Introduction

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The cyanoguanidine and the 1,1-diamino-2-nitroethylene functions are considered bioisosteres of the urea and thiourea functions (Thornber, 1979). The substitution of the thiourea moiety of metiamide, initially developed as H₂ receptor antagonist, or the substitution of the urea group of its derivatives by a cyanoguanidine or a 1,1-diamino-2 nitroethylene function led to the discovery of powerful inhibitors of gastric acid secretion such as cimetidine (Durant et al., 1977) and ranitidine (Brodgen et al., 1979), respectively. The latter compounds exhibited a higher H₂-blocking activity than the parent molecule metiamide. The inverse strategy was conducted from pinacidil, an antihypertensive cyanoguanidine, and generated thioureas as potent as pinacidil on vascular smooth muscle (Manley and Quast, 1992). Moreover, the cyanoguanidine bioisosteres of phenytoin, an antiepileptic acylurea, and torasemide, a diuretic sulfonylurea, have also been prepared (Masereel et al., 1995; Lambert et al., 1996). These compounds, unfortunately, appeared to be less active than their parent molecules (Masereel et al., 1995; Lambert et al., 1996).

In an attempt to improve the potency of hypoglycemic sulfonylureas, we recently applied a similar strategy by replacing their urea function with a sulfonylcyanoguanidine or a sulfonamidonitroethylene (Masereel et al., 1996, 1997). Among the different compounds synthesized, two isosteres of glibenclamide (Fig. 1); namely, BM 208 (N-[4-(5-chloro2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano-N"-cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonamido]-1-cyclohexylamino-2-nitroethylene), stimulated insulin release from incubated rat pancreatic islets (Masereel et al., 1997).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of glibenclamide, BM 208, and BM 225.

The aim of the present study was to further document the effects of BM 208 and BM 225 on the secretory activity of rat pancreatic B cells. In addition, we determined whether the capacity of the glibenclamide isosteres to stimulate insulin release was related to changes in ionic movements and cytosolic Ca²⁺ concentrations.

	Materials and Methods

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Animals. All experiments were performed with pancreatic islets isolated by the collagenase method from fed female albino rats.

Measurements of ⁸⁶Rb, ⁴⁵Ca Outflow, and Insulin Release From Perifused Pancreatic Islets. The methods used to measure ⁸⁶Rb (⁴²K substitute) outflow, ⁴⁵Ca outflow, and insulin release from perifused islets have been described previously (Lebrun et al., 1982a, 1985, 1996). Groups of 100 islets were incubated for 60 min in a bicarbonate-buffered medium (115 mM NaCl, 5 mM KCl, 2.56 mM CaCl₂, 1 mM MgCl₂, 24 mM NaHCO₃) containing 16.7 mM glucose and either ⁸⁶Rb (0.15-0.25 mM; 50 µCi/ml) or ⁴⁵Ca (0.02-0.04 mM; 100 µCi/ml). After incubation, the islets were washed four times with a nonradioactive medium and then placed in a perifusion chamber. The perifusate was delivered at a constant rate (1.0 ml/min). From minute 31 to min 90 of perifusion, the effluent was continuously collected over successive periods of 1 min each. An aliquot of the effluent (0.5 ml) was used for scintillation counting while the remainder was stored at 20°C for insulin radioimmunoassay (Leclercq-Meyer et al., 1985). At the end of the perifusion, the radioactive content of the islets was also determined. The outflow of ⁸⁶Rb or ⁴⁵Ca (cpm/min) was expressed as a fractional outflow rate (percentage of instantaneous islet content/min; FOR). The validity of ⁸⁶Rb as a tracer for the study of K⁺ handling in the islets has been previously assessed (Malaisse et al., 1978).

Patch-Clamp Measurements. All electrophysiological studies were performed on primary cultured rat islet cells prepared as described previously (Findlay et al., 1985). Cells were maintained using standard tissue culture conditions for 18 to 72 h in RPMI 1640 medium (GIBCO, UK) supplemented with 10% FCS and 100 IU/0.1 mg/ml penicillin/streptomycin, respectively. Single-channel currents were recorded using the cell-free, inside-out patch-clamp configuration (Hamill et al., 1981). Micropipettes were filled with an Na⁺-rich solution of the following ionic composition: 140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl₂, 2.5 mM CaCl₂, 2.5 mM glucose, 10 mM HEPES, pH 7.4. The internal face of membrane was bathed with a K⁺-rich solution containing 140 mM KCl, 10 mM NaCl, 1.13 mM MgCl₂, 1 mM EGTA, 2.5 mM glucose, and 10 mM HEPES, pH 7.2. Unitary current events from K_ATP⁺ channels were recorded at 0-mV voltage-clamp. Under these experimental conditions, with the intracellular Ca²⁺ buffered to below 10 nM, openings of Ca²⁺- and voltage-gated K⁺ channels were not observed (Harding et al., 1994; Lebrun et al., 1996). Experimental data were stored on digital tape for subsequent replay and analysis. Modulation of K_ATP⁺ channels was described in terms of a change in the channel open-state probability. These values were quantified from prerecorded stretches of data lasting between 30 and 40 s and were expressed as a fraction of the immediate precontrol value (Lebrun et al., 1996).

Measurements of Fura-2 Fluorescence From Single Islet Cells. Pancreatic islets were disrupted in a Ca²⁺-deprived medium and then centrifuged through an albumin solution to remove debris and dead cells. Cells were seeded onto glass coverslips and maintained in tissue culture for 72 h. Islet cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% newborn calf serum and containing penicillin (100 IU/ml) and streptomycin (0.1 mg/ml). The cells were then incubated with the fluorescent Ca²⁺ indicator fura-2 AM (2 µM) for 1 h, and after washing, the coverslips with the cells were mounted as the bottom of an open chamber (1 ml) placed on the stage of the microscope. The medium used to perfuse the cells contained 115 mM NaCl, 5 mM KCl, 2.56 mM CaCl₂, 1 mM MgCl₂, 24 mM NaHCO₃, and 2.8 mM glucose, and was gassed with 95% O₂/5% CO₂. fura-2 Fluorescence of single-loaded cells was measured by use of dual-excitation microfluorimetry with a Spex photometric system (Optilas, Alphen aan den Rijn, Holland). The excitation and emission wavelengths were set at 340/380 and 510 nm, respectively. [Ca²⁺]_i was calculated as described previously (Lebrun et al., 1996). Individual experiments were repeated at least four times, on different cell populations.

Drugs. In some experiments, extracellular Ca²⁺ was eliminated by the omission of CaCl₂ from the physiological medium and the addition of 0.5 mM EGTA (Sigma Chemical Co., St. Louis, MO). The medium also contained (as required) glucose (Merck, Darmstadt, Germany), glibenclamide (Hoechst Roussel, Belgium), verapamil hydrochloride (Knoll A.G., Germany), BM 208, and BM 225. BM 208 and BM 225 were synthesized at the Department of Medicinal Chemistry, University of Liège, Belgium. Purity and stability were checked by thin-layer chromatography and elemental analyses (C, H, N, S). Both drugs were revealed to be stable compounds as powders as well as in solution. Glibenclamide, BM 208, and BM 225 were dissolved in dimethyl sulfoxide, which was added to both control and test media at final concentrations not exceeding 0.1% (v/v). At this concentration, dimethyl sulfoxide fails to affect islet function (Lebrun and Atwater, 1985; Lebrun et al., 1996).

Calculations. Results are expressed as the mean ± S.E.M. The basal value for ⁸⁶Rb, ⁴⁵Ca outflow, or insulin release was computed from minute 40 to 44 of perifusion inclusive. The inhibitory effect of drugs on ⁸⁶Rb outflow was taken as the difference between the mean value for ⁸⁶Rb outflow recorded in each individual experiment between the 40 and 44 min and 60 and 68 min of perifusion. The magnitude of the increase in ⁸⁶Rb, ⁴⁵Ca outflow, or insulin release was estimated in each individual experiment from the integrated outflow of ⁸⁶Rb, ⁴⁵Ca, or insulin release observed during stimulation (minute 45 to 68) after correction for basal value (40-44 min). The statistical significance of differences between mean data was assessed by using Student's t test.

	Results

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Effects of BM 208 and BM 225 on ⁸⁶Rb, ⁴⁵Ca Outflow, and Insulin Release From Perifused Rat Pancreatic Islets. BM 208 (10 µM) and BM 225 (10 µM) provoked a rapid and sustained inhibition of ⁸⁶Rb outflow from pancreatic islets perifused throughout in the presence of 2.8 mM glucose and extracellular Ca²⁺ (Fig. 2, top). The capacity of 10 µM BM 208 and 10 µM BM 225 to inhibit the ⁸⁶Rb outflow rate was identical. Thus, the paired difference in ⁸⁶Rb FOR before (minute 40-44) and during (minute 60-68) exposure to the drugs averaged 1.83 ± 0.10 %/min after the addition of BM 208 and 2.00 ± 0.09 %/min after the addition of BM 225 (P > .05). The inhibitory effect of BM 208 on ⁸⁶Rb FOR was slowly reversible, whereas the latter effect persisted for at least 20 min after removal of BM 225 from the perifusate.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of BM 208 (10 µM; ) and BM 225 (10 µM; ) on 86Rb (top), 45Ca outflow (middle), and insulin release (bottom) from perifused pancreatic islets. Basal media contained 2.8 mM glucose and extracellular Ca2+. Mean values (±S.E.) refer to five to eight individual experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of BM 208 (25 µM; left) and BM 225 (25 µM; right) on 86Rb (top), 45Ca outflow (middle), and insulin release (bottom) from perifused pancreatic islets. Basal media contained 2.8 mM glucose and extracellular Ca2+ () or 2.8 mM glucose without extracellular Ca2+ (). Mean values (±S.E.) refer to four to six individual experiments.

Effects of BM 208 and BM 225 on Individual K_ATP⁺ Channel Events. The effects of the glibenclamide isosters were further characterized using the inside-out configuration of the patch-clamp technique. As described previously (Harding et al. 1994; Lebrun et al., 1996), the addition of ATP (1 mM) on the inside face of the B cell membrane inhibited K⁺ channel activity (Fig. 4A). Removal of ATP was followed by an immediate increase in K_ATP⁺ channel opening. Under these experimental conditions (no ATP in the bathing medium), the addition of BM 208 (25 µM) inhibited K_ATP⁺ channel activity (Fig. 4A). Similar effects were noticed with BM 225 (25 µM; Fig. 4B). A quantitative analysis revealed that the K_ATP⁺ channel open-state probability averaged 17.7 ± 3.6% after the addition of BM 208 (P < .05) and 24.7 ± 6.1% after addition of BM 225 (P < .05) (Fig. 4C). The effects of BM 208 and BM 225 were sustained and not readily reversible (Fig. 4C).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Effect of BM 208 and BM 225 on KATP+ channels. Data were obtained at 0 mV voltage-clamp with upward deflections from the base line representing outward currents. A, effect of the addition and removal of either ATP (1 mM) or BM 208 (25 µM). B, data from the same patch recorded before (top), during the application of BM 225 (25 µM; middle), and after removal of the drug (bottom). C, quantitative analysis of the effects of BM 208 (25 µM) and BM 225 (25 µM) on KATP+ channels. The third column refers, in each case, to recovery values. Average values (±S.E.) from six patches in each case.

Effects of BM 208 and BM 225 on Intracellular Ca²⁺ Concentration. Both BM 208 (25 µM) and BM 225 (25 µM) increased the fluorescence intensity of fura-2-loaded islet cells perifused in the presence of 2.8 mM glucose (Fig. 5). Although the addition of BM 208 or BM 225 caused an immediate 2-fold increase in cytosolic Ca²⁺ concentration, the effect of BM 208 declined rapidly, whereas the enhancing effect of BM 225 was more sustained (Fig. 5). After the addition of BM 225 (25 µM), the initial peak of intracellular Ca²⁺ concentration ([Ca²⁺]_i) was followed by a slowly declining plateau that was higher than prestimulation values and was maintained for at least 10 min (Fig. 5, right).

View larger version (7K): [in this window] [in a new window]   Fig. 5.   Effect of BM 208 (25 µM; left) and BM 225 (25 µM; right) on the cytosolic Ca2+ concentration of single pancreatic B cells. Basal media contained 2.8 mM glucose and extracellular Ca2+. Each graph is a representative experiment conducted on a single cell.

View larger version (7K): [in this window] [in a new window]   Fig. 6.   Effect of verapamil (50 µM) on BM 208 (25 µM; left) and BM 225 (25 µM; right)-induced increases in cytosolic Ca2+ concentration. Basal media contained 2.8 mM glucose and extracellular Ca2+. Each graph is a representative experiment conducted on a single cell.

	Discussion

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Our data indicate that at a low glucose concentration (2.8 mM), BM 208 and BM 225 cause a sustained decrease in ⁸⁶Rb outflow from prelabeled and perifused rat pancreatic islets. This feature is reminiscent of the inhibitory effect of hypoglycemic sulfonylureas on ⁸⁶Rb FOR and is compatible with the view that the newly synthesized isosteres of glibenclamide provoke the closure of K_ATP⁺ channels (Malaisse and Lebrun, 1990; Lebrun and Malaisse, 1992). Such an hypothesis was confirmed by single patch-clamp recordings that revealed that BM 208 and BM 225 reduced K_ATP⁺ channel open-state probability. By either directly measuring K⁺ channel activity or by monitoring ⁸⁶Rb outflow, we found that the actions of both compounds were poorly reversible. This feature is reminiscent of the previously reported effects of glibenclamide (Malaisse and Lebrun, 1990; Lebrun and Malaisse, 1992).

The inhibition of ⁸⁶Rb outflow brought about by BM 208 and BM 225 coincided with an increase in ⁴⁵Ca outflow. The latter phenomenon was abolished in islets exposed to Ca²⁺-free medium. Thus, the BM-induced increases in ⁴⁵Ca outflow are likely to reflect a stimulation of ⁴⁰Ca inflow into the islet cells with resulting release of labeled ⁴⁵Ca from intracellular sites (Lebrun et al., 1982a, b). The ability of BM 208 and BM 225 to enhance Ca²⁺ entry is substantiated by the observation that both compounds increase the [Ca²⁺]_i in islet cells incubated under the same experimental conditions. Moreover, the BM-induced increases in [Ca²⁺]_i were reduced by verapamil; a phenylalkylamine Ca²⁺ entry blocker (Lebrun et al., 1982a, 1997; Godfraind et al., 1986; Plasman et al., 1991).

At first glance, these results recorded at a low concentration of D-glucose would suggest that the newly synthesized isosteres of glibenclamide decrease K⁺ permeability by closing K_ATP⁺ channels, depolarize the plasma membrane, activate the voltage-sensitive Ca²⁺ channels, increase Ca²⁺ inflow, and, eventually, promote insulin release.

However, a closer examination of the data revealed further features of the cationic and secretory responses to glibenclamide isosteres. Indeed, BM 208 and BM 225 inhibited ⁸⁶Rb outflow and K_ATP⁺ current to the same extent, whereas the stimulatory effect of BM 225 on ⁴⁵Ca outflow, [Ca²⁺]_i, and insulin output was more marked than that of BM 208. Moreover, for both compounds, a poor reversibility of the ⁸⁶Rb response contrasted with a more or less rapid return of ⁴⁵Ca outflow rate and insulin release to basal values. Taken as a whole, these data suggest that the BM-induced inhibition of K_ATP⁺ channels cannot fully account for the modifications in Ca²⁺ inflow and insulin output.

Although a distal effect in the sequence of events leading to insulin release cannot be absolutely excluded (Eliasson et al., 1996), it is more likely that the insulinotropic effect of the new compounds is attributable to their capacity to affect transmembrane Ca²⁺ movements.

Different K_ATP⁺-independent modalities can be considered to account, at least in part, for the BM-induced Ca²⁺ inflow across the B cell membrane. It can be hypothesized that the bioisosteres of glibenclamide facilitate Ca²⁺ inflow through voltage-insensitive Ca²⁺ channels. Previous studies indeed suggested that the B cell might be equipped with a voltage-insensitive pathway of Ca²⁺ entry (Lebrun et al., 1982a; Rojas et al., 1990). Alternatively, and perhaps more likely, it is conceivable that part of the effect of BM compounds on Ca²⁺ inflow might correspond to a drug-induced facilitation of Ca²⁺ transport as mediated by an ionophoretic process. This interpretation is consistent with the knowledge that hypoglycemic sulfonylureas may behave as ionophoretic agents and enhance an ionophoretic modality of Ca²⁺ transport across the B cell plasma membrane (Couturier and Malaisse, 1980). Incidentally, the latter effects can be inhibited by organic compounds such as verapamil (Couturier and Malaisse, 1980; Malaisse et al., 1981). Thus, the demonstration of an inhibitory effect of verapamil on the BM-induced increases in [Ca²⁺]_i could also support the idea that the BM isosteres mediate an ionophoretic modality of Ca²⁺ inflow (Lebrun et al., 1982b). Last, this ionophoretic hypothesis is also supported by the finding that BM 225 was more potent than BM 208 at increasing Ca²⁺ inflow. Indeed, the more pronounced effect of BM 225 on Ca²⁺ movements coincides with its higher capacity to penetrate the plasma membrane (Masereel et al., 1997).

In conclusion, the present study indicates that two newly synthesized isosteres of glibenclamide provoke a concentration-dependent increase in insulin output from pancreatic islets perifused at low glucose concentration (2.8 mM). Under these experimental conditions, the secretory capacity of BM 225 was more marked than that of BM 208. The stimulation of insulin release induced by both compounds results, at least in part, from the inhibition of K_ATP⁺ channels with subsequent opening of voltage-sensitive Ca²⁺ channels. The work further reveals that the time courses of the cationic, in terms of ⁸⁶Rb outflow and ⁴⁵Ca outflow, and secretory responses exhibited obvious dissociations. Such features suggest that the modifications in Ca²⁺ handling induced by the glibenclamide bioisosteres are not exclusively dependent on changes in the activity of the K_ATP⁺ channels. It is speculated that the insulinotropic action of both compounds could also be mediated, at least in part, by a Ca²⁺ ionophoretic process.