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NEUROSCIENCE |
1 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
2
Division of Urology, Department of Surgery, Tri-Service General Hospital, no. 325, Sec. 2, Chenggong Road, Neihu, Taipei 114, Taiwan, ROC
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Abstract |
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(Received 21 August 2008;
accepted after revision 12 October 2008;
first published online 20 October 2008)
Corresponding author J. S. Young: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. Email: john.s.young{at}gmail.com
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.
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Methods |
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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 M
). 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 M) 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 (
F/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:
A is an arbritrary constant. The time of initiation (to) 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 
-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.
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Results |
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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 ,β-methylene ATP (,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-,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 ,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).
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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, -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).
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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 ,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 ,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 ,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).
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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).
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= –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, > 0.41 and P < 0.05 for each of n = 3 experiments; Fig. 7).
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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).
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Discussion |
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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 ,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 -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 ,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 M) 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.
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| (1) |
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| (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 (tmax; found when dC/dt = 0) is:
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| (3) |
The peak Ca2+ concentration (Cmax) is hence found (by substituting tmax into eqn (2)) to be:
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| (4) |
So, for constants k1 and k2, the peak Ca2+ concentration (Cmax) is proportional to the number of P2X1 receptors opened at t = 0 (as this is proportional to Ao).
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