Introduction

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In urinary bladder smooth muscle, ATP-sensitive potassium (K_ATP) channels play important roles in the regulation of cellular excitability. K_ATP channels are inhibited by intracellular ATP and couple cellular metabolism to membrane excitability. These channels are inhibited by sulfonylurea analogs such as glyburide and are activated by structurally diverse K_ATP channel openers (KCOs), including cromakalim and pinacidil (Edwards and Weston, 1993; Gopalakrishnan et al., 1993; Quayle et al., 1997).

Over the past decade, K_ATP channel openers have been increasingly investigated as potential therapeutic agents for the treatment of smooth muscle disorders, including overactive bladder. Cromakalim, pinacidil, ZM244085, ZD6169, YM934, Y26763, and WAY-133537 have been shown to evoke concentration-dependent relaxation of isolated bladder smooth muscle strips precontracted by a variety of stimuli, including electrical field, carbachol, or high external [K⁺] (Fovaeus et al., 1989; Fujii et al., 1990; Seki et al., 1992; Li et al., 1995; Masuda et al., 1995; Wojdan et al., 1999; Buckner et al., 2000). Electrophysiological studies have shown that ()-cromakalim, ZD-6169, and WAY-133537 can evoke glyburide- and ATP-sensitive K⁺ currents in guinea pig (Bonev and Nelson, 1993a; Heppner et al., 1996) and rat bladder smooth muscle cells (Wojdan et al., 1999). Thus, relaxation of mechanical responses to neurotransmitters and other stimulants by KCOs is primarily due to opening of K_ATP channels that lead to membrane hyperpolarization and attenuated Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (Gopalakrishnan et al., 1993; Quayle et al., 1997).

Although opening of K_ATP channels by KCOs can relax smooth muscle by membrane hyperpolarization, the functional contribution of K_ATP channels to smooth muscle relaxation remains unclear. Previous studies have shown that at concentrations that evoke muscle relaxation, K_ATP channel activity is not significantly altered as measured by ⁸⁶Rb⁺ efflux or current responses (Quast et al., 1993). The concentrations of KCOs required to relax smooth muscle are lower than those required for activation of K_ATP currents (for review, see Quast, 1993; Quayle et al., 1997). In addition, it has been shown that activation of muscarinic receptors in guinea pig bladder smooth muscle cells can inhibit ~40 to 60% of K_ATP currents through a protein kinase C pathway (Bonev and Nelson, 1993b), although muscle relaxation still occurs under these conditions in the presence of KCOs. Based on these observations, it could be hypothesized that opening a fraction of K_ATP channels may be sufficient to relax smooth muscle contraction by hyperpolarization. However, whether urinary bladder smooth muscle has "spare" K_ATP channels remains to be demonstrated.

Both guinea pig and pig bladders have been widely used as in vitro and in vivo models with potential for treatment of bladder instability for characterization of KCOs (Seki et al., 1992; Howe et al., 1995; Masuda et al., 1995; Hashitani et al., 1996). Although the electrophysiological properties of guinea pig bladder smooth muscle cells have been characterized (Bonev and Nelson, 1993a; Heppner et al., 1996), similar studies have not been undertaken using pig bladder smooth muscle cells. In the present study, we have characterized K_ATP channel opener-activated currents in pig and guinea pig bladder smooth muscle and correlated changes in K_ATP currents and membrane potential responses evoked by KCOs to demonstrate the existence of spare K_ATP channels in bladder smooth muscle. Preliminary results of this study have been previously reported in an abstract (Shieh et al., 1999).

	Materials and Methods

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Cell Isolation. Urinary bladders were removed from anesthetized guinea pigs (Hartley; Charles River, Wilmington, MA) or pigs (Landrace) and transferred directly into preoxygenated physiological saline solution containing 137 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.42 mM KH₂PO₄, 4.17 mM NaHCO₃, 10 mM HEPES, 10 mM glucose, pH 7.4, with NaOH. After mucosa and connective tissues were removed, bladder smooth muscles were cut into pieces (~1 × 5 mm). Pieces of bladder smooth muscle were incubated with collagenase (Worthington type 2; 1 mg/ml for guinea pig and 2 mg/ml for pig) at 36°C for 60 min in a dissociation solution containing 80 mM sodium glutamate, 55 mM NaCl, 6 mM KCl, 2 mM MgCl₂, 0.2 mM CaCl₂, 10 mM HEPES, 11 mM glucose, pH 7.4, with NaOH. The solution was gently agitated by bubbling with 95% O₂ and 5% CO₂ during the cell isolation. Single smooth muscle cells were obtained by triturating using a fire-polished large-bore Pasteur pipette and stored at 4°C before use.

Electrophysiology. Whole-cell patch-clamp technique was used to measure ionic currents and changes in membrane potentials from bladder smooth muscle cells. Fire-polished patch electrodes had a resistance of 2 to 5 M. The intracellular pipette solution contained 107 mM KCl, 1.2 mM MgCl₂, 1 mM CaCl₂, 10 mM EGTA, 5 mM HEPES, 0.1 mM ATP (pH 7.2 with KOH; total K ~140 mM). The bath solution contained 5 mM KCl, 135 mM NaCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 5 mM HEPES (pH 7.4 with NaOH). For whole-cell recordings in bath solutions containing 60 mM [K⁺], an equimolar concentration of NaCl was substituted. After a tight seal was formed, the membrane was ruptured and the capacitance transient was integrated on-line to estimate cell capacitance as a measure of cell size. Uncompensated series resistance was typically 3 to 10 M. The resting input resistance in the solution containing 60 mM [K⁺] was 2 to 5 G. The whole-cell currents were amplified using an Axopatch-200B amplifier (Axon Instruments, Foster City, CA) and low-pass filtered at 5 kHz (3 dB, four pole Bessel filter) before digitization by Digidata 1200B at a sampling rate of 10 kHz.

Isolated Smooth Muscle Studies. Urinary bladders from pig or guinea pig were removed and immediately placed in Krebs-Ringer-bicarbonate solution containing 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.5 mM MgSO₄, 1.2 mM KH₂PO₄, 20 mM NaHCO₃, 11 mM dextrose, equilibrated with 5% CO₂,95% O₂, pH 7.4 at 37°C. Muscle strips, 3 to 5 mm in width and 10 mm in length, were prepared from the bladder tissue by cutting in a circular manner. One end of the strip was fixed to a stationary glass rod, and the other was attached to a Grass FT03 transducer at a basal preload of 1.0 g. This preload proved to be the best condition for a steady-state baseline and reproducible responses to field stimulation. Two parallel platinum electrodes were included in the stationary rod to provide field stimulation (0.05 Hz for pig or 0.1 Hz for guinea pig, 0.5 ms at 20 V). Tissues were allowed to equilibrate for at least 60 min before the assay. Cumulative concentration-response curves were generated for each tissue, and each tissue was exposed to only one test compound as previously described (Gopalakrishnan et al., 1999).

Compounds. All potassium channel openers were obtained as previously described (Gopalakrishnan et al., 1999). Stock solutions of compounds were prepared in 100% dimethyl sulfoxide and diluted in buffer before use. Collagenase was purchased from Worthington Biochemical Co. (Lakewood, NJ). Glyburide, iberiotoxin, and all other chemicals were purchased from Research Biochemicals International/Sigma Chemical Co. (St. Louis, MO).

Data Analysis. Electrophysiological data were analyzed using pClamp 6.0 (Axon Instruments). The concentration-response curves of changes in the membrane potential were best fit with the equation V = V_max/[1 + (EC₅₀/[P1075])ⁿ, where V is membrane potential, V_max is maximal hyperpolarization, and n is Hill coefficient. The concentration-dependent increases in membrane current induced by P1075 was best fit with the equation I = I_max/[1 + (EC₅₀/[P1075])ⁿ, where I is membrane current and I_max is maximal current. Data are expressed as mean ± S.E.M. Comparisons of current densities between pig and guinea pig bladder smooth muscle cells were made using an independent-sample t test. Results were deemed significant at P < 0.05.

	Results

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K_ATP Currents in Urinary Bladder Smooth Muscle Cells

Although KCOs have been shown to relax the pig bladder smooth muscle (Foster et al., 1989b; Masuda et al., 1995), the properties of ATP-sensitive K⁺ currents in these cells have not been characterized. In the presence of 10 µM P1075, a cyanoguanidine KCO, an increase in inward current from a basal value of 46 ± 9 to 613 ± 118 pA (n = 16) was recorded under conditions where cells were bathed in the solution containing 60 mM K⁺ and voltage-clamped at 80 mV with patch pipette containing 140 mM K⁺ and 0.1 mM ATP. Upon addition of glyburide (5 µM), the P1075-evoked inward current was reduced to 110 ± 17 pA (Fig. 1A), which can be further reduced to the basal values when glyburide application was prolonged more than 5 min (data not shown). Under identical conditions, P1075 evoked an inward current in guinea pig bladder smooth muscle cells from a basal value of 31.4 ± 5.1 to 94.2 ± 12.0 pA (n = 13), which was then reduced to 31.0 ± 5.5 pA after application of 5 µM glyburide (Fig. 1B). To compare the K_ATP current density between pig and guinea pig bladder, current amplitudes evoked by P1075 (10 µM) were normalized to the cell capacitance. It was found that pig and guinea pig cells had an average cell capacitance of 16.6 ± 1.4 and 20.6 ± 2.7 pF, respectively. The normalized current density values were about 9-fold higher in the pig bladder smooth muscle cells (39.6 ± 11.8 pA/pF, n = 9) compared with those of the guinea pig (4.5 ± 0.6 pA/pF, n = 15) (Fig. 1C).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   P1075-induced increase in whole-cell currents in urinary bladder smooth muscle cells. P1075 evoked a glyburide-sensitive current in pig (A) and guinea pig (B) bladder. Cells were voltage-clamped at 80 mV and changes in membrane currents were measured in bath solution containing 60 mM K+. Pig and guinea pig cells had an average cell capacitance of 20.6 ± 2.7 and 16.6 ± 1.4 pF, respectively. KATP current densities obtained by normalizing the current amplitudes to cell capacitance (C) are 39.6 ± 11.8 pA/pF (n = 9) and 4.5 ± 0.6 pA/pF (n = 15) for pig and guinea pig cells, respectively. *Indicates significant (P < 0.05) differences in current densities.

To further examine the properties of K_ATP channels, several KCOs were evaluated for their effects on low-frequency-stimulated contractions of pig and guinea pig bladder muscle strips. Besides P1075, pinacidil, (±)-cromakalim and its active enantiomer ()-cromakalim, Bay X 9228 and its enantiomer Bay X 9227, ZD6169, ZM244085, and diazoxide all suppressed field-stimulated twitch responses with full efficacy in a concentration-dependent manner in bladder strips from both pig and guinea pig (Fig. 2). The rank order potencies (EC₅₀) for relaxation of low-frequency-stimulated pig detrusor strips showed an excellent correlation (r = 0.94) with the potencies to relax muscle strips from the guinea pig (Fig. 2). It has been suggested that KCOs might exert inhibitory effects on acetic acid-induced bladder hyperactivity by targeting the afferent nerves innervating the bladder (Yu and de Groat, 1998). To examine whether presynaptic inhibition on neurotransmitter release by KCOs is involved in the suppression on field-stimulated muscle contraction, we tested the effect of KCOs on suppression of exogenous carbachol-induced muscle contraction. P1075 completely inhibited pig bladder smooth muscle contractions evoked by 0.1 µM carbachol and by low-frequency electrical-field stimulation with comparable EC₅₀ values (0.036 ± 0.020 µM, n = 10, carbachol; 0.086 ± 0.004 µM, n = 12, field stimulation). Similar results were observed in guinea pig bladder smooth muscle (data not shown). Thus, the postsynaptic inhibition entirely accounts for the suppression of field-stimulated muscle contraction by KCOs.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Correlation of the potencies of KCOs to suppress low-frequency-stimulated contractions in pig and guinea pig bladder smooth muscle. The solid line represents linear regression through the points (Y = 0.119X + 1.06, r = 0.94 ± 0.34). Data shown are means ± S.E.M. (n = 3-4).

Taken together, these results suggest that the pharmacological properties of K_ATP channels in both pig and guinea pig bladder smooth muscle cells are similar, although the measured K_ATP current density in pig bladder smooth muscle is about 9-fold greater than that of the guinea pig. These findings suggest that the majority of K_ATP channels in pig bladder smooth muscle cells might be spare and that opening a fraction of channels might be sufficient for muscle relaxation.

Analysis of Spare K_ATP Channels in Urinary Bladder Smooth Muscle

It has been reported that, following the muscarinic receptor activation, K_ATP channels are inhibited by ~40 to 60% through a protein kinase C pathway in urinary bladder (Bonev and Nelson, 1993b) and tracheal smooth muscle (Nuttle and Farley, 1997). Although muscarinic receptor activation can significantly inhibit K_ATP channels in bladder smooth muscle cells, KCOs are still capable of preventing carbachol- or electrical-field stimulus-evoked muscle contractions (Foster et al., 1989b; Buckner et al., 2000). One explanation is that only a small fraction of K_ATP channel opening is sufficient for membrane hyperpolarization. This was examined in the following set of studies.

Assessment of Membrane Potential and K_ATP Currents in Single Bladder Smooth Muscle Cells. Changes in membrane current and potential evoked by P1075 were recorded successively from single pig smooth muscle cells bathed at 60 mM [K⁺]_ext. As shown in Fig. 3A, when recorded in the voltage-clamp mode (at 80 mV), application of 10 µM P1075 increased membrane current to 484.7 ± 45.4 (n = 5). During the 10-min exposure to P1075, the evoked current amplitude progressively declined by 30.5 ± 7.7% (n = 5). When recorded in the current-clamp mode, P1075 reduced the membrane noise and hyperpolarized the membrane potential toward 18.2 ± 0.3 mV (n = 5), which is close to the K⁺ equilibrium potential of 20 mV (Fig. 3B). Although membrane current declined about 30.5% of the initial current amplitude during 10-min exposure, P1075-evoked membrane potential response remained unaffected during this period. This clear dissociation of KCO-evoked changes in membrane current and membrane potential observed in the same single smooth muscle cell demonstrates that a fraction (~70%) of K_ATP channel opening is sufficient to evoke membrane hyperpolarization.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Dissociation of P1075-induced changes in membrane currents and potentials. Changes in membrane currents (A, by voltage-clamp at 80 mV) and potentials (B, by current-clamp) in the presence of P1075 were measured from the same pig bladder smooth muscle cell in bath solution containing 60 mM K+. During the 10-min exposure to P1075, the evoked current amplitude declined by 30.5 ± 7.7% (n = 5); however, the elicited membrane hyperpolarization remained steady at 18.2 ± 0.3 mV (n = 5), which is close to the K+ equilibrium potential of 20 mV under the current recording conditions where the intracellular K+ concentration and [K+]ext are 140 and 60 mM, respectively. Representative traces are shown (n = 5).

Effects on Membrane Hyperpolarization, K_ATP Current Activation, and Bladder Smooth Muscle Relaxation. Next, the concentration dependence of P1075-evoked changes in membrane potential, current responses, and smooth muscle relaxation were investigated. Under normal physiological recording conditions (5 mM [K⁺]_ext), bladder smooth muscle cells from guinea pig have a resting membrane potential of 25.5 ± 1.4 mV (n = 7) and exhibit spontaneous spike potential discharges, a possible mechanism contributing to spontaneous contractions of the bladder (Fujii et al., 1990; Herrera et al., 2000). Addition of low concentrations of P1075 (100 nM) reduced the spike potential with little or no changes in the resting membrane potential (Fig. 4A). Increases in P1075 concentrations (>100 nM) subsequently hyperpolarized the membrane potential to 56.8 ± 1.2 mV (n = 7), which is comparable to the maximum hyperpolarization evoked by pinacidil (62 mV) in guinea pig bladder smooth muscle strips reported by Seki et al. (1992) and approximately 50 mV evoked by Y-26763 (Hashitani et al., 1996). The calculated EC₅₀ value for P1075-elicited membrane hyperpolarization was 0.20 ± 0.02 µM (slope = 1.6 ± 0.2, n = 3; Fig. 5). This potency is similar to that observed for inhibition of bladder smooth muscle contraction evoked by electrical field stimulation (EC₅₀ = 0.11 ± 0.02 µM; n = 4; Fig. 5), supporting the notion that KCO-evoked membrane hyperpolarization underlies relaxation of bladder smooth muscle tone (Fujii et al., 1990; Wammack et al., 1994).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of P1075-evoked changes in membrane potential and current responses. A, concentration-dependent modulation of membrane excitability by P1075 in 5 mM [K+]ext. Changes in membrane excitability from single guinea pig bladder smooth muscle cells were measured by whole-cell current clamp. P1075 induced concentration-dependent changes in membrane hyperpolarization. Reduction in spontaneous spike activities with minimal changes in membrane hyperpolarization occurred in the low concentrations of P1075 (<0.3 µM). Hyperpolarization occurred at high P1075 concentrations. Shown are representative responses (n = 4). The inset shows that 0.1 µM P1075 reduced spontaneous spike activities with no changes in membrane potential. The recording shown in the inset was obtained from a different smooth muscle cell. B, concentration dependence of P1075-evoked increases in membrane current. Single smooth muscle cells from guinea pig were voltage-clamped at 80 mV in 60 mM [K+]ext. P1075 activated membrane currents in a concentration-dependent manner. Both changes in membrane current and potential were sensitive to application of glyburide.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves of P1075-indcued changes in muscle relaxation, changes in membrane potentials, and KATP currents from guinea pig bladder smooth muscle. All values are normalized and expressed as a percentage of the response evoked by 10 µM P1075. Each data point represents the mean ± S.E.M. of three to four determinations. The EC50 values for P1075-elicited muscle relaxation, hyperpolarization, and evoked membrane currents were 0.11 ± 0.01 µM (Hill coefficient, nH = 1.6 ± 0.2), 0.20 ± 0.02 µM (nH = 1.6 ± 0.2), and 0.73 ± 0.14 µM (nH = 1.7 ± 0.4), respectively.

Effect of KCOs on Acetylcholine-Evoked Changes in Membrane Excitability

As shown previously, KCOs can hyperpolarize and relax bladder smooth muscle precontracted by carbachol- or electrical field stimulus (Buckner et al., 2000), although it is also known that muscarinic receptor activation can significantly inhibit K_ATP currents (Bonev and Nelson, 1993b). Addition of 10 µM ACh resulted in depolarization of the single smooth muscle cell to 28.6 ± 7.5 mV (n = 5) from the resting potential of 34.5 ± 2.3 mV (n = 5; Fig. 6). This ACh-induced depolarization was antagonized by addition of 10 µM P1075, which repolarized the membrane potential to 62.8 ± 2.4 mV (n = 5) in the presence of 10 µM ACh. The hyperpolarizing effects of P1075 were reversed by glyburide (5 µM) to the membrane potential of 30.6 ± 4.8 mV. This suggests that opening of K_ATP channels by P1075 could antagonize the excitatory effects of ACh in bladder smooth muscle cells.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Membrane hyperpolarization by P1075 in the presence of acetylcholine in bladder smooth muscle cells. Addition of ACh (10 µM) depolarized membrane potential to approximately 10 mV, which was repolarized to 55 mV by the addition of 10 µM P1075. The hyperpolarizing effects of P1075 were reversed by glyburide (5 µM), suggesting that opening KATP channels antagonized excitatory effects of ACh. Changes in membrane potentials were measured by whole-cell current-clamp under normal physiological conditions (5 mM [K+]ext). The trace plotted is the representative of responses recorded from five pig bladder smooth muscle cells. Similar results were also obtained from guinea pig bladder smooth muscle cells (n = 3; data not shown).

	Discussion

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By whole-cell patch-clamp recording, the present study shows that ATP-sensitive K⁺ channels represent a major class of K⁺ channels in both pig and guinea pig bladder smooth muscle cells that can be activated by KCOs such as P1075 and that are sensitive to inhibition by glyburide. Both pig and guinea pig have been widely used in in vivo and in vitro models for characterization of KCOs for the treatment of overactive bladder. Analysis of KCO-evoked changes in membrane current and membrane potential demonstrate that a fraction of K_ATP channel opening is sufficient to evoke membrane hyperpolarization.

Although the measured K_ATP current density in pig bladder smooth muscle is about 9-fold greater than that of the guinea pig, the pharmacological properties of K_ATP channels in pig and guinea pig appear to be similar, as indicated by the similar rank order potencies and efficacies of KCOs to suppress field-stimulated contractions. This is also supported by recent molecular analysis of inward rectifiers and sulfonylurea isoforms, which suggest that K_ATP channels in guinea pig detrusor are composed of inward rectifying K⁺ channel Kir6.2 and sulfonylurea receptor SUR2B, while SUR1 subunit also exists (Gopalakrishnan et al., 1999). A similar sulfonylurea receptor profile has been elucidated in the pig bladder smooth muscle as well (Buckner et al., 2000).

Previous studies have shown that KCOs such as cromakalim or pinacidil can suppress spontaneous contractions in human (Wammack et al., 1994), and guinea pig (Fujii et al., 1990) and rat bladders with instability (Malmgren et al., 1989). The suppression of spontaneous mechanical responses was attributed to the fact that KCOs inhibit spontaneous electrical discharge from smooth muscle cells (Fujii et al., 1990; Seki et al., 1992; Wammack et al., 1994; Hashitani et al., 1996). The results of the present study in single smooth muscle cells confirm this notion (Fig. 5). The spontaneous spike discharge of single smooth muscle cells was diminished by a low concentration of P1075 (100 nM), whereas membrane hyperpolarization was observed at higher concentrations. These two modes of alterations in membrane excitability by P1075, viz., suppression of spontaneous spike discharges and membrane hyperpolarization, are consistent with the effects reported for K_ATP channel openers such as pinacidil (Seki et al., 1992) and Y-26763 (Hashitani et al., 1996) in bladder muscle strips. Based on studies using muscle strips, it was previously proposed that selective suppression of activities in pace-making cells by KCOs may be responsible for inhibition of spike discharges (Hamilton et al., 1986). However, the present studies show that KCOs can suppress spike discharge in single bladder smooth muscle cells and that this phenomenon may not be restricted to pace-making types of cells. Several mechanisms including Ca²⁺ channel inhibition, reduction in inositol 1,4,5-triphosphate levels, or changes in Ca²⁺ sensitivity of contractile proteins have been proposed as mechanisms for smooth muscle relaxation by KCOs (Ito et al., 1991, 1992). The effects of P1075 on both spike discharge and membrane hyperpolarization were reversed by glyburide, clearly demonstrating that both these effects of KCOs involve activation of K_ATP channels.

The concentration-response curve for suppression of field-stimulated muscle contraction by P1075 overlaps with that of hyperpolarization effects measured from single smooth muscle cells by patch clamp, consistent with the notion that membrane hyperpolarization mediates smooth muscle relaxation. In addition, our study shows a dissociation of KCO effects on membrane conductance and hyperpolarization in single bladder smooth muscle cells from guinea pig (Fig. 5). The EC₅₀ value for hyperpolarization is about 6-fold lower than that necessary for increases in membrane conductance. Since the K_ATP current density is 9-fold greater in pig than in guinea pig bladder smooth muscle cells, the dissociation between the EC₅₀ value for hyperpolarization and increases in membrane conductance would be expected to be more than 6-fold. A similar separation in the potencies of KCOs for relaxation or binding to K_ATP channels and to activate ⁸⁶Rb⁺ effects has been previously reported in rat vascular smooth muscle (Bray and Quast, 1992; Quast et al., 1993), although it has been suggested that ⁸⁶Rb⁺ is not as permeant an ion as K⁺ (Foster et al., 1989a).

It should be noted, however, that the comparison of hyperpolarization with contraction would best be done by measuring hyperpolarization in intact muscle or in cells with physiological levels of intracellular ATP. For example, the intracellular ATP concentration of 0.1 mM used in whole-cell experiments may allow more channels to be available for activation compared with the normal state of 2 to 3 mM ATP in intact cells. This may alter the fraction of channels available for activation in intact muscle to evoke relaxation.

Our analysis shows that bladder smooth muscle cells from pig and guinea pig have a resting input resistance of 2 to 5 G, which is close to the values of 11 to 12 G measured previously in guinea pig bladder cells (Bonev and Nelson, 1993a) and from arterial smooth muscle cells with values of 5 to 15 G (Nelson and Quayle, 1995). Thus, a small application of current would lead to large changes in membrane potential. It was estimated by single channel analysis that the number of K_ATP channels in guinea pig bladder cells was ~425/cell (Bonev and Nelson, 1993a). With the characteristic high-input resistance of the muscle cells, it was previously suggested that very few K_ATP channels need to be open to contribute to membrane conductance (Bonev and Nelson, 1993a; Nelson and Quayle, 1995; Quayle et al., 1997). As noted earlier, comparison of the rank order potencies and efficacies of KCOs for relaxation of pig and guinea pig bladder showed a good 1:1 correlation, although the pig smooth muscle cells have channel density 9-fold greater than that from guinea pig. This observation is in agreement with the notion that few K_ATP channel openings are sufficient to contribute to changes in membrane potential (Bonev and Nelson, 1993a; Quayle et al., 1997). Our results also demonstrate the presence of spare K_ATP channels in bladder smooth muscle cells and suggest that there are more spare K_ATP channels available in pig than in guinea pig bladder smooth muscle cells. Studies with pancreatic -cells and cardiac myocytes have suggested that activation of a fraction of K_ATP channels can influence cellular excitability consistent with the spare K_ATP channels hypothesis (Cook et al., 1988; Findlay and Faivre, 1991). Using high-affinity ligand-binding assays, previous studies have shown the existence of spare muscarinic receptors in bovine airway smooth muscle cells (Grandordy et al., 1986) and rat parotid cells (Dai and Baum, 1993) as well as spare ₁-adrenergic receptors in rat vas deferens (Minneman and Abel, 1984). However, the lack of high-affinity KCO ligands together with nucleotide dependencies of binding activity and relatively low expression levels of K_ATP channels in native tissues currently limit such studies.

K_ATP channel openers such as cromakalim, YM934, ZD6169, and WAY-133537 have been shown to reduce frequency and amplitude of contractions evoked by acetylcholine (Howe et al., 1995; Li et al., 1995; Martin et al., 1997; Pandita and Andersson, 1999; Wojdan et al., 1999). However, it has been shown that muscarinic receptor activation could suppress 40 to 70% of K_ATP currents in smooth muscle cells from bladder (Bonev and Nelson, 1993b), esophageal muscularis mucosae (Hatakeyama et al., 1995), and trachea (Nuttle and Farley, 1997) through protein kinase C pathways. Similar inhibition by protein kinase C-mediated pathways of ⁸⁶Rb⁺ efflux and vasorelaxation induced by P1075 was also observed in vascular smooth muscle (Linde et al., 1997). Our studies demonstrate that, in the presence of acetylcholine, KCOs are still capable of causing membrane hyperpolarization in both pig and guinea pig bladder smooth muscle cells. By measuring P1075-evoked changes in current and membrane potential from the same single smooth muscle cell, it was found that hyperpolarization remained constant, whereas P1075-activated current ran down by ~30.5% (Fig. 3), a condition mimicking muscarinic suppression of K_ATP currents. The availability of spare K_ATP channels may serve as an underlying mechanism by which KCOs are capable of suppressing muscle contraction triggered by carbachol or electrical field stimulation.