Involvement of ryanodine receptors in muscarinic receptor-mediated membrane current oscillation in urinary bladder smooth muscle

Shunichi Kajioka,1 Shinsuke Nakayama,2 Haruhiko Asano,3 and Alison F. Brading1

1University Department of Pharmacology, Oxford, United Kingdom; and 2Department of Cell Physiology and 3Department of Molecular Medicine and Clinical Science, Nagoya University Graduate School of Medicine, Nagoya, Japan

Submitted 25 March 2004 ; accepted in final form 13 August 2004


    ABSTRACT
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 ABSTRACT
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The urinary bladder pressure during micturition consists of two components: an initial, phasic component and a subsequent, sustained component. To investigate the excitation mechanisms underlying the sustained pressure, we recorded from membranes of isolated detrusor cells from the pig, which can be used as a model for human micturition. Parasympathomimetic agents promptly evoke a large transient inward current, and subsequently during its continuous presence, oscillating inward currents of relatively small amplitudes are observed. The two types of inward current are considered to cause the phasic and sustained pressure rises, respectively. Ionic substitution and applications of channel blockers revealed that Ca2+-activated Cl channels were responsible for the large transient and oscillating inward currents. Furthermore, the inclusion of guanosine 5'-O-(2-thiodiphosphate) in the patch pipette indicates that both inward currents involve G proteins. However, applications of heparin in the patch pipette and of xestospongin C in the bathing solution suggest a signaling pathway other than inositol 1,4,5-trisphosphate (IP3) operating in the inward current oscillations, unlike the initial transient inward current. This IP3-independent inward current oscillation system required both sustained Ca2+ influx from the extracellular space and Ca2+ release from the intracellular stores. These two requirements are presumably SKF-96365-sensitive cation channels and ryanodine receptors, respectively. Experiments with various Ca2+ concentrations suggested that Ca2+ influx from the extracellular space plays a major role in pacing the oscillatory rhythm. The fact that distinct mechanisms underlie the two types of inward current may help in development of clinical treatments of, for example, urinary incontinence and residual urine volume control.

G proteins; micturition; oscillation; carbachol; SKF-96365


URINARY INCONTINENCE afflicts 15–30% of older individuals at home and 50% of those in nursing homes (30), leading to increasing demands for improved medical and social treatments for geriatric urinary incontinence and voiding dysfunction (25). Activation of the pelvic parasympathetic nerves causes excitation of detrusor smooth muscle during micturition. Excitatory neurotransmissions activate voltage-sensitive Ca2+ channels in detrusor cells connected via gap junctions and consequently produce whole bladder contraction (4, 7). The detrusor contractions induced by this nervous control are characterized by an initial phasic component and a subsequent sustained component (3). Correspondingly, cystometry shows an initial high bladder pressure at the start of voiding urine and a subsequent sustained elevation of pressure that can last long enough to complete voiding.

Comparative physiological and pharmacological studies have revealed that in guinea pig and rabbit, detrusor smooth muscle contractions induced by parasympathetic nerve stimulation contains a large atropine-resistant component of excitatory neurotransmission (5, 6). This component is nonadrenergic and noncholinergic. On the other hand, in the normal human detrusor, there is no atropine-resistant contraction, and in the pig, there is only a small nonadrenergic and noncholinergic response to nerve stimuli, although these smooth muscles clearly possess purinergic receptors in the plasma membrane (8, 13, 15, 35). These results suggest that humans and pigs have similar excitation-contraction (E-C) coupling mechanisms in controlling micturition.

Lines of evidence have been provided showing that Ca2+-activated Cl channels play important roles in E-C coupling and myogenic spontaneous rhythmicity in various smooth muscles (18, 21, 22, 33). Recently, we found that ETA receptor agonists promptly cause a large inward current and that during their continuous application, oscillatory inward currents with relatively small amplitudes persist. Furthermore, Cl channels seem to be responsible for both the initial transient and subsequent oscillatory inward currents (16). In this article, we report that similar inward currents are mediated by muscarinic receptors, which play a major role in human micturition. We propose that distinct intracellular Ca2+ release channels, i.e., inositol 1,4,5-trisphosphate (IP3) receptors and ryanodine receptors, contribute to activating the two inward currents. This fact might provide a new therapeutic target to control micturition in a phase-dependent manner in functional diseases of lower urinary tracts. The emphasis of the present study is especially placed on the latter, sustained component, which is necessary to complete voiding urine.


    METHODS
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 METHODS
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Preparation of cells. Pig urinary bladders were obtained from a local abattoir. The procedures for detrusor cell isolation were essentially the same as described previously (17). After the urothelium was removed, smooth muscle samples were cut into small pieces (1- to 2-mm cubes). The pieces were pretreated with 0.05% papain for 8 min and subsequently incubated with 0.15% collagenase and 0.05% trypsin inhibitor for 15 min in a Ca2+-free, Mg2+-free solution (at 36°C). Finally, after being washed with Ca2+-free, Mg2+-free solution (containing no enzymes), the digested pieces in a test tube were gently tapped until single cells were yielded. The single cells were kept in a low (0.5 mM) Ca2+-containing solution with 0.5% bovine serum albumin (BSA) and stored at 5°C. All experiments were performed within 3 h of the digestion.

Membrane current recording. Whole cell membrane currents were recorded using a standard patch-clamp technique (11). A patch-clamp amplifier (EPC-7; List, Darmstadt, Germany) was operated through a Macintosh computer equipped with an analog-to-digital/digital-to-analog converter (ITC-16; Instrutech). The resistance of the patch pipette was 3–4 M{Omega} when a Cs+-rich pipette solution was used. After rupture of the cell membrane, the series resistance was normally <10 M{Omega}. Smooth muscle cells used had a membrane capacitance of 61.6 ± 11.7 pF (n = 25). The capacitive surge was electrically compensated. A cut-off frequency of 10 kHz was applied to reduce the noise. Unless otherwise described, the cell membrane was clamped at –60 mV (holding potential). The experiments were carried out at room temperature (20–25°C).

Solutions and chemicals. The normal extracellular solution had the following composition (in mM): 140 NaCl, 6 KCl, 2.4 CaCl2, 12 glucose, and 5 HEPES; pH was adjusted to 7.4 with Tris base. This solution was also used as normal solution for cell isolation. Isosmotic substitution was used for all other changes in the ionic composition of the extracellular medium, including solutions used for cell isolation. In the Ca2+-free, Mg2+-free solution used for cell isolation, the osmotic pressure was also adjusted by modifying the NaCl concentration. In low extracellular Cl (low [Cl]out) solution, Cl was reduced to a concentration of 115 mM by substitution with equimolar aspartate.

The composition of the normal pipette solution was (in mM) as follows: 144 CsCl, 2 MgCl2, 0.05 EGTA, 2 ATP, and 10 HEPES-Tris (pH 7.2). The ionized Ca2+ concentration was set to ~100 nM by adding 25 µM CaCl2. This solution results in the blocking of all of the K+ channels in the membrane. In some experiments, 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S) or 5 mg/ml heparin were added to the pipette solution to suppress G protein action or antagonize IP3 receptors, respectively. In low [Cl] pipette (low [Cl]in) solution, Cl was reduced to 78 mM by substitution with equimolar aspartate.

With the normal extracellular and pipette solutions (a quasi-equivalent Cl condition), the reversal (equilibrium) potential (Erev) calculated for Cl is –0.5 mV (at 25°C). When the low [Cl]out solution with normal pipette solution or the low [Cl]in solution with normal extracellular solution was used, the Erev calculated for Cl was +6.5 or –16.9 mV, respectively.

The following chemicals, drugs, and enzymes were used in the present study: ATP (disodium salt), N-methyl-D-glucamine (NMDG), nifedipine, niflumic acid, GDP{beta}S (trilithium salt), EGTA (free acid), trypsin inhibitor (type 1-S), collagenase (type H), papain, carbamyl choline chloride (carbachol, CCh), and atropine, obtained from Sigma (St. Louis, MO); SKF-96365, obtained from Tocris (Ballwin, MO); and cyclopyazonic acid (CPA) and xestospongin C, obtained from Calbiochem (Nottingham, UK).

The volume of the recording chamber was ~0.2 ml. The extracellular solution was changed quickly (within a second) using a tear-drop device.

Statistics. Numerical data are expressed as means ± SD. The averaged amplitude of the inward current oscillation was calculated from the peaks of four to six currents when the amplitude of the oscillations had become stable. Differences between means were evaluated using t-tests or ANOVA; when a significant difference was permitted by ANOVA in the same category of groups, differences between groups were evaluated using the Bonferroni-Dunn test. A P value <0.05 was normally taken as a statistically significant difference.


    RESULTS
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Muscarinic agonist-induced inward currents. The effects of CCh application were observed on isolated pig detrusor cells, with outward K+ channel currents blocked by including Cs+ in the patch pipette. Throughout the following experiments, a holding potential of –60 mV was used, and the concentration of EGTA in the pipette was 50 µM. Under these conditions, inward currents were observed upon CCh application. In the majority of the cells, 10 µM CCh induced a single transient inward current (63%, n = 102), as shown in Fig. 1A. This current was completely blocked by the muscarinic receptor antagonist atropine (1 µM, n = 5). On the other hand, in ~40% of responding cells (n = 60), the initial transient inward current was followed by oscillating inward currents that continued throughout the application of CCh (10 µM). These oscillatory inward currents also were inhibited by 1 µM atropine (n = 5) (Fig. 1B). Thus both types of CCh-induced inward current were caused by activation of muscarinic receptors. During continuous application of CCh, the resting membrane current was not obviously affected.



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Fig. 1. Examples of the 2 types of response to carbachol (CCh) seen in pig urinary bladder cells. A: 10 µM CCh induced a transient inward current, and this response was blocked by 1 µM atropine. B: 10 µM CCh induced an initial large inward current and subsequent oscillating inward currents, and these responses were both blocked by atropine. Holding potential was –60 mV in A and B. Insets show the same traces on an expanded time scale. Arrows define the amplitude and duration of the inward currents. C: the relationships between amplitude and duration of the initial large inward current (a) and the subsequent oscillating inward current (b) are plotted for a number of cells. Data are shown as means ± SD (n = 5). Pairs of data showing a statistically significant difference are indicated (Bonferroni-Dunn test). D: 10 µM CCh was repeatedly applied. The interval was ~7 min.

 
The features of initial and oscillatory inward currents induced by 10 µM CCh application are compared with CCh-induced single inward current in Fig. 1C. The amplitude of the CCh-induced oscillatory currents (237 ± 189 pA, n = 38) was usually smaller than that of the initial inward current (475 ± 295 pA, n = 38). The duration of the initial inward current ranged widely (1.5–27.5 s; 8.0 ± 5.3 s), whereas that of the oscillatory inward current varied less (2.3–6.5 s; 4.1 ± 2.1 s). The mean durations were statistically significantly different (P = 0.0013), suggesting that distinct mechanisms were operating for the two types of inward currents. On the other hand, there were no significant differences in either amplitude or duration between CCh-induced initial inward current and single inward current.

Figure 1D shows repetitive applications of CCh. The first and second applications of 10 µM CCh induced essentially similar transient inward currents. Furthermore, in eight detrusor cells that showed oscillating inward currents, effects of prolonged application of 10 µM CCh were examined for up to 30 min. In all of the eight cells, inward current oscillation lasted until washout of CCh.

The effects of changing the CCh concentration and membrane potential are shown in Fig. 2. Figure 2A shows a typical tracing of the effects of 10 and 100 µM CCh. The frequency of the inward current oscillations [(no. of inward currents observed)/(duration of exposure to a particular CCh concentration)] significantly increased in a dose-dependent manner (Fig. 2Ba: 0.020 ± 0.002 Hz at 1 µM, 0.044 ± 0.008 Hz at 10 µM, and 0.086 ± 0.021 Hz at 100 µM; ANOVA, P = 0.0041), whereas the amplitude did not (Fig. 2Bb: 103 ± 26 pA at 1 µM, 283 ± 122 pA at 10 µM, and 244 ± 108 pA at 100 µM, n = 5). Figure 2C shows a typical current trace examining the voltage dependence of the inward current oscillations. During continuous exposure to 10 µM CCh, the holding potential was altered from –80 to –20 mV (n = 6). The frequency of the inward current oscillations increased with increasing negativity of the membrane potential (Fig. 2Da: 0.063 ± 0.029 Hz at –80 mV, 0.049 ± 0.012 Hz at –60 mV, 0.034 ± 0.006 Hz at –40 mV, and 0.017 ± 0.009 Hz at –20 mV). The oscillation frequency at –80 mV was more than three times greater than that at –20 mV. The amplitude of the inward current was maximal at –60 mV (Fig. 2Db: 314 ± 213 at –80 mV, 372 ± 233 pA at –60 mV, 276 ± 190 pA at –40 mV, and 128 ± 97 pA at –20 mV).



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Fig. 2. Effects of changing CCh concentration (A and B) and membrane potential (C and D) on inward current oscillation. A: oscillatory inward currents induced by 10 and 100 µM CCh in a typical cell. Holding potential was –60 mV. B: graphs showing effects of CCh concentration on frequency (a) and amplitude (b) of oscillatory inward currents. C: oscillatory inward currents in a typical cell observed under various membrane potentials. The extracellular solution contained 10 µM CCh. D: graphs showing effects of membrane potential on frequency (a) and amplitude (b) of oscillatory inward currents. Data are shown as means ± SD (B, n = 5; D, n = 6). Pairs of data showing a statistically significant difference are indicated (Bonferroni-Dunn test).

 
Ca2+ dependence of inward current oscillation. The ionic dependence of the CCh-induced inward current oscillations was examined by performing ion substitution experiments in the presence of 10 µM CCh. In the absence of extracellular Ca2+ (replaced with Na+), application of CCh (10 µM) evoked a transient inward current but did not produce oscillatory inward currents. After the extracellular Ca2+ concentration was returned to 2.4 mM, CCh induced oscillatory inward currents preceded by an initial inward current (Fig. 3A).



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Fig. 3. Extracellular Ca2+ dependence of CCh-induced inward current oscillation. A: after a membrane current response to 10 µM CCh was observed in the absence of extracellular Ca2+, the same concentration of CCh was applied in the presence of Ca2+ (2.4 mM). B: during exposure to 10 µM CCh, extracellular Na+ and Ca2+ were substituted with N-methyl-D-glucamine (NMDG), and subsequently, Ca2+ alone was reapplied. Note that CCh induced oscillatory inward currents in the absence of extracellular Na+. C: 10 µM CCh-induced oscillating inward currents observed in a typical cell in the presence of various concentrations of extracellular Ca2+. D: graphs summarizing Ca2+-dependent changes in frequency (a) and amplitude (b) of 10 µM CCh-induced oscillatory inward currents (n = 6). Holding potential was –60 mV.

 
As shown in Fig. 3B, extracellular Na+ and Ca2+ were substituted with NMDG during exposure to 10 µM CCh. This treatment greatly reduced the frequency of the inward current oscillations; however, the subsequent readmission of Ca2+ alone restored it. On the other hand, the current oscillations were not affected by simply increasing the extracellular Mg2+ concentration up to 10 mM in the presence of extracellular Ca2+ (2.4 mM) (data not shown). Also, when the intracellular Ca2+-buffering capacity was raised by increasing the EGTA concentration of the pipette solution from 50 µM to 2–10 mM, application of CCh caused neither the initial transient inward current nor subsequent spontaneous oscillatory inward currents (n = 15, data not shown). These results suggest that rises in transient Ca2+ in the cytoplasm mediate the inward current oscillations observed during CCh application and that Ca2+ influx from the extracellular space is necessary to maintain the intracellular Ca2+ oscillations.

The Ca2+ dependence of the inward current oscillation was further examined. As shown in Fig. 3C, the extracellular Ca2+ concentration was changed during continuous application of CCh (10 µM) (n = 6). The frequency of the inward current oscillations was significantly increased by increasing the extracellular Ca2+ concentration in a dose-dependent manner (Fig. 3Da: 0.0095 ± 0.0005 Hz at 0 mM, 0.016 ± 0.003 Hz at 1.2 mM, 0.044 ± 0.019 Hz at 2.4 mM, and 0.064 ± 0.024 Hz at 4.8 mM; ANOVA, P = 0.0087). However, the amplitude of the inward current was less sensitive to extracellular Ca2+ (Fig. 3Db: 295 ± 144 pA at 0 mM, 265 ± 132 pA at 1.2 mM, 308 ± 161 pA at 2.4 mM, and 207 ± 37 at 4.8 mM; ANOVA, P = 0.4495).

Effects of channel blockers. The effects of blockers for plasmalemmal ion channels and modulators for intracellular Ca2+ release channels were next examined. Oscillatory inward currents were induced by 10 µM CCh. Nifedipine (100 nM), which would cause complete suppression of voltage-sensitive (L-type) Ca2+ channel current in detrusor smooth muscle (17, 27), had little affect on the CCh-induced current oscillation (control: 0.035 ± 0.010 Hz, 560 ± 383 pA; 100 nM nifedipine: 0.033 ± 0.020 Hz, 540 ± 333 pA, n = 4) (Fig. 4 Aa). This result suggests that voltage-sensitive Ca2+ channels are not essentially required.



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Fig. 4. Effects of various channel blockers on CCh-induced oscillating inward currents. Nifedipine (100 nM; A), SKF-96365 (30 µM; B), niflumic acid (100 µM; C), ryanodine (30 µM; D), and caffeine (10 mM; E) were applied during inward current oscillation induced by 10 µM CCh. Holding potential was –60 mV. Aa: ramp pulses were applied before application of 10 µM CCh ({circ}) and during an inward current after addition of CCh, in the presence of 100 nM nifedipine ({bullet}). Ab: instantaneous I-V relationships obtained using a pair of ramp pulses. The patch pipette contained normal pipette solution when the current traces shown in Aa and B–E were recorded. In these experiments, the extracellular Cl ([Cl]out) and pipette ([Cl]in) solutions were quasi-equivalent. Ac: I-V relationship of CCh-induced oscillating inward current was plotted by subtracting membrane current {circ} from {bullet}. The I-V relationships obtained under low [Cl]out (115 mM) or low [Cl]i (78 mM) conditions are superimposed.

 
To further characterize the properties of CCh-induced oscillating inward currents, we applied ramp pulses in the presence of nifedipine (Fig. 4Aa). The instantaneous current-voltage (I-V) relationship (Fig. 4Ab) consequently obtained during the oscillatory inward current was nearly linear over –100 to +40 mV and much steeper than that of the control current recorded in the absence of CCh. Subtraction of the membrane current shows the I-V relationship of the CCh-induced inward current (Fig. 4Ac) (under a quasi-equivalent Cl condition; see METHODS). The Erev was 0.9 ± 1.0 mV (n = 5). The same ramp pulse protocol was applied under low [Cl]out (115 mM Cl in the bathing solution) or low [Cl]in (78 mM Cl in the pipette solution) conditions. Typical I-V relationships (after subtraction) for low [Cl]out and low [Cl]in conditions are superimposed in Fig. 4Ac. The Erev was +7.2 ± 0.8 mV under low [Cl]out conditions (n = 4) and –16.8 ± 1.5 mV under low [Cl]in conditions (n = 4). The shifts in Erev suggest that Cl channels are responsible for CCh-induced oscillatory currents.

SKF-96365, a known general channel blocker that would suppress Ca2+ influx through transient receptor potential (TRP) homolog channels (12, 28, 31, 38), significantly reduced the frequency and amplitude of the oscillating inward currents at 10 µM (to 0.68 ± 0.24 and 0.66 ± 0.05 of control, respectively, n = 4) and completely suppressed them at 30 µM (Fig. 4B). Similar inhibitory effects of this drug were observed in all cells examined (n = 5). Niflumic acid, a known blocker of Ca2+-activated Cl channels (10), significantly suppressed the oscillatory inward currents at a concentration of 100 µM, as shown in Fig. 4C (n = 8).

Figure 4D shows that ryanodine (30 µM) completely and irreversibly inhibited the CCh-induced oscillatory inward currents (n = 4). Caffeine is also known to deplete intracellular Ca2+ stores in smooth muscle (14). Figure 4E shows that application of 10 mM caffeine in the presence of CCh (10 µM) abolished oscillatory inward currents and that subsequent washout completely restored them (n = 6). In addition, after caffeine was washed out in the presence of CCh (10 µM), the frequency of the oscillation increased (3.44 ± 0.98 times, n = 6) and the amplitude decreased for 2–4 min (0.26 ± 0.14 times, n = 5). These results suggest that Ca2+ release from the intracellular stores plays an important role in generating the inward current oscillations seen during CCh application.

Assessment of IP3 signaling pathway. In detrusor smooth muscle, muscarinic agonists are known to release Ca2+ from the intracellular stores via G protein activation and subsequent IP3 formation (26, 36). Figure 5 shows the effects of the IP3 receptor antagonists heparin and xestospongin C (cell membrane-impermeable and -permeable IP3 receptor antagonists, respectively) (9, 32). In Fig. 5Aa, the patch pipette contained 5 mg/ml heparin. Approximately 15 min after rupture of the cell membrane, application of 10 µM CCh still caused oscillatory inward currents, and 10 mM caffeine completely abolished them. Note that this heparin treatment caused remarkable changes in the features of the first CCh-induced inward current. The latency (time until the first inward current observed after application of CCh) was significantly prolonged from 10.5 ± 4.7 (control, n = 9) to 28.4 ± 8.8 s (heparin, n = 5) (Fig. 5Ab). Furthermore, the amplitude of the first inward current (Fig. 5Ac) was decreased from 2.83 ± 1.46 (control, n = 9) to 1.26 ± 0.18 (heparin, n = 5) relative to the mean amplitude of the following oscillatory inward currents.



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Fig. 5. Effects of IP3 receptor and G protein blockers. Aa: the patch pipette contained 5 mg/ml heparin. CCh (10 µM) still caused oscillating inward currents. Subsequent application of caffeine (10 mM) abolished them, after causing a transient inward current. Ab and Ac: effects of heparin on latency (Ab) and amplitude (Ac) of the first transient inward current induced by CCh are compared. Amplitude of the first inward current was normalized to the mean value of the oscillating inward currents. B: after a control CCh-induced inward current oscillation was observed, CCh was applied again in the presence of 10 µM xestospongin C. C: the patch pipette contained 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S). CCh did not induce any inward currents. Three subsequent procedures were then applied to evoked inward currents: transient depolarization to 0 mV (100 ms) (a) and applications of 100 µM ATP (b) and 10 mM caffeine (c). Insets show expansion of membrane currents induced by the procedures. All experiments were carried out at a holding potential of –60 mV.

 
The effect of xestospongin C is shown in Fig. 5B. After a control response to 10 µM CCh had been observed, CCh was again applied in the presence of 10 µM xestospongin C. Current oscillation was again induced by CCh, but the amplitude of the first inward current was smaller in the presence of xestospongin C. Together, the results shown in Fig. 5, A and B, suggest that the initial transient inward currents seen in control experiments have actually been abolished by heparin or xestospongin C and that IP3 receptors do not play an essential role in the sustained CCh-induced oscillatory inward currents presumably caused by periodic rises in intracellular Ca2+ concentration ([Ca2+]i).

In Fig. 5C, the pipette solution contained 1 mM GDP{beta}S, which would completely inhibit the dissociation of {alpha}- and {beta}{gamma}-complexes of GTP-binding proteins. Application of 10 µM CCh caused neither the initial transient inward current nor the subsequent inward current oscillation (n = 25). In the detrusor cell study shown in Fig 5C, three procedures that would increase the [Ca2+]i independently of G proteins were subsequently carried out: 1) step depolarization to 0 mV (Ca2+ influx through voltage-sensitive Ca2+ channels), 2) application of 100 µM ATP (activation of Ca2+-permeable nonselective cation channels) (17), and 3) application of 10 mM caffeine (Ca2+ release from intracellular Ca2+ stores). Inward currents induced by the three procedures showed that Ca2+-activated ionic conductances, responses to P2X receptors, and Ca2+ release from intracellular stores are preserved even in the presence of GDP{beta}S. These results suggest that G proteins play an essential role in generating intracellular Ca2+ oscillations during continuous application of CCh.

In addition, we characterized intracellular signaling of the CCh-induced single transient inward current seen in the majority of detrusor cells. As shown in Fig. 6A, 10µM CCh evoked a transient inward current even in the absence of extracellular Ca2+ (amplitude: 317 ± 275 pA; duration: 9.35 ± 8.57, n = 4). Furthermore, after observing that 10 µM CCh failed to induce any inward current in the presence of heparin (5 mg/ml) in the pipette, we examined the effect of caffeine, which would cause a transient rise in [Ca2+]i. As shown in Fig. 6B, subsequent application of 10 mM caffeine evoked a transient inward current. Together, the results suggest that the single transient inward current is due to Ca2+ release from IP3 receptors.



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Fig. 6. Intracellular Ca2+ dependence of single transient inward current. A: applications of 10 µM CCh evoked a transient inward current regardless of extracellular Ca2+. B: the patch pipette contained 5 mg/ml heparin. CCh (10 µM) did not, but 10 mM caffeine did, cause a transient inward current. Holding potential was –60 mV.

 

    DISCUSSION
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 ABSTRACT
 METHODS
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Muscarinic receptors play a dominant role in the voiding contractions of detrusor smooth muscle in both the human and pig, whereas in other, smaller animals, such as the guinea pig and rabbit, nonadrenergic, noncholinergic neurotransmission is significant. In the present study, we thus used pig detrusor cells to investigate the underlying mechanisms of parasympathetic nerve activation-induced contractions (1, 17). In pig detrusor cells, application of CCh promptly evoked not only a large inward current but also, in about one-third of the cells, inward current oscillations, which persisted during application of CCh. It is generally accepted that these inward current oscillations reflect oscillations of [Ca2+]i (34). Our study provides evidence to suggest that the currents flow through Ca2+-activated Cl channels: 1) the currents were observed in Na+-free solution; 2) the currents were never observed in the presence of a high concentration of Ca2+ chelator in the pipette (4–10 mM EGTA; data not shown, n = 15); 3) the current Erev was ~0 mV, which was very close to the equilibrium potential of Cl, and furthermore, the Erev of the CCh-induced oscillating currents was shifted in a [Cl] gradient-dependent manner by lowering [Cl]out or [Cl]in; and 4) the inward currents were inhibited by the Ca2+-activated Cl channel inhibitor niflumic acid. Therefore, it is unlikely that this inward current oscillation occurs through nonselective cation channels operated by intracellular signals relating to Ca2+ stores. However, as discussed below, Ca2+ entry, presumably through this type of nonselective cation channel, appears to be the primary cause generating the inward current oscillation in the pig detrusor cells.

The present study suggests differences in the mechanisms involved in the production of the initial inward current evoked by CCh application and the subsequent oscillatory inward currents observed during CCh application. The initial inward current, but not the oscillatory inward currents, was observed even in the Ca2+-free solution (Figs. 3A and 6A). The amplitude and duration of the initial inward current were larger than those of oscillatory inward currents (Fig. 1C). Inclusion of the IP3 receptor antagonist heparin in the patch pipette resulted in failure of CCh to induce a larger initial inward current than the following oscillatory inward currents (Fig. 5). In addition, CCh could not induce any inward current in the presence of GDP{beta}S in the patch pipette. These results suggest that the initial inward current was mainly due to G protein-coupled IP3 formation, with the resultant Ca2+ release from the intracellular stores activating Cl channels. Similar membrane depolarizations coupled to a G protein-related mechanism have been reported in various types of smooth muscles (19, 34).

In contrast to the initial inward current, the oscillatory inward currents subsequently observed during continuous applications of muscarinic agonists show rather unique features, although the inward current itself flows through the same Ca2+-activated Cl channels. First, inclusion of heparin in the patch pipette and bath application of xestospongin C did not affect inward current oscillations (Fig. 5, A and B), suggesting a signaling pathway other than IP3 formation. However, when the G protein was inactivated by GDP{beta}S in the patch pipette, CCh could never induce oscillatory inward currents (Fig. 5C). Second, the CCh-induced inward current oscillations require Ca2+ influx from the extracellular space, although nifedipine-sensitive Ca2+ channels do not seem to be involved (Fig. 4Aa). Consistent with this feature, the oscillation frequency depends on the membrane potential (Fig. 2, C and D) and extracellular Ca2+ concentration (Fig. 3).

As shown in Fig. 2Db, the amplitude of oscillating inward current was decreased by hyperpolarizing the cell membrane to –80 mV, at which potential the driving force of Cl was the largest among the membrane potentials applied in the experiment. This may be explained by interaction between the frequency and amplitude of the oscillating inward current. At –80 mV, inward current oscillation is most frequent (Fig. 2Da). It is likely that frequent Ca2+ release from the sarcoplasmic reticulum (SR) would decrease the amount of Ca2+ to be released if the frequency were to exceed the ability for Ca2+ uptake into the SR. This hypothesis is also compatible with the fact that CCh applications from 1 to 100 µM consistently increased the frequency of oscillating inward current, while the amplitude decreased at 100 µM (Fig. 2, A and B).

Various oscillating membrane currents have been reported in other smooth muscles. We were able to compare the properties of membrane current oscillation seen in the present study with those previously described. Application of nifedipine affected neither amplitude nor frequency of the inward current oscillations induced by CCh in the present study, indicating that voltage-sensitive Ca2+ channels were not a major pathway in the Ca2+ influx maintaining the Ca2+ oscillations in pig detrusor cells. Similar muscarinic agonist-induced oscillating inward currents have previously been reported in several smooth muscles, for instance, pig tracheal and mouse anococcygeus smooth muscle cells (23, 34). Pharmacological examinations have revealed that the oscillating inward currents seen in pig tracheal and mouse anococcygeus muscles are due to Ca2+-activated Cl channels driven by rhythmic Ca2+ release from the intracellular Ca2+ stores. Ca2+ influx from the extracellular space is also required for these oscillations. However, unlike in pig detrusor cells, Ca2+ influx through voltage-sensitive Ca2+ channels does not make a major contribution to muscarinic agonist-induced inward current oscillation in these muscles. Furthermore, in the presence of IP3 receptor monoclonal antibodies or heparin in the patch pipette, muscarinic agonists did not induce any inward current, suggesting that IP3 plays an important role in those smooth muscles (24, 34). A different type of IP3-dependent oscillating inward currents has been reported in intestinal smooth muscle (20, 37). This oscillating inward current is ascribed to nonselective cation channels, and Ca2+ influx through voltage-sensitive Ca2+ channels is involved in generating this oscillation.

In pig detrusor cells, we found unique features of muscarinic agonist-induced current oscillations. CCh-induced oscillating inward currents are not affected by the IP3 receptor inhibitors heparin and xestospongin C but are terminated by ryanodine and caffeine. It is known that smooth muscles express ryanodine as well as IP3 receptors on the SR membranes and that these two receptors share common structural and functional features (2, 14). Therefore, it is considered that rather than the IP3 receptors previously reported in other smooth muscles, periodic Ca2+ release from ryanodine receptors produces the inward current oscillations seen in pig detrusor cells during CCh application. In relation to ryanodine receptors, it has been reported that subcellular Ca2+ sparks evoke spontaneous transient inward currents (STICs) in tracheal smooth muscle cells (39). The STICs seen in normal solution are sensitive to niflumic acid and ryanodine, but the amplitude (several tens of pA) is much smaller than that of the CCh-induced oscillatory inward currents in pig detrusor cells. It is speculated that more global intracellular Ca2+ events occur via muscarinic receptor activation in detrusor cells (see Supplemental Fig. 1, available online).1

SKF-96365 is often used to block the TRP homologs that are considered to form receptor-operated nonselective cation channels and store-operated Ca2+ channels in the plasma membrane (12, 28, 31, 38). (We have detected trp1 and trp6 in RT-PCR examinations of pig detrusor muscle; see Supplemental Fig. 2.) In the present study, applications of SKF-96365 completely abolished CCh-induced inward current oscillations, although the basal membrane currents suppressed by this drug were often below detectable levels. This result suggests that Ca2+ entry through this type of cation channel is the primary cause of the generation of the CCh-induced inward current oscillations in pig detrusor cells. The fact that CCh-induced basal inward current was too small to be observed in pig detrusor cells makes it tempting to speculate that SKF-sensitive Ca2+-permeable channels, presumably TRP channels, are closely coupled to ryanodine receptors in superficial SR and can efficiently activate Ca2+-induced Ca2+ release and/or that Ca2+-induced Ca2+ release is facilitated by some G protein-related mechanisms. Also, these channels may be highly Ca2+ selective, like ICRAC (Ca2+ release-activated Ca2+ current) (29). Furthermore, if the SKF-sensitive channels are store-operated type Ca2+ channels, there may be some factors linking the SKF-sensitive channels and the SR, where ryanodine receptors are present. In support of this hypothesis, the frequency of inward current oscillations just after the removal of caffeine was faster than that before application of caffeine (Fig. 4E), indicating that some unknown signal could be transmitted from the depleted Ca2+ store to this channel. Further investigation of this finding is necessary.

CCh-induced inward current oscillations were resistant to nifedipine. It should be noted that the present experiments were carried out under voltage-clamp conditions to clearly demonstrate the importance of SKF-sensitive Ca2+-influx pathways. In intact tissues, it is possible that Ca2+ influx through voltage-sensitive Ca2+ channels interferes with the mechanisms yielding inward current oscillation. Spontaneous inward currents are, however, indispensable in activating voltage-sensitive Ca2+ channels. The frequency of CCh-induced inward current oscillations is considered to be an index of the electrical excitability (not indicating the frequency of the action potentials in intact tissue directly).

In conclusion, the present experiments have demonstrated that application of parasympathomimetic agents produces an initial transient large inward current and subsequent relatively small oscillating inward currents through distinct mechanisms (Fig. 7). The initial inward current is mainly ascribed to IP3-induced Ca2+ release from the SR. On the other hand, the latter CCh-induced inward current oscillation is caused by IP3-independent mechanisms. Nifedipine-insensitive Ca2+ pathways, presumably nonselective cation channels activated by a G protein-related mechanism, seem to be a primary factor in pacing the oscillation. Also, ryanodine receptors are essential in periodically activating Cl channels to generate oscillatory inward currents.



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Fig. 7. Schematic diagram showing putative underlying mechanisms for the initial inward current induced by activation of muscarinic receptors and subsequent inward current oscillation. ACh, acetylcholine; RyR, ryanodine receptors; IP3R, IP3 receptors; PLC, phospholipase C; ChSKF, SKF-96365-sensitive channels.

 
Ca2+ oscillations may be a better way to transduce extracellular signals than a prolonged rise in Ca2+, because cells could avoid the toxic effects of high [Ca2+]i, general fatigue, and receptor desensitization, even in response to strong stimuli. It is generally believed that IP3-related mechanisms are indispensable for agonist-induced membrane current oscillations linked with intracellular Ca2+ rises, as has been reported for many cells and tissues (2), including smooth muscles. In this study, we have demonstrated a unique mechanism of muscarinic receptor-mediated membrane current oscillations that require ryanodine receptors but not IP3 receptors. If this is specific to the urinary bladder, it is worthy of further study, because the presence of two mechanisms (IP3 dependent and independent) contributing to the phasic and tonic contractions of urinary bladder might enable development of more selective drugs for the treatment of incontinence. For instance, selective suppression of the large initial, phasic contraction may prevent the involuntarily initiation of micturition.


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 ABSTRACT
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This work was supported by a Medical Research Council Project Grant from the United Kingdom. S. Nakayama was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Nobuoki Eshima (Oita University) for valuable advice on statistical analysis.


    FOOTNOTES
 

S. Nakayama, Dept. of Cell Physiology, Nagoya Univ. Graduate School of Medicine, Nagoya 466-8550, Japan (E-mail: h44673a{at}nucc.cc.nagoya-u.ac.jp)


Address for reprint requests and other correspondence: A. F. Brading, University Department of Pharmacology, Oxford OX1 3QT, UK (E-mail: alison.brading{at}pharm.ox.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supplemental figures for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00161.2004/DC1. Back


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