Introduction

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The maintenance of blood glucose concentration is an integrated process regulated primarily by the antihyperglycemic hormone insulin. When blood glucose rises, uptake of glucose into the -cell leads to an elevation in the ATP/ADP ratio and closure of the K_ATP channels. The closure of K_ATP channels and the resultant membrane depolarization lead to the increase of Ca²⁺ influx through voltage-gated Ca²⁺ channels, which triggers exocytosis and insulin release. In addition to glucose, many agents are capable of blocking K_ATP channels in pancreatic -cells, thus inducing insulin secretion and having an antidiabetic effect. Among them, glibenclamide, a sulfonylurea, has been used for >30 years in the treatment of type 2 diabetes (Loubatiéres, 1977; Sturgess et al., 1985; Dunne et al., 1987). Repaglinide, a benzoic acid derivative of the meglitinide family, is reportedly a more potent insulinotropic agent than glibenclamide and other sulfonylureas (Frøkjær-Jensen et al., 1992; Gromada et al., 1995; Malaisse, 1995; Fuhlendorff et al., 1998). Nateglinide (N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine; A-4166) is a phenylalanine derivative (nonsulfonylurea) reported to have a similar mechanism of action to glibenclamide and repaglinide (Akiyoshi et al., 1995; Fujita et al., 1996; Ikenoue et al., 1997). Glibenclamide and repaglinide cause long-lasting hypoglycemic action under both normoglycemic and hyperglycemic conditions in animal models (Mark and Grell, 1997; DeSouza et al., 1997). Nateglinide, although less potent, appears to differ from the other two agents in several respects: 1) preferential first-phase insulin release effect due to rapid onset, 2) no sustained hypoglycemia and reduced total insulin secretion due to its short duration of action, and 3) enhanced activity under hyperglycemic conditions due to glucose-sensitive action (Akiyoshi et al., 1995; Sato et al., 1995; Ikenoue et al., 1997; DeSouza et al., 1997). Although these in vivo differences may be due to differential effects on absorption, distribution, and elimination of the various compounds, we have carried out receptor-binding and K_ATP channel activity measurements to distinguish the molecular mechanism of nateglinide from the mechanism of the sulfonylureas and other agents such as repaglinide. The results of these experiments indicate that the interactions of nateglinide with its receptor are unique compared with glibenclamide and repaglinide. Moreover, the nature of this interaction at the K_ATP channel also may be relevant to its shorter duration of action, reduction in excessive insulin release, and reduced risk of hypoglycemia.

	Materials and Methods

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Cell Culture and Preparation of RIN-m5F Membranes. RIN-m5F cells (passages 23-32) [CRL-2058; American Type Culture Collection, Manassas, VA; (Bhathena et al., 1984)] were cultured in T175 flasks in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD) and penicillin (100 U/ml)/streptomycin(100 µg/ml) (Life Technologies). Cells, at 85% confluence, were washed once with PBS, then scraped into 10 ml of PBS. Cells were collected and stored as a pellet at 80°C. Membranes were prepared by the method of Müller et al. (1994) and stored at 80°C until use. Protein determinations were made with the Coomassie stain method (Pierce, Rockford, IL) with BSA as the protein standard.

Binding Assays. The binding assays with [³H]glibenclamide (DuPont/NEN, Boston, MA) with RIN-m5F cell membranes were performed in buffer A [50 mM 3-(N-morpholino)propanesulfonic acid, pH 7.4, and 0.1 mM CaCl₂] in a total volume of 1.0 ml/tube at 23-25°C. The RIN-m5F cell membranes (0.1 mg/ml) were incubated with [³H]glibenclamide at a concentration range of 0.2 to 30 nM. Nonspecific binding was determined in the presence of 2 µM unlabeled glibenclamide and was <20% of total binding. The protein concentration used in the binding assay was in the range in which there was a linear ligand-binding response with protein concentration. Glibenclamide and glipizide were obtained from Research Biochemicals International (Natick, MA). Tolbutamide was obtained from the Upjohn Company (Kalamazoo, MI). Nateglinide, [³H]nateglinide, repaglinide, and glimepiride were prepared in-house. The L-isomer of nateglinide was obtained from Ajinomoto Co. Inc. (Yokohama, Japan). Compound stock solutions were prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide (2%) did not affect binding. The binding assay proceeded for 2 h with orbital shaking and was terminated by rapid filtration through Whatman GF/F 25-mm-diameter glass microfiber filters (presoaked in buffer A) followed by 5 × 5-ml washes with cold 0.1 M NaCl. The 2-h incubation time was sufficient to ensure that a steady state in binding was achieved over the range of glibenclamide concentrations used in the assay. The wet filters were placed in 10 ml of Formula 989 scintillation fluid (Packard, Meriden, CT) and radioactivity was determined by liquid scintillation counting after overnight incubation. The binding assays with the HEK.EBNA[human SUR1] membranes were carried out in a 96-well format with Millipore multiScreen-FC opaque plates with 1.2-µm glass fiber type C filters. Binding assays with [³H]glibenclamide (DuPont/NEN) were performed in buffer A in a total volume of 250 µl at 23-25°C. HEK.EBNA[human SUR1] membranes (25 µg/well) were incubated with [³H]glibenclamide over a concentration range of 0.25 to 80 nM. The assay conditions and compound preparation were as described above. The assay was terminated by rapid filtration followed by 5 × 250-µl washes with ice-cold 0.1 M NaCl. Wallac Optiphase Hi Safe 3 scintillation cocktail was added per well (200 µl) and plates were counted in a Wallac Microbeta liquid scintillation counter. The competitive binding assays were carried out in the presence of 2.0 nM [³H]glibenclamide (K_d = 1.8 ± 0.002 nM for [³H]glibenclamide with human SUR1). The nonspecific binding was <5% of total binding under these assay conditions. All competitive binding experiments were repeated at least three times. Specific [³H]glibenclamide binding was not observed with membrane preparations from HEK-293 cells that were not transfected with the human SUR1 expression plasmid (pOriP/ZeoSUR1.hum).

Enzymatic Isolation of Rat Pancreatic -Cells. Male Sprague-Dawley rats weighing 250 to 275 g were anesthetized with sodium pentobarbital i.p. at 250 mg/kg before the operative procedure. After a midline abdominal incision was performed, the distal end of the bile duct was clamped with a hemostat to occlude it adjacent to the duodenum. The upper portion of the duct was nicked with a retina scissor and cannulated with a PE-50 polyethylene catheter near the hilus of liver. The acinar tissue was disrupted by injection of 20 ml of HEPES saline into the common bile duct and the pancreas was dissected from the stomach, duodenum, and spleen. The pancreas was cleaned free of fat, connective tissue, and blood vessels, and chopped into small pieces (1 × 1 mm). The pancreas slurry was transferred to a jar filled with 10 ml of HEPES saline containing librase at 0.5 mg/ml (Boehringer Mannheim, Indianapolis, IN) and placed on a submersible stirrer in a 37°C water bath for 25 min. The digest was then washed several times by centrifugation. The supernatant was discarded and the final sediment resuspended in HEPES saline for Ficoll (type 400 DL; Sigma Chemical Co., St. Louis, MO) gradient purification.

Electrophysiological Recording of K_ATP Currents. Experiments were performed at 22°C with the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981) in the primary culture of rat pancreatic -cells. Whole-cell current was used instead of single-channel current in a membrane patch as the end product of the measurement was to maximally maintain the cell integrity and preserve cytosolic nucleotides.

Data Analysis. IC₅₀ values for the binding studies were calculated from a dose-response curve with the four-parameter logistic equation. F tests were used to compare models, i.e., single-site versus two-site binding models and single- versus double-exponential decay kinetics, for data fitting. For comparison of the Hill coefficients (slope values of the four-parameter logistic equation), statistical significance was determined with a t test. Groups with P values <.05 were considered significantly different. The K_i values for the competitive ligands were estimated from the experimental IC₅₀ values with the Cheng-Prusoff equation, i.e., K_i = IC₅₀/(1 + ([Glib]/K_i^Glib)) (Titeler, 1989).

	Results

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Steady-State Binding of [³H]Glibenclamide to Membranes from RIN-m5F Cells and HEK.EBNA[Human SUR1] Cells. The saturation-binding curve for glibenclamide with RIN-m5F cell membranes indicates that there are two binding sites present in the membrane preparations (data combined from seven separate experiments; data not shown). Both high-affinity (K_d = 0.27 ± 0.07 nM) and low-affinity (K_d = 25 ± 13 nM) binding sites were observed. In the competitive binding and dissociation kinetic experiments described below, low concentrations of [³H]glibenclamide are used such that only binding at the high-affinity site would be measured. The saturation-binding experiments also were carried out with membrane preparations from HEK.EBNA[humanSUR1] cells (data obtained from five separate experiments; data not shown). In this case, a single binding site was observed with a K_d = 1.8 ± 0.002 nM. This value compares favorably with the reported value of 2 nM for 5-iodo-2-hydroxyglibenclamide binding to recombinant rat-SUR1 transfected into COSm6 cells (Aguilar-Bryan et al., 1995). 5-Iodo-2-hydroxyglibenclamide binds with a 2-fold lower affinity than glibenclamide to HIT-T15 cell SUR1 (Aguilar-Bryan et al., 1990)

Competitive-Binding Experiments with [³H]Glibenclamide. A series of compounds was tested for their ability to directly compete with 0.5 nM [³H]glibenclamide for binding to RIN-m5F cell membranes. Displacement of 0.5 nM [³H]glibenclamide should reflect binding primarily to the high-affinity sites, i.e., the sites involved with K_ATP channel activity and insulin release. Table 1 summarizes the IC₅₀, K_i, and slope (Hill coefficients) values determined in these competitive binding experiments. These results indicate that the relative order of potency is glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-isomer of nateglinide > tolbutamide. The K_i values were estimated from the Cheng-Prusoff equation (see Data Analysis).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Comparison of steady-state binding constants for various ligands that bind to RIN-m5F SUR1 and human SUR1. The steady-state binding constants were determined in competitive binding experiments with [3H]glibenclamide with cell membranes from either RIN-m5F -cells or HEK.EBNA[human SUR1] cells. The line represents a linear regression fit of the data with all compounds except repaglinide. Data points were collected in duplicate and combined from three separate experiments.

[³H]Glibenclamide Dissociation Kinetics. One approach to determine whether a displacing ligand, such as nateglinide, binds to the same or a different site as glibenclamide on SUR1 is to measure the effect of nateglinide on the dissociation kinetics of [³H]glibenclamide. If nateglinide binds to a site separate from glibenclamide (but prevents its binding through a conformation change on the receptor), a change in the rate of dissociation of [³H]glibenclamide should occur. Therefore, the rate of [³H]glibenclamide dissociation was determined by preincubating the RIN-m5F cell membranes with [³H]glibenclamide. An excess (2 µM) of unlabeled glibenclamide was then added and the amount of bound [³H]glibenclamide was determined at various time points over a 4-h period. Figure 2 illustrates such an experiment. Note that the dissociation kinetics fit best to a double exponential decay model. It also should be noted that the observed biphasic kinetics occurred under conditions (low [³H]glibenclamide concentrations) that reflect only high-affinity glibenclamide binding. The concentration of [³H]glibenclamide that was used in the initial preincubation conditions was 2.0 nM [³H]glibenclamide. At this concentration, only the high-affinity sites should have [³H]glibenclamide bound. However, the experiments also were carried out at lower concentrations of [³H]glibenclamide during the preincubation phase of the experiment and in all cases biphasic dissociation kinetics were observed. Even varying the concentration of [³H]glibenclamide over the 20-fold concentration range had little effect on the dissociation kinetic parameters for [³H]glibenclamide (data not shown). Therefore, 0.5 nM [³H]glibenclamide was preincubated with the RIN-m5F cell membranes and then either nateglinide, repaglinide, or glibenclamide was added during the dissociation phase of the experiments. Table 3 summarizes the results of these experiments. Note that neither nateglinide nor repaglinide had any effect on the dissociation kinetic parameters for [³H]glibenclamide. This result is consistent with nateglinide and repaglinide binding directly to the glibenclamide-binding site on the sulfonylurea receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   [3H]Glibenclamide dissociation kinetics. RIN-m5F cell membranes were preincubated with 0.5 nM [3H]glibenclamide for 90 min at 25°C. At t = 0, 1 µM unlabeled glibenclamide was added and portions of the incubation mixture were removed at various times and assayed by the filtration-binding assay procedure. Data were fit to single- and double-exponential decay kinetic equations. An F test of these two models indicated that the double-exponential decay equation described the data significantly better than a single-exponential decay equation. The kinetic parameters are listed in Table 3. Data points were collected in duplicate and combined from three separate experiments.

1

1

Attempts to Measure [³H]Nateglinide Binding to RIN-m5F Cell Membranes. The experiments described thus far indicate that nateglinide can compete with [³H]glibenclamide binding to RIN-m5F and HEK.EBNA[human SUR1] cell membranes. This finding suggests that [³H]nateglinide should bind to RIN-m5F cell membranes and that glibenclamide should compete with the binding. However, all attempts to measure specific binding of [³H]nateglinide to -cell membranes have been unsuccessful in our laboratory as well as in other laboratories (Fujita et al., 1996). A plausible explanation is that [³H]nateglinide dissociated from its binding site during the workup procedure of the filtration-binding assay. A method that has been used to measure weak binding ligands is a centrifugation assay (see Materials and Methods). However, specific [³H]nateglinide binding could not be detected with this assay, whereas specific [³H]glibenclamide binding was measured (data not shown). Consequently, it appears that even under conditions of rapid separation (~5 s; Bennett, 1978), specific binding with [³H]nateglinide to the RIN-m5F cell membranes is not observed.

Glucose Dependence of K_ATP Currents in -Cells. In pancreatic -cells, the effect of glucose on insulin secretion is mediated via its influence on cellular ATP levels. A rise in intracellular ATP after hyperglycemia results in the closure of K_ATP channels, which initiates a sequence of electrical events to induce insulin release. We first assessed the influence of ambient glucose on whole-cell K_ATP current in pancreatic -cells. Figure 3 shows that the K_ATP currents in -cells were dependent on extracellular glucose concentration such that their amplitude reduced with increasing concentrations of glucose. Normalized with the maximal current without glucose, the magnitude of the currents at various glucose concentrations fit reasonably well with a single exponential equation, in which the amplitude of K_ATP currents reduces by e-fold with every 3.7 mM increase in extracellular glucose. These results demonstrate that the pancreatic -cells used in the electrophysiological experiments are metabolically active and respond appropriately to changes in external glucose levels.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Concentration-response curve for glucose-dependence of the KATP current in -cells. The data were fit with a single exponential equation: Y = 0.98 exp[(X)/3.7 mM]. Y is the normalized KATP currents and X is the glucose concentration (millimolar). The points are the mean of five separate experiments.

Inhibition of K_ATP Currents by Nateglinide, Glibenclamide, and Repaglinide. The ability to block the K_ATP current in -cells by the antidiabetic agents nateglinide, glibenclamide, and repaglinide was evaluated. Given that the basal level of K_ATP currents in -cells is rather low, we applied 100 µM diazoxide, a known opener of the K_ATP channels in insulin-secreting cells, to activate K_ATP currents before the administration of the blockers. The data in Fig. 4A demonstrate that nateglinide inhibited K_ATP currents in a concentration-dependent manner. Glibenclamide and repaglinide produced inhibitory effects on K_ATP currents in a manner similar to that observed for nateglinide (Fig. 4, B and C). The effects of all agents were examined in 5 mM extracellular glucose to mimic a normoglycemic condition and concentration-response curves were formed as shown in Fig. 5. The IC₅₀ values and the slope coefficients were obtained from the nonlinear fitting of the data with least-squares analysis. The IC₅₀ values for K_ATP-blocking effect in normal glucose are 5.0 ± 1.4 nM for repaglinide, 16.6 ± 0.3 nM for glibenclamide, and 7.4 ± 0.2 µM for nateglinide. In a rank order of repaglinide > glibenclamide > nateglinide, the potencies for closure of K_ATP channels were commensurate with those shown to stimulate insulin release from isolated islets (Malaisse, 1995; Ikenoue et al., 1997).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Representative recordings of the inhibition of KATP currents by nateglinide (NAT), glibenclamide (GLY), and repaglinide (REP). Experiment was performed in 5 mM glucose. KATP currents are shown in control, in 100 mM diazoxide (DIA), and in diazoxide with nateglinide at various concentrations as indicated. Dotted lines indicate zero current levels. The amplitude of current at 300 ms from the beginning of the pulse (~90 mV) was measured to performed the quantitative analysis. All currents were recorded from the same cell.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves for the inhibition of KATP currents by nateglinide (), glibenclamide (), and repaglinide () in 5 mM glucose. Points are the amplitudes of KATP currents at 90 mV, averaged from six to eight experiments. The y-axis values are the KATP currents in the presence of drugs relative to the maximal values activated by diazoxide (as percentages). The x-axis indicates the concentration of the compounds (molar concentration) presented in a logarithmic scale. The slope coefficients were 0.7, 0.9, and 0.7, respectively, for repaglinide, glibenclamide, and nateglinide.

Time Course of K_ATP Channel-Blocking Actions. We studied the time-dependence of the K_ATP channel-blocking effects of nateglinide and compared it with that of glibenclamide and repaglinide. All compounds were tested at an equipotent concentration, i.e., 2-fold of their respective IC₅₀ values. To circumvent cell-cell variability, the time courses of nateglinide and one of the other two agents were investigated sequentially in the same cell. Figure 6, A and B, show, respectively, the time course of nateglinide with glibenclamide and nateglinide with repaglinide. The effect by nateglinide on K_ATP channels had a rapid onset and was largely or completely reversed shortly after the drug was removed. In addition, the action of nateglinide could be seen with repeated application without a sign of desensitization. In contrast, the duration of K_ATP channel blocking action by glibenclamide and repaglinide was longer lasting. This was especially true with repaglinide, whose action often outlasted the duration of drug presence by severalfold. In most cases, a complete recovery of repaglinide's effect was not seen within ~3 h (Fig. 6B). A summary of the time to a half-maximal inhibition [t_1/2(on)] and the time to a half-recovery from maximal inhibition [t_1/2(off)] is given in Table 4.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Time course of the KATP-blocking effects by nateglinide (NAT) versus glibenclamide (GLY) (A) and nateglinide versus repaglinide (REP) (B). In each figure, data were recorded from the same cell and expressed as the fraction of their maximal values activated by 100 µM diazoxide. The duration of compound applications are marked at the top of the figures.

	Discussion

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The high-affinity glibenclamide binding site (K_d = 0.27 ± 0.07 nM) is most likely involved in controlling K_ATP channel activity and insulin release because the effect on insulin release by glibenclamide occurs at low nanomolar concentrations after correction for ligand binding to BSA (Gaines et al., 1988). Moreover, Aguilar-Bryan et al. (1992) have observed a correlation between the number of high-affinity receptors and K_ATP channel density with HIT-T15 cells at low- and high-passage number. The low-affinity sites (K_d = 25 ± 13 nM) may have resulted from damage to the receptor during the membrane isolation procedure because binding experiments with intact RIN-m5F -cells reveal only a single receptor population (data not shown; K_d = 2.4 ± 0.27 nM). With intact HIT-T15 -cells, a single binding site with a K_d = 0.29 nM for [³H]glibenclamide has been reported (Ikenoue et al., 1997). Gaines et al. (1988) also reported a single high-affinity binding site with HIT-T15 cell membranes (K_d = 0.76 ± 0.04 nM). However, Niki et al. (1989) observed both high- and low-affinity binding sites (K_d = 1.1 and 140 nM) with membrane preparations from HIT-T15 cells. The relative magnitude of the differences between the high- and low-affinity sites is similar, i.e., 130-fold difference versus the 90-fold difference observed in the present experiments.

As shown in Table 1 glibenclamide is 440-fold more potent than nateglinide and nateglinide is 160-fold more potent than tolbutamide in binding to RIN-m5F cell membranes. In addition, nateglinide is 25-fold more potent than the L-isomer of nateglinide. These results compare favorably with the results of Fujita et al. (1996) and Ikenoue et al. (1997) with HIT cell membranes and intact HIT cells. The 25-fold reduced potency of the L-isomer in the membrane-binding assay is also consistent with the 60-fold reduced in vivo biological activity for the L-isomer of nateglinide (Shinkai et al., 1989). The relative order of efficacy for displacement of [³H]glibenclamide is consistent with a mode of action involving competitive binding at SUR1. Moreover, there is also a correlation between the relative potencies for binding to SUR1 and the ability of the compounds to inhibit the K_ATP channel and/or stimulate insulin secretion (Sugita et al., 1981; Schmid-Antomarchi et al., 1987; Gaines et al., 1988).

It is of interest to note that all of the compounds tested with the RIN-m5F cell membranes, with the exception of repaglinide and glimepiride, have Hill coefficients near unity (>0.85). The Hill coefficient for repaglinide is 0.60 ± 0.009 and for glimepiride is 0.58 ± 0.001. These values are significantly different from the Hill coefficients of the other compounds tested based on t tests of the Hill coefficients determined in the individual competitive binding experiments. A plausible interpretation of this result is that there is heterogeneity in the high-affinity receptor population (McPherson, 1989), i.e., there are two forms of the high-affinity SUR1 in RIN-m5F membranes or SUR1 can exist in two interchangeable states. Repaglinide and glimepiride (but not the other compounds) bind to the two forms or states of the receptor with different affinities. Additional experiments, described below, in which [³H]glibenclamide dissociation kinetics is measured also support a two-state/two-receptor model. It should be noted that an analog of repaglinide, AZ-DF 265, also exhibits a reduced Hill coefficient (0.51 ± 0.04) in competitive binding experiments with RIN-m5F cells (Ronner et al., 1992). The reduced Hill coefficients for repaglinide and glimepiride in the competitive binding experiments with RIN-m5F cell membranes (Table 1) distinguish the interactions of nateglinide, repaglinide, and glimepiride because nateglinide binds to both putative receptor states with equal affinity (Hill coefficient = 0.98 ± 0.06), whereas repaglinide and glimepiride bind with different affinity (Hill coefficients = 0.60 ± 0.009 and 0.58 ± 0.001, respectively). However, these differences in Hill coefficients are not observed with membranes from HEK.EBNA[human SUR1] cells (Table 2). This would suggest that the heterogeneity observed with RIN-m5F cell membranes, i.e., reduced Hill coefficients, is either artifactual, e.g., some of the receptor was "damaged" during its preparation, or that another membrane component (such as the inward rectifier component of the -cell K_ATP channel, K_ir6.2, which is present in RIN-m5F cells but not in the HEK-293[human SUR1] cells) is required to observe the two putative states of SUR1.

These compounds also were tested in direct competition studies with 2 nM [³H]glibenclamide and membranes from HEK.EBNA[human SUR1] cells. In general, the inhibitors bind more weakly (5- to 17-fold for all compounds except repaglinide) to human SUR1 than to RIN-m5F SUR1. The greatest difference is seen with repaglinide, which binds ~130-fold more weakly to human SUR1 than to RIN-m5F SUR1. Figure 1 displays the relationship for the binding of the seven compounds to RIN-m5F SUR1 and human SUR1. These results indicate that although the ligand-binding site is similar for RIN-m5F SUR1 and human SUR1, there are differences that become most apparent with repaglinide binding. Substantial differences in the receptor/enzyme ligand binding sites of human and other species is often observed (LoGrasso et al., 1994).

Neither nateglinide nor repaglinide had any effect on the dissociation kinetic parameters for [³H]glibenclamide (Table 3). This is consistent with nateglinide and repaglinide binding directly to the glibenclamide-binding site on SUR1. The biphasic release kinetics observed with membranes from both RIN-m5F cells and HEK.EBNA[human SUR1] cells can be explained by heterogeneity in the high-affinity binding site. One possible explanation for the heterogeneity is a two-state model for SUR1, i.e., the binding site exists in two interconvertable states. Consistent with this possibility, Aguilar-Bryan et al. (1998) have proposed at least two states for SUR1 and suggest that SUR1 may function to regulate the transition between the silent and bursting states of the K_ATP channel. However, for human SUR1 this heterogeneous (biphasic) behavior in [³H]glibenclamide dissociation kinetics is not reflected in the Hill coefficients for the steady-state binding of the various ligands, all of which have values near unity (Table 2).

The inability to measure [³H]nateglinide binding in both the filtration and centrifugation binding experiments is consistent with a large k_off value for [³H]nateglinide binding to SUR1. The estimated dissociation half-life for nateglinide is ~1 s (k_off = ~34 min¹), which was calculated assuming that the k_on value is 2 × 10⁸ min¹ M¹. This is a reasonable estimate of the k_on value because Müller et al. (1994) has reported k_on values of 1.7 × 10⁸ min¹ M¹ for glibenclamide and 4.9 × 10⁸ min¹ M¹ for glimepiride binding to RIN-m5F cell membranes. This value is also in the range of association rates reported for enzyme-substrate interactions. For example, the association rate of tyrosine with tyrosyl-tRNA synthetase is 1.4 × 10⁸ min¹ M¹ (Ferst, 1985). This rapid dissociation of nateglinide from its binding site would explain the inability to directly measure [³H]nateglinide binding with the filtration and centrifugation assay methods. Biphasic dissociation kinetics were observed for [³H]glibenclamide with dissociation half-lives of 2.9 min (k_off = 0.23 min¹) and 63 min (k_off = 0.011 min¹). The value for the fast phase is in good agreement with the value reported by Müller et al. (1994) for the rate of [³H]glibenclamide dissociation from RIN-m5F cell membranes (half-life = 2.8 min; k_off = 0.25 min¹). The estimated dissociation half-life for repaglinide is ~2 min (k_off = ~0.36 min¹; calculated assuming that k_on = 2 × 10⁸ min¹ M¹). The estimated dissociation rates for nateglinide, glibenclamide, and repaglinide are consistent with the kinetic effects observed on K_ATP channel activity after removal of the compounds in the whole-cell patch-clamp experiments (see below).

Table 4 summarizes the kinetic effects of the compounds on K_ATP channel activity. The t_1/2(on) of nateglinide (4.1 min) was similar to that of glibenclamide (4.2 min), although nateglinide exhibited a considerably earlier in vivo insulinotropic action (Ikenoue et al., 1997). The discrepancy is most likely attributed to their different pharmacokinetic profiles, with nateglinide being absorbed more rapidly than glibenclamide. In our experiments, the duration of compound application was in the range between 15 and 20 min because all three compounds reached their steady-state maximal effect in <20 min. The t_1/2(off) values of glibenclamide and repaglinide, being considerably >20 min, suggested that a majority of K_ATP channels remained in a closed state long after the compounds were removed. Consequently, these agents would cause a prolonged insulinotropic action in vivo. Thus, the mechanism-based slow recovery of the action by glibenclamide and repaglinide appears to be a crucial factor that contributes to the long-lasting hypoglycemic action of these compounds.

The kinetic data on the recovery of K_ATP channel activity after removal of the compound is consistent with the reported data on the reversibility of the effect of these agents on insulin release with perifused pancreatic islets. Thus, the effect of nateglinide on insulin release with perifused islets is completely reversed within 10 min after removal of the compound (Jijakli et al., 1997). However, both glibenclamide and repaglinide show little or no change in insulin release after removal of the compounds (Lebrun and Malaisse, 1992; Jijakli et al., 1996).

	Conclusions

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Both nateglinide and repaglinide bind competitively with glibenclamide to rat (RIN-m5F) and human SUR1. Differences in binding are most apparent with repaglinide, which binds 130-fold more weakly to human SUR1 than to RIN-m5F SUR1. The six other ligands bind, on average, 9-fold more weakly to human SUR1 than to RIN-m5F SUR1. Nateglinide rapidly dissociates from the RIN-m5F SUR1 with an estimated half-life of ~1 s, whereas the half-life for glibenclamide is 2.9 and 63 min (biphasic dissociation kinetics) and ~2 min for repaglinide (estimated value). The effect of nateglinide on K_ATP channel activity with intact -cells is more rapidly reversed compared with the effects of glibenclamide and repaglinide. Table 5 compares nateglinide, glibenclamide, and repaglinide with respect to receptor binding, K_ATP channel activity and insulin secretion. Finally, the receptor-binding results and intact -cell K_ATP channel activity measurements are consistent with the more rapid in vivo and ex vivo onset and shorter duration of action for nateglinide relative to repaglinide and sulfonylureas. These characteristics may contribute to the low hypoglycemic potential for this compound and reduction in excessive insulin release.