Introduction

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ZD6169, the S-enantiomer of the racemic compound N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide recently discovered by Zeneca Pharmaceuticals Group (fig. 1), was reported to reduce the spontaneous mechanical activity in isolated guinea pig bladder detrusor (Li et al., 1995) and to inhibit contraction of normal and unstable bladder in rat and dog (Howe et al., 1995). These actions are considered potentially beneficial for the treatment of urinary urge incontinence, which is closely associated with instability or hyperreflexia of bladder detrusor smooth muscle. The results from electrophysiological and several functional studies demonstrated that ZD6169 selectively opened the K_ATP channels in smooth muscle cells, which led to membrane hyperpolarization and inhibition of the contractile force of the bladder muscle (Li et al., 1995; Trivedi et al., 1995b).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of ZD6169 (S-enantiomer) and racemic ZD6169.

Multiple types of K⁺ channel, including K_ATP, K_V and BK_Ca channels, are present in bladder detrusor cells (Klockner and Isenberg, 1985; Bonev and Nelson, 1993; Trivedi et al., 1995a; Heppner and Nelson, 1995). The BK_Ca channel in spontaneously active bladder detrusor, unlike in many other types of vascular and nonvascular smooth muscle tissue, plays a predominant role in modulation of membrane excitability and myogenic contractility. As evidence of this notion, several reports showed that the application of BK_Ca channel blockers like chTX, ibTX and tetraethylammonium (TEA) drastically augmented the basal as well as stimulated contractile activity (Suarez-Kurtz et al., 1991; Zografos et al., 1992; Shetty et al., unpublished data). Relative to the BK_Ca channel, K_ATP channel in bladder detrusor contributes little to the muscle excitability, because glyburide, a specific K_ATP channel blocker, modifies neither the spontaneous myogenic activity nor ⁸⁶Rb efflux in the absence of K_ATP channel openers (Zografos et al., 1992; Shetty et al., unpublished data). In light of the unique regulatory role of BK_Ca channel in bladder function in combination with the reported in vivo bladder selectivity of ZD6169 compared with cromakalim (Howe et al., 1995), it is speculative that ZD6169 may also interact with BK_Ca channel. To this end, the present study attempted to establish a profile of the electrophysiological actions of ZD6169 and its racemic compound on individual components of K_ATP and BK_Ca currents in freshly isolated guinea pig detrusor muscle cells. The results revealed a complex mode of action of this compound on K_ATP channel and a direct opening activity on the BK_Ca channel. ZD6169 thus appears to be a dual K⁺ channel opener.

	Methods

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Cell isolation. Male guinea pigs (~300 g) were sacrificed by exposure to CO₂ for 2 min. The urinary bladder was removed and rinsed in a Ca⁺⁺-free medium composed of 135 mM NaCl, 5.4 mM KCl, 2 mM MgSO₄, 5 mM glucose, 10 mM HEPES and pH 7.3 adjusted with NaOH. The detrusor muscle was cut and cleaned free of connective tissue and blood vessels. Pieces of tissue were then incubated for two periods of 35 min at 36°C in an enzyme medium containing 130 mM K glutamate, 20 mM taurine, 5 mM pyruvate, 5 mM creatine, 10 mM HEPES and 0.5 mM CaCl₂ complemented with 1 mg/ml collagenase (Sigma, C2139), 0.2 mg/ml pronase (Sigma, P5147), 1 mg/ml fatty acid-free albumin. During incubation, tissue pieces were gently stirred to assure a full exposure to the enzymes. After a total of 70 min enzyme digestion, single bladder smooth muscle cells were obtained by gentle agitation of the preparation through a Pasteur pipette into the "KB" solution (Isenberg and Klockner, 1982). Single cells were stored at 4°C for 1 h before recording.

Experimental setting and current recording. Experiments were performed at 22°C with the whole-cell or inside-out configurations of the patch-clamp technique (Hamill et al., 1981) in enzymatically dispersed smooth muscle cells from guinea pig bladder detrusor. Patch electrodes were pulled from Kimax-51 capillary tubes (Kimble Products, Vineland, NJ). The resistance of electrodes after fire polishing was around 2 megohm for the whole-cell mode to increase the rate of solution dialysis and around 5 megohm for the inside-out mode to reduce the number of channels recorded. Whole-cell currents were amplified by a List EPC-7 amplifier (Adams & List Assoc., Darmstadt, Germany), digitized at 4 kHz with a TL-1-125 DMA interface (Axon Instruments) and stored on a Compaq DeskPro/66 M microcomputer for later analysis with pClamp version 6.03 (Axon Instruments, Foster City, CA). The inside-out single-channel recordings were first low-pass-filtered at 2 kHz by an 8-poles Bessel filter (Frequency Devices 902LPF) and video-taped with a Toshiba Pulse-Code Modulation data recorder (DX-900). After each experiment, the recordings were played back through a window discriminator (AI2020, Axon Instruments) at a sampling rate of 4 kHz and stored on the computer for analysis. The junction potential between the electrodes and the bath solution was compensated by the DC offset on the amplifier. No leak subtraction was applied. The cell capacitance was 34.8 ± 2.5 pF (n = 16).

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	Results

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Concentration-dependent dual effect of ZD6169 on K_ATP current. The effect of ZD6169 on the K_ATP current was investigated when cells were bathed in symmetrical K⁺ (140 mM) solutions to increase inward current and clamped at 50 mV to minimize activation of other voltage-dependent K⁺ currents at negative membrane potentials. Current was elicited by a ramp protocol ranging from 110 mV to +110 mV over a period of 1.5 s. With only 0.5 mM ATP in the pipette solution, the steady-state holding current was presumably composed of leakage current and a basal activity of K_ATP channels. In the absence of ZD6169, small inward current, basically K_ATP channel in isolation, was detected at potentials negative to 0 mV (fig. 2A). To better display the modification of inward K_ATP current by the drug, a high-current magnification was used in figure 2, in which the sizable outward rectifying current at the positive voltages was largely out of scale. The inward K_ATP current was increased by ZD6169 at 0.5 µM (fig. 2B) and 5 µM (fig. 2C) in a concentration-dependent manner. As the concentration of ZD6169 further increased to 50 µM, the stimulatory effect on the K_ATP current diminished and a significant inhibitory effect manifested instead (fig. 2D). This dual effect on the K_ATP channel was observed in 10 of 12 cells studied. Additional studies in which the concentration increment was made smaller showed that a maximal activation of K_ATP channel by ZD6169 occurred between 5 and 10 µM, depending on individual cell. The effects on K_ATP channel were fully reversible upon washout of ZD6169 (fig. 2E). The current activated by ZD6169 (5 µM) was completely prevented (fig. 3, A-C) and reversed (fig. 3, A, C and D) by 1 µM glyburide, a specific K_ATP channel blocker, which indicated that ZD6169-activated current was, by nature, the K_ATP current.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent dual action of ZD6169 on KATP channel. These recordings are representative of the whole-cell currents in response to a 1.5-s voltage ramp from 110 to +110 mV (as shown at the top) in control (A), in the presence of ZD6169 at 0.5 µM (B), 5 µM (C) and 50 µM (D), and after washout of the drug (E). The cell was bathed in symmetrical 140 mM K+ solutions and held at 50 mV. Calcium concentration was 103 and 108 M in bath and pipette solution, respectively. The pipette solution contained 0.5 mM ATP to facilitate the activation of KATP channels. A high magnification is used to better display the changes in the inward KATP current, whereas a large portion of current at positive potential, rectifying outwardly, is out of scale. Dashed lines indicate the zero current level. The scale of a 0.5 nA reference current is shown at the lower left corner.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   ZD6169-activated current is sensitive to glyburide. Whole-cell currents were elicited in response to a 1.5-s voltage ramp from 110 mV to +30 mV (as shown at the top) in control (A), in 5 µM ZD6169 with 1 µM glyburide (B), in 5 µM ZD6169 alone (C) and again in 5 µM ZD6169 and 1 µM glyburide (D). The cell was bathed in symmetrical 140 mM K+ solution and held at 50 mV. The pipette solution contained 0.5 mM ATP. Dashed lines indicate the zero current level. The scale of a 0.5 nA reference current is shown at the lower left corner.

Activation of BK_Ca current by ZD6169. The effect of ZD6169 on the BK_Ca current was investigated when cells were bathed in physiological solutions ([K⁺]_i 140 mM/[K⁺]_o 5 mM) and clamped at 0 mV to inactivate other voltage-dependent K⁺ currents, allowing the demonstration of BK_Ca channel alone in response to a series of voltage steps (Olesen et al., 1994). The pipette solution contained 2 mM ATP, a concentration considerably higher than 40 to 50 µM, the concentration generally found to half-maximally inhibit the K_ATP channel. Voltage steps ranging between 30 mV and +75 mV with an increment of 15 mV from a holding potential of 0 mV elicited a family of noisy, time-dependent and noninactivating currents (fig. 4, A and D), of which more than 85% were blockable by 2 mM TEA (fig. 4, B and C) and 80 nM ibTX (fig. 4, E and F), which indicated a predominant contribution of the BK_Ca channel to the current. Figure 5 shows the effects of ZD6169 at concentrations of 0.5, 5 and 50 µM on the whole-cell BK_Ca currents in a typical experiment. In 13 of 14 cells tested, significant activation of the BK_Ca currents by ZD6169 was observed only at concentrations equal to or higher than 20 µM (fig. 5D). However, in 4 of 13 responsive cells ZD6169 caused a dose-dependent augmentation of the BK_Ca currents at concentrations ranging between 0.5 and 50 µM. The BK_Ca channel-activating effect had a rapid onset and offset, and was fully reversible within 1 to 2 min (fig. 5E).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Whole-cell BKCa currents are sensitive to TEA and ibTX. A family of whole-cell BKCa currents were elicited by a voltage-step protocol ranging from 30 mV to +75 mV with an increment of 15 mV from a holding potential of 0 mV in control (A), in 2 mM TEA (B) and the current-voltage relationship of A (circles) and B (squares). Data in A, B and C were obtained from the same cell. Results of a parallel experiment from another cell in control (D), in 80 nM ibTX (E) and the current-voltage curves for D (triangles) and E (diamonds). The voltage-step protocol had a duration of 1 s. The cell was bathed in physiological K+ solutions ([K+]i 140 mM/[K+]o 5 mM). The pipette solution contained 2 mM ATP.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   ZD6169 activates BKCa current. Whole-cell currents were elicited by a voltage-step protocol ranging from 30 mV to +75 mV with an increment of 15 mV and a duration of 1 s (as shown at the top) in control (A), during subsequent exposure to ZD6169 at 0.5 (B), 5 (C) and 50 µM (D), and after washout of the drug (E). A holding potential of 0 mV was used to inactivate other voltage-dependent K+ currents. The cell was bathed in physiological K+ solutions ([K+]i 140 mM/[K+]o 5 mM). The pipette solution contained 2 mM ATP. The scale of a 5 nA reference current is shown at the lower left corner.

Voltage dependence of the effects of ZD6169. The instantaneous current-voltage relationship in the absence and presence of ZD6169 was assessed in a conventional way by use of the ramp protocol used in figure 2; the results are shown in figure 6. The K_ATP current (most visible in the inward direction) underwent a reversible stimulation and a suppression as the concentration increased (fig. 6, A-E). The current at potentials positive to 0 mV, rectifying in an outward direction, was largely composed of the current through the BK_Ca channels. A reversible augmentation of this current component by ZD6169 at 50 µM is readily visible in figure 6D. Figure 6F illustrates the overall effects of ZD6169 on K_ATP and BK_Ca currents. The increase (from the control) in the amplitude of the maximum inward K_ATP current measured at 110 mV in the ramp experiments was 15.0 ± 6.3%, 52.1 ± 12.7% and 29.6 ± 4.1% (n = 12); whereas the increase (from the control) of the BK_Ca current at +60 mV in the step experiments was 10.0 ± 2.3%, 15.2 ± 3.2% and 47.5 ± 3.3% (n = 14), respectively, in the presence of 0.5, 5 and 50 µM of ZD6169.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of ZD6169 on current-voltage relationship. Whole-cell currents were recorded with a voltage ramp ranging from 110 mV to +110 mV over a period of 1.5 s (as shown at the top) in control (A), in ZD6169 at 0.5 µM (B), 5 µM (C) and 50 µM (D), and after washout of the drug (E). The holding potential was 50 mV. The cell was bathed in symmetrical 140 mM K+ solutions. The pipette solution contained 0.5 mM ATP. Dashed lines indicate the zero current level. The scale of a 2 nA reference current is shown at the lower left corner. Panel F illustrates the changes (from the control) in the maximum inward KATP current measured at 110 mV in the ramp voltage-clamp experiments (hatched bars) and in the BKCa current measured at +60 mV in step voltage-clamp experiments (filled bars) induced by ZD6169 at 0.5, 5 and 50 µM. The abscissa gives the concentration of ZD6169 (µM), and the ordinate denotes changes of the current, which were calculated according to the formula: % change = (IK in ZD6169  IK in control)/(IK in control).

Activation of BK_Ca by ZD6169 (50 µM) is independent of Ca⁺⁺. The results in figure 6F demonstrated that ZD6169 at 50 µM caused simultaneously an inhibition of the K_ATP current and an activation of the BK_Ca current. The former effect would presumably lead to membrane depolarization followed by the opening of the voltage-dependent Ca⁺⁺ channels. It was therefore speculated that the stimulation of the BK_Ca channel might be secondary to the inhibition of K_ATP channels via an increase in intracellular free Ca⁺⁺. We investigated whether Ca⁺⁺ signaling bridged these two events by scrutinizing the Ca⁺⁺ dependence of ZD6169-induced BK_Ca channel activation. It was found that ZD6169 stimulated the whole-cell BK_Ca current by a similar magnitude in the presence and absence of Ca⁺⁺ in the bath (data not shown), which indicated that the effect is independent of extracellular Ca⁺⁺ concentration. The effect was also unaffected by the addition of up to 12 µM nifedipine, a Ca⁺⁺ channel blocker, to the bath. Figure 7 shows the effect of 50 µM ZD6169 on the whole-cell BK_Ca currents elicited by depolarization steps in the absence (fig. 7, A-C) and in the presence (fig. 7, D-F) of 1 µM nifedipine. Apparently, the activation of the BK_Ca currents by ZD6169 persisted regardless of blockade of the voltage-dependent Ca⁺⁺ influx. Thus, the effect was independent of the change in [Ca⁺⁺]_i resulting from Ca⁺⁺ influx.

View larger version (46K): [in this window] [in a new window]   Fig. 7.   Effect of ZD6169 on BKCa currents is independent of Ca++ influx. Whole-cell BKCa currents were augmented by 50 µM ZD6169 in the absence (A, B and C) and presence of 1 µM nifedipine (D, E and F). Currents were elicited by a voltage-step protocol ranging from 30 mV to +75 mV with an increment of 15 mV from a holding potential of 0 mV (as shown at the top). The bath and pipette solutions contained, respectively, 103 and 108 M Ca++. The BKCa currents in control (A) were activated during exposure to 50 µM ZD6169 (B), and reversed upon washout of ZD6169 (C). Current recordings of a parallel experiment but in the presence of 1 µM nifedipine throughout were made in another cell (D, E and F). The ordinates in A and D indicate the current scales. The scales in B and C are the same as in A, and those in E and F are the same as in D.

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View larger version (25K): [in this window] [in a new window]   Fig. 8.   ZD6169 activates single BKCa channels in an inside-out patch. Representative single BKCa recordings (from top to bottom) in control, in the presence of ZD6169 at 0.5, 5 and 50 µM, and after washout of the drug. Recordings were made from an inside-out patch held at +50 mV and exposed to symmetrical K+ (140 mM) solutions with a free Ca++ concentration of 108 M in bath solution. In all panels, two current traces are consecutive recordings with a total duration of 20 s. Upward deflections are the opening events of the channel, whose closed state is indicated by the short lines at the left of the traces. Currents were filtered at 2 kHz.

Effects of racemic ZD6169 on K_ATP and BK_Ca channels. ZD6169 was reported to display a stereoselectivity. Being the active S-enantiomer, ZD6169 is about 30-fold more potent than the R-enantiomer in mechanoinhibitory activity (Li et al., 1995). The racemic ZD6169 was tested in experiments parallel with those with ZD6169. Analogous to the experiment with ZD6169 (S-enantiomer) in figure 2, figure 9 shows a typical concentration-dependent dual effect of the racemic ZD6169 on inward K_ATP current evoked by a voltage ramp. Likewise, the opening effect on the BK_Ca current elicited by a step voltage protocol is demonstrated in figure 10. It is obvious that the effects of the racemic ZD6169 on the K_ATP and the BK_Ca currents were not dissimilar to those of ZD6169. All effects were readily reversible.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Concentration-related dual action of racemic ZD6169 on KATP current. These recordings are representative of the whole-cell current in response to a 1.5-sec voltage ramp from 110 to +30 mV (as shown at the top) in control (A), in the presence of ZD6169 at 0.5 µM (B), 5 µM (C) and 50 µM (D). The cell was bathed in symmetrical 140 mM K+ solutions and held at 50 mV. Calcium concentration was 103 and 108 M in bath and pipette solution, respectively. The pipette solution contained 0.5 mM ATP. Dashed lines indicate the zero current level. A 0.5 nA reference current is shown at bottom left of the figure.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Racemic ZD6169 activates BKCa current. Whole-cell currents were elicited by a voltage-step protocol ranging from 30 mV to +75 mV with an increment of 15 mV and a duration of 1 s (as shown at the top) in control (A), during subsequent exposure to ZD6169 at 0.5 (B), 5 (C), and 50 µM (D), and after washout of the drug (E). The holding potential was 0 mV. The cell was bathed in physiological K+ solutions ([K+]i 140 mM/[K+]o 5 mM). The pipette solution contained 2 mM ATP. A 2 nA reference current is shown at lower left corner.

	Discussion

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Zeneca ZD6169, an anilide tertiary carbinol, represents a new structural class of K⁺ channel openers and is currently under the development by Zeneca Pharmaceuticals Group as an intervention therapy for urinary urge incontinence. As a bladder smooth muscle relaxant agent, its mechanism of action has recently been investigated. Results from several functional and electrophysiological assays provided evidence showing that ZD6169 exerts its mechanoinhibitory action in bladder detrusor by opening the K_ATP channels (Li et al., 1995; Trivedi et al., 1995b). The present study has shown that the pharmacology of ZD6169 is more complex than originally described, and the drug possesses multiple excitatory and inhibitory effects on multiple types of K⁺ channel.

In this study, the K_ATP current was measured with voltage ramp protocols at voltages negative to 0 mV after optimizing the conditions for the detection of the current. Cells were bathed in symmetrical K⁺ (140 mM) solutions to increase inward current and clamped at 50 mV to minimize activation of other voltage-dependent K⁺ currents (Clapp et al., 1994). The intracellular ATP concentration was reduced to 0.5 mM to facilitate the activation of the K_ATP channel. At voltages negative to 0 mV where BK_Ca channel and delayed rectifier K⁺ current were minimal, the inward K_ATP current was visibly detected (figs. 2 and 3). After the application of ZD6169 at lower concentrations (0.5-10 µM, fig. 2), significant inward current was activated in a concentration-dependent manner. The activated current had little voltage dependence and was completely abolished by 1 µM glyburide (fig. 3), which indicated that ZD6169 acted to open the K_ATP channel. Our observation is consistent with the electrophysiological data reported by Li et al. (1995). They showed that 10 µM ZD6169 induced a sustained increase in whole-cell K_ATP current. A novel and unexpected finding was the significant inhibition of K_ATP current by ZD6169 at concentrations higher than 20 µM. The mechanism(s) underlying the transition from a stimulatory to an inhibitory action is not clear at the present time and will be the subject of future study. Collectively, the concentration-response curve of the effects of ZD6169 on the K_ATP current had a bell shape with a maximum activation reached at a concentration of approximately 10 µM.

The chTX- and ibTX-sensitive BK_Ca channel has been reported to play a predominant role in controlling cell excitability and contractility in guinea pig bladder muscle (Suarez-Kurtz et al., 1991; Zografos et al., 1992; Shetty et al., unpublished data). Although the presence of the BK_Ca channel in guinea pig bladder smooth muscle cells was explored earlier (Klockner and Isenberg, 1985; Trivedi et al., 1995a), direct recording of the isolated BK_Ca current is however lacking. In the present study, the whole-cell BK_Ca current was demonstrated in isolation by use of voltage-step protocol with a holding potential of 0 mV, at which the other voltage-dependent K⁺ current was inactivated and by use of 2 mM ATP in the pipette solution to inhibit the K_ATP current (Olesen et al., 1994; Edwards et al., 1994). The single BK_Ca channel in guinea pig bladder cells was found to be highly dependent on membrane potential and intracellular Ca⁺⁺ with a voltage and Ca⁺⁺ sensitivity similar to those of the conventional BK_Ca channels in various types of cells (Marty, 1983; Singer and Walsh, 1987). The single-channel conductance of 209 pS in symmetrical K⁺ (140 mM) solutions was also in a good agreement with the conductances of typical BK_Ca channels in other tissues (Lattore et al., 1989). To our best knowledge, the BK_Ca currents at whole-cell as well as single-channel level in guinea pig bladder muscle cells, for the first time, were measured directly, and the effects of ZD6169 on the channels were assessed.

An important observation of the present study was that over a similar concentration range (>20 µM), ZD6169 not only inhibited the K_ATP current, but also significantly enhanced the BK_Ca current. This type of multiple inhibitory and stimulatory actions is not unique to ZD6169 but is also shared by reported BK_Ca channel openers NS004 (Xu et al., 1994; Hu et al., 1994) and NS1619 (Edwards et al., 1994). In theory, a closing of the K_ATP channel and an opening of the BK_Ca channel over a similar concentration range could be causally related, because the inhibition of K_ATP channel would lead to membrane depolarization, activation of Ca⁺⁺ influx and hence stimulation of the BK_Ca channel. However, this sequence of events did not apply to the effects of ZD6169. We found that the augmentation of the whole-cell BK_Ca current by ZD6169 was unaffected by the removal of extracellular Ca⁺⁺ or by the presence of Ca⁺⁺ channel blocker, which suggested that the effect did not involve changes in [Ca⁺⁺]_o or [Ca⁺⁺]_i subsequent to Ca⁺⁺ influx. In inside-out detached patches, ZD6169 was capable of opening the channel when the cytosolic Ca⁺⁺ concentration was held constant. In addition, the magnitude of stimulation did not significantly differ whether [Ca⁺⁺]_i was 10⁹ or 10⁸ M. Thus, the effect was independent of the change in [Ca⁺⁺]_i. In settings like detached patches, in which the activities of intracellular biochemical or biophysical pathways are usually disrupted because of a lack of substrates or cellular organelles, a second messenger (e.g., IP₃) mediated process for the effect of ZD6169 on the BK_Ca channel was most unlikely, although there was a report on the preservation of functional intracellular Ca⁺⁺ store sites in detached patches of smooth muscle cells from rabbit portal vein (Xiong et al., 1992). It is therefore conceivable that the stimulatory effect of ZD6169 on the BK_Ca channel is a direct interaction between the drug and the channel gating, and the inhibition of K_ATP channel is not causally linked to the activation of BK_Ca channel through Ca⁺⁺ signaling.

Based on the reported in vitro data that the mechanoinhibitory effect of ZD6169 was antagonized by glyburide (Li et al., 1995), an opening of the K_ATP channel is likely to be the primary mechanism of relaxant action. Our data in figure 6 showed that ZD6169 is capable of augmenting the BK_Ca channel with a magnitude comparable to the magnitude of the activation of the K_ATP channel. Considering the unique role of the BK_Ca channel in bladder muscle excitability, the ZD6169-induced the BK_Ca channel activation would also be an obligate mechanism underlying its bladder relaxant effect. Although glyburide was reported to cause a rightward shift of the concentration-response curve of ZD6169-induced relaxation of guinea pig detrusor strips (fig. 4; Li et al., 1995), the shift was not strictly parallel when the concentration of ZD6169 was 10 µM or greater. A possible explanation could be that the relaxation induced by ZD6169 at higher concentrations is not primarily caused by an opening of the K_ATP channel but of the BK_Ca channel.

The in vivo pharmacology of ZD6169 has recently been assessed in comparison with cromakalim, a reference K_ATP channel opener (Howe et al., 1995). The results showed that ZD6169 produced a bladder inhibitory profile similar to that of cromakalim, but a significantly weaker cardiovascular activity than cromakalim. It remains to be established whether this in vivo selectivity derives from pharmacokinetic factors or ZD6169 is able to exploit any possible difference between K_ATP channels in different tissues. Nevertheless, the diverse activities of ZD6169 on multiple types of K⁺ channels might, in part, be responsible for the in vivo selectivity. The diminished stimulatory effects on K_ATP channel as the drug concentration increases may contribute to the weaker effects on heart rate and blood pressure, whereas a simultaneous activation of BK_Ca channel may ameliorate the inhibitory effect on bladder because of the pivotal role of the BK_Ca channel (generally absent in cardiac tissues) in bladder contractility (Suarez-Kurtz et al., 1991; Zografos et al., 1992).

In summary, Zeneca ZD6169 exerts diverse actions on multiple types of K⁺ channels in smooth muscle cells from guinea pig bladder detrusor. Depending on the concentration, it produces a dual stimulatory and inhibitory effect on K_ATP channel and a stimulatory effect on the BK_Ca channel. An agent with such an electrophysiological profile may be potentially beneficial for the treatment of urinary urge incontinence with less undesired cardiovascular side effects.