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 Ca(2+) and electrical signals induced by nerve-released ACh. Photolysis of caged inositol 1,4,5-trisphosphate (IP(3)) evoked Ca(2+) release from the sarcoplasmic reticulum. Electrical field stimulation (20 Hz) induced Ca(2+) waves within the smooth muscle that were present only during stimulus application. Ca(2+) waves were blocked by inhibition of muscarinic ACh receptors (mAChRs) with atropine and depletion of sarcoplasmic reticulum Ca(2+) stores with cyclopiazonic acid (CPA), and therefore likely reflect activation of IP(3) receptors (IP(3)Rs). Electrical field stimulation also increased excitability to induce action potentials (APs) that were accompanied by Ca(2+) flashes, reflecting Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCCs) during the action potential. The evoked Ca(2+) flashes and APs occurred as a burst with a lag time of approximately 1.5 s after onset of stimulation. They were not inhibited by blocking IP(3)-mediated Ca(2+) 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 IP(3)-mediated Ca(2+) signaling with cyclopiazonic acid or inhibition of PLC with U73122. These results indicate that ACh released from nerve varicosities induces IP(3)-mediated Ca(2+) 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 Ca(2+) entry through VDCCs during APs.

Fig. 1.

Depletion of sarcoplasmic reticulum (SR) Ca²⁺ stores abolishes inositol 1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ release. A: fluo-4-loaded urinary bladder smooth muscle (UBSM) strip with regions of interest (ROIs, colored boxes). B: increase in Ca²⁺ recorded from the ROIs shown in A following photolysis of iso-Ins (1,4,5)P₃-PM (iso-IP₃). C: color-coded F/F₀ recordings showing the increase in cytosolic Ca²⁺ after photolysis of iso-IP₃. Warmer color temperatures indicate higher concentration of Ca²⁺. D: F/F₀ increased by 1.10 ± 0.34 following photolysis of iso-IP₃. In tissue not loaded with iso-IP₃, exposure to UV flash did not trigger any Ca²⁺ release. Data are means ± SD. E: depletion of SR Ca²⁺ stores by cyclopiazonic acid (CPA) prevented IP₃-induced Ca²⁺ release. Inhibition of voltage-dependent Ca²⁺ channels (VDCCs) by diltiazem for 15 min had no effect. Significance by paired t-test; data are means ± SE.

Fig. 2.

Stimulation of muscarinic receptors causes Ca²⁺ waves followed by Ca²⁺ flashes. A: fluo-4 loaded UBSM strip with ROIs (colored boxes) in which F/F₀ (B and C) was measured. The line scan (D) was performed offline along the yellow line. B: representative recording of F/F₀ during a Ca²⁺ wave. Note the sequential rise in F/F₀ in the 3 ROIs indicating propagation. C: representative recording of F/F₀ during Ca²⁺ flashes. Note the simultaneous rise in F/F₀ in all three ROIs. D: line scan showing the different Ca²⁺ events evoked by nerve-released ACh. Ca²⁺ waves were characterized by propagation (sloped line), whereas Ca²⁺ flashes did not show any propagation (vertical lines). E: average intensity measured between the red lines in D; a Ca²⁺ wave is followed by 9 Ca²⁺ flashes. F: histogram showing the lag time of Ca²⁺ events. Ca²⁺ waves occurred during stimulation, whereas Ca²⁺ flashes followed in a burst ∼1–6 s after the onset of stimulation (n = 145 waves and 521 flashes from 21 strips). G: blocking Ca²⁺ flashes with diltiazem (50 μM) revealed that Ca²⁺ wave activity stops with cessation of stimulation (n = 24 waves from 7 strips). H: Ca²⁺ waves were sensitive to atropine (10 μM) and CPA (10 μM), but not to diltiazem (50 μM). I: Ca²⁺ flashes were blocked by atropine and diltiazem, but not by CPA. Significance by paired t-test; data are means ± SE.

Fig. 3.

Nerve-released ACh increases urinary bladder smooth muscle excitability to evoke action potentials independently of SR Ca²⁺. A: representative recording of membrane potential in mouse UBSM. Electrical field stimulation (EFS) in the presence of α,β-methylene ATP (α,β-meATP; 10 μM) triggered a burst of APs. B: histogram showing the distribution of nerve-induced APs over time. Most APs occurred between 1 and 6 s after the onset of EFS (n = 751 APs from 72 strips). C: summary data showing the lag time of APs after onset of EFS (n = 751 APs from 72 strips). D: inhibition of muscarinic ACh receptors (mAChRs) with atropine (10 μM) abolished nerve-induced APs. E: blockade of VDCCs with diltiazem (50 μM) prevented nerve-induced APs. F: depletion of SR Ca²⁺ stores by CPA (10 μM) did not inhibit nerve-induced APs. G: summary data of the effects of atropine, diltiazem, and CPA on the number of evoked action potentials. Significance by paired t-test; data are means ± SE. H: in strips treated with diltiazem, EFS caused a membrane depolarization (measured 2 s after onset of EFS and compared with hypothetical mean 0; 1-sample t-test) that was absent in strips treated with atropine.

Fig. 4.

Ca²⁺ influx through VDCC is crucial for contraction. A: representative recordings of contractions evoked by EFS with increasing frequency in the presence of α,β-meATP (10 μM) before (A, a) and after (A, b) application of atropine (10 μM). Contractions in response to 20 Hz stimulation are shown on a faster time scale (A, c). Atropine significantly reduced force amplitude (A, d), force integral (A, e), and half-amplitude duration (A, f). B: representative recordings of nerve-evoked contractions in the presence of α,β-meATP (10 μM) before (B, a) and after (B, b) application of diltiazem (50 μM). Contractions in response to 20 Hz stimulation are shown on a faster time scale (B, c). Diltiazem significantly reduced force amplitude (B, d), force integral (B, e), and half-amplitude duration (B, f). *P < 0.05 and ***P < 0.001 by two-way repeated-measures ANOVA; data are means ± SE.

Fig. 5.

SR Ca²⁺ release does not promote contractility. A: representative recordings of nerve-evoked contractions in the presence of α,β-meATP (10 μM) before (A, a) and after (A, b) application of CPA (10 μM). Contractions in response to 20 Hz stimulation are shown on a faster time scale (A, c). CPA had no effect on force amplitude (A, d), but significantly increased force integral (A, e) and half-amplitude duration (A, f). B: contractions evoked by 20 Hz stimulation in the presence of diltiazem alone and diltiazem with CPA (B, a). Combined inhibition of SR Ca²⁺ release and Ca²⁺ influx was not different from blocking only Ca²⁺ influx. Note that the graphs for force amplitude (B, b) and force integral (B, c) coincide (P > 0.05 by two-way ANOVA). C: representative recordings of nerve-evoked contractions in the presence of α,β-meATP (10 μM) before (C, a) and after (C, b) application of U73122 (10 μM). Contractions in response to 20 Hz stimulation are shown on a faster time scale (C, c). U73122 slightly increased force amplitude at 2, 3.5, and 5 Hz stimulation frequency (C, d), but reduced force integral by maximally ∼15% (C, e) and half-amplitude duration (C, f) at stimulation frequencies > 20 Hz and 12.5 Hz, respectively. *P < 0.05, **P < 0.01 and *** P < 0.001 by two-way repeated-measures ANOVA; data are means ± SE.

Fig. 6.

Illustration of proposed muscarinic signaling. Nerve-released ACh activates mAChRs to 1) rapidly activate PLC to produce IP₃ and release Ca²⁺ from SR via IP₃ receptors (IP₃Rs) (Ca²⁺ wave), and 2) with a slower onset, increase excitability to promote opening of VDCCs (Ca²⁺ flash/AP). Both events occur independently of each other. Ca²⁺ waves do not contribute to excitability and contractility; their function remains unknown. Ca²⁺ flashes/APs, on the other hand, are crucial for contraction. PIP₂, phosphatidylinositol-4,5-bisphosphate; Gα_q, a G protein.