Spontaneous purinergic neurotransmission in the mouse urinary bladder

Abstract

Spontaneous purinergic neurotransmission was characterized in the mouse urinary bladder, a model for the pathological or ageing human bladder. Intracellular electrophysiological recording from smooth muscle cells of the detrusor muscle revealed spontaneous depolarizations, distinguishable from spontaneous action potentials (sAPs) by their amplitude (< 40 mV) and insensitivity to the L-type Ca²⁺ channel blocker nifedipine (1 μm) (100 ± 29%). Spontaneous depolarizations were abolished by the P2X₁ receptor antagonist NF449 (10 μm) (frequency 8.5 ± 8.5% of controls), insensitive to the muscarinic acetylcholine receptor antagonist atropine (1 μm) (103.4 ± 3.0%), and became more frequent in latrotoxin (LTX; 1 nm) (438 ± 95%), suggesting that they are spontaneous excitatory junction potentials (sEJPs). Such sEJPs were correlated, in amplitude and timing, with focal Ca²⁺ transients in smooth muscle cells (measured using confocal microscopy), suggesting a common origin: ATP binding to P2X₁ receptors. sAPs were abolished by NF449, insensitive to atropine (126 ± 39%) and increased in frequency by LTX (930 ± 450%) suggesting a neurogenic, purinergic origin, in common with sEJPs. By comparing the kinetics of sAPs and sEJPs, we demonstrated that sAPs occur when sufficient cation influx through P2X₁ receptors triggers L-type Ca²⁺ channels; the first peak of the differentiated rising phase of depolarizations – attributed to the influx of cations through the P2X₁ receptor – is of larger amplitude for sAPs (2248 mV s⁻¹) than sEJPs (439 mV s⁻¹). Surprisingly, sAPs in the mouse urinary bladder, unlike those from other species, are triggered by stochastic ATP release from parasympathetic nerve terminals rather than being myogenic.

In most mammals, excitation of the detrusor muscle of the urinary bladder is achieved through the parasympathetic activation of both purinergic and cholinergic neurotransmission, leading to voiding of the bladder. The brief, initial phase of contraction is atropine insensitive (Henderson & Roepke, 1934) and purinergic (Burnstock et al. 1978); the second phase, of longer duration, is cholinergic (Krell et al. 1981). The balance between purinergic and cholinergic neurotransmission observed experimentally varies widely. This variation is often attributed to the following three factors. (i) Species used. Detrusor contraction is almost entirely controlled by purinergic neurotransmission in cat (Nergårdh, 1981) and rabbit (Sibley, 1984), whereas cholinergic neurotransmission dominates in the Old World primates (Craggs & Stephenson, 1985). (ii) Age. For example, there is an increase with age in the contribution of purinergic neurotransmission to bladder voiding in the human (Yoshida et al. 2001) and rat (Saito et al. 1991). (iii) Experimental protocol. Single pulse or low frequency, repetitive transmural electrical stimulation evokes an atropine-insensitive contraction (Ambache & Zar, 1970); high frequency repetitive stimulation evokes excitation that comprises a much larger proportion of cholinergic than purinergic neurotransmission (Sibley, 1984).

Normal voiding of the human bladder is attributed to the actions of acetylcholine as the primary excitatory neurotransmitter. The administration of the muscarinic cholinergic receptor antagonist atropine to healthy subjects resulted in difficulty in initiating, and pain during, micturition (Cullumbine et al. 1955) and in some in vitro studies, atropine was shown to abolish electrically evoked contraction (Sjörgen et al. 1982; Sibley, 1984). There are other in vitro studies, however, which suggest a role for purinergic neurotransmission in normal voiding; Cowan & Daniel (1983), for example, demonstrated that atropine abolished only 50% of stimulated contractions, and others (e.g. Elmér et al. 1986; Hoyle et al. 1989; Luheshi & Zar, 1990) have shown atropine-resistant contractions, albeit comprising a smaller proportion of excitatory neurotransmission than is attributed to the actions of acetylcholine. The wide variation in reported contributions of purinergic neurotransmission to bladder voiding is partly explained by the methodological difficulty of evoking neural stimulation without also evoking potentially masking direct muscle stimulation (Hoyle et al. 1989). It is, however, generally agreed that purinergic neurotransmission is up-regulated in bladder dysfunction; Sjörgen et al. (1982) demonstrated, for example, that while atropine abolished contractions in preparations from normal bladders, preparations from pathological bladders exhibited contractions that were up to 50% atropine resistant.

It has been demonstrated that ATP, released from parasympathetic nerve terminals, binds to P2X receptors (Burnstock & Kennedy, 1985; Howson et al. 1988) on urinary bladder smooth muscle cells (SMCs), producing an excitatory junction potential (EJP) that triggers the opening of L-type Ca²⁺ channels and the subsequent initiation of an action potential (Hoyle & Burnstock, 1985; Howson et al. 1988). Heppner et al. (2005) recently identified purinergic focal Ca²⁺ transients, corresponding to the influx of Ca²⁺ through P2X receptors upon ATP binding in rat urinary bladder smooth muscle.

In summary, although purines may not be the major excitatory neurotransmitters responsible for human bladder voiding, their up-regulated role in pathological states and with ageing makes purinergic neurotransmission a key area of study. The aims of the present study are to: (i) use simultaneous electrophysiological recording of membrane potential and high-speed imaging of Ca²⁺ fluorescence to determine whether a relationship exists between spontaneous purinergic Ca²⁺ transients and spontaneous depolarizations in mouse urinary bladder smooth muscle, and (ii) to characterize spontaneous depolarizations and, in particular, to determine the mechanisms underlying their generation.

Methods

General

Mice of the C57BL/6 strain, of either sex, weighing 18–30 g, were killed by head concussion followed by cervical dislocation. Efforts were made to minimize the number of animals used and their suffering; all experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and European Communities Council Directive 86/09/EEC.

The urinary bladder was removed and its ventral wall was opened longitudinally from the bladder neck (posterior) to the top of the dome (anterior). The urothelium was removed by careful dissection using iris scissors; care was taken not to stretch or damage the underlying detrusor muscle. Tissue strips, which contained a few bundles of smooth muscle, 6–8 mm long and 1–2 mm wide, were cut along the craniocaudal axis of the dorsal surface of the detrusor. Strips were pinned out on a Sylgard (Dow Corning Corporation, Midland, MI, USA)-lined plate at the bottom of a shallow chamber (volume, approximately 1 ml), which was mounted on the stage of an upright microscope. Preparations were superfused with warmed (35°C) physiological saline solution (PSS) (composition, mm: NaCl, 120; KCl, 5.9; MgCl₂, 1.2; CaCl₂ 2.5; NaHCO₃, 15.5; NaH₂PO₄, 1.2 and glucose, 11.5; gassed with 95% O₂ and 5% CO₂) at a constant flow rate (100 ml h⁻¹), maintaining a pH of 7.2–7.3 (Hashitani & Brading, 2003). Preparations, when pinned, were allowed to equilibrate (i.e. to accommodate under tension) for at least 30 min before electrophysiological recording or Ca²⁺ imaging.

Electrophysiological recordings

Individual bladder smooth muscle cells in muscle bundles were impaled with glass capillary microelectrodes, filled with 0.5 m KCl (tip resistance, 100–300 MΩ). Membrane potential changes were recorded using a high input impedance amplifier (Axoclamp-2B, Axon Instruments, Inc., Sunnyvale, CA, USA) and displayed on a digital oscilloscope (DSO 1602, Gould, Ilford, UK). Membrane potential changes were digitized using PowerLab/4SP (ADInstruments, Chalgrove, UK) at 4 kHz and stored on computer for later analysis.

Recordings were made from a minimum of six cells, for at least 5 min each, in the absence of drugs (‘control’ measurements). One long, continuous recording was then made, which included a control period (typically 10 min), and then a period (up to 1 h) in the presence of the drug. Recordings were subsequently made from a further six cells (minimum), for at least 5 min each. Subsequent analysis of the effect of the superfused reagents compared the median frequency for all cells recorded in the control period with the median frequency of events in the presence of the reagent, in a paired comparison.

Ca²⁺ imaging and analysis

Each detrusor strip was exposed to 10 μm Oregon Green 488 BAPTA-1 AM (Invitrogen, Paisley, UK) in 1% DMSO–0.2% Pluronic F-127 (Sigma-Aldrich, St Louis, MO, USA) in physiological salt solution (PSS) for 70 min at 36°C. The tissue was rinsed in PSS, bubbled with 95% O₂–5% CO₂, for at least 10 min. Tissues were pinned flat, serosal side up, in a Sylgard-lined organ bath, and mounted on the stage of an upright confocal microscope (Leica SP2 upright confocal microscope; Leica Microsystems, Milton Keynes, UK).

A series of 200 frames was captured at 13.5 Hz to generate one image set. Such sets were acquired once every minute. Ten sets were generated for each region of the preparation, with at least three regions sampled per drug treatment and/or preparation.

Microelectrodes used for simultaneous confocal recordings (tip resistances of 200–300 MΩ) were filled with a potassium salt of Oregon Green BAPTA-1 (100 μm in 0.5 m potassium acetate; weight 10 kDa; Invitrogen). The microelectrode tip (or part of it) was always within the field of view of the confocal image.

Image analysis was performed using plug-ins for ImageJ (http://rsb.info.nih.gov/ij/download.html) that were custom-written by Dr R. J. Amos to detect focal increases in fluorescence of the Ca²⁺ indicator. Data were exported to Excel (Microsoft, Redmond, WA, USA) for formatting and then to Spike 2 (Cambridge Electronic Design, Cambridge, UK) for analysis in conjunction with electrophysiological recordings.

Analysis of correlations between focal Ca²⁺ transients and spontaneous depolarizations

The peak amplitudes of focal Ca²⁺ transients and spontaneous depolarizations were calculated using a custom-written script for Spike 2. The threshold for determining focal Ca²⁺ transients varied between recordings in different SMCs. The threshold was adjusted until it provided specificity comparable to manual counting (ΔF/F range of 0.06–0.1).

Occasionally, > 1 coincident focal Ca²⁺ transient was observed within the impaled cell. In this instance, the amplitudes of spontaneous depolarizations and focal Ca²⁺ transients were not included in the subsequent analysis.

In order to estimate the time of initiation of focal Ca²⁺ transients, experiments were performed in which Ca²⁺ fluorescence was measured along the long axis of a SMC at a high acquisition rate (2 kHz) and the amplitudes and timing of focal Ca²⁺ transients were correlated with the membrane potential of the imaged cell, simultaneously measured using the previously described electrophysiological recording technique. Ca²⁺ fluorescence was measured in a 20 μm-wide window around each focal Ca²⁺ transient for the duration of each acquisition period (approx. 2 s) using ImageJ. Using a custom-written script for R (http://www.r-project.org) a curve was then fitted, comprising:

• y=C_o (the resting fluorescence), before the initiation of the focal Ca²⁺ transient (i.e. t < t_o), and

• A is an arbritrary constant.

The time of initiation (t_o) was that giving the minimum residual sum of squares for this curve fit. This function arises from a simple model of Ca²⁺ influx through P2X₁ receptors, with Ca²⁺ then cleared by a first-order process (see Appendix).

Drug sources, preparation and application

Tetrodotoxin (Tocris, Bristol, UK), nifedipine (Bayer, Leverkusen, North Rhine-Westphalia, Germany) and atropine sulphate (VWR; Lutterworth, UK) were stored frozen (−20°C) at a concentration of 10 mm in DMSO. NF449 (at 1 mm; Tocris, Bristol, UK) and α-latrotoxin (at 300 nm; Sigma-Aldrich) were stored frozen in distilled water. Drugs were diluted to their final concentration in PSS and applied to the preparation by switching the superfusate from standard PSS.

Statistical analysis

The normality and homogeneity of variance was tested prior to further statistical analysis using Kolmogorov–Smirnov and Levene's tests, respectively (SPSS version 11, SPSS Inc., Chicago, IL, USA). Unless otherwise stated, statistical comparisons are Student's two-tailed paired t test. The term n, used in the presentation of statistical analyses throughout, refers to the number of animals used.

Results

Pharmacology of spontaneous depolarizations

Intracellular recording of smooth muscle cells in the mouse urinary bladder revealed both spontaneous depolarizations (sDeps) and spontaneous action potentials (sAPs), as previously described. sAPs can be distinguished from sDeps by their greater amplitude, the presence of an after-hyperpolarization or after-depolarization and their sensitivity to the voltage-dependent Ca²⁺ channel (VDCC) antagonist nifedipine (Meng et al. 2008).

In order to determine the mechanisms underlying the generation of sDeps, the contributions of purinergic and cholinergic neurotransmission were explored through the application of specific antagonists. The purinergic P2X₁ antagonist NF449 (10 μm) greatly reduced the frequency of sDeps. The frequency at which sDeps were detected was reduced from 8.0 ± 3.6 min⁻¹ in control cells to 0.7 ± 0.3 min⁻¹ following 1 h of superfusion with NF449 (post-NF449, 8.5 ± 8.5% of controls; P < 0.01, n= 4; Fig. 1Aa and B), with the amplitude of the remaining events reduced to 75 ± 6% of controls (P < 0.05, n= 4). Superfusion of the P2X₁-desensitizing agonist α,β-methylene ATP (α,β-mATP; 1 μm) reduced the frequency with which sDeps were detected from 11.3 ± 2.0 min⁻¹ in control cells to 1.4 ± 0.6 min⁻¹ (post-α,β-mATP, 11.4 ± 0.6%; P < 0.01, n= 4; Fig. 1Ab and B) with the amplitude of remaining sDeps reduced to 55 ± 10% of controls (P < 0.05, n= 4). In contrast to NF449 and α,β-mATP, the muscarinic acetylcholine receptor antagonist atropine (1 μm) did not affect the frequency of sDeps, which was 74 ± 31 min⁻¹ in control cells and 76 ± 31 min⁻¹ following 1 h application (post-atropine, 103.4 ± 2.6% of controls; P= not significant (NS), n= 4; Fig. 1Ac and B).

Figure 1

Spontaneous depolarizations (sDeps) are purinergic in origin
A, comparing the effects of the superfusion of the P2X₁ antagonist NF449 (10 μm) (a), the P2X₁-desensitizing agent α,β-methylene ATP (α,β-mATP; 1 μm) (b), the muscarinic acetylcholine receptor antagonist atropine (1 μm) (c), a blocker of Na⁺ channels, tetrodotoxin (TTX, 1 μm) (d) and an antagonist of L-type Ca²⁺ channels, nifedipine (1 μm) (e) on the membrane potential of SMCs. Note the longer time-scale of Ab. A depolarization and increase in the sAP frequency induced by α,β-mATP (Ab) is initially noted (consistent with its action as an agonist), before the membrane potential returns towards its resting level and the sAPs decline in frequency (presumably as the receptors desensitize). B, a comparison of the frequency of sDeps for each drug application to normalized control frequencies, with paired t test statistical comparisons (see text for details).

In the urinary bladder of the guinea pig, it has been reported that some large-amplitude sEJPs result from spontaneous nerve terminal action potentials (Bramich & Brading, 1996). In the present work in mouse urinary bladder, however, TTX (1 μm) did not abolish large-amplitude (> 10 mV) sDeps (range, control: 1.7–35.0 mV; TTX, following superfusion for 1 h: 1.4–31.2 mV, n= 4). TTX (1 μm) also did not affect the frequency of sDeps, which was 4.3 ± 0.5 min⁻¹ in control cells, and 7.1 ± 1.1 min⁻¹ following TTX (post-TTX, 166 ± 55% of controls; P= NS; Fig. 1Ad and B).

In order to determine whether blocking L-type Ca²⁺ channels affected the frequency at which sDeps were detected, the effect of nifedipine was assessed. Nifedipine (1 μm) did not affect the frequency of sDeps, which was 6.6 ± 2.3 min⁻¹ in control cells, and 6.5 ± 2.0 min⁻¹ following nifedipine superfusion for 1 h (post-nifedipine, 100 ± 29% of controls; P= NS; Fig. 1Ae and B).

In order to determine whether the ATP that initiates sDeps comes from nerves, experiments were performed in the presence of an extract of the black widow spider venom, α-latrotoxin (LTX), which increases the rate of spontaneous neurotransmitter release from nerve terminals. Superfusion of LTX (1 nm) for 10 min produced a 438 ± 95% increase in sDep frequency (to 35.3 ± 8.3 min⁻¹) with respect to the control period (9.3 ± 2.6 min⁻¹) (P < 0.05, n= 4; Fig. 2A). The superfusion of atropine (1 μm), in addition to LTX (1 nm), produced a similarly large augmentation of sDeps, to 460 ± 320% (63 ± 13 min⁻¹) of control frequency (13.8 ± 2.8 min⁻¹) (P < 0.05, n= 4; Fig. 2B). By contrast, when LTX (1 nm) was superfused in addition to NF449 (10 μm) sDeps were completely abolished within 15 min (n= 4; Fig. 2C).

Figure 2

Spontaneous depolarizations are neurogenic
A, α-latrotoxin (LTX; 1 nm), which serves to increase the rate of spontaneous neurotransmitter release, caused an increase in the frequency of sDeps and spontaneous action potentials (sAPs). B, similarly, the superfusion of LTX and atropine (1 μm) produced a marked increase in the frequency of sDeps and sAPs. C, superfusion of LTX in combination with NF449 (10 μm), however, abolished all depolarizations. Data shown (i.e. control, then following 15 min drug superfusion) are excerpts from continuous recordings.

Pharmacology of spontaneous action potentials

Spontaneous action potentials (sAPs; of amplitude: 53.4 ± 2 mV) were observed in 38% of SMCs (i.e. 11 of 29, from n= 4) where they typically occurred at a low frequency (0.77 ± 0.20 min⁻¹, range: 0.2–2.6). Following superfusion of the P2X₁ antagonist NF449 (10 μm) for 1 h, sAPs were not observed in any SMC (i.e. 28 of 28, from n= 4). To confirm that NF449 had no direct effect on L-type Ca²⁺ channels, SMC membrane properties were examined with a series of brief current injections. In control cells and those superfused with NF449 (10 μm) for 1 h, current injections ≥+0.09 nA stimulated active currents consistent with normal opening of L-type Ca²⁺ channels (see online Supplemental material, Fig. 1).

Superfusion of the P2X₁-desensitizing agent α,β-mATP (1 μm) reduced the number of SMCs in which sAPs were observed from 17 of 25 (from n= 4) in control cells to 4 of 20 following α,β-mATP superfusion (Fisher's exact test P < 0.05). The frequency of sAPs occurring in cells that had at least one sAP was reduced from 1.17 ± 0.44 min⁻¹ in control cells to 0.141 ± 0.009 min⁻¹ (P < 0.05, n= 4).

By contrast to the effects of NF449 and α,β-mATP, the muscarinic acetylcholine receptor antagonist atropine (1 μm) did not affect the frequency of sAPs, which was 0.54 ± 0.37 min⁻¹ in control cells and 0.89 ± 0.58 min⁻¹ following 1 h application (post-atropine, 126 ± 39% of controls; P= NS, n= 4).

In order to determine the contribution of spontaneous activation of nerve branches or spontaneous nerve terminal action potentials to sAPs, the frequency of sAPs was compared following superfusion of TTX (1 μm). The frequency of sAPs was similar in control cells (0.52 ± 0.3 min⁻¹, range: 0.1–2.1; observed in 6 of 28 SMCs, from n= 4) to those superfused with TTX for 1 h (0.73 ± 0.40 min⁻¹, range: 0.1–2.1; observed in 7 of 28 SMCs, from n= 4) (P= NS, Student's t test). The amplitudes of sAPs were unaffected by TTX superfusion (control: 50.1 ± 3.0 mV, post-TTX: 51.5 ± 2.0 mV; P= NS, Student's t test).

In order to investigate whether sAPs result from nerve-released ATP, experiments were performed in the presence of LTX. Following the superfusion of LTX (1 nm) for 10 min, sAP frequency was increased to 930 ± 450% of control (i.e. 1.9 ± 0.5 min⁻¹ to 11.0 ± 3.1 min⁻¹; P < 0.05, n= 4). sAPs were also increased in frequency following superfusion of LTX and atropine (1 μm) (600 ± 370% of control; from 0.4 ± 0.1 min⁻¹ to 2.8 ± 1.8 min⁻¹; P= NS, n= 3), but sAPs were abolished by LTX and NF449 (10 μm) (9 ± 5% of control; from 1.5 ± 0.6 min⁻¹ to 0.1 min⁻¹; P < 0.001, n= 4).

The relationship between sDeps and sAPs

To determine whether sDeps might intermittently initiate sAPs, their kinetics were compared. Both sDeps and sAPs occurred with variable rising phases, with times-to-peak ranging from 5 to 89 ms for sDeps (median: 30 ms, no. of events = 4755, e.g. Fig. 3A) and 11–49 ms for sAPs (median: 19 ms, no. of events = 369, e.g. Fig. 3B). In order to test the hypothesis that the more prolonged rise times of sDeps indicate a lower peak current after ATP binding to P2X₁ receptors, the rising phases of both sDeps and sAPs were differentiated and compared. The differentiated rising phases of sDeps usually contained one peak, which varied in amplitude with a positively skewed distribution from 0.4 to 4.5 V s⁻¹ (Fig. 3A). By contrast, the differentiated rising phase of sAPs always contained two determinable peaks and the broad amplitude frequency distribution (0.6–6.3 V s⁻¹) of the first peak is consistent with the highly variable time-to-peak of sAPs (Fig. 3B). Comparing the first peak of the differentiated rising phase of the depolarization revealed that the amplitude of the first derivative was much greater for sAPs (median: 2248 mV s⁻¹, range: 454–4913) than sDeps (median: 439 mV s⁻¹, range: 334–950) (P < 0.0001, n= 12, Wilcoxon signed rank test; Fig. 3C).

Figure 3

Peaks in the differentiated rising phases of sAPs are of, on average, larger amplitude than the differentiated peaks of sDeps
A, sDeps (grey trace) occurred with variable rising phases. When differentiated (black trace), peaks (arrows) occurred with a positively skewed amplitude distribution. B, sAPs (grey trace), which occurred with similarly varying waveforms as sDeps, reveal two peaks (arrows) when differentiated (black trace); the amplitude of the first peak of which has a broad amplitude distribution. Data shown for A and B are from a single, long-duration recording. Bin sizes for the amplitude distributions of first derivatives are 0.125 V s⁻¹. Note that the scale bars associated with the raw data of A and B are of differing magnitudes, and that the differentiated sAPs (B) are truncated at –1 V s⁻¹. C, the median amplitude of the first peak of the differentiated sAP is greater than the amplitude of the differentiated sDep. Data are median, quartiles and range (n= 12). See text for details of statistical comparison.

The L-type Ca²⁺ channel antagonist nifedipine (1 μm) abolished sAPs (Fig. 4A), and thus removed all depolarizations > 40 mV (Fig. 4B). We wanted to test the hypothesis that a subpopulation of large-amplitude depolarizations may trigger sAPs. If so, nifedipine should reveal a subpopulation of large-amplitude sDeps. No such subpopulation of large amplitude (e.g. 30–40 mV) sDeps was observed (e.g. data from a single, long-duration recording. Control, median amplitude: 8.8 mV, interquartile range: 6–13 mV; post-nifedipine: 8.1 mV, 6–14 mV; no. of sDeps was 996 and 355 for the control and post-nifedipine periods, respectively. P= NS, Mann–Whitney U test) (Fig. 4B).

Figure 4

Blocking sAPs, through the application of nifedipine, reveals a subpopulation of sDeps with an especially rapid rising phase
A, superfusion of nifedipine (1 μm; 15 min superfusion) abolished sAPs in murine detrusor smooth muscle. A spontaneous depolarization with a following prolonged hyperpolarization was incidentally observed in the trace in the presence of nifedipine; such events were also occasionally observed in control cells (not shown) and have not been further characterized. Data shown are from a continuous recording. B, the amplitude frequency distribution post-nifedipine does not reveal a subpopulation of large amplitude sDeps; only the loss of depolarizations > 40 mV in amplitude. The threshold for detecting sEJPs was 2 mV. C, differentiated rising phases of sDeps reveal an increase in the frequency of larger amplitude events following nifedipine (1 μm) superfusion, compared to a control period.

However, the peak inward current through P2X₁ receptors might provide a more sensitive measure of transmitter release than the amplitude of sDeps. To assess the inward current through P2X₁ receptors, the differentiated rising phases of only sDeps, pre- and post-superfusion with nifedipine (1 μm), were compared. The amplitude of the first derivative of the sDep increased from 237 mV s⁻¹ in control cells (median amplitude; range: 182–359) to 268 mV s⁻¹ (range: 196–422) following nifedipine superfusion (i.e. to 113 ± 3% of controls; Student's one-tailed paired t test, P < 0.05, n= 4) (data shown for one experiment, Fig. 4C). In control cells of these experiments, sAPs represented 12% (range: 3–21) of all depolarizations.

The relationship between spontaneous depolarizations and focal Ca²⁺ transients

Loading detrusor smooth muscle with the Ca²⁺ indicator Oregon Green BAPTA-1 AM revealed three types of spontaneous Ca²⁺ transient (whole-cell Ca²⁺ flashes, Ca²⁺ waves and focal Ca²⁺ transients), as reported by Meng et al. (2008). Simultaneous electrophysiology and confocal imaging of Ca²⁺ indicator fluorescence was used to determine the relationship between spontaneous depolarizations (sDeps) of the membrane potential of SMCs, with spontaneous focal, transient increases in fluorescence. Focal Ca²⁺ transients (Fig. 5A) were coincident with sDeps (Fig. 5B and C).

Figure 5

Spontaneous focal Ca²⁺ transients are coincident with spontaneous depolarizations (sDeps) in mouse urinary bladder smooth muscle
A, a region of a smooth muscle cell (SMC) in a mouse isolated urinary bladder (loaded with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM) during an intracellular recording. Images, acquired at 13.5 Hz, are 4 consecutive frames of a 200 frame series. A spontaneous focal Ca²⁺ transient occurred on frame ii (arrowhead). B, intracellular recording of a period that includes the same four frames that compose A, showing simultaneous recordings of membrane potential (black line) and whole-cell fluorescence (grey dots). C, simultaneous Ca²⁺ imaging and electrophysiology demonstrate the coincidence of focal Ca²⁺ transients and sDeps during a longer period.

The relationship between these two events was explored with higher temporal resolution (2 kHz), by correlating line-scans taken along the long axis of a SMC (Fig. 6A), with a simultaneous measure of the membrane potential, recorded with an intracellular electrode impaled in the imaged cell. A focal Ca²⁺ transient occurred as a local event along the x-axis of the line-scan (Fig. 6B). A measure of the intensity of the Ca²⁺ fluorescence for this region revealed a rapid rise in [Ca²⁺] over a period of approximately 100 ms, and then a slow decline to baseline (Fig. 6C). For the same period of time and in the same cell, a sDep was observed that exhibits a rise-to-peak amplitude and time course of decay that was much more rapid than the concurrent focal Ca²⁺ transient (459 ms; median half-decay time: 568 ms, range 329–921; n= 3). Expanding the scale (Fig. 6D), the rise in [Ca²⁺] starts approximately 8 ms after the initiation of the sDep. The latency of rise in [Ca²⁺] relative to the initiation of a sDep was calculated for 12 events occurring in preparations from three animals. The median latency was 10.4 ms (range: 4.3–25.8 ms).

Figure 6

Simultaneous Ca²⁺ imaging and electrophysiological measurement at a higher temporal resolution of imaging reveal the temporal relationship between focal Ca²⁺ transients and sDeps
A, a region of a smooth muscle cell (SMC) in a mouse isolated urinary bladder (loaded with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM) during an intracellular recording. Line scans were taken along the long axis of an impaled SMC, the position of which is denoted by the dashed white line. B, line scan, acquired at 2 kHz, of the SMC (A) showing the occurrence of a focal Ca²⁺ transient (arrow). C, a measure of the Ca²⁺ fluorescence for a 20 μm region of the SMC in which the focal Ca²⁺ transient occurs (grey) compared to the membrane potential of the same cell for the same period of time (black). D, the occurrence of the focal Ca²⁺ transient (grey) and sDep (black) shown on an expanded time scale. The dark grey line of Ca²⁺ fluorescence (C and D) is a curve fitted using a custom-written script; see Methods for details.

The model fit to the Ca²⁺ signal (e.g. Fig. 6D) is based on an exponentially declining calcium influx, which is then removed by a first-order process. The change in membrane potential is proportional to the number of P2X receptors open (i.e. the conductance change) and hence to the rate of Ca²⁺ influx (see Appendix). The sEJP fall time (median: 40 ms, interquartile range: 30–44 ms, calculated for 12 events which occurred in preparations from 3 animals) was similar to the estimate of the rate of Ca²⁺ influx, 1/k₂ (median: 42 ms, 30–75 ms). There was no significant difference in the median time course of the sEJP and the modelled Ca²⁺ influx (1/k₂) (P= NS, Mann–Whitney U test), although these two measures were not correlated on a pair-wise basis (Spearman rank correlation, ρ=−0.23, and P= NS).

In addition to the temporal correlation, there was also a correlation in the amplitudes of focal Ca²⁺ transients and sDeps, both for large and small amplitude events (Pearson product moment correlation, ρ > 0.41 and P < 0.05 for each of n= 3 experiments; Fig. 7).

Figure 7

Focal Ca²⁺ transients and concurrent spontaneous depolarizations are correlated in amplitude
Correlations are shown for three recordings in which there were sufficient events to draw comparison of amplitudes.Focal Ca²⁺ transient amplitude =, where ΔF is the change in fluorescence, F_o the resting fluorescence, and ‘area’ is the area over which the fluorescent signal is measured (in pixels) divided by 32; this is hence an arbitrary unit.

Whole-cell Ca²⁺ flashes and Ca²⁺ waves were also observed during simultaneous recordings, but both events occurred infrequently. Whole-cell Ca²⁺ flashes were observed to be concurrent with sAPs but Ca²⁺ waves did not correlate with any changes of the membrane potential of the impaled SMC.

In order to establish whether focal Ca²⁺ transients were associated with nerve terminal varicosities, parasympathetic neurones were visualized using the fluorescent dye, 3,3-diethyloxardicarbocyanine iodide (DiOC₂(5); Yoshikami & Okun, 1984). Focal Ca²⁺ transients were observed close to nerve terminal varicosities (e.g. online Supplemental movie).

Discussion

Purinergic neurotransmission in the detrusor muscle of the human urinary bladder is up-regulated in pathological states (Palea et al. 1993) and with ageing (Yoshida et al. 2001). The mouse is an ideal model for the study of purinergic neurotransmission due to the relative ease with which transgenic animals can be produced (e.g. Vial & Evans, 2000) and because purinergic neurotransmission is a major component of detrusor excitation in this species (Holt et al. 1985; Meng et al. 2008). Thus, the aim of the current study was to characterize further spontaneous purinergic neurotransmission in the mouse urinary bladder, by first determining the origin of frequent ‘spontaneous depolarizations’ and spontaneous action potentials in smooth muscle cells (SMCs), and second to investigate their relationship to ‘purinergic Ca²⁺ transients’ (Heppner et al. 2005) identified in rat urinary bladder smooth muscle cells.

Spontaneous depolarizations are neurogenic, purinergic spontaneous excitatory junction potentials

ATP, released from nerve terminals, acts on P2X₁ receptors to cause frequent spontaneous depolarizations (sDeps) in the mouse urinary bladder. First, sDeps recorded using intracellular electrophysiology from SMCs were demonstrated to be purinergic in origin, consistent with the study of Meng et al. (2008). In the present study, sDeps were shown to be the result of ATP binding to P2X₁ receptors, as they were almost abolished by the specific P2X₁ receptor antagonist NF449 and the desensitizing agonist α,β-mATP (Fig. 1B). This conclusion is supported by the study of Vial & Evans (2000), in which ATP-induced inward currents were not present in bladder SMCs isolated from P2X₁ receptor-deficient mice. Also consistent with Meng et al. (2008) was the insensitivity of sDeps to atropine (1 μm; Fig. 1B). Second, the ATP that causes sDeps was shown to be released from nerve terminals. Following superfusion of α-latrotoxin (LTX; 1 nm), an agent which stimulates spontaneous neurotransmitter release (Ceccarelli & Hurlbut, 1980), the frequency of sDeps increased markedly (Fig. 2A). LTX-stimulated sDeps were abolished by NF449 (Fig. 2C) but were insensitive to atropine (Fig. 2B), suggesting that LTX did not stimulate acetylcholine release that, in turn, stimulated ATP release from, for example, SMCs.

The possibility that sDeps could be generated by spontaneous transient inward currents (‘STICs’) seems unlikely given that the time course of sDeps (49 ± 26 ms; Meng et al. 2008) is an order of magnitude briefer than stretch-induced STICs in isolated SMCs (approx. 500 ms; Ji et al. 2002). The detection of the sDeps before the onset of local Ca²⁺ transients (by about 10 ms), also argues against the Ca²⁺ transient activating a local ion conductance to generate a depolarization.

It should also be noted that there is unlikely to be any influence from urothelium-derived ATP (produced, for example, in response to stretch; Burnstock, 2001) as experiments were performed on muscle strips following careful removal of the urothelium, and recordings were made from the serosal surface.

The observations that sDeps were (i) increased in frequency by LTX, (ii) unaffected by TTX, and (iii) that their time course is very different to that of STICs recorded from isolated myocytes, imply that sDeps are the result of spontaneous neurotransmitter release from nerve terminals and are therefore spontaneous excitatory junction potentials (sEJPs).

Spontaneous action potentials (sAPs) are neurogenic and purinergic

Spontaneous action potentials (sAPs) were demonstrated to have a similar origin to sEJPs, i.e. nerve terminal-released ATP. sAPs, present in 38% of control cells sampled, were not observed in any cells following the superfusion of NF449 and were almost abolished following P2X₁ receptor desensitization with α,β-mATP. By contrast, atropine (1 μm) did not affect the frequency of sAPs. These findings suggest that sAP frequency at rest is driven by the action of ATP on P2X₁ receptors rather than by acetylcholine.

sAP frequency was not influenced by spontaneous activation of nerve branches or spontaneous nerve terminal action potentials, as the frequency of sAPs was similar in control cells (0.52 min⁻¹; observed in 6 of 28 SMCs) to those superfused with TTX for 1 h (0.73 min⁻¹; observed in 7 of 28 SMCs). In order to investigate whether sAPs result from nerve terminal-released ATP, experiments were performed in the presence of LTX. Superfusion of LTX (1 nm) for 10 min increased sAP frequency to 930 ± 450% of control.

During the present series of experiments, the sAPs had a broad range of rise times with a prominent after-hyperpolarization, similar to the pacemaker-type sAPs previously described (Meng et al. 2008). Very few action potentials with only an after-depolarization (previously described as spike-type) were observed; such a difference might have arisen because the shallow angle of incidence of the electrode used in present work might select more superficial cells than the more vertical electrode approach used by Meng, Young and Brading (J. S. Young, personal observation). The shallow angle of electrode approach is necessary for the simultaneous Ca²⁺-imaging experiments, so simultaneous recordings of a significant number of spike-type sAPs with Ca²⁺ measurements presents an unconquered technical hurdle.

The relationship between sEJPs and sAPs

It is likely that the EJP is the basis of urinary bladder smooth muscle action potentials; weak electrical stimulation evokes only an EJP, while stronger stimulation evokes an action potential (e.g. Creed et al. 1983; Brading & Mostwin, 1989).

Both sEJPs and sAPs occur with highly variable times-to-peak, ranging from 5 to 89 ms for sEJPs and 11–49 ms for sAPs. The peak amplitude of the first-time derivative of the rising phase is proportional to the peak current into the cell (Young et al. 2007b), thus in order to test the hypothesis that sAPs arise when the P2X₁ receptor current is high, the rising phases of both sEJPs and sAPs were differentiated for comparison. For a sAP, the differentiated rising phase revealed two peaks; the first attributed to the influx of cations through the P2X receptor, and second, the influx of Ca²⁺ ions through VDCCs (Fig. 3B). Thus comparing only the first peak of the differentiated rising phase of the depolarization revealed that the amplitude of the first derivative was much greater for sAPs than sEJPs. To further assess whether VDCC opening is correlated with the peak current into the cell through P2X₁ receptors, the differentiated rising phases of sEJPs were compared pre- and post-VDCC blockade. There was a significant increase in the amplitude of the first derivative following nifedipine (1 μm) superfusion. The magnitude of the increase in first-derivative amplitudes following nifedipine superfusion was, however, relatively small (13 ± 3%), but this is consistent with sAPs representing only a small proportion (12%) of the depolarizations observed.

An increase in the median first-derivative amplitude following VDCC blockade, taken together with a greater amplitude of the first derivative for sAPs than sDeps, suggests that sAPs are generated when a large-amplitude post-junctional response to spontaneous ATP release from nerve terminals depolarizes the smooth muscle membrane potential to the voltage threshold for L-type Ca²⁺ channels.

Focal Ca²⁺ transients are concurrent with sEJPs in mouse urinary bladder SMCs

Confocal imaging of Ca²⁺ fluorescence identified, for the first time, frequent, spontaneous focal Ca²⁺ transients in SMCs of the mouse urinary bladder (Fig. 5A). These events were similar in appearance and time course to purinergic Ca²⁺ transients of SMCs of mouse vas deferens (Brain et al. 2002), rat mesenteric arteries (Lamont & Wier, 2002) and the rat urinary bladder (referred to there as ‘localized Ca²⁺ transients’; Heppner et al. 2005). To determine the relationship between the focal Ca²⁺ transients of the mouse urinary bladder and sEJPs, simultaneous intracellular electrophysiology and confocal imaging of Ca²⁺ fluorescence were performed. A temporal correlation between focal Ca²⁺ transients and sEJPs (Fig. 5B and C) was further examined at a high (2 kHz) resolution, which revealed a 10.4 ± 3.3 ms delay between the onset of a focal Ca²⁺ transient and a sEJP recorded from the same SMC (Fig. 6D). This delay is of similar magnitude to the delay at neuroeffector junctions of the mouse vas deferens (6 ms; Brain et al. 2002) and rat urinary bladder (12–16 ms; Heppner et al. 2005). Binding of ATP to P2X₁ receptors generates an influx of Na⁺ ions and Ca²⁺ ions (Isenberg et al. 1992; Evans et al. 1996), but it is unlikely that these movements are genuinely separated by 10 ms in SMCs of the mouse urinary bladder. During the first few milliseconds, the Ca²⁺ entering the cell is located close to the P2X receptors; the high local Ca²⁺ concentration saturates the high-affinity Ca²⁺ indicator so that the mean fluorescent signal underestimates the mean Ca²⁺ concentration during the initial period of Ca²⁺ entry. This non-linearity might account for at least part of the estimated delay. High endogenous Ca²⁺ buffering close to the plasma membrane would also induce such an effect. It is also possible that calcium-induced calcium release (CICR) augments the Ca²⁺ that enters the cell through the P2X₁ receptor upon ATP binding, causing the delay between the onsets of the sEJP and the focal Ca²⁺ transient. CICR has been demonstrated to contribute to purinergic Ca²⁺ transients in SMCs of the guinea-pig urinary bladder (Imaizumi et al. 1998; Hashitani et al. 2000) and mouse vas deferens (Brain et al. 2003), but plays little role in SMCs of the mesenteric arteries (Lamont & Wier, 2002) or urinary bladder (Heppner et al. 2005) of rat. The potential contribution by CICR to focal Ca²⁺ transients in mouse urinary bladder SMCs awaits further experiments to inhibit CICR.

Focal Ca²⁺ transients correlate in amplitude with concurrent sEJPs

In addition to temporal coupling, focal Ca²⁺ transients and sEJPs were also correlated in amplitude throughout the amplitude range (Fig. 7). It has been suggested that spontaneous focal transients only arise from the release of the contents of a subpopulation of synaptic vesicles, giant dense-cored vesicles (Blair et al. 2003), rather than all vesicle types. The correlation of focal Ca²⁺ transients with even low-amplitude sEJPs, also observed in another smooth muscle organ, the mouse vas deferens (Young et al. 2007a), argues against this hypothesis. As argued elsewhere (Young et al. 2007a), an amplitude correlation of focal Ca²⁺ transients and sEJPs is contrary to the hypothesis that the variable amplitude of sEJPs results from the attenuation of depolarizations originating from distant release sites on neighbouring SMCs (Tomita, 1970; Purves, 1976). The hypothesis that the variable sEJP amplitudes represent the effects of ATP on only the impaled cell (Young et al. 2007a) is supported by the high input resistances of SMCs (388 ± 83 MΩ) in this tissue (Meng et al. 2008).

The observation that sEJP fall time (40 ms) was similar to the estimate of the rate of Ca²⁺ influx, 1/k₂ (42 ms) argues that the focal Ca²⁺ transients and sEJPs within a SMC are determined by a common mechanism – ion conductance through P2X₁ receptors.

As the focal Ca²⁺ transients described in the present study were shown to be correlated in timing and amplitude with sEJPs, themselves abolished by the P2X₁ receptor antagonist NF449, it is likely that the focal Ca²⁺ transients are purinergic in origin. It is unlikely that the focal Ca²⁺ transients observed are sparks, as their time course (half-decay time, 568 ms) was similar to purinergic Ca²⁺ transients of rat urinary bladder smooth muscle (112 ± 9 ms; Heppner et al. 2005) and much greater than that of sparks in urinary bladder smooth muscle (34 ± 5 ms, Heppner et al. 2005), mouse urinary bladder myocytes (69 ± 9 ms, Fritz et al. 2007) or vascular smooth muscle (range from 27 ms, Mironneau et al. 1996; to 61 ms, Jaggar et al. 1998).

Whole-cell Ca²⁺ flashes correlate with sAPs

The present work confirms a correlation between whole-cell Ca²⁺ flashes and sAPs. The whole-cell Ca²⁺ flashes seem to represent the same type of event described as ‘global Ca²⁺ flashes’ in rat urinary bladder smooth muscle cells (Heppner et al. 2005), as they have similar spatial and temporal characteristics, are abolished when L-type Ca²⁺ channels are blocked, and decreased in frequency by P2X receptor inhibition or desensitization. In both cases these Ca²⁺ transients arise as a consequence of smooth muscle action potentials, commonly initiated by the action of ATP on P2X receptors.

Conclusions

We have characterized spontaneous purinergic neurotransmission in mouse urinary bladder smooth muscle, demonstrating frequent sEJPs and sAPs that result from nerve-released ATP binding to P2X₁ receptors, sAPs occurring when the P2X₁ current is sufficient to trigger L-type Ca²⁺ channel opening. sEJPs correlate in timing and amplitude with focal Ca²⁺ transients, suggesting that both events result from the same quanta of ATP binding to P2X₁ receptors and providing further evidence for a variable effective post-junctional response to ATP binding in electrically well-coupled smooth muscle. The present study demonstrates that sAPs and sEJPs are triggered by the stochastic release of ATP from parasympathetic nerve terminals.

Appendix

In SMCs of many organs the time course of the sEJP is determined by the rate at which P2X₁ channels inactivate (Åstrand et al. 1988). To model the Ca²⁺ kinetics in a focal Ca²⁺ transient, consider the P2X₁ receptors opening rapidly at t= 0 and then closing stochastically with a time constant of k₂. It is hypothesized that the P2X₁ receptor is the primary, or only, conductance for Ca²⁺ under these conditions. Hence, the Ca²⁺ influx also falls exponentially with a rate constant of k₂. Assume that Ca²⁺ is removed from the cells by a first-order process with a rate constant of k₁, then the change in Ca²⁺ concentration (C) is given by:

(1)

where A_o is a constant proportional to Ca²⁺ influx at t = 0. This has the solution (given the limiting case of C= 0 as t goes to infinity):

(2)

This can be checked by substitution.

Fitting this function to experimentally obtained line-scanning confocal microscopy data of smooth muscle Ca²⁺ during a focal Ca²⁺ transient allows k₂ to be calculated and compared with the known sEJP time constant.

The time at the peak Ca²⁺ concentration (t_max; found when dC/dt= 0) is:

The peak Ca²⁺ concentration (C_max) is hence found (by substituting t_max into eqn (2)) to be:

So, for constants k₁ and k₂, the peak Ca²⁺ concentration (C_max) is proportional to the number of P2X₁ receptors opened at t= 0 (as this is proportional to A_o).

Appendix

Acknowledgements

Dr R. J. Amos (formerly of Department of Pharmacology, University of Oxford, UK) for providing the custom-written plug-ins for image analysis. The Tri-Service General Hospital, Taipei, Taiwan, ROC, for a DPhil studentship, to E.M. The Wellcome Trust provided financial support (069768 to T.C.C.; 074128 Research Fellowship to K.L.B). We are grateful to Mr Ben Wild (College of Richard Collyer, West Sussex) for mathematical checking of the Appendix.