with a lag time of ∼1.5 s after onset of stimulation. They were not inhibited by blocking IP3-mediated Ca2+ waves, but by blockers of mAChRs (atropine) and VDCCs (diltiazem). Nerve-evoked contractions of UBSM strips were greatly reduced by blocking VDCCs, but not by preventing IP3-mediated Ca2+ signaling with cyclopiazonic acid or inhibition of PLC with U73122. These results indicate that ACh released from nerve varicosities induces IP3-mediated Ca2+ waves during stimulation; but contrary to expectations, these signals do not appear to participate in contraction. In addition, our data provide compelling evidence that UBSM contractions evoked by nerve-released ACh depend on increased excitability and the resultant Ca2+ entry through VDCCs during APs.