No abstract

Prolonged stimulation of insulin secretion by depolarization and Ca2؉ influx regularly leads to a reversible state of decreased secretory responsiveness to nutrient and nonnutrient stimuli. This state is termed "desensitization." The onset of desensitization may occur within 1 h of exposure to depolarizing stimuli. Desensitization by exposure to sulfonylureas, imidazolines, or quinine produces a marked cross-desensitization against other ATP-sensitive K ؉ channel (K ATP channel)-blocking secretagogues. However, desensitized ␤-cells do not necessarily show changes in K ATP channel activity or Ca 2؉ handling. Care has to be taken to distinguish desensitization-induced changes in signaling from effects due to the persisting presence of secretagogues. The desensitization by depolarizing secretagogues is mostly accompanied by a reduced content of immunoreactive insulin and a marked reduction of secretory granules in the ␤-cells. In vitro recovery from a desensitization by the imidazoline efaroxan was nearly complete after 4 h. At this time point the depletion of the granule content was partially reversed. Apparently, recovery from desensitization affects the whole lifespan of a granule from biogenesis to exocytosis. There is, however, no direct relation between the ␤-cell granule content and the secretory responsiveness. Even though a prolonged exposure of isolated islets to depolarizing secretagogues is often associated with the occurrence of ultrastructural damage to ␤-cells, we could not find a cogent link between depolarization and Ca 2؉ influx and apoptotic or necrotic ␤-cell death. Diabetes 53 (Suppl. 3)

Essential role of the imidazoline moiety in the insulinotropic effect but not the K ATP channel-blocking effect of imidazolines; a comparison of the effects of efaroxan and its imidazole analogue, KU14R Abstract Aims/hypothesis: Imidazolines are a class of investigational antidiabetic drugs. It is still unclear whether the imidazoline ring is decisive for insulinotropic characteristics. Materials and methods: We studied the imidazoline efaroxan and its imidazole analogue, KU14R, which is currently classified as an imidazoline antagonist. The effects of both on stimulus secretion-coupling in normal mouse islets and beta cells were compared by measuring K ATP channel activity, plasma membrane potential, cytosolic calcium concentration ([Ca 2+ ] c ) and dynamic insulin secretion. Results: In the presence of 10 mmol/l but not of 5 mmol/l glucose, efaroxan (100 μmol/l) strongly enhanced insulin secretion by freshly isolated perifused islets, whereas KU14R (30, 100 or 300 μmol/l) was ineffective at both glucose concentrations. Surprisingly, the insulinotropic effect of efaroxan was not antagonised by KU14R. K ATP channels were blocked by efaroxan (IC 50 8.8 μmol/l, Hill slope −1.1) and by KU14R (IC 50 31.9 μmol/l, Hill slope −1.5). Neither the K ATP channelblocking effect nor the depolarising effect of efaroxan was antagonised by KU14R. Rather, both compounds strongly depolarised the beta cell membrane potential and induced action potential spiking. However, KU14R was clearly less efficient than efaroxan in raising [Ca 2+ ] c in single beta cells and whole islets at 5 mmol/l glucose. The increase in [Ca 2+ ] c induced by 10 mmol/l glucose was affected neither by efaroxan nor by KU14R. Again, KU14R did not antagonise the effects of efaroxan. Conclusions/interpretation:The presence of an imidazole instead of an imidazoline ring leads to virtually complete loss of the insulinotropic effect in spite of a preserved ability to block K ATP channels. The imidazole compound is less efficient in raising [Ca 2+ ] c ; in particular, it lacks the ability of the imidazoline to potentiate the enhancing effect of energy metabolism on Ca 2+ -induced insulin secretion.

The contribution of ATP-sensitive K ϩ channel (K ATP channel)-dependent and -independent signaling to the insulinotropic characteristics of imidazolines was explored using perifused mouse islets and ␤-cells. Up to a concentration of 100 M efaroxan had no insulinotropic effect in the presence of a basal glucose concentration, but enhanced the effect of a stimulatory concentration of glucose or nonglucidic nutrients (ketoisocaproate plus glutamine). The secretion by a non-nutrient (40 mM KCl) was not enhanced. At 500 M, efaroxan stimulated insulin secretion when glucose was basal. Likewise, at 0.1 to 10 M RX871024 [2-(imidazolin-2-yl)-1-phenylindole] showed a purely enhancing effect, but at 100 M it elicited a strong KCl-like secretory response in the presence of basal glucose. At 0.1 and 1 M RX871024 did not significantly depolarize the ␤-cell membrane. However, at a purely enhancing drug concentration (10 M RX871024 or 100 M efaroxan) K ATP channel activity was strongly reduced, the membrane was depolarized, and the cytosolic Ca 2ϩ concentration was elevated in the presence of basal glucose. Insulin secretion by sulfonylurea receptor (SUR)1 knockout (KO) islets, which have no functional K ATP channels, was not increased by efaroxan (100 or 500 M) or by 10 M RX871024 but was increased by 100 M RX871024. The imidazolines phentolamine and alinidine (100 M) were also ineffective on SUR1 KO islets. It is concluded that a significant K ATP channel block is compatible with a purely enhancing effect of the imidazolines on nutrient-induced insulin secretion. Only RX871024 has an additional, nondepolarizing effect, which at a high drug concentration is able to elicit a K ATP channel-independent secretion.The insulinotropic effect of the prototypical imidazoline phentolamine was originally believed to be due to an antagonism at ␣ 2 -adrenoceptors of the ␤-cell (Robertson and Porte, 1973; Efendic et al., 1975). The observation that the insulinotropic effect of this compound was not shared by other antagonists at ␣ 2 -adrenoceptors but was shared by other compounds with imidazoline moieties (Ostenson et al., 1988;Schulz and Hasselblatt, 1989) led to the hypothesis that the insulinotropic effect was mediated by a ␤-cell-specific subtype of the imidazoline receptor (Chan et al., 1994). In other tissues, at least two nonadrenergic imidazoline binding sites have been identified, and consequently the hypothetical ␤-cell subtype was named I 3 receptor (Eglen et al., 1998;Morgan, 1999). In contrast to the I 1 and I 2 sites where binding occurs with nanomolar affinity, binding to I 3 sites requires micromolar concentrations of the imidazoline (Rustenbeck et al., 1997).The demonstration that phentolamine and other imidazolines block ATP-sensitive K ϩ channels (K ATP channels) in pancreatic ␤-cells offered an explanation for their insulinotropic property (Plant and Henquin, 1990;Chan and Morgan, 1990). However, it remained unclear how the effect on the K ATP channel was related to the nonadrenergic imidazoline binding sites on...

The glucose dependence of the insulinotropic action of K ATP channel-blocking imidazoline compounds was investigated. Administration of 100 mol/l phentolamine, but not 100 mol/l efaroxan, markedly increased insulin secretion of freshly isolated mouse islets when the perifusion medium contained 5 mmol/l glucose. When the glucose concentration was raised to 10 mmol/l in the continued presence of either imidazoline, a clear potentiation of secretion occurred as compared with 10 mmol/l glucose alone. In the presence of efaroxan, a brisk first-phase-like increase was followed by a sustained phase, whereas a more gradual increase resulted in the presence of phentolamine. Administration of 100 mol/l phentolamine was somewhat more effective than 100 mol/l efaroxan to inhibit K ATP channel activity in intact cultured ␤-cells (reduction by 96 vs. 83%). Both compounds were similarly effective to depolarize the ␤-cells. When measured by the perforated patch-technique, the depolarization by efaroxan was often oscillatory, whereas that by phentolamine was sustained. In perifused cultured islets, both compounds increased the cytosolic calcium concentration ( I midazolines are a group of investigational antidiabetic drugs. Originally, an antagonism at ␣-adrenoceptors was believed to be the mechanism of action of the prototypical imidazoline, phentolamine (1,2). When it became clear that the insulinotropic effect of this compound was not due to an ␣-antagonism but was related to its imidazoline moiety (3,4), it was hypothesized that this effect was mediated by a ␤-cell imidazoline receptor, as had been described previously for other pharmacological actions of imidazoline compounds (5).The demonstration that phentolamine and other imidazolines block K ATP channels in pancreatic ␤-cells (6,7) offered an explanation for their insulinotropic property. However, it remained unclear how the effect on the K ATP channel was related to the hypothetical ␤-cell imidazoline receptor (8). The demonstration that the imidazoline RX 871024 increased insulin secretion not only by blocking K ATP channels, but also by acting at a site distal to calcium influx, led to the alternative hypothesis that imidazolines have effects at multiple independent sites of the pancreatic ␤-cell (9). The concept of a ␤-cell imidazoline receptor mediating a multitude of effects was rendered less likely by the observation that the K ATP channel block by imidazolines was exerted directly at Kir6.2, the pore-forming subunit of the channel (10,11).Thus, it could be expected that by structural modification it should be possible to separate the blocking effect of the imidazolines on K ATP channels from their more direct effect on exocytosis. An imidazoline devoid of a K ATP channel-blocking activity was expected to stimulate secretion only in the presence of high but not basal glucose concentrations. In fact, several newly synthesized imidazoline compounds were shown to stimulate insulin secretion at concentrations at which they did not affect K ATP channel activity (12,13...