The role of Ni2+-sensitive T-type Ca2+ channels in the regulation of spontaneous excitation in detrusor smooth muscles of the guinea-pig bladder

Authors


Yoshimasa Yanai, Department of nephro-urology, Nagoya City University Medical School, Nagoya 467–8601, Japan.
e-mail: y-yanai@med.nagoya-cu.ac.jp

Abstract

OBJECTIVE

To explore the role of Ni2+-sensitive T-type Ca2+ channels in the generation of spontaneous excitation of detrusor smooth muscles.

MATERIALS AND METHODS

In isolated detrusor smooth muscle bundles of the guinea-pig bladder, changes in the membrane potential and muscle tension were measured using intracellular microelectrodes and isometric tension recording. Changes in the intracellular Ca2+ concentration were recorded from bundles loaded with the fluorescent dye fura-PE3.

RESULTS

Detrusor smooth muscles had two types of spontaneous electrical activity, i.e. individual and bursting action potentials. Ni2+ (30 µm), a blocker for T-type Ca2+ channels, reduced the frequency of individual action potentials without changing their amplitude. Higher concentrations of Ni2+ (100–300 µm) converted individual action potentials into the bursts, as did apamin (0.1 µm), a blocker of small-conductance Ca2+-activated K+ channels (SK). They also increased the amplitudes of spontaneous Ca2+ transients and corresponding contractions whilst reducing their frequencies. In preparations which generated bursting action potentials, nifedipine (1 µm) converted action potentials into spontaneous transient depolarizations (STDs), and subsequent applications of Ni2+ (100 µm) abolished STDs. Gadolinium (100 µm) and SKF96365 (10 µm), blockers for nonselective cation channels, and niflumic acid (100 µm), a blocker for Ca2+-activated Cl– channels, had no effect on either the amplitude or frequency of spontaneous action potentials.

CONCLUSIONS

The T-type Ca2+ channel may have dual roles in generating spontaneous excitation in detrusor smooth muscles. First, activity of these channels may account for the preceding depolarizations that lead to action potentials. Second, Ca2+ influx through T-type Ca2+ channels may couple functionally to SK channels, contributing to the stability of the resting membrane potential in detrusor smooth muscle. Thus, pharmacological manipulation of T-type Ca2+ channels in detrusor smooth muscles could be of potential value for treating the overactive bladder.

Abbreviations
IC

interstitial cells of Cajal

BK channels

large-conductance Ca2+-activated K+ channels

R340/380

the ratio of the emission fluorescence

SK channels

small-conductance Ca2+-activated K+ channels STD, spontaneous transient depolarization.

INTRODUCTION

Detrusor smooth muscle strips from human and other mammalian bladders show spontaneous phasic contractions [1]. Underlying these contractions are spontaneous action potentials, which result from the opening of L-type Ca2+ channels [2], and corresponding Ca2+ transients [3]. In the normal bladder, spontaneous contractions of detrusor smooth muscles may account for micromotion of the bladder wall [4]. Regardless of whether spontaneous activity occurs as localized contractions or propagating waves of contraction, it does not initiate sufficiently synchronized contractions of the whole bladder to cause large increases in intravesical pressure [5]. However, spontaneous activity is thought to contribute to the ‘myogenic’ contractions that are often seen in patients with an overactive bladder [6]. Consistently, enhanced autonomous contractions have been reported in the isolated rat bladder after BOO [7].

In the gastrointestinal tract, interstitial cells of Cajal (IC) are now thought to play a fundamental role in both pacemaking of smooth muscles and neuromuscular signal transmission [8]. IC have also been identified in the urinary tract by their c-kit immunoreactivity or morphological criteria [9]. In the bladder, IC are seen in both suburothelial and detrusor smooth muscle layers, and are closely associated with intramural nerves [10–12], suggesting that they might also contribute to the generation of spontaneous excitation of the bladder.

In tissues where IC act as pacemaker cells, Ca2+ release from internal stores seems to play a key role in generating spontaneous excitation [13–15]. In such smooth muscles, both spontaneous depolarizations and corresponding inward currents are readily blocked by chemicals that disrupt the function of intracellular Ca2+ stores, e.g. cyclopiazonic acid, ryanodine or BAPTA-AM, regardless of their varied sensitivity to Ca2+-channel blockers. Conversely, spontaneous action potentials in detrusor smooth muscles persist under such conditions, whilst being abolished by L-type Ca2+-channel blockers, indicating that Ca2+ release from internal stores is not a fundamental process for initiating spontaneous action potentials in detrusor smooth muscles [16]. Therefore, voltage- and time-dependent changes in ionic conductance may play a primary role in generating spontaneous action potentials.

There is no doubt about the central role of L-type Ca2+ channels, which open to generate the rapid rising phase of detrusor action potentials [2,17]. During the action potentials, both depolarization and increases in intracellular calcium concentration ([Ca2+]i) activate large-conductance Ca2+-activated K+ (BK) channels to repolarize the membrane [18]. Ca2+-dependent inactivation of l-type Ca2+ channels may also contribute to the repolarization. Further openings of BK channels by Ca2+-induced Ca2+ release from intracellular stores then generate an after-hyperpolarization [16]. However, the mechanisms of the preceding depolarization which triggers the opening of L-type Ca2+ channels are unknown. It was shown that both L-type and T-type Ca2+ channels, which are activated around the resting membrane potential in detrusor smooth muscle, i.e. at − 50 to − 40 mV, have a physiological role in initiating excitation in the bladder [19]. Other candidates for the preceding depolarization could be nonselective cation channels or chloride channels, but their contribution to the generation of spontaneous action potentials also remains to be established.

In the present study, the effects of micromolar concentrations of NiCl2 on spontaneous action potentials, [Ca2+]i transients and contractions were examined. The results are interpreted through the effect of NiCl2 at these concentrations acting on T-type Ca2+ channels. The effects on spontaneous action potentials of blockers for nonselective cation channels and Ca2+-activated Cl channels were also studied.

MATERIALS AND METHODS

The procedures used were approved by the animal experimentation ethics committee at Nagoya City University Medical School. Male guinea-pigs (400–600 g) were killed by a blow to the head followed by cervical dislocation. The urinary bladder was removed and its ventral wall opened longitudinally from the bladder neck to the top of the dome. The mucosal layer, connective tissues and several smooth muscle layers were then removed, leaving underlying single smooth muscle bundles attached to the serosal layer. A serosal sheet, which contained one or a few single bundles of smooth muscle, 2–3 mm long and 0.2–0.7 mm wide, was then prepared, and experiments were carried out on the outer layer muscle bundles.

For the intracellular recordings, preparations were pinned on a silicone elastomer plate (Sylgard, Dow Corning Corp., Midland, MI, USA) at the bottom of the recording chamber (volume ≈ 1 mL), which was mounted on the stage of an inverted microscope. The preparations were superfused with warmed (35 °C) physiological saline at a constant flow rate (2 mL/min). Individual bladder smooth muscle cells in the muscle bundles were impaled with glass capillary microelectrodes filled with 0.5 m KCl (tip resistance, 120–250 MΩ). Membrane potential changes were recorded using a high input impedance amplifier (Axoclamp-2B, Axon Instruments, Inc., Foster City, CA, USA), and displayed on a cathode-ray oscilloscope (SS-5702, Iwatsu, Tokyo, Japan). After low-pass filtering (threshold frequency, 1 kHz), membrane potential changes were digitized using either Digidata 1322 or Digidata 1200 interfaces (Axon Instruments) and stored on a personal computer for later analysis.

For isometric tension recordings, detrusor muscle strips similar to those used for intracellular recordings were maintained in 0.5-mL organ baths containing physiological saline at 35 °C. Threads were was tied around each end of the muscle strips; one was attached to an isometric force transducer connected to a bridge amplifier (SEN-6102, Nihonkohden, Japan), and the other was attached to the bottom of the organ bath. Data were recorded using a PenRecorder (Mathushita, Japan). A tension of ≈ 1 mN was applied to preparations that were then left to equilibrate for 60–90 min until spontaneous phasic contractions were generated that were stable in both amplitude and frequency.

To measure changes in [Ca2+]i, detrusor smooth muscle preparations were pinned on the bottom of a recording chamber similar to that used for electrical and mechanical recordings. After a 30-min incubation with warmed (35 °C) physiological saline, spontaneous contractions of the tissues were generated, and then the preparations were loaded with the fluorescent dye fura-PE3 by incubating in low-Ca2+ physiological saline (1 mm Ca2+) containing 10 µm fura-PE3 AM for 1 h at room temperature. After loading, preparations were superfused with dye-free, warmed (35 °C) physiological saline at a constant flow (≈ 2 mL/min) for 30 min, and [Ca2+]i determined ratiometrically. Briefly, preparations loaded with fura-PE3 were illuminated with two periods of ultraviolet light, at 340 and 380 nm, alternating at >40 Hz. The ratio of the emission fluorescence (R340/380) in a desired size of rectangular window was measured through a barrier filter of 510 nm (sampling interval <200 ms), using a microphotoluminescence measurement system (ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan).

The composition of physiological saline was (in mM): Na+ 137.5, K+ 4.7, Ca2+ 2.5, Mg2+ 1.2, HCO3 15.5, H2PO4 1.2, Cl 134 and glucose 15. The solution was bubbled with 95% O2 and 5% CO2, and the pH of the solution was adjusted at 7.2–7.3 to maintain the pH of recording bath at ≈ 7.4.

Drugs used were fura-PE3 AM (Calbiochem-Novabiochem Ltd, San Diego, CA, USA), nickel chloride (Katayama Chemical Industry Co. Ltd, Osaka, Japan), gadolinium chloride (Aldrich Chemical Co., Inc., Milwaukee, WI, USA), α,β methylene-ATP (Me-ATP), apamin, atropine sulphate, nifedipine, niflumic acid, SKF96365, and tetrodotoxin (Sigma, St Louis, MO, USA). All but nifedipine were dissolved in distilled water; nifedipine was dissolved in 100% ethanol. The final concentration of these solvents in the physiological saline did not exceed 1 : 1000.

Measured values were expressed as the mean (sd) and were compared using paired t-tests, with P < 0.05 considered to indicate significant differences from the control. When drug effects were studied, the numbers of preparations refer to all the successful experiments carried out for each investigation.

The following variables of action potentials were measured: peak amplitude, measured as the value from the resting membrane potential to the action potential peak; leading dV/dt (dV/dtL), measured as the slope at 20–80% of the peak amplitude of events on the raising phase; half-width, measured as the time between 50% peak amplitude on the rising and falling phases; and trailing dV/dt (dV/dtT), measured as the slope at 20–80% of the peak amplitude of events on the falling phase.

For isometric tension changes, the peak amplitude, measured as the value from the basal tension level to the peak of phasic contractions, and frequency, defined as an average of 5-min recordings, were measured. For [Ca2+]i measurements, the following variables were recorded: peak amplitude, measured as the value from the basal [Ca2+]i level to the peak of [Ca2+]i transients (which was defined as an average of 2 ms on either side of the maximum point); half-width, measured as the time between 50% peak amplitude on the rising and falling phases; and frequency, defined as an average of 5-min recordings.

RESULTS

In all preparations examined, bladder smooth muscle cells showed spontaneous action potentials. The mean (sd, range) resting membrane potential determined at the most stable negative potential between each action potential was − 43.0 (4.9, − 34.7 to − 53.1) mV (n = 23). These values were similar to those reported previously [8]. In ≈80% of detrusor smooth muscle preparations individual action potentials were spontaneously generated, and their generation was prevented by nifedipine (1 µm, n = 4).

To investigate the possible contribution of T-type Ca2+ channels to the generation of spontaneous excitation, the effects of Ni2+, a blocker of these channels, on action potentials were assessed. Ni2+ (30 µm) reduced the frequency of spontaneous action potentials, from 19.5 (6.1)/min in controls, to 14.8 (7.2)/min (n = 7, P < 0.05) without changing their characteristics (Fig. 1Ab). Briefly, in the presence of Ni2+ (30 µm), action potentials had an amplitude of 50.2 (9.8) mV vs 51.8 (8.3) in the controls (P > 0.05) and a dV/dtL of 1.1 (0.58) mV/ms, vs 1.2 (0.79) in the controls (P > 0.05). Higher concentrations of Ni2+ (100–300 µm) clustered individual action potentials so that bursts of action potentials were generated (Fig. 1Bb). However, Ni2+ (100–300 µm) did not change the characteristics of the action potentials. For example, in the presence of 300 µm Ni2+, action potentials had an amplitude of 49.1 (9.84) mV, vs 50.8 (4.6) in the control (P > 0.05) and a dV/dtL of 1.9 (1.2) mV/ms, vs 0.9 (0.4) in the control (P > 0.05).

Figure 1.

Effects of Ni2+ on spontaneous action potentials recorded from detrusor smooth muscles of the guinea-pig. In control conditions, detrusor smooth muscles had spontaneous action potentials with an amplitude of ≈50 mV and a frequency of ≈20 min−1 (Aa). Ni2+ (30 µm) decreased the frequency of spontaneous action potentials without reducing their amplitude (Ab). In another preparation, individual action potentials were generated (Ba). A higher concentration of Ni2+ (100 µm) converted individual action potentials into the bursts (Bb). Resting membrane potential − 50 mV in A and − 48 mV in B.

As higher concentrations of Ni2+ converted individual action potentials into bursts, their effects on spontaneous action potentials were compared to those of apamin, a blocker for small-conductance Ca2+-activated K+ (SK) channels. Apamin (0.1 µm) converted individual action potentials into bursting action potentials without changing the characteristics of action potentials (Fig. 2Aa,b). In the presence of apamin, bursts were generated at 2.4 (0.48)/min, and each burst had a mean duration of 6.5 (0.72) s and was composed of 13.6 (2.8) action potentials (n = 6). In the presence of Ni2+ (300 µm), bursts were at 1.9 (0.74)/min (n = 6), and each burst had a duration of 7.5 (4.3) s and was composed of 4.9 (2.8) action potentials, while the action potential characteristics were unchanged (Fig. 2Ba,b). Thus, the effects of 300 µm NiCl2 and 0.1 µm apamin were similar on the pattern of spontaneous generation of action potentials.

Figure 2.

A comparison of the effects of apamin and Ni2+ on spontaneous action potentials recorded from detrusor smooth muscles of the guinea-pig bladder. In control conditions, detrusor smooth muscles had spontaneous action potentials (Aa). Apamin (0.1 µm) converted individual action potentials into bursts without affecting either action potentials or after-hyperpolarizations (Ab). In another preparation, in which individual action potentials were generated (Ba), Ni2+ (300 µm) clustered individual action potentials into bursts (Bb). In the presence of Ni2+, the characteristics of individual action potential were no different from those in the control condition (Bb). Resting membrane potential − 43.6 mV in A and − 47.3 mV in B.

In ≈ 20% of preparations, bursting action potentials were spontaneously generated in control solution. Unlike individual action potentials, which were invariably abolished by 1 µm nifedipine, bursting action potentials were converted into spontaneous transients depolarizations (STDs) with nifedipine (1 µm, n= 10, Fig. 3Aa, B).

Figure 3.

Effects of Ni2+ on STDs recorded from detrusor smooth muscles of the guinea-pig. In control conditions, detrusor smooth muscle cells had bursting action potentials (Aa). Nifedipine (1 µm), converted bursting action potentials into STDs and depolarized the membrane by ≈ 5 mV (Aa). Subsequent application of Ni2+ (100 µm) further depolarized the membrane and prevented the generation of STDs (Ab). On a fast time scale, STDs had a much slower time course than that of action potentials (B). The resting membrane potential was −45.3 mV.

In the presence of 1 µm nifedipine, the mean (sd, range) resting membrane potential was −38.9 (2.2, − 42 to – 35) mV (n = 10). STDs had a frequency of 14.1 (4.2, 8–22)/min, a peak amplitude of 35.2 (4.0, 29–41) mV, a leading dV/dt (dV/dtL) of 0.18 (0.07, 0.1–0.28) mV/ms, a half-width of 114.3 (48.1, 45–201) ms and trailing dV/dt (dV/dtT) of − 0.15 (0.94, − 0.1 to −0.19) ms.

Subsequent applications of Ni2+ (100 µm) initially reduced the amplitude and frequency of STDs, and then abolished them (n = 5, Fig. 3Ab). STDs were also abolished by switching from physiological saline to nominally Ca2+-free solutions (n = 3), suggesting that STDs result from the opening of T-type Ca2+ channels.

As Ni2+ modulated the generation of spontaneous action potentials, the effects of Ni2+ on spontaneous contractions were examined. Ni2+ (30 µm) reduced the frequency of contractions from 8.3 (2.9) to 3.0 (1.3)/min (n = 5, P < 0.05) without changing their amplitude, at 58.1 (22.2) mN in the control and 74.7 (41.2) mN in Ni2+ (P < 0.05, n = 5, Fig. 4B). A higher concentration of Ni2+ (100 µm) reduced the frequency of spontaneous contractions, from 8.3 (2.9)/min in the control and 2.9 (0.8)/min in Ni2+ (n = 5, P < 0.05) and increased their amplitude from 58.1 (22.2) mN in the control and 162.2 (113.6) mN in Ni2+ (n = 5, P < 0.05, Fig. 4C). Ni2+ (300 µm) further reduced the frequency of the contractions from 8.3 (2.9)/min in the control and 1.7 (0.5)/min in Ni2+ (n = 5, P < 0.05) and greatly increased their amplitude, from 58.1 (22.2) mN in the control to 385.3 (215.1) mN in Ni2+ (n = 5, P < 0.05, Fig. 4D).

Figure 4.

Effects of Ni2+ on spontaneous contractions in detrusor smooth muscles of the guinea-pig. Spontaneous contractions were recorded in the presence of tetrodotoxin (1 µm), atropine (1 µm) and α,β methylene-ATP (10 µm). In control conditions, a detrusor smooth muscle strip had spontaneous contractions (A). Ni2+ (30 µm) decreased the frequency of spontaneous contractions without changing their amplitude (B). A higher concentration of Ni2+ (100 µm) reduced the frequency of spontaneous contractions and increased their amplitude (C). Ni2+ (300 µm) further decreased the frequency and increased the contractions (D).

To further investigate cellular mechanisms of the Ni2+-induced modulation of spontaneous contractions, the effects of Ni2+ on spontaneous Ca2+ transients and corresponding contractions were examined. In control conditions, spontaneous Ca2+ transients had a frequency of 15.2 (2.8)/min and an amplitude of 0.10 (0.03) R340/380 and a half-width of 500 (97.2) ms (n = 5, Fig. 5A). Corresponding contractions were generated at the same frequency and had an amplitude of 0.76 (0.20) mN and a half-width of 1080 (180) ms (n = 5). Ni2+ (100 µm) reduced the frequency of spontaneous Ca2+ transients from 5.8 (1.6)/min (P < 0.05) and tended to increase their amplitude and half-width, at 0.12 (0.03) R340/380 and 660 (154.6) ms (n = 5, P < 0.05, Fig. 5Bb). It also reduced the frequency of corresponding contractions and increased their amplitude and half-width to 0.85 (0.23) mN and 1120 (250) ms (n = 5, P < 0.05, Fig. 5Ba). A higher concentration of Ni2+ (300 µm) further reduced the frequency of Ca2+ transients to 2.3 (1.1)/min (P < 0.05) and increased their amplitude and half-width to 0.16 (0.03) R340/380 and 1308 (285.4) ms (n = 5, P < 0.05, Fig. 5Cb). It also reduced the frequency of corresponding contractions and increased their amplitude and half-width at 2.5 (0.95) mN and 1570 (250) ms (n = 5, P < 0.05, Fig. 5).

Figure 5.

Effects of Ni2+ on the correlation between spontaneous Ca2+ transients and contractions. In a detrusor smooth muscle preparation, changes in [Ca2+]i (b) were simultaneously recorded with isometric muscle tension (a). In control conditions, spontaneous Ca2+ transients were invariably associated with spontaneous phasic contractions (A). Ni2+ (100 µm) increased the amplitude of Ca2+ transients and contractions, and reduced their frequency (B). A higher concentration of Ni2+ (300 µm) further increased the amplitude of Ca2+ transients and corresponding contractions, and reduced their frequency (C). All traces were recorded from the same preparation.

Ca2+-activated Cl channels are reported to contribute to endothelin-induced oscillations of the membrane potential in isolated detrusor smooth muscle cells of the pig [20], and could be responsible for the preceding depolarizations. Niflumic acid (0.1 mm), a blocker for Ca2+-activated Cl channels, did not change either the frequency or amplitude of action potentials in the isolated detrusor smooth muscle of the guinea-pig. In the presence of 0.1 mm niflumic acid, action potentials had an amplitude of 50.9 (4.3) mV vs 52.3 (3.2) in the controls (n = 3) and their frequency was 13.3 (7.40)/min vs 12.9 (6.8) in the controls (n = 3, not significantly different).

Non-selective cation channels have been identified in isolated detrusor smooth muscle cells of the guinea-pig and mice [20,21], and may account for the preceding depolarizations. SKF96365 (0.1 mm), a blocker for nonselective cation channels, did not change either the frequency, at 18.0 (12.1)/min in control and 18.7 (2.6) in SKF (n = 3, P > 0.05) or amplitude of action potentials, at 56.8 (6.1) mV in control and 54.2 (7.8) in SKF (n = 3, P > 0.05). Gadolinium, another blocker for stretch-activated cation channels, at (0.1 mm) also failed to significantly change either the frequency, at 14.9 (9.4)/min in the control and 15.4 (9.3) in gadolinium (n = 3) or amplitudes of spontaneous action potentials, at 57.6 (5.8) mV in the control and 58.3 (4.4) in gadolinium (n = 3).

DISCUSSION

The possible role of T-type Ca2+ channels in regulating spontaneous activity was investigated, the interpretation being derived from the use of micromolar concentrations of NiCl2, as this has been shown by others to have a predominant action on the blockade of T-type Ca2+ channels (see below). NiCl2 reduced the frequencies of spontaneous action potentials, Ca2+ transients, and associated contractions without reducing their amplitude. Higher concentrations of Ni2+ converted individual action potentials into bursts. Consistently, they also increased the amplitudes of spontaneous Ca2+ transients and associated contractions, whilst reducing their frequency.

In detrusor smooth muscles taken from humans and other mammals, L-type Ca2+ channels play a critical role in generating spontaneous excitation [22,23]. T-type Ca2+ channels have also been identified in isolated detrusor smooth muscle cells of both human and guinea-pigs, and their role in the initiation of spontaneous action potentials proposed [19]. T-type Ca2+ channels are activated at more negative membrane potentials than L-type Ca2+ channels, i.e. at − 60 to − 40 mV, and therefore they may contribute to the preceding depolarization to activate the L-type Ca2+ channels which play a dominant role in spontaneous excitation [24]. It was reported that Ni2+ reduced the frequency of spontaneous action potentials in the rabbit urethra without affecting their amplitude [25]. Consistent with those observations, in the present study, a relatively low concentration of nickel (30 µm) reduced the frequency of spontaneous action potentials without affecting their characteristics, suggesting that T-type Ca2+ channels may contribute to the preceding depolarizations, and thus determine the action potential frequency.

Besides inhibiting T-type Ca2+ channels, Ni2+ has been reported to affect several other ionic conductances, e.g. L-type Ca2+ channels, nonselective cation channels and Na+/Ca2+ exchange [26]. T-type Ca2+ channels are composed of different subtypes of α1-subunit of varying Ni2+ sensitivity [27]. As the T-type Ca2+ currents in detrusor smooth muscle cells of the guinea-pig bladder are reported to be fully blocked by 100 µm NiCl2[19], the subunit appears to be α1H. Therefore, the effects of Ni2+ on T-type Ca2+ channels should be separated from those on other ionic conductance. In fact, Ni2+ did not change either the amplitude or dV/dtL of action potentials, and thus it did not block L-type Ca2+ channels. Furthermore, as neither gadolinium nor SKF96365 [28] altered the frequency of spontaneous action potentials, it is again unlikely that Ni2+ inhibited these channels. It was reported that the level of resting [Ca2+]i in detrusor smooth muscle cells of the guinea-pig bladder is not determined by Na+/Ca2+ exchanger [29], and that millimolar concentrations of Ni2+ are needed to block Na+/Ca2+ exchanger [30]. Therefore, the Ni2+-induced modulation of spontaneous excitation is unlikely to result from inhibition of Na+/Ca2+ exchange.

As shown above, nifedipine converted bursting action potentials into STDs whilst preventing the generation of individual action potentials [22]. STDs were abolished either by Ni2+ or by nominally Ca2+-free solution, suggesting that T-type Ca2+ channels may contribute to their generation. As isolated detrusor smooth muscle cells are capable of generating action potentials, STDs might originate from a population of cells other than detrusor smooth muscle cells, such as IC. Indeed, nifedipine-insensitive Ca2+ transients have been recorded from IC in the guinea-pig bladder [9,31]. Furthermore, a c-kit inhibitor was reported to alter the pattern of spontaneous action potentials in detrusor smooth muscle of the guinea-pig [32]. Although we have not yet established the physiological role of IC in the bladder, they are probably not the primary pacemaker for detrusor smooth muscle excitation, as Ca2+ transients recorded from IC occurred independently from those of smooth muscle cells. Nevertheless, they may modulate the pattern of action potentials in larger preparations or whole bladders, where they compose an extensive network. Alternatively, there might be a different situation in an inner smooth muscle layer, which has a tight connection with suburothelial IC network [10].

Higher concentrations of Ni2+ converted individual action potentials into bursts; as a consequence of the clustering action potentials, the amplitude of spontaneous Ca2+ transients and corresponding contractions was increased but their frequency was reduced, indicating that the changes in Ca2+ and contractile responses are closely associated with alternations in the electrical responses. Apamin, a blocker for SK channels, also changed individual action potentials into bursts without altering the characteristics of action potentials [16,22]. Consistently, apamin also increased the amplitude and duration of spontaneous contractions but decreased their frequency [1,16]. As T-type Ca2+ channels activate at potentials around the resting membrane potentials, the continuous Ca2+ influx through these channels may contribute to the sustained activation of SK channels to stabilize the membrane potential. Indeed there is a substantial window current around this potential [19]. In dopaminergic neurones, selective coupling between T-type Ca2+ channels and SK channels has indeed been reported [33]. Therefore, inhibition of the putative coupling between T-type Ca2+ channels and SK channels may induce bursting action potentials. Relatively low concentrations of Ni2+ might block the regenerative activation of T-type Ca2+ channels required to generate the depolarizations without affecting SK channel activity. On the other hand, higher concentrations of Ni2+ may suppress SK channels activity as a consequence of the reduction of Ca2+ influx through T-type Ca2+ channels.

In the present study, other ionic channels that might regulate spontaneous excitability of detrusor smooth muscles were also examined. Ca2+-activated Cl channels are involved in the initiation of excitation in many varieties of smooth muscles [34–36]. For example, the spontaneous opening of Ca2+-activated Cl channels caused inward currents underlying STDs in the urethra [14,37]. In pig detrusor smooth muscles, Ca2+-activated Cl channels have been reported to be responsible for endothelin-induced oscillations in the membrane potential [20], although these channels could not be identified in isolated detrusor smooth muscle cells of the guinea-pig bladder. Niflumic acid, a known blocker for Ca2+-activated Cl channels, failed to modulate spontaneous action potentials even at relatively high concentration, suggesting that these channels do not contribute at least to spontaneous excitation in the guinea-pig bladder.

In relation to the importance of nonselective cation channels for the activation of l-type Ca2+ channels, it was reported that a tonically active, Na+-permeable current causes sustained depolarization of the membrane to facilitate action potential generation and thereby maintenance of bladder wall tone [38]. However, in the present study neither gadolinium nor SKF96365, blockers for nonselective cation channels, changed spontaneous action potentials, suggesting the physiological significance of these channels in regulating spontaneous excitations is minor, at least under our experimental conditions. As inhibiting these channels reduced the force produced by carbachol, they may be important either during muscarinic stimulations or in depolarized conditions [39].

In conclusion, T-type Ca2+ channels may cause dual modulation of the spontaneous excitation of detrusor smooth muscles of the guinea-pig bladder. Increasing activation of T-type Ca2+ channels would produce a depolarization to trigger the opening of L-type Ca2+ channels and initiate spontaneous excitation. Putative coupling between T-type Ca2+ channels and SK channels may contribute to the stability of the resting membrane potential of detrusor smooth muscles. Further investigations of the physiological significance of T-type Ca2+ channels might open up a new route for the pharmacological management of the overactive bladder.

ACKNOWLEDGEMENTS

The authors thank Prof A.F. Brading for her critical reading on the manuscript. This work was supported by a grant from Japan Society for the Promotion of Science (No.15591704, No.17390443) to H.H.

CONFLICT OF INTEREST

None declared.

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