The involvement of L-type Ca²⁺ channels in the relaxant effects of the ATP-sensitive K⁺ channel opener ZD6169 on pig urethral smooth muscle

Abstract

1. The effects of ZD6169, a novel ATP-sensitive K⁺ channel (K_ATP channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle.

2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (⩽3 μM) was completely suppressed by additional application of glibenclamide (1 μM). In contrast, glibenclamide (1 – 10 μM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (⩾10 μM).

3. Bay K8644 (1 μM) reduced the maximum relaxation produced by ZD6169 (⩾10 μM).

4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba²⁺ currents in a concentration- and voltage-dependent manner, and at 100 μM, shifted the steady-state inactivation curve of the voltage-dependent Ba²⁺ currents to the left at a holding potential of −90 mV.

5. In cell-attached configuration, open probability of unitary voltage-dependent Ba²⁺ channels (27 pS, 90 mM Ba²⁺) was inhibited by 100 μM ZD6169 and by 10 μM nifedipine.

6. Reverse transcriptase-polymerase chain reaction (RT – PCR) analysis revealed the presence of the transcript of the α_1C subunit of L-type Ca²⁺ channels in pig urethra.

7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K_ATP channels at lower concentrations and inhibition of voltage-dependent Ca²⁺ channels at higher concentrations (about 10 μM).

Introduction

It has been reported that ZD6169, the S-enantiomer of the racemic compound N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide, increases the capacity of the urinary bladder following oral administration in a dose which produces minimal cardiovascular effects in normal conscious rats and dogs (Howe et al., 1995). Several results from electrophysiological and functional studies suggest that ZD6169 may activate ATP-sensitive K⁺ channels (i.e. K_ATP channels) in detrusor smooth muscle, hyperpolarize the membrane and inhibit the contractile force (Li et al., 1995; Trivedi et al., 1995). Thus, a general agreement seems to have been reached that ZD6169 is a detrusor-selective K_ATP channel opener.

Recently, by use of single-channel recordings, we have been able to confirm that a glibenclamide-sensitive K_ATP channel is indeed the target K⁺ channel for ZD6169, and further demonstrate that it has a dual effect (activation and inhibition) on the activity of K_ATP channels in pig urethra (Teramoto et al., 2001a). We suggested that ZD6169 may be useful for the treatment of incontinence, not only abolishing unstable contractions of detrusor muscle, but also inhibiting the activation of K_ATP channels in the urethra at relatively high concentrations (⩾30 μM). However, the effects of ZD6169 on the resting urethral tone still remain to be elucidated. The purpose of the present study was to compare the relaxing potency of ZD6169 on the urethral tone with that of levcromakalim a known potent K_ATP channel opener, by use of tension recordings. Furthermore, in the present experiments, we found that glibenclamide partially inhibited the muscle relaxation induced by higher concentration of ZD6169 (⩾10 μM) and that Bay K8644 (1 μM) reduced the relaxing action of ZD6169. Therefore, we have investigated the effects of ZD6169 on voltage-dependent Ba²⁺ macroscopic and unitary currents which demonstrate electrophysiological and molecular properties of voltage-dependent Ca²⁺ channels in pig urethra. We found a glibenclamide-insensitive ZD6169-induced urethral relaxation which may be caused by inhibition of voltage-dependent Ca²⁺ channels in pig urethra. A preliminary account of the present results has been communicated to the 74th annual meeting of the Japanese Pharmacological Society (Teramoto et al., 2001b).v

Methods

Cell dispersion

Fresh urethras from female pigs were collected from a local abattoir. Pig urethral myocytes were freshly isolated by the gentle tapping method as described previously (Teramoto & Brading, 1998). Briefly, thin strips of smooth muscle (10 – 15 mm×2 – 4 mm) were dissected from the fresh proximal urethral wall and stored in nominally Ca²⁺-free solution (mM): Na⁺ 140, K⁺ 5, Mg²⁺ 0.5, Cl⁻ 146, glucose 10, HEPES 10/Tris, titrated to pH 7.35 – 7.4, containing papain (17 u mg⁻¹ protein, 0.3 – 0.4 mg ml⁻¹) bubbled with O₂ at 4 – 6°C for 20 min. The digested strips were washed in Ca²⁺-free solution containing 1 mg ml⁻¹ bovine serum albumin (BSA), and preincubated in Ca²⁺-free solution at 35°C for 5 – 6 min. The strips were then incubated in Ca²⁺-free solution containing 0.3 – 0.4 mg ml⁻¹ collagenase (Type I, Sigma Chemical K.K., Tokyo, Japan) at 35°C for 10 – 15 min. Relaxed spindle-shaped cells, with length varying between 400 and 500 μm, were isolated and stored at 4°C. The dispersed cells were used within 2 h for experiments.

Tension measurement and data analysis

For isometric tension recording, fine strips were prepared as described previously (Teramoto & Ito, 1999). An initial tension equivalent to 1 g weight was applied to each strip, which was then allowed to equilibrate for approximately 1 – 1.5 h until the basal urethral tone became stable (37°C). To prevent both noradrenaline outflow from sympathetic nerve terminals and ß-adrenoceptor stimulation, 3 μM guanethidine and 0.3 μM propranolol were present throughout the experiments. Data were recorded on a Macintosh computer, through ‘MacLab 3.5.6' (ADInstruments Pty Ltd., Castle Hill, Australia). The tension was expressed as g mg⁻¹ of tissue.

Patch-clamp experiments recording procedure

Patch-clamp experiments were performed at room temperature (21 – 23°C) as described previously (Teramoto et al., 2001a). Junction potentials between bath and pipette solutions were measured with a 3 M KCl reference electrode and were <2 mV, so that correction for these potentials was not made. Capacitance noise was kept to a minimum by maintaining the test solution in the electrode as low as possible. At the beginning of each experiment, the series resistance was compensated.

Drugs and solutions

For tension measurement, modified Krebs solution was used (mM): 137 Na⁺, 5.9 K⁺, 1.2 Mg²⁺, 2.5 Ca²⁺, 133.7 Cl⁻, 15.4 HCO₃⁻, 1.2 H₂PO₄⁻ and 11.5 glucose which was bubbled with 97% O₂ and 3% CO₂. Occasionally, a proportion of external Ca²⁺ in the above modified Krebs solution was replaced with equimolar Na⁺, and 2 mM EGTA (pH 7.3) was added. For recording voltage-dependent Ba²⁺ currents in whole-cell configuration, high caesium pipette solution contained (mM): Cs⁺ 130, tetraethylammonium (TEA⁺) 10, Mg²⁺ 2, Cl⁻ 144, glucose 5, EGTA 5, ATP 5, HEPES 10/Tris (pH 7.35 – 7.40). Ba²⁺ 10 mM bath solution contained (mM): Ba²⁺ 10, TEA⁺, 135, Cl⁻ 155, glucose 10, HEPES 10/Tris (pH 7.35 – 7.40). For recording unitary Ba²⁺ current (cell-attached configuration), a pipette was filled with high Ba²⁺ solution (mM): Ba²⁺ 90, TEA⁺ 10, glucose 10, Cl⁻ 190, HEPES 10, titrated to pH 7.35 – 7.40 with Tris base. In some experiments, 1 μM Bay K 8644 (methyl 1,4-dihydro-2,6-dimethyl -3 -nitro -4 -(2 -trifluoromethylphenyl)-pyridine-5-carboxylate) was included in the above pipette solution. High K⁺ bath solution contained (mM): K⁺ 142, glucose 10, Cl⁻ 142, EGTA 5, HEPES 10, titrated to pH 7.35 – 7.40 with Tris base. The bath solution was superfused by gravity throughout the experiments at a rate of 2 ml min⁻¹. The following chemicals were used: ATP, Bay K 8644, collagenase (type I), dimethylsulphoxide (DMSO), EGTA, glibenclamide, HEPES, papain, TEACl and Tris (Sigma Chemical K.K., Tokyo, Japan). Levcromakalim (SmithKline Beecham Pharmaceuticals, Harlow, U.K.), ZD6169 (AstraZeneca Pharmaceuticals, Cheshire, U.K.) and glibenclamide were prepared daily as 100 mM stock solutions in DMSO. Bay K 8644 was dissolved to 1 mM in DMSO. The final concentration of DMSO was less than 0.3%, and this concentration was shown not to affect urethral tone and membrane currents in pig urethra.

Data analysis and statistics

The whole-cell recording data were low-pass filtered at 500 Hz by an 8 pole Bessel filter (E-3201B, NF Electronic Instruments, Yokohama, Japan), sampled at either 1 ms (voltage-dependent Ba²⁺ currents) or 25 ms (glibenclamide-sensitive membrane currents) intervals and analysed on a computer (Macintosh PowerPC G3, Apple Computer Japan Limited, Tokyo, Japan) by the commercial software ‘MacLab 3.5.6' (ADInstruments Pty Ltd., Castle Hill, Australia). Statistical analyses were performed with Student's t-test for paired values. Changes were considered significant at P<0.01. Data are expressed as mean with the standard deviation (s.d.).

RNA preparation and reverse transcription-polymerase chain reaction analysis

For RNA isolation, total RNA from pig urethral smooth muscle was isolated using TRIzol reagent according to the manufacturer's instructions (RNeasy Mini Kit, QIAGEN K.K., Tokyo, Japan). First-strand synthesis of cDNA using random hexamers was prepared as follows: total RNA isolated from tissues was incubated with random hexamers at 70°C for 10 min and then with PCR buffer (20 mM Tris/HCl, pH 8.4, 50 mM KCl), 2.5 mM MgCl₂, 0.5 mM deoxynucleoside-5′-triphosphate, and 10 mM dithiothreitol at 25°C for 5 min. RT – PCR was initiated by the addition of Superscript II RT (200 u) at 25°C for 10 min followed by incubation at 42°C for 50 min. The reaction was terminated by incubation at 70°C for 15 min, before chilling on ice. PCR was performed using 1 μl of cDNA in 20 μl reaction mixture containing 0.5 μM concentration of each primer, 200 μM concentration of each deoxynucleoside-5′-triphosphate, and 0.5 units of Taq polymerase (Takara Co. Ltd., Osaka, Japan). The cycling conditions were 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 40 cycles. An aliquot (10 μl) of the RT – PCR product was analysed on a 2% Tris-borate-EDTA polyacrylamide gel. Since no sequence information is available regarding the α_1C subunit in pig, generic subunit-specific primers were designed based on information from rabbit sequences (GenBank DNA sequence X15539; Mikami et al., 1989). The sequences of the primers for amplification of the novel α_1C subunit were as follows: 5′-GCTGTGGACCCCCTTCATCAAGTCC-3′ (forward) and 5′-ACCCTTGGCTTCAGGGTCATA-3′ (reverse). Control reactions were carried out where samples in the absence of reverse transcriptase were amplified to ensure that the detected product was not the result of possible DNA contamination and by use of corresponding templates as positive controls to ensure that the primers were annealing successfully. These primers gave products of the expected sizes that were confirmed by DNA sequence analysis.

Results

The effects of ZD6169 on the resting urethral tone

Figure 1a shows that cumulative application of ZD6169 produced a concentration-dependent relaxation of the resting urethral tone. After washing out the drug (⩽10 μM), the urethral tone recovered to the control level. Subsequently, 10 μM levcromakalim was applied in order to obtain the maximum relaxation in the urethral strips. Since the ZD6169 (⩾30 μM)-induced relaxation did not completely recover to the control level, on some occasions, 10 μM levcromakalim was applied before application of higher concentrations of ZD6169. Figure 1b shows the concentration-response curve of ZD6169, expressed relative to the maximum relaxation to 10 μM levcromakalim. The EC₅₀ value for the ZD6169-induced relaxation was 1.6 μM.

Figure 1.

The relaxing effects of ZD6169 on the resting tone of pig urethral smooth muscle strips. The dashed line indicates the mean resting tone. (a) The effects of cumulative addition of ZD6169 (0.1 – 10 μM) and after recovery, 10 μM levcromakalim. (b) Relationships between the relative value of ZD6169-induced urethral relaxation and the concentration of ZD6169. The peak amplitude of the 10 μM levcromakalim-induced relaxation was normalized as one. The curve was drawn by fitting the equation using the least-squares method,

where K, D and n_H are dissociation constant, concentration of glibenclamide (nM) and Hill's coefficient, respectively. The following values were used for the curve fitting: K=1.6 μM, n_H=1.2 (n=6 – 9). Each symbol indicates mean with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol. The urethral relaxing curve with the broken line is for the effects of levcromakalim (K=0.3 μM, n_H=2.1, n=18), and is taken from Teramoto & Ito (1999).

Identification of the glibenclamide-sensitive component of the ZD6169-induced relaxation

As shown in Figure 2a, ZD6169 (3 μM) produced a slow relaxation which reached a level similar to the maximal relaxation produced by 10 μM levcromakalim, and this relaxation was completely suppressed by application of 1 μM glibenclamide (n=10). However, the relaxation produced by higher concentrations of ZD6169 (⩾10 μM) were not completely suppressed by subsequent application of 1 μM glibenclamide. Figure 2b shows that glibenclamide caused a partial inhibition of the relaxation induced by 30 μM ZD6169 (0.41±0.12, n=6) and had no effect on the 100 μM ZD6169-induced relaxation (0.02±0.03, n=6, Figure 2c). Note that even application of higher concentration of glibenclamide (⩾10 μM) did not change the level of the 100 μM ZD6169-induced relaxation (data not shown). When tissues were pretreated with 1 μM glibenclamide for 11 min (Figure 2d), the time taken to reach peak relaxation in 100 μM ZD6169 was noticeably slower than that in the absence of 1 μM glibenclamide (in the absence of glibenclamide, 3 min 53 s±1 min 34 s, n=5, vs. in the presence of glibenclamide, 10 min 52 s±2 min 44 s, n=5). In contrast, 10 μM levcromakalim induced a much quicker relaxation which reached maximum in 2 min 15±59 s (n=10).

Figure 2.

Glibenclamide-sensitive and -insensitive components of the ZD6169-induced urethral relaxation. The dashed line indicates the mean resting urethral tone. (a), (b), (c) The effects of 1 μM glibenclamide on the relaxation induced by 3 μM, (a); 30 μM, (b); 100 μM, (c); ZD6169. (d) ZD6169 (100 μM)-induced relaxation after 11 min pretreatments with 1 μM glibenclamide.

Bay K8644, a selective voltage-dependent L-type Ca²⁺ channel agonist, caused a dramatic increase in resting tone (Figure 3a), and the maximum level of relaxation achieved by 10 μM levcromakalim showed no significant difference in the presence and absence of 1 μM Bay K8644. In contrast, the relative ratio of the ZD6169-induced relaxation to the 10 μM levcromakalim-induced relaxation was significantly reduced in the presence of 1 μM Bay K8644 (0.78±0.08, n=4, 30 μM ZD6169; 0.71±0.02, n=4, 100 μM ZD6169) in comparison with that in the absence of Bay K8644 (0.95±0.09, n=6, 30 μM ZD6169; 1.02±0.02, n=6, 100 μM ZD6169) in Figure 3b. Figure 3b also shows the relationships between the relative ratio of the glibenclamide-sensitive ZD6169-induced relaxation and the concentration of ZD6169, resulting in a bell-shaped curve and that at higher concentrations relaxations to ZD6169 (⩾30 μM) is insensitive to 1 μM glibenclamide. When extracellular Ca²⁺ was removed and 2 mM EGTA was added in order to obtain zero Ca²⁺, the urethral tone was reversibly reduced (Figure 3c, n=16). Application of 1 μM nifedipine caused an irreversible urethral relaxation (Figure 3d, n=6). These results suggest that concentrations of ZD6169 (⩾30 μM) are likely to cause glibenclamide-insensitive urethral relaxation in pig urethra which may be related to voltage-dependent Ca²⁺ channels and that voltage-dependent Ca²⁺ channels may play an important role in regulating the resting tone of pig urethra.

Figure 3.

The relaxing effects of ZD6169, levcromakalim and nifedipine on the urethral tone. (a) The effects of ZD6169 and levcromakalim on the urethral tone in the absence and presence of 1 μM Bay K8644. The dashed line indicates the increased urethral tone in the presence of Bay K8644. (b) Relationships between the relative value of the glibenclamide-sensitive ZD6169-induced relaxation and the concentration of ZD6169. The peak amplitude of the 10 μM levcromakalim-induced relaxation was taken as one. Each symbol indicates mean with s.d. shown by vertical lines (n=4 – 7). The line was drawn by eye. The curve with the broken line (ZD6169) is obtained from Figure 1(b). *Significantly different from the relative value in the absence of Bay K8644 (t-test, P<0.01). (c) When extracellular Ca²⁺ was removed (and 2 mM EGTA added), the resting urethral tone was reversibly reduced. The dashed line indicates the mean resting urethral tone. (d) Applying 1 μM nifedipine caused an irreversible urethral relaxation. The dashed line indicates the mean resting urethral tone.

Actions of ZD6169 on voltage-dependent Ba²⁺ currents in pig urethra

In order to study further the mechanisms involved in the glibenclamide-insensitive relaxation, the effects of ZD6169 on voltage-dependent Ca²⁺ currents were investigated by use of conventional whole-cell configurations. When the bath solution was PSS, the peak amplitude of inward Ca²⁺ current was too small to analyse (less than 20 pA at 0 mV). In order to enhance the inward currents for reasonable analysis and to isolate voltage-dependent inward currents through Ca²⁺ channels by inhibiting other Ca²⁺-activated mechanisms (such as Ca²⁺-activated K⁺ currents and Ca²⁺-activated Cl⁻ currents etc.), 10 mM Ba²⁺ bath solution containing 135 mM TEA⁺ was used and the pipette was filled with a Cs⁺ – TEA⁺ solution containing 5 mM EGTA. Figure 4a shows the time course of the effects of ZD6169 (100 μM) on the Ba²⁺ inward current evoked by a depolarizing pulse of +10 mV from a holding potential of −50 mV. The depolarizing pulses were applied every 15 s. When the peak amplitude of the Ba²⁺ inward current just before application of ZD6169 (control) was taken as one, ZD6169 reduced the peak amplitude of the Ba²⁺ inward current (0.2±0.1, n=8). On removal of ZD6169, the peak amplitude gradually recovered, but not to the control level. Subsequently, application of 100 μM calciseptine, a peptide which is a selective L-type Ca²⁺ channel blocker (Teramoto et al., 1996), greatly inhibited the Ba²⁺ inward currents. Similarly, 10 μM nifedipine completely suppressed the Ba²⁺ inward currents. The block produced by ZD6169 showed some use dependence. As shown in Figure 4a, when a depolarizing pulse was applied after an interval of 4 min in the presence of 100 μM ZD6169, the peak amplitude of the Ba²⁺ inward current was smaller than that observed before application of ZD6169 but consistently larger than that recorded at 4 min with repetitive application of the depolarizing pulses (single pulse, 0.79±0.09, n=6 vs repetitive pulse 0.32±0.1, n=4). Similar experiments were performed at three different holding potentials (−90 mV, 0.85±0.09, n=4; −100 mV, 0.87±0.05, n=4; −120 mV, 0.89±0.06, n=3). A depolarizing pulse to +10 mV produced the same amplitude of voltage-dependent Ba²⁺ currents in pig urethra at holding potentials more negative than −80 mV when the peak amplitude of voltage-dependent Ba²⁺ currents at −120 mV was taken as one (−90 mV, 0.98±0.03, n=4; −100 mV, 0.99±0.02, n=4). Figure 4b shows the relationships between the relative peak amplitude of Ba²⁺ inward currents and concentrations of ZD6169 at two different holding potentials (−50 mV and −90 mV). The Ba²⁺ inward currents were evoked by a depolarizing pulse to 10 mV applied every 15 s. ZD6169 inhibited the peak amplitude of the Ba²⁺ inward currents in a concentration-dependent manner (K_i; 55 μM, −50 mV; 122 μM, −90 mV).

Figure 4.

Effects of 100 μM ZD6169, 100 nM calciseptine and 10 μM nifedipine on voltage-dependent Ba²⁺ currents in pig urethra. Whole-cell recording, pipette solution Cs⁺ – TEA⁺ solution containing 5 mM EGTA and the bath solution 10 mM Ba²⁺ containing 135 mM TEA⁺. (a) The time course of the effects of application of the three antagonists on the relative amplitude of the voltage-dependent Ba²⁺ current evoked by repetitive depolarizing pulses to +10 mV from a holding potential of −50 mV. The peak amplitude of the voltage-dependent Ba²⁺ current just before application of ZD6169 was normalized as one (control). The inset below shows individual traces as indicated by the numbers in the graph. On some occasions no pulses were applied for the initial four min after application of 100 μM ZD6169. The solid symbols show the size of the mean value of the peak amplitude of the voltage-dependent Ba²⁺ current evoked by the first depolarizing pulse after this four min from two holding potentials (−50 mV, 0.79±0.09, n=6; −90 mV, 0.85±0.09, n=4). Time 0 indicates the time when 100 μM ZD6169 was applied to the bath. (b) Relationships between relative inhibition of the peak amplitude of Ba²⁺ current and the concentration of ZD6169 at two holding potentials (−50 mV and −90 mV). The peak amplitude of the Ba²⁺ current elicited by a step pulse to +10 mV from the holding potential just before application of ZD6169 was normalized as one. The curves were drawn by fitting the following equation using the least-squares method:

where K_i, D and n_H are the inhibitory dissociation constant, concentration of ZD6169 (μM) and Hill's coefficient, respectively. The following values were used for the curve fitting: −50 mV, K_i=55 μM, n_H=2.3; −90 mV K_i=122 μM, n_H=2.2. Each symbol indicates the mean of 4 – 6 observation with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol.

Electrophysiological properties of the voltage-dependent Ba²⁺ currents in pig urethra

In order to further characterize the electrophysiological properties of Ba²⁺ inward currents in pig urethra, depolarizing step pulses (10 mV increment from −40 to +40 mV for 1 s duration, every 15 s, whole-cell recording) were applied from a holding potential of −90 mV. At potentials more positive than −30 mV, an inward Ba²⁺ current was evoked (Figure 5a) which reached a peak and then gradually decayed. The maximum peak amplitude was obtained at approximately +10 mV and the amplitude was reduced at more positive potentials, demonstrating a voltage-dependency. At a holding potential of −40 mV, both the peak amplitude and the amplitude at the end of the command pulse were smaller, but the time course of the current decay was identical at both holding potentials (Figure 5a).

Figure 5.

Inward Ba²⁺ currents recorded by application of depolarizing pulses at two different holding potentials (−90 and −40 mV). Whole-cell recording, pipette solution Cs⁺ – TEA⁺ solution containing 5 mM EGTA and the bath solution 10 mM Ba²⁺ containing 135 mM TEA⁺. (a)(i) Inward Ba²⁺ currents at each indicated depolarizing potential from both holding potentials superimposed. (ii) Inward Ba²⁺ current from (i) scaled to match their peak amplitudes and superimposed. (b) Current-voltage relationships of inward Ba²⁺ current obtained at −40 and −90 mV. The current amplitude was measured as the peak amplitude of inward Ba²⁺ current in each condition. The lines were drawn by eye. (c) RT – PCR detection of voltage-dependent Ca²⁺ channel α_1C subunit mRNA. RT – PCR was performed as described in the Methods and size makers were used to indicate the size of the experimental fragments (lane 1). RT – PCR yielded visible amounts of α_1C subunit (484 bp fragment) in mRNAs from pig urethra (lane 2) and rabbit cDNA of α_1C subunit (lane 3).

Molecular expression of α_1C subunit in pig urethra

The distribution of the α_1C subunit in voltage-dependent Ca²⁺ channels in the tissue was examined by use of RT – PCR technique. Specific primers for the amplification of α_1C subunit mRNA were designed to produce a cDNA fragment of 484 bp. As shown in Figure 5c, the α_1C subunit mRNA was expressed in pig urethra and rabbit α_1C cDNA with the expected fragment size (484 bp, see Methods). Similar observations were obtained in seven other urethral tissues. Expression of α_1B or α_1G subunit mRNAs were not detectable in pig urethra using set of specific primers (data not shown).

Voltage-dependent inhibitory effects of ZD6169 on voltage-dependent Ba²⁺ currents

Figure 6b shows the current-voltage relationships in the absence and presence of 50 μM ZD6169. ZD6169 inhibited the peak amplitude of the Ba²⁺ currents evoked by depolarizing pulses (1 s duration) from a holding potential of −50 mV at levels more positive than −30 mV, and the inhibition showed some voltage-dependency (Figure 6c). This voltage-dependency was investigated before and after application of 100 μM ZD6169 using the experimental protocol shown in Figure 7 (conditioning pulse duration, 8 s; holding membrane potential, −90 mV). In the absence of ZD6169 (control), inactivation of the Ba²⁺ current occurred with depolarizing pulses positive to −50 mV. After application of 100 μM ZD6169 (approximately 5 min later), the voltage-dependent inactivation curve in the same cells was shifted to the left (Figure 7).

Figure 6.

Effects of ZD6169 on voltage-dependent Ba²⁺ inward currents at a holding membrane potential of −50 mV in pig urethra. The pipette solution was Cs⁺ – TEA⁺ solution containing 5 mM EGTA and the bath solution was 10 mM Ba²⁺ containing 135 mM TEA⁺. (a) (i) Original current traces before (control) and after application of 50 μM ZD6169 at the indicated pulse potentials. (ii) Inward Ba²⁺ current from (i) scaled to match their peak amplitudes and superimposed. (b) Current-voltage relationships obtained in the absence (control) or presence of 50 μM ZD6169. The current amplitude was measured as the peak amplitude of the Ba²⁺ inward current in each condition. The lines were drawn by eye. (c) Relationship between the test potential and relative value of the Ba²⁺ inward currents inhibited by 50 μM ZD6169, expressed as a fraction of the peak amplitude of the Ba²⁺ inward current evoked by various amplitude of the depolarizing pulse in the absence of ZD6169. Each symbol indicates the mean of four observations with±s.d. shown by vertical lines. The line was drawn by eye.

Figure 7.

Effects of ZD6169 (100 μM) on the voltage-dependent inactivation of the Ba²⁺ inward currents in pig urethra. Whole-cell recording, pipette solution Cs⁺ – TEA⁺ solution containing 5 mM EGTA and the bath solution 10 mM Ba²⁺ containing 135 mM TEA⁺. The holding potential was −90 mV. Conditioning pulses of various amplitudes were applied (up to +30 mV, 8 s duration) before application of the test pulse (to +10 mV, 1 s duration). An interval of 20 ms was allowed between these two pulses to estimate possible contamination of the capacitive current. The peak amplitude of Ba²⁺ current evoked by each test pulse was measured before and after application of 100 μM ZD6169. The curves with the solid line; the peak amplitude of Ba²⁺ inward current in the absence and presence of ZD6169 without application of any conditioning pulse was normalized as one. The curve with the broken line was normalized to the current at +10 mV upon stepping from −90 mV in 100 μM ZD6169. The lines were drawn by fitting the data to the following equation in the least-squares method,

where I, I_max, V, V_half, k and C are the relative amplitude of Ba²⁺ inward currents observed at various amplitudes of the conditioning pulse (I) and observed with application of the conditioning pulse of −90 mV (I_max), amplitude of the conditioning pulse (V), and that where the amplitude of Ba²⁺ inward current was reduced to half (V_half), slope factor (k) and fraction of the non-inactivating component of Ba²⁺ inward current (C). The curves in the absence or presence of ZD6169 were drawn using the following values: (control), I_max=1.0, V_half=−30.7, k=8.5 and C=0.14; (ZD6169, 100 μM), I_max=0.69, V_half=−47.2, k=8.5 and C=0.04. Each symbol indicates the mean of four observations with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol.

Effects of ZD6169 and nifedipine on unitary Ba²⁺ currents in pig urethra

Single-channel recordings in the cell-attached configuration (pipette solution, 90 mM Ba²⁺; bath solution, 142 mM K⁺) were used to investigate further the inhibitory effects of ZD6169 on voltage-dependent Ba²⁺ currents. At potentials more positive than −40 mV, depolarizing pulses (400 ms duration) from a holding potential of −90 mV elicited a unitary inward current. The current-voltage relationship for this current was linear as shown in Figure 8b. The conductance, obtained from the amplitude of the Ba²⁺ currents, was 27.3±1.9 pS (n=8) measured from the all-points amplitude histograms. Application of 10 μM nifedipine suppressed the unitary Ba²⁺ current activity at all of the depolarizing membrane potentials (n=5, data not shown). Bay K8644 (1 μM, included in the pipette solution) enhanced the channel activity. Under these experimental conditions, on some occasions (11 out of 31 patches), channel openings of a 1.9 pA unitary Ba²⁺ current were continuously observed at potentials more negative than −30 mV in cell-attached configuration (Figure 9a, NP_o value, 0.055). Note that the channel activity could be observed for more than 20 min (n=4) and that the NP_o value increased in a voltage-dependent manner (−40 mV, 0.02±0.01, n=7; −30 mV, 0.06±0.02, n=7). Application of 100 μM ZD6169 inhibited the channel activity to an NP_o value of 0.005 without changing the amplitude of the unitary current (control, 1.9 pA; ZD6169 100 μM, 1.9 pA, n=5, in Figure 9b). When the NP_o value just before application of ZD6169 (control) was taken as one, the relative NP_o value was 0.11±0.05 (n=5) in the presence of 100 μM ZD6169, and 0.62±0.12, n=5 in the presence of 50 μM ZD6169 (Figure 9c). The channel activity did not recover to the control level on removal of ZD6169. Approximately 3 min later, bath application of nifedipine (10 μM) abolished the channel activity.

Figure 8.

Unitary Ba²⁺ currents recorded in cell-attached patches of pig urethral myocytes. The pipette was filled with 90 mM Ba²⁺ solution and 142 mM K⁺ solution was superfused in the bath. (a) The unitary Ba²⁺ currents were obtained using a depolarizing pulse (400 ms duration, 10 s interval) from a holding potential of −90 mV to the indicated membrane potential (from −40 mV to +20 mV). The average currents from 10 null traces (the nearest five traces before and after the event in which the channel did not open) were subtracted. The dashed line indicates the current base line where the channel is not open. (b) Current-voltage relationships of the unitary Ba²⁺ current obtained from −40 mV to +20 mV. The amplitude of the Ba²⁺ channel currents was taken from the all-points amplitude histograms at each depolarizing potential. The line was fitted by the least-squares method. The channel conductance was 27 pS (27.3±1.9 pS, n=8).

Figure 9.

Effects of ZD6169 on the unitary Ba²⁺ current in pig urethra at a holding potential of −30 mV. High K⁺ (142 mM) solution was superfused in the bath and high Ba²⁺ (90 mM) solution in the pipette. Bay K8644 (1 μM) had been included in the pipette solution. (a) Application of 100 μM ZD6169 (5 min duration) reduced the activity of the 1.9 pA Ba²⁺ channel. After removal of ZD6169, 3 min later, application of nifedipine (10 μM) abolished channel activity. Lower traces show expansions of the upper trace (1 kHz filtration; 80 μs digital sampling interval). The open horizontal bars in the absence and presence of 100 μM ZD6169 indicate the duration (2 min) of the traces analysed in (b). The dashed line indicates the current when the channel is not open. (b) All-point amplitude histograms in the absence (obtained during the last 2 min just before application of ZD6169) and presence of 100 μM ZD6169 (obtained during 2 min of a 5 min application). Continuous lines in the histograms are theoretical curves fitted with the Gaussian distribution, by the least-squares method. The abscissa scales show the amplitude of the current (pA) and the ordinate scales show the percentage value of the probability density function (per cent) for the recording period (2 min). (c) Each column shows the relative level of the activity of the 1.9 pA Ba²⁺ channel (mean value with+s.d.) when the mean open probability of the channel activity was normalized as one just before application of each concentration of ZD6169 (n=5).

Discussion

Potency of ZD6169 in urethral smooth muscle

In the present experiments, we have been able to evaluate the relaxing efficacy of ZD6169 on spontaneous tone of urethra in the absence of either excess [K⁺]_o (high KCl test protocol) or agonists (such as acetylcholine, serotonin etc.), comparing ZD6169 with levcromakalim (EC₅₀ value; 1.6 μM, ZD6169 versus 0.3 μM, levcromakalim). The potency of levcromakalim was clearly greater than ZD6169, as has been observed in detrusor smooth muscles (guinea-pig, Gopalakrishnan et al., 1999; pig, Buckner et al., 2000). However, the potencies of each K_ATP channel opener in pig urethra were slightly less than in urinary bladder. In cell-attached configuration, at the same concentration (10 μM), ZD6169 was again less potent than levcromakalim in inducing channel activity in pig urethra (Teramoto et al., 1999; 2001a). This coincided well with the present observations on urethral tension measurements. However, in guinea-pig detrusor, Li et al. (1995) reported that there was no significant difference between the peak amplitude of the 10 μM levcromakalim-induced currents and that of the 10 μM ZD6169-induced currents, suggesting that the potency of ZD6169 is the same as that of levcromakalim. We are not certain whether this significant difference of the potency of these K_ATP channel openers may be due to different experimental conditions or to species difference.

In patch-clamp experiments, we have recently reported that ZD6169 possesses a dual effect (i.e. agonistic and antagonistic action) on the activity of the K_ATP channel in pig urethra (Teramoto et al., 2001a). We have also reported that continuous application of ZD6169 (⩾30 μM) suppressed the activity of K_ATP channels after their initial activation (Teramoto et al., 2001a). However, in the present tension experiments, application of ZD6169 caused a stable and concentration-dependent urethral relaxation between 100 nM – 100 μM. Furthermore, the ZD6169 (⩾30 μM)- induced urethral relaxation was partially but not completely inhibited by application of 1 μM glibenclamide. These results strongly suggest that other relaxing mechanisms may be involved in the ZD6169-induced urethral relaxation in addition to the activation of K_ATP channels. Indeed, in the present experiments, although the ability of the levcromakalim-induced relaxation was not significantly different in the presence and absence of 1 μM Bay K8644, the ZD6169-induced relaxation was significantly reduced in the presence of Bay K8644 in comparison with the control. These results suggest that the glibenclamide-insensitive relaxation induced by ZD6169 may involve voltage-dependent Ca²⁺ channels in pig urethra.

Physiological roles of voltage-dependent Ca²⁺ channels in the smooth muscle cells of pig urethra

In tension measurement, when extracellular Ca²⁺ was removed and 2 mM EGTA was added, the resting urethral tone was reversibly reduced, suggesting that Ca²⁺ influx may play an important role in regulating the resting tone. Although application of 1 μM nifedipine also relaxed the smooth muscle tone, 1 μM BAY K8644 caused a dramatic increase in resting tone. These results suggest that Ca²⁺ entry through voltage-dependent Ca²⁺ channels may play a major role in generating the resting tone.

Voltage-dependent Ca²⁺ currents have been demonstrated in urethral myocytes by use of whole-cell recordings (sheep, Cotton et al., 1997; pig, Teramoto & Brading, 1998). The inward Ba²⁺ currents studied in the present study in pig urethra probably flow through L-type voltage-dependent Ca²⁺ channels due to the following electrophysiological and molecular properties; (1) There was no hump or second peak at less positive membrane potential level in the current-voltage relationships. (2) When the holding membrane potential was changed from −40 to −90 mV, the threshold potential to produce inward Ba²⁺ current did not shift. (3) There was no difference in the decay of inward currents recorded at two different holding membrane potentials (−40 and −90 mV). (4) Application of nifedipine, one of the most common dihydropyridine (DHP) Ca²⁺ channel antagonists, suppressed the amplitude of the inward Ba²⁺ current at a holding potential of −50 mV (K_i=31 nM; Teramoto & Brading, 1998). (5) Calciseptine suppressed the inward Ba²⁺ currents. (6) RT – PCR studies show that α_1C subunit mRNA was expressed in pig urethra with the expected fragment size for rabbit α_1C subunit. (7) By use of cell-attached mode, only one class of the voltage-dependent Ba²⁺ channel (27 pS voltage-dependent Ba²⁺ channel, 90 mM Ba²⁺ solution) which was suppressed by subsequent application of 10 μM nifedipine was recorded from −40 to +20 mV. These results suggest that the L-type Ca²⁺ channel is probably the only type of voltage-dependent Ca²⁺ channel present in pig urethra. Other smooth muscles may also possess only a single type of voltage-dependent Ca²⁺ channel (reviewed by Bolton et al., 1999).

Inhibitory effects of ZD6169 on the voltage-dependent Ba²⁺ currents in pig urethra

The same amplitude of voltage-dependent Ba²⁺ currents was produced when holding at −80 mV or more negatively, suggesting that all of the voltage-dependent Ca²⁺ channels at these potentials may be in the resting state. The ability of 100 μM ZD6169 to suppress the peak amplitude of the Ba²⁺ currents evoked by a depolarizing pulse from three different holding potentials (−90, −100 and −120 mV) were not significantly different, suggesting that at these negative holding potentials ZD6169 may inhibit the voltage-dependent Ba²⁺ currents in a voltage-independent manner (resting state block). When the holding potential was elevated to −50 from −90 mV, voltage-dependent inhibition by ZD6169 was seen and the concentration response curve was shifted to the left. The voltage-dependent inactivation curve was also shifted to the left after application of 100 μM ZD6169. These results suggest the voltage-dependent inhibitory actions of ZD6169 occur at the inactivated state of voltage-dependent Ba²⁺ channels in pig urethra (voltage-dependent block). The dissociation constant for drug binding to the channel to the inactivated state could be estimated from the shift of the voltage-dependent inactivation curve and the concentration response curve obtained at the resting state by use of the following equation (Uehara & Hume, 1985),

where ΔV_half is the amplitude of the shift of the voltage-dependent inactivation curve, k is a slope factor for the inactivation curve and [D] is the concentration of drug applied. K_inact and K_rest are dissociation constants of ZD6169 for the inactivated and the resting states of voltage-dependent Ba²⁺ channels, respectively. In the present experiments, the K_rest value was estimated to be 122 μM from the concentration response curve at a holding potential of −90 mV. When ΔV_half value was obtained from the results using 8 s conditioning pulses, the estimated K_inact value was 9 μM. Given this, we suggest that ZD6169 may bind to the inactivated state with approximately 14 times higher affinity than to the resting state in pig urethra. In single-channel recordings, we have demonstrated that ZD6169 irreversibly inhibited the activity of the unitary Ba²⁺ current which was also suppressed by 10 μM nifedipine. From these results, we suggest that ZD6169 may inhibit the voltage-dependent Ba²⁺ currents through the blocking of the unitary 27 pS Ba²⁺ channel in pig urethra.

Multiple effects of ZD6169 on the membrane currents in pig urethra

Although various types of K_ATP channel openers have been reported to possess multiple effects on cellular functions in smooth muscle (such as Ca²⁺ antagonist-like effects, internal Ca²⁺ reuptake actions, NO releaser etc., reviewed by Quayle et al., 1997), we have been able to demonstrate at least two different effects of ZD6169 on two significantly different ion channels by use of single-channel recordings in an independent manner. Namely, at lower concentrations (⩽10 μM), ZD6169 seems to cause an urethral relaxation mainly through activation of K_ATP channels. In contrast, at higher concentrations (30 μM ⩾), ZD6169 causes a significant and stable relaxation, by inhibiting the voltage-dependent Ca²⁺ channels (after initially activating K_ATP channels). ZD6169 may thus be termed not only a K_ATP channel opener but also a voltage-dependent Ca²⁺ channel blocker, depending on its concentration. In the present experiments, 100 μM ZD6169 was able to induce a similar level of urethral relaxation in the presence and absence of 1 μM glibenclamide, although the maximum level of relaxation was achieved much more slowly in the presence of glibenclamide.

In conclusion, we have been able to demonstrate an inhibitory effect of ZD6169 on the voltage-dependent L-type Ca²⁺ channels in pig urethra, causing a significant ZD6169-induced urethral relaxation. ZD6169 is not selective for K_ATP channel in urethral smooth muscle.

Abbreviations

Bay K 8644

methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate

DHP

dihydropyridine

DMSO

dimethylsulphoxide

k

slope factor for the inactivation curve

K_ATP channels

ATP-sensitive K⁺ channels

K_inact

dissociation constant for the inactivated state of voltage-dependent Ba²⁺ channels

K_rest

dissociation constant for the resting state of voltage-dependent Ba²⁺ channels

NO

nitric oxide

PSS

physiological salt solution

RT – PCR

reverse transcriptase-polymerase chain reaction

TEA⁺

tetraethylammonium