The role of plasma membrane depolarization as a determinant of the initial phase of insulin secretion was investigated. NMRI mouse islets and beta-cells were used to measure the kinetics of insulin secretion, ATP and ADP content, membrane potential, and cytosolic free Ca(2+) concentration ([Ca(2+)](i)). The depolarization of metabolically intact beta-cells by KCl corresponded closely to the theoretical values. In contrast to physiological (glucose) or pharmacological (tolbutamide) ATP-sensitive K(+) (K(ATP)) channel block, KCl depolarization did not induce action potential spiking. The depolarization by 15 mM K(+) (21 mV) corresponded to the plateau depolarization by 50 or 500 microM tolbutamide; that by 40 mM K(+) (41 mV) corresponded to the action potential peaks. Nifedipine and diazoxide abolished action potentials but not KCl depolarization, suggesting that the depolarizing strength of 15, but not 40 mM K(+) corresponds to that of K(ATP) channel closure. K(+) (40 mM) induced a massive secretory response in the presence of 5 mM glucose, whereas 15 mM K(+), like 50 microM tolbutamide, was only slightly effective, even though a marked increase in [Ca(2+)](i) was produced. Raising glucose from 5 to 10 mM in the continued presence of 15 mM K(+) resulted in a strongly enhanced biphasic response. The depolarization pattern of this combination could be mimicked by combining basal glucose with 15 mM K(+) and 50 microM tolbutamide; however, the secretory response to these nonnutrients was much weaker. In conclusion, the initial secretory response to nutrient secretagogues is largely influenced by signaling mechanisms that do not involve depolarization.

The rates of granule arrival at and departure from the submembrane space changed in parallel and were two orders of magnitude higher than the release rates, suggesting a back-and-forth movement of the granules as the primary determinant of the submembrane granule number. The effect of 15 mM KCl resembled that of 40 mM but did not achieve significance. Both 15 and 40 mM KCl evoked a [Ca 2+ ] i increase, which was antagonized by 10 μM nifedipine. Nifedipine also antagonized the effect on secretion and on granule number and mobility. In conclusion, during KCl depolarization L-type Ca 2+ channels seem to regulate two processes, insulin granule turnover in the submembrane space and granule exocytosis.

Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X-Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.

Willenborg M, Belz M, Schumacher K, Paufler A, Hatlapatka K, Rustenbeck I. Ca 2ϩ -dependent desensitization of insulin secretion by strong potassium depolarization.

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...