Introduction

Summary Introduction Materials & Methods Results Discussion References

I_K-ATP, which were first described in the heart, couple cellular metabolic status to the electrical excitability of the plasma membrane. Patch-clamp studies have also demonstrated their presence on pancreatic , skeletal, smooth muscle, and brain cells (1). Antagonist and agonist drugs have been described that interact with these channels. Thus, the hypoglycemic sulfonylureas, such as glyburide, act as specific antagonists and inhibit I_K-ATP opening induced by metabolic inhibition or KCOs. KCOs represent a structurally diverse class of compounds that open I_K-ATP and thus have broad therapeutic potential. However, effective therapies for nonvascular indications are likely to require the introduction of tissue-selectivity because vasorelaxation and hypotension would produce unacceptable side effects. Indeed, cardioprotective KCOs structurally related to cromakalim have recently been described that have the cardioprotective potency of cromakalim but exhibit markedly diminished vascular activities (2). The KCO diazoxide also seems to be somewhat unique because it activates pancreatic  cell I_K-ATP but functions, like tolbutamide, to inhibit cardiac channels (3). The basis for this tissue specificity is currently unknown but may relate to differences in I_K-ATP isoforms.

Skeletal muscle contains a large abundance of I_K-ATP, which can be activated by KCOs such as levcromakalim (4) and pinacidil (5). Treatment with pinacidil mimicked the effects of ischemic preconditioning (6), which suggests that I_K-ATP activation may be a protective mechanism during skeletal muscle anoxia. Treatment with the KCOs cromakalim and P1075 improved force recovery of rat skeletal muscle exposed to anoxia and reperfusion (7). Patch-clamp studies of mammalian skeletal muscle cells led to the development of models of I_K-ATP that contain stimulatory and inhibitory nucleotide regulation sites (5, 8). Pinacidil is thought to open skeletal muscle I_K-ATP by interaction with these sites (5). To examine the biochemical interaction of KCOs with skeletal muscle sites, we conducted binding studies using the potent KCO [³H]P1075. Bray and Quast (9) and Quast et al. (10) used this radioligand to define a sulfonylurea-sensitive KCO binding site in endothelium-denuded rat aortic rings, which correlated with sites mediating vasorelaxation. The challenge of the current work was to define conditions that allowed KCO binding to skeletal muscle membranes because homogenization of cells or tissues results in loss of specific binding sites (9, 11). Thus, binding studies, to date, have necessitated the use of tissue rings (9, 10) or large numbers of viable cells (11).

I_K-ATP are thought to consist of a complex of proteins, including ATP binding cassette protein or proteins (SUR) and inward rectifier protein or proteins (12). They are modulated by nucleotides (13) and phosphorylation states (14). In the current study, we examined the effects of these parameters on KCO binding and describe the pharmacological characteristics of a [³H]P1075 binding site on rabbit skeletal muscle membranes.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Skeletal muscle membrane preparations. Rabbits (2.5-3 kg) were anesthetized with sodium pentobarbital, and their hindlimbs were dissected and weighed. Tissue was minced with scissors into 5 volumes (w/v) of 20 mM NaHCO₃ and 0.1 mM phenylmethylsulfonyl fluoride and homogenized using a Brinkmann Instruments Polytron homogenizer (4 × 10 sec, setting 7; Westbury, NY). The homogenate was centrifuged at 3,500 × g for 15 min at 4°, and the supernatant was filtered through cheesecloth. The filtrate was recentrifuged at 100,000 × g for 60 min, and the pellet was washed into 50 mM Tris·HCl, pH 7.4. The membranes were resuspended in 25 ml of 50 mM Tris·HCl, pH 7.4, at a protein concentration of 8-11 mg/ml and stored frozen in aliquots at 80°.

Enzyme marker assays. Analysis of Na⁺/K⁺-ATPase, Ca²⁺/K⁺-ATPase, and azide-sensitive-ATPase was performed according to Jones and Besch (15). Membranes used in the Na⁺/K⁺-ATPase assay were preincubated with 0.9 mg alamethicin/mg of membrane protein for 20 min at room temperature to unmask latent Na⁺/K⁺-ATPase activity. ATP blanks and phosphate standards were included to assess contaminating ATP in the reaction solutions and generate a standard curve.

[³H]P1075 binding assay. Assays were conducted in 12 × 75-mm tubes in a total volume of 1 ml. The assay buffer consisted of 240 mM sucrose, 3 mM sodium orthovanadate, 2 mM MgCl₂, and 50 mM Tris·HCl, pH 7.4 The nucleotides (sodium or trilithium salts), [³H]P1075 (14 nM, 51-61 Ci/mmol), and inhibitors were incubated with membranes (700 µg of rat brain, 50-200 µg of rabbit skeletal muscle, 500-700 µg of canine cardiac) for 60 (canine cardiac and rat brain) or 90 (skeletal muscle) min. Nonspecific binding was defined with 10 µM P1075. Bound and free radioligands were separated by filtration through a GF/C glass-fiber filter on a Brandel (Montreal, Quebec, Canada) cell harvester with three 2-ml washes of 50 mM Tris, pH 7.4, at 4°. The filters were transferred to 6-ml plastic scintillation vials, soaked overnight in 5 ml of scintillation fluid (Opti-fluor; Packard, Meriden, CT), and counted in a Packard liquid scintillation counter.

Nucleotide metabolism assay. Skeletal muscle membranes (7 mg/ml in 50 mM Tris/240 mM sucrose/4 mM MgCl₂) were incubated at 25° in 1 ml of buffer (50 mM Tris·HCl, pH 7.4, 240 mM sucrose, 4 mM MgCl₂, 2 mM ATP, 3 mM sodium orthovanadate) or an ATP regenerating system (1 mM dithiothreitol, 20 mM phosphocreatine, 10 units/ml creatine kinase, pH 7.4) or a system that removed enzymatically formed ATP (20 mM glucose, 15 units/ml hexokinase). The assay was initiated with the addition of membranes and stopped at appropriate times with the addition of 160 µl of 3 M HClO₄ to achieve a final acid concentration of 3%. Each sample was then placed in ice and homogenized at 4° for 10 sec and centrifuged at 3000 rpm for 20 min at 4°. The supernatant was removed and neutralized with 1 ml of 2 M KHCO₃. The neutralized samples were centrifuged at 3000 rpm for 20 min at 4°. The supernatants were removed, placed on ice, and filtered through a Millipore (Bedford, MA) 0.45-µm HV filter. Separation of the nucleotides was achieved by reversed-phase ion-pair chromatography on a Hewlett-Packard 1090 high performance liquid chromatograph equipped with a photodiode array detector and a Hewlett-Packard workstation computer interface. Samples (250 µl) were applied to a C₁₈ 218TP54 (250 × 4.6 mm) 5-µm column equilibrated in 30 mM KH₂PO₄, pH 5, 5 mM tetrabutylammonium dihydrogen phosphate, and 4% CH₃CN (solvent A) at 1.5 ml/min. Nucleotides were eluted during a linear gradient of 0-35% solvent B (50% CH₃CN) over 35 min. The nucleotides were detected at 254 nm, and their elution times were identified using ATP, ADP, AMP, UTP, UDP, and UMP standards. Nucleotide peaks, calculated as the integrated area under the curve, were obtained as in milliabsorption units and converted to micromoles from comparison of standard curves. There was base-line-to-base-line separation of the nucleotides, and as little as 50 nmol was readily detected. The identity of the compounds in the peaks was also confirmed by mass spectrometry analysis. Analysis of ATP, ADP, UTP, and UDP incubated without membranes indicated there was no appreciable degradation of the compounds during the time course of the experiment.

Drugs. [³H]P1075 was prepared in-house by catalytic tritiation of an olefinic precursor. Two tritium atoms were incorporated; the P1075 molecule and the resultant radioligand had a current specific activity of 51-61 Ci/mmol. The BMS compounds BMS-182264 [4-[[9-cyanoimino)[(1,2,2-trimethylpropyl)amino]methyl]amino]benzonitrile], BMS-180448 [3S-trans)-N-(4-chlorophenyl)-N-cyano-N-(6-cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl)guanidine], and BMS-212345 [cis-1-(6-cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl)-3-phenylurea] were also synthesized in-house.

Calculations. K_i values of competing drugs was calculated for IC₅₀ values according to the equation of Cheng and Prusoff (16). Competition curves and saturation isotherms were analyzed to a four-parameter logistic equation using Sigma plot curve fitter, which fits data to nonlinear functions using the Marquardt-Levenberg algorithm. Goodness of fit to a one- or two-site binding model was determined by F test, in which F = [SS₁  SS₂)/(df₁  df₂)]/SS₂/df₂, where SS is the sum of squares of residuals with one- versus two-site models and df is degrees of freedom of the corresponding models. Association rate constants (k₊₁) were determined from pseudo-first-order plots using the equation k₊₁ = k_obs  k₁/L, where k_obs is the observed association rate constant, and L is the radioligand concentration. Dissociation rate constants were calculated from the first-order plot of the dissociation reaction: k₁·t = ln[X/X_eq], where X is the specific radioligand bound at time t, and X_eq is the radioligand bound at equilibrium. The half-time values for dissociation reaction were calculated after linear regression analysis of this plot.

	Results

Summary Introduction Materials & Methods Results Discussion References

Nucleotide regulation. Specific [³H]P1075 binding (displaceable by 10 µM P1075) to rabbit skeletal muscle membranes was observed in the presence of sodium vanadate and MgATP. Vanadate alone was unable to support binding, and in the presence of vanadate and MgATP, binding was little different from binding with MgATP alone. These data suggest that MgATP was the key factor for supporting [³H]P1075 binding. Membranes incubated with the catalytic subunit of PKA and MgATP had no greater binding than membranes incubated with MgATP alone (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of an ATP regeneration system on [3H]P1075 binding to rabbit skeletal muscle membranes and on adenine nucleotide levels. A, [3H]P1075 binding to membranes was determined in the absence () or presence () of an ATP regeneration system (20 mM phosphocreatine, 10 units/ml creatine kinase). All samples contained 4 mM MgCl2 and 1 mM dithiothreitol. Binding has been calculated as percent of control values in the absence of a regenerating system (mean = 63 ± 5 fmol/mg of protein at 14 nM [3H]P1075). Curves, the computer-derived fits of mean data (± standard error) from three experiments using one-site analysis. B, Adenine nucleotide metabolism by skeletal muscle membranes. Membranes were incubated with 2 mM ATP for the stated times under standard binding assay conditions. Formation of ADP () and AMP () from added ATP () was determined using techniques described in Materials and Methods. C, Nucleotide metabolism was determined in the presence of an ATP-regeneration system, as described above.

Equilibrium, kinetics, and pH dependence. Specific [³H]P1075 binding (14 nM) to rabbit skeletal muscle membranes at 25° reached equilibrium after 60 min. The association rate constant was 0.00110 ± 0.0004 nM/min (three experiments). Binding was reversible, and after the addition of excess P1075, the radioligand dissociated with a half-life of 16 ± 1.5 min (five experiments). The K_d value calculated as the ratio of the dissociation and association rate constants was 29 ± 4 nM (three experiments); this compared favorably with the K_d value calculated from saturation binding isotherms, which was 37 ± 3 nM (four experiments). The binding site maxima calculated from these experiments was 280 ± 14 fmol/mg of protein.

Subcellular distribution. [³H]P1075 binding to membrane fractions of rabbit skeletal muscle membranes was examined. Table 2 shows that binding was enriched in the P3 (100,000 × g) membrane fraction, which had high levels of plasma membrane (Na⁺/K⁺-ATPase) and SR (Ca²⁺-ATPase) markers. [³H]P1075 binding was not correlated with the activity of azide-sensitive ATPase or oligomycin-sensitive ATPase, a more specific mitochondrial marker (data not shown). Moreover, there was no enrichment of binding to a mitochondrial preparation of rabbit heart (data not shown). These data suggest that the membrane [³H]P1075 binding sites were unlikely to be associated with mitochondria.

Regional distribution. Specific [³H]P1075 binding was also observed with guinea pig skeletal muscle, canine and rat cardiac P2/P3 membrane preparations, and rat brain and liver. In contrast to rabbit skeletal muscle, vanadate increased binding to canine cardiac membranes in the presence of 2 mM MgATP by ~75%. Dissociation constants of [³H]P1075 for canine cardiac (28 ± 5 nM) and rat brain (22 ± 15 nM) were similar to those of skeletal muscle, but B_max values were lower (73 ± 8 and 26 ± 10 fmol/mg of protein, respectively). Intermediate levels of [³H]P1075 binding were observed in rat kidney, rabbit aorta, and sheep choroid plexus cells. Low or barely detectable levels were found in membranes from hamster insulinoma tumor and rat aortic smooth muscle cells and rat pancreas.

Competition by KCOs, antagonists, and fluoresceins. The specificity of the binding sites was examined by competition analyses. There was no significant inhibition of [³H]P1075 binding by 100 µM concentration of verapamil, diltiazem, nifedipine, dofetilide, ryanodine, spermine, or spermidine or 10 mM tetraethylammonium. The amphipathic drugs propranolol and trifluoperazine (100 µM) inhibited [³H]P1075 binding by only 31% and 48%, respectively.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]P1075 binding to rabbit skeletal muscle membranes by fluorescein derivatives. [3H]P1075 (14 nM) was incubated with rose bengal (), phloxine B (), ethyl eosin (), eosin maleimide (), fluorescein (), or dichlorofluorescein () in the presence of MgATP (2 mM), and binding was assessed as described in Materials and Methods. Results are mean ± standard error values of three experiments.

Kinetic studies of binding inhibition. We examined the effect of rose bengal on [³H]P1075 dissociation from rabbit skeletal muscle membranes. After the attainment of equilibrium, the addition of excess P1075 dissociated [³H]P1075 in a first-order reaction with a half-life of 14 ± 2 min (two experiments). Dissociation induced by rose bengal (10 µM) was markedly faster, and ~90% of binding dissociated at the earliest time point (1 min). These data suggest that the inhibition of [³H]P1075 binding to membranes by fluoresceins was not competitive but was consistent with an allosteric or noncompetitive interaction.

1

1

1

1

1

1

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Dissociation of [3H]P1075 binding by KCOs and glyburide. [3H]P1075 (15 nM) was incubated with rabbit skeletal muscle membranes until equilibrium was reached. Dissociation of [3H]P1075 was initiated by P1075 (, 1.35 µM), BMS-180448 (, 65 µM), or 10 µM P1075 and 0.1 () or 6.5 () µM glyburide, and binding was determined at the indicated times. Results are plotted as a first-order plot (see Materials and Methods). Results are representative of three experiments.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

This study defined conditions that allowed [³H]P1075 binding to be observed in membranes from skeletal muscle tissue. Previous [³H]P1075 binding studies have used smooth muscle tissue (9, 10) but preliminary data indicate that viable cells may also be used (11). All studies to date report the loss of specific binding on homogenization of cells or tissue. The current work indicates that nucleotides are major regulators of KCO binding and their presence allows membrane [³H]P1075 binding sites to be revealed. Initial studies showed that a "phosphorylation cocktail," containing PKA, vanadate, and MgATP, provided conditions that supported [³H]P1075 binding to membranes. Further experiments established that PKA was unnecessary and the contribution of the phosphatase inhibitor sodium vanadate varied with the tissue source. These findings indicated that the key component for support of [³H]P1075 binding to membranes was MgATP, and our previous inability to observe [³H]P1075 binding to membranes may have resulted from rapid metabolism of added ATP by nucleotidases. Previous studies have also demonstrated the importance of MgATP for regulating the interaction of KCOs with SUR/I_K-ATP . Thus, inhibition of [³H]glyburide binding by pinacidil was enhanced by ATP (18), and [³H]P1075 binding to rat aortic rings was dependent on the metabolic status of the tissue, notably, the ATP levels (10).

When ATP metabolism was inhibited by a regenerating system, the EC₅₀ value for support of [³H]P1075 binding was 30 µM. This potency is similar to that for ATP-mediated inhibition of I_K-ATP activity (1). However, in contrast to the ATP inhibitory site, which can also be occupied by nonhydrolyzable analogs of ATP, support of [³H]P1075 binding required a hydrolyzable form of ATP. Moreover, the rank order of potency for nucleotide inhibition of I_K-ATP [ATP  ADP > AMP (13)] did not correlate with that for nucleotide interaction with the [³H]P1075 sites. These data suggest that the nucleotide sites that supported [³H]P1075 binding were unlikely to be the ATP inhibitory site of I_K-ATP . Nucleotide phosphates have been shown to inhibit [³H]glyburide binding (18, 19) with a pharmacological profile that was similar to that for support of [³H]P1075 binding. Thus, in the presence of Mg²⁺, nucleotide triphosphates were equipotent to NDPs and monophosphates, and nonhydrolyzable forms of ATP and GTP were inactive (19). These findings suggest similarities in the nucleotide regulatory sites coupled to SUR and [³H]P1075 binding.

[³H]P1075 binding to membranes could have resulted from ATP-dependent phosphorylation and/or nucleotide binding to regulatory sites. Because the presence of alkaline phosphatase eliminated binding, it is likely that phosphorylation state is important. However, binding was supported by a broad range of nucleotide triphosphates, which contrasts with the high specificity of kinases for ATP. The ability of ATP to support binding seemed irreversible because added ATP was essentially degraded during the course of the incubation, and we noted no time-dependent decrease in binding. Our metabolism studies indicated marked interconversion of nucleotides during the course of the incubation. Thus, the marked degradation of ATP to ADP and AMP probably occurred via the action of an ADPase (20). Because we were unable to control this activity, the contribution of NDPs to [³H]P1075 binding could not be accurately determined. Indeed, at least some portion of ADP-supported binding was likely due to its interconversion to ATP. In contrast, UDP was not transphosphorylated to a triphosphate but was degraded to an inactive monophosphate (data not shown). Therefore, it is likely that UDP supported [³H]P1075 binding via its interaction with UDP/NDP recognition sites. Indeed, NDPs such as UDP have been shown to open Ca²⁺-inactivated I_K-ATP in skeletal muscle (4).

Fluorescein analogs have been shown to label active sites of Na⁺/K⁺-ATPase, perhaps by interaction with adenine recognition sites (21). They also modulate I_K-ATP and SURs in brain and insulinoma cells (22, 23). Thus, fluoresceins such as rose bengal inhibited active I_K-ATP and reactivated I_K-ATP that had rundown (23), which suggests that they interact with activator and inhibitor nucleotide sites coupled to I_K-ATP. Fluoresceins also inhibited [³H]glyburide binding to insulinoma and brain membranes, which suggests that their target sites are the SUR (22, 23). The basis for this inhibition seems to be an allosteric interaction because rose bengal increased the [³H]glyburide dissociation rate (22). Based on these data, we were not surprised to discover that [³H]P1075 binding to skeletal muscle membranes was inhibited by fluoresceins. Rose bengal and phloxine B were relatively potent inhibitors, with K_i values of 280 and 190 nM, respectively. Halogenated fluoresceins were generally more potent inhibitors of [³H]P1075 binding, which correlated with their greater inhibition of ⁸⁶Rb efflux in HIT cells (23). Inhibition of [³H]P1075 binding by rose bengal also seems to involve an allosteric interaction. Thus, specific [³H]P1075 binding to skeletal muscle membranes was dissociated by ~90% within 1 min. This rate of dissociation was significantly greater than that observed with excess P1075, which suggests that fluoresceins were potent allosteric or noncompetitive regulators of [³H]P1075 binding.

In the presence of MgATP, the [³H]P1075 binding sites exhibited characteristics of a drug/receptor interaction. Thus, binding was saturable, reversible, pH and trypsin sensitive, and highly specific for interaction with KCOs. The K_d value for [³H]P1075 binding to membranes was ~30 nM, which was higher than the K_d values of 3-6 nM reported for binding to smooth muscle cells and tissue (9, 11). The [³H]P1075 sites displayed many characteristics expected of a K_ATP-associated binding site, including stereoselectivity for the (3S,4R)-enantiomer of cromakalim and inhibition by the sulfonylurea glyburide. The KCO BMS-180448 has been shown to protect the myocardium in a glyburide-sensitive manner (2). In this study, we demonstrated that BMS-180448 increased the K_d value of [³H]P1075 for skeletal muscle membranes without effects on the B_max value. These findings suggest that BMS-180448 competed directly for the [³H]P1075 sites. In addition, BMS-180448, cromakalim, and BMS-182264 dissociated [³H]P1075 from skeletal muscle membranes with similar rates to P1075, which suggests that these structurally diverse KCOs interacted directly, and competitively, with the labeled pyridinocyanoguanadinium recognition sites.

KCOs are generally assumed to target plasmalemmal I_K-ATP, although they have also been suggested to exert intracellular effects on Ca²⁺ regulation (for a review, see Ref. 24). These data imply there may be intracellular sites of action for P1075; however, examination of the subcellular distribution of [³H]P1075 binding suggested little evidence of an intracellular location. Binding was enriched in P3 fractions and not associated with purified mitochondrial or SR preparations. In contrast, [³H]P1075 binding was enriched in fractions high in cholesterol, which is a marker for T tubules. Therefore, it is likely that the observed skeletal muscle [³H]P1075 sites are associated with T tubules. Skeletal muscle T tubules are in close proximity to the terminal cisternae of the SR and are involved in propagation of the action potential and Ca²⁺ release from the SR. These organelles also play a major role in the transport of ions into cells.

Glyburide has been reported to inhibit [³H]P1075 binding to aortic smooth muscle, with an apparent K_i value of 400 nM, and significantly increase the [³H]P1075 dissociation rates relative to that induced by P1075 (9). These data are consistent with allosteric interaction of glyburide with smooth muscle [³H]P1075 sites. We obtained similar data with membrane preparations of skeletal muscle. Glyburide competed with [³H]P1075 binding with an apparent K_i value of 130 nM. However, in contrast to the data of Bray and Quast (9), in which glyburide inhibition curves were steep, competition curves obtained with membranes had slopes less than unity, which could represent heterogeneous binding sites or negative heterotropic cooperative interactions. Dissociation experiments indicated that glyburide increased the [³H]P1075 dissociation rate more significantly than the increase induced by excess P1075 and that high concentrations generated biphasic dissociation curves. The effect of glyburide to increase the dissociation rate of [³H]P1075 occurred at relatively low glyburide concentrations (0.1-6 µM) in contrast to the high concentrations of glyburide required to produce such effects in rat aortic rings (9). These data support the concept that glyburide modulates KCO binding over physiologically relevant concentration ranges. Thus, patch-clamp studies have shown glyburide inhibited skeletal muscle I_K-ATP with an apparent K_i value of 190 nM (8).