Introduction

Abstract Introduction Methods Results Discussion References

Transmembrane currents play a fundamental role in the activation and functioning of excitable tissues. In urinary bladder smooth muscle, depolarization and excitation-contraction depend on the activation of voltage-gated calcium channels (Klöckner and Isenberg, 1985a, b; Montgomery and Fry, 1992). In isolated guinea pig bladder, the calcium current has been characterized as that carried by dihydropyridine sensitive, L-type channels (Nakayama and Brading et al., 1993; Sheldon and Argentieri, 1995). The current underlying repolarization in detrusor smooth muscle is carried through several ion channels, virtually all of which use potassium as the charge carrier. These include a transient, 4-aminopyridine-sensitive current (Fujii et al., 1990), a delayed rectifier (Klöckner and Isenberg, 1985b), an ATP-dependent current (Bonev and Nelson, 1993; Trivedi et al., 1994) and a charybdotoxin-sensitive current consistent with the BK_Ca (Zografos et al., 1992). Several of these channels have been the target of compounds and drugs aimed at modulating the physiology and functioning of smooth muscle and other tissues (Edwards and Weston, 1995). Recent reports have indicated that NS-1619 selectively increases the conductance of the BK_Ca in several tissue types including vascular and airway smooth muscle (Edwards et al., 1994; Macmillan et al., 1995) and neuronal tissues (Sellers and Ashford, 1994). Edwards et al. (1994) demonstrated that NS-1619 activated a charybdotoxin-sensitive current in isolated rat portal vein smooth muscle cells and produced a relaxation of KCl-contracted aortic ring preparations. In a preliminary report, Green et al. (1995) demonstrated an NS-1619-induced increase in outward current in rat detrusor myocytes.

Several mechanisms can be targeted for inhibition of smooth muscle contraction, including inhibition of depolarizing (calcium; I_Ca) current, or enhancement of repolarizing (potassium) current. The purpose of the present study was the determine the underlying mechanism of action of NS-1619 on inhibiting in vitro bladder smooth muscle contractions. With use of isolated guinea pig detrusor strips and patch-clamp analysis of isolated guinea pig detrusor myocytes, we examined the effects of NS-1619 on both contractile properties and transmembrane calcium and BK_Ca currents. A preliminary report of these findings has been presented previously (Sheldon et al., 1996).

	Methods

Abstract Introduction Methods Results Discussion References

Detrusor strip contraction studies. Male Hartley guinea pigs (400-600 g) were euthanized by CO₂ inhalation and exsanguination. Their urinary bladders were rapidly removed and placed in 37°C physiological salt solution (PSS) that contained the following (mM): NaCl, 118.4; KCl, 4.7; CaCl₂, 2.5; MgSO₄, 1.2; KH₂PO₄, 1.2; NaHCO₃, 24.9; and D-glucose, 11.1, gassed with 95%/5% CO₂/O_2, to achieve a pH of 7.4. The dome of the bladder was isolated from the trigon region and the mucosa was removed. This tissue was then cut into 4- to 5-mm-wide by 10-mm-long strips. One end was secured to the bottom of a water-jacketed tissue bath and the other to a Grass isometric force transducer (Grass Instruments, Quincy, MA). Tissues were pretensioned (0.25-0.5 g) and after 30 min of equilibration were contracted with an additional 15 mM KCl and again allowed to equilibrate. Compounds were administered directly into the tissue baths as sequential concentrations.

Isolation of guinea pig detrusor cells. Male Hartley guinea pigs (400-600 g) were euthanized by CO₂ inhalation and exsanguination. Their urinary bladders were rapidly removed and placed in 37°C physiological solution with the following composition (mM): Na glutamate, 80.0; NaCl, 54.7; KCl, 5.0; NaHCO₃, 25.0; MgCl₂·2H₂O, 2.5; D-glucose, 11.8; and CaCl₂, 0.2, gassed with 95%/5% CO₂/O₂ for a final pH of 7.4. The dome of the bladder was isolated from the trigon region and the mucosa was removed. This tissue was then cut into 2- to 3-mm-wide strips and placed in fresh buffer for 1 hr. Tissues were then transferred into 7.5 ml of an isolation buffer containing the above-mentioned composition plus collagenase type VIII (1.0 mg/ml) and pronase (0.5 mg/ml). After 10 min the isolation buffer was replaced with fresh isolation buffer for an additional 10 min. The tissue was then washed three times in fresh collagenase and pronase-free solution and stored at room temperature until studied. Cells for study were prepared by triturating two to three pieces of detrusor tissue in 7.5 ml of fresh isolation buffer for 5 min with a polished Pasteur pipette (tip diameter ~1.5 ml) attached to a modified Harvard Respirator pump (Harvard Apparatus, Southnatic, MA) at a rate of 20 times/min with an approximate volume of 5 ml. Cells were then filtered through a 100-µm polypropylene screen and placed on a microscope stage in a temperature-regulated tissue bath at 32.5°C and continually superfused with PSS.

Voltage-clamp recordings. Whole cell recordings were made with use of a List-Medical EPC-7 patch-clamp amplifier (Adams & List Assoc., Westbury, NY). The pipette solution for BK_Ca recording contained the following (mM): KCl, 126.0; MgCl₂·6H₂O, 4.5; ATP Mg salt, 4.0; GTP tris salt, 0.3; creatine PO₄, 14.0; D-glucose, 9.0; EGTA, 9.0; HEPES, 9.0. The pH was adjusted to 7.4 with KOH. The pipette solution for recording calcium currents contained Cs⁺ to block potassium currents and was composed of the following (mM): CsMeSO₄, 126.0; MgCl₂·6H₂O, 4.5; ATP Mg salt, 4.0; GTP tris salt, 0.3; creatine PO₄, 14.0; D-glucose, 9.0; EGTA, 9.0; HEPES, 9.0. The pH was adjusted to 7.4 with CsOH. Electrodes had tip resistances of 2 to 4 megohm. Currents were evoked with the voltage-clamp protocols described in the figure legends. Signals were acquired (3 kHz high-frequency cut-off) and analyzed by a 486-based personal computer and pClamp (Axon Instruments, Foster City, CA) software.

Data analysis. Because the spontaneous bladder contractions were irregular in amplitude and frequency, we evaluated the area under the contraction curve as a measure of contractility. Signals were digitized (12-bit resolution) and analyzed on-line by a 386-based computer and custom software.

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	Results

Abstract Introduction Methods Results Discussion References

Detrusor Strip Contraction

Isolated detrusor strips exposed to 20 mM KCl contract spontaneously with irregular amplitude and frequency. Exposure to NS-1619 (0.3-30 µM) produced a concentration-dependent inhibition of contraction as measured by the decrease in area under the contraction curve (fig. 1A). The average IC₅₀ (n = 6-12 animals) was 12.2 ± 3.2 µM (mean ± S.E.M.). In all preparations, the BK_Ca channel blocker, Ibtx (100 nM; Galvez et al., 1990) produced a complete recovery from inhibition. In addition to Ibtx, inhibition of contraction could be reversed in all preparations by exposure to 200 nM carbachol (231 ± 51%, n = 4), but not 6 µM glyburide (data not shown). Figure 1B shows the average dose-response curve to NS-1619 alone, and in the presence of 100 and 300 nM Ibtx (n = 4 each). The concentration-response data were expressed as percent of maximal contraction above zero (pretension weight, 0.25-0.5 g). NS-1619 produced a concentration-dependent decrease in contraction relaxing the tissue beyond the starting pretensioned weight. In the presence of Ibtx, there was a decrease in the intrinsic activity of NS-1619 (i.e., decrease E_max for both concentrations), as well as a significant shift to the right in the concentration-response curve (IC₅₀ = 31.1 ± 9.4 µM; 300 nM).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   (A) A representative tracing from an isolated guinea pig detrusor strip. Tissues contracted spontaneously after exposure to 20 mM KCl. Contractions were quantified by determining the area under the contraction curve. NS-1619 caused a concentration-dependent decrease in the area under the contraction curve. In the continued presence of NS-1619, the BKCa channel blocker Ibtx reversed the effects of NS-1619. The entire trace represents approximately a 2-hr period. The chart recorded speed was increased between concentrations to better resolve individual contractions. (B) Concentration-response curve for NS-1619 before (open circles) and after pre-exposure to 100 (filled circles) and 300 nM (triangles). The curve through the data points is the best fit to a Michaelis-Menten logistic. The average IC50 was 12.2 ± 3.2 µM. After pre-exposure to 300 nM Ibtx, the average IC50 shifted to 31.1 ± 9.4 µM (*P < .05; two population Student's t test)

Outward Currents

Voltage steps. Isolated cells were typically 10 to 15 µm wide and 100 to 200 µm long. The average resting potential (5 mM external potassium) recorded with pipettes containing 140 mM potassium was 45.3 ± 2.7 mV (n = 10). The cells were typically quiescent and did not fire spontaneous action potentials. These characteristics agree closely with those originally reported by Klöckner and Isenberg (1985a) for guinea pig detrusor myocytes.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Whole-cell voltage-clamp tracings from an isolated guinea pig detrusor myocyte. Cells were held at 10 mV and stepped in +10-mV increments for 1000 msec. NS-1619 increased both net outward current as well as the amplitude of current fluctuations. Ibtx blocked this effect.

Voltage ramp. Quasi steady-state transmembrane currents were assessed by holding the cell at 50 mV, then ramping the command voltage from 60 to 40 mV at a rate of 3.34 mV/sec. NS-1619 exposure produced an increase in steady-state outward current between 20 and 40 mV (fig. 3A). The control steady-state current also demonstrated a small inward calcium "window" current between 40 and 0 mV. In addition to the increase in outward current, NS-1619 also appeared to inhibit this window current. Exposure to Ibtx reversed the effect of NS-1619 on outward current, but not the window current (data not shown). The steady-state NS-1619 sensitive current is shown in figure 3B.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   (A) Ramp-clamp tracing. Cells were held at 50 mV and ramped from 60 to 40 mV. A large increase in outward current is evident at positive potentials. Shown are control (bottom trace) and the effects of 30 µM NS-1619 (upper trace). Calcium "window" current underlies the inward deflection in the control trace between 40 and 0 mV. (B) Difference current from control and NS-1619 ramp clamp in figure 3A. The tracing illustrates both the increase in outward current above 0 mV and the inhibition of calcium "window" current between 40 and 0 mV.

Single-channel recordings. Single-channel recordings from cell-attached patches with microelectrodes containing 140 mM potassium were performed on five cells. Figure 4A shows tracings from a cell-attached patch before and after exposure to NS-1619. In control, single-channel openings (upward events) are observed between relatively long closed periods. After 30 µM NS-1619, the open channel probability increased from 0.089 ± 0.032 to 0.229 ± 0.074, which resulted in an increase in the number of open channels. The all-points histogram in figure 4B illustrates a large decrease in the number of closed channels and an increase in the number of single-channel openings after NS-1619. There was no evidence of an increase in single-channel conductance or single-channel open time.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   (A) Cell-attached patch-clamp tracing. The holding potential was 50 mV. Channel openings are shown as upward deflections. The control trace shows single-channel openings with long closed periods between, and the NS-1619 trace shows single-channel openings in the same patch after 30 µM NS-1619 was applied to the bath. An increase in the number of channel openings is evident. Single-channel conductance and open time remained unchanged. The single-channel open probability increased from 0.089 ± 0.032 to 0.229 ± 0.074 (n = 5). (B) All points (events) histogram for single-channel openings before (Control) and after (NS-1619) bath exposure to 30 µM NS-1619. Cell was held at 50 mV. After NS-1619 there was a decrease in the amount of time that the patch spent in the closed state, as well as an increase in the number of simultaneously opened channels.

Calcium Current

Calcium currents were elicited by holding the cells at 40 mV and stepping to 50 mV in 5-mV increments for msec. A typical "family" of calcium currents generated between 40 and 0 mV is displayed in figure 5A. Previous studies (Sheldon and Argentieri, 1995) have demonstrated that guinea pig detrusor myocyte calcium currents are stable during the recording period (15-20 min, 0.1% dimethyl sulfoxide). After a 15-min exposure to 30 µM NS-1619 (n = 5), a decrease in both peak current and noninactivating current was evident. Figure 5B shows the current voltage relationship for peak calcium current before and after 30 µM NS-1619. The curve through the data points represents the best fit for equation 1. Approximately 50% of the maximal peak current was blocked (504 ± 207 to 280 ± 153 pA, P < .05, paired t test). Fitting the data points to equation 1 revealed a significant decrease (P < .05, paired t test) in the maximum conductance from 10.5 ± 3.8 to 6.2 ± 2.9 nS (n = 5). There was no change in the calcium current reversal potential (49.4 ± 2.1 and 47.6 ± 4.8 for control and NS-1619, respectively). The effects on peak calcium current were partially restored on washout of NS-1619 (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   (A) Representative calcium current tracings in the whole-cell patch-clamp configuration, before (upper traces) and after (lower traces) exposure to NS-1619. Cells were held at 40 mV and pulsed to depolarizing potentials (Vtest) for 180 msec. (Shown are tracings from 40 to 0 mV in 10 mV steps.) NS-1619 (30 µM) produced approximately a 50% decrease in peak calcium current (n = 5). (B) Current-voltage relationship for peak calcium currents before (filled circles) and after (open circles) exposure to 30 µM NS-1619. Currents were generated with the protocol described in the text. The line through the data points is the best fit to equation 1 (n = 5).

The voltage dependence of activation (d, n = 5) of I_Ca is shown in figure 6. Peak calcium currents were divided by the driving force (V_m  E_rev) and expressed as a fraction of the maximal conductance. Peak currents were well fit by the Boltzmann distribution described in equation 2. Plotted are the control values and those after 15 min exposure to 30 µM NS-1619. The Boltzmann parameters are summarized in table 1. There was a significant decrease in the fractional maximal conductance (g/g_max; 0.98 ± 0.04 to 0.50 ± 0.06), but no significant change in the half-activation voltage (V_1/2) or slope factor (k).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Voltage dependence of calcium channel activation (d, open symbols) and inactivation (f, closed symbols), before (squares) and after (circles) exposure to 30 µM NS-1619 (n = 5). Plotted are the fractional conductances (g/gmax) as a function of activation or inactivation test potential (Vtest). The lines through the data points are the best fits to equations 2 and 3 for peak calcium current activation and inactivation, respectively. NS-1619 decreased the fractional conductance and shifted the half-inactivation voltage (V1/2) to more negative voltages. See table 1 for summary of derived Boltzmann parameters.

The voltage dependence of inactivation (f, n = 5) of I_Ca is also shown in figure 6. Both the control and NS-1619 data are well fit by the Boltzmann distribution given in equation 3. After 30 µM NS-1619, there was a significant (P < .05) decrease in the fraction of maximal conductance and the half-inactivation voltage (V_1/2) was significantly (P < .05) shifted to the left by approximately 4 mV (table 1).

Calcium "window" current is a measure of the time-independent current that remains after inactivation of the peak calcium current. The theoretical window current was estimated by integrating the product of the activation and inactivation curves [_{100  +40 mV} (d·f)·g_max·driving force]. After 30 µM NS-1619, there was a significant decrease in the area under the curve (P < .05) that represented a 70.8 ± 15.1% decrease in the theoretical window current.

	Discussion

Abstract Introduction Methods Results Discussion References

In the United States alone, more than 13 million people suffer from some form of UI. Of the various types of UI, bladder instability associated with urge incontinence represents the underlying etiology for most UI cases (Resnick, 1995). Present therapy for the treatment of urge UI includes anticholinergic and antispasmodic agents that also possess calcium channel-blocking properties. Although efficacy with these agents is reasonably good, patient compliance is poor chiefly because of anticholinergic side effects. In addition, a significant portion of the patient population with urge UI have an impaired ability to generate a sustained bladder contraction, despite having bladder instability (Resnick and Yalla, 1985). In these patients, agents that suppress contractility can cause urinary retention and overflow incontinence (Blaivas et al., 1980). Theoretically, an agent that "normalizes" diseased bladder through hyperpolarization of the detrusor smooth muscle would inhibit abnormal instability without affecting normal contractility and the ability of the bladder to empty. Recently Trivedi et al. (1995) published findings on a compound (ZD-6169) designed to activate the ATP-dependent potassium channel. Evidence suggests that this compound will stabilize the bladder and thereby increase the urine volume necessary to evoke the micturition reflex (Howe et al., 1995). NS-1619 is a compound that was developed as a BK_Ca channel agonist aimed at hyperpolarizing excitable tissues. Several other laboratories have reported effects on repolarization in various smooth muscle types and in neuronal tissue. To date, however, there have been no reports on the effects of NS-1619 on bladder mechanical activity. In addition, the activity of NS-1619 on BK_Ca channels in bladder smooth muscle has not been fully characterized, nor have there been any reports indicating that NS-1619 inhibits calcium currents in these cells. In the present study, we examined the effects of NS-1619 on guinea pig bladder strip function and determined its effects on BK_Ca currents and calcium currents in isolated guinea pig detrusor myocytes.

Intact tissue studies. Our data in the isolated guinea pig detrusor strips indicate that NS-1619 produced a concentration-dependent inhibition of KCl-induced spontaneous contractions with an IC₅₀ of 12.2 ± 3.2 µM. The concentration response curve was affected by pre-exposure to Ibtx, which suggests that the inhibition of contraction involves, at least in part, the activation of BK_Ca channels. The fact that 6 µM glyburide did not reverse the effect of NS-1619 suggests that activation of the ATP-dependent potassium channel is not involved in relaxation. These findings are consistent with previous reports in which NS-1619 inhibited contractions in isolated rat portal vein and aorta preparations (Edwards et al., 1994).

BK_Ca studies. NS-1619 produced an increase in the amplitude of both net outward current and current fluctuations in isolated guinea pig detrusor myocytes. This was demonstrated with both the voltage step and the steady-state ramp protocols. In addition to the increase in outward current, inhibition of an inward current that correlated with the calcium "window" current was also observed. Both the increase in net outward current and current fluctuations were inhibited by 100 nM Ibtx even in the continued presence of NS-1619. Ibtx has been shown to be a selective blocker of BK_Ca channels (Galvez et al., 1990), and therefore it would appear likely that NS-1619 increases outward current in guinea pig detrusor myocytes by increasing BK_Ca current. In a preliminary report, Green et al. (1995) demonstrated a NS-1619-induced increase in BK_Ca currents from isolated rat detrusor myocytes. Other investigators have reported similar findings in other tissues including tracheal smooth muscle (Macmillan et al., 1995), vascular smooth muscle (Edwards et al., 1994; Olesen et al., 1994) and neuronal tissue (Sellers and Ashford, 1994; Lee et al., 1995). Edwards et al. (1994) also reported an inhibition of the potassium-delayed rectifier current in isolated rat portal vein cells. Although we did not specifically assess this property, we saw no strong evidence for block of this current. The increase in BK_Ca as the underlying mechanism for the increase in net outward current was confirmed at the single-channel level. In cell-attached patches, 30 µM NS-1619 increased the number of open channels in the patch without causing a significant increase in single-channel conductance or open time. Thus, NS-1619 appears to activate BK_Ca channels by increasing the open-channel probability. Olesen et al. (1994) reported similar single-channel results with NS-1619 in bovine aortic smooth muscle. The outside pore of the BK_Ca channels was protected from exposure to NS-1619 because they were inside the patch electrode. It is therefore likely that NS-1619 activates channels from the inside. This is not surprising because of the lipophilic nature of the compound that enables rapid permeation of the membrane. The mechanism by which NS-1619 increases BK_Ca current is not clear. Olesen et al. (1994) found that the NS-1619 response was not modulated by changing the level of ATP exposure with inside-out patches, which suggests that channel activation did not involve a channel phosphorylation process. The same group also reported that 30 µM NS-1619 could not activate the BK_Ca channel in the absence of internal calcium, which indicates that the compound cannot substitute for Ca⁺⁺ as a channel activator.

Calcium channel studies. Calcium currents recorded from isolated Cs⁺-loaded detrusor myocytes are dihydropyridine sensitive and characterized as L-type currents. In this study NS-1619 caused a significant decrease in calcium channel conductance relative to control. The voltage dependence of calcium channel activation and inactivation in the absence and presence of NS-1619 was well described by a Boltzmann distribution. Approximately an equal fractional decrease in current was observed at all activation potentials, which suggests that block of current activation is not voltage dependent. Except for conductance (g/g_max), there were no changes in any of the other activation Boltzmann parameters. Exposure to NS-1619 resulted in a decrease in the fractional conductance and a negative shift of V_1/2 in the calcium current inactivation curve. Although the shift in V_1/2 was small (4 mV), it was very consistent and suggests that NS-1619 block shows some preference for block of the open state of the channel. The decrease in channel conductance resulted in a 71% decrease in the theoretical calcium "window" current. This derived decrease correlated well with the decrease in dihydropyridine-sensitive (Sheldon and Argentieri, 1995) inward current observed with the voltage-clamp ramps between 40 and 0 mV shown in figure 3, A and B. An NS-1619-induced decrease in peak calcium current in isolated rat portal vein cells was also reported by Edwards et al. (1995).

Conclusions. BK_Ca channels have been shown to associated with several tissue types and play a significant role in the modulation of cell electrophysiology. NS-1619 is an organic small molecule that activates BK_Ca channels in a variety of tissues including bladder smooth muscle. In this study we demonstrated that NS-1619 inhibits bladder smooth muscle contractions in vitro in a concentration-dependent manner and this effect is partially antagonized by Ibtx. Patch-clamp data confirmed an activation of BK_Ca channels that resulted in an increase in an Ibtx-sensitive current. Single-channel analysis showed an increase in the open-channel probability with NS-1619. In addition to the activation of BK_Ca channels, NS-1619 inhibited peak inward calcium current amplitude and shifted the calcium current inactivation curve to the left. NS-1619 also inhibited the time-independent calcium window current. It is concluded that NS-1619 effectively inhibits guinea pig detrusor smooth muscle involving both activation of BK_Ca channels and inhibition of calcium channels.