Selective Enhancement of Nutrient-Induced Insulin Secretion by ATP-Sensitive Kϩ Channel-Blocking Imidazolines

0022-3565/09/3313-1033–1041$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 152751/3533565 JPET 331:1033–1041, 2009 Printed in U.S.A. Selective Enhancement of Nutrient-Induced Insulin Secretion by ATP-Sensitive Kϩ Channel-Blocking Imidazolines Kathrin Hatlapatka, Antje Wienbergen, Claudia Ku¨hne, Anne Jo¨rns, Michael Willenborg, and Ingo Rustenbeck Institute of Pharmacology and Toxicology, Technical University of Braunschweig, Braunschweig, Germany (K.H., A.W., C.K., M.W., I.R.); and Institute of Anatomy, Hannover Medical School, Hannover, Germany (A.J.) Received February 27, 2009; accepted September 9, 2009 Downloaded from ABSTRACT ϩ ␮ ␮ The contribution of ATP-sensitive K channel (KATP channel)- M RX871024 or 100 M efaroxan) KATP channel activity was dependent and -independent signaling to the insulinotropic strongly reduced, the membrane was depolarized, and the characteristics of imidazolines was explored using perifused cytosolic Ca2ϩ concentration was elevated in the presence of mouse islets and ␤-cells. Up to a concentration of 100 ␮M basal glucose. Insulin secretion by sulfonylurea receptor efaroxan had no insulinotropic effect in the presence of a basal (SUR)1 knockout (KO) islets, which have no functional KATP jpet.aspetjournals.org glucose concentration, but enhanced the effect of a stimulatory channels, was not increased by efaroxan (100 or 500 ␮M) or by concentration of glucose or nonglucidic nutrients (ketoisocap- 10 ␮M RX871024 but was increased by 100 ␮M RX871024. The roate plus glutamine). The secretion by a non-nutrient (40 mM imidazolines phentolamine and alinidine (100 ␮M) were also KCl) was not enhanced. At 500 ␮M, efaroxan stimulated insulin ineffective on SUR1 KO islets. It is concluded that a significant ␮ secretion when glucose was basal. Likewise, at 0.1 to 10 M KATP channel block is compatible with a purely enhancing RX871024 [2-(imidazolin-2-yl)-1-phenylindole] showed a purely effect of the imidazolines on nutrient-induced insulin secretion. enhancing effect, but at 100 ␮M it elicited a strong KCl-like Only RX871024 has an additional, nondepolarizing effect, at ASPET Journals on March 10, 2015 secretory response in the presence of basal glucose. At 0.1 and which at a high drug concentration is able to elicit a KATP 1 ␮M RX871024 did not significantly depolarize the ␤-cell mem- channel-independent secretion. brane. However, at a purely enhancing drug concentration (10 The insulinotropic effect of the prototypical imidazoline ing occurs with nanomolar affinity, binding to I3 sites re- phentolamine was originally believed to be due to an antag- quires micromolar concentrations of the imidazoline (Rust- ␣ ␤ onism at 2-adrenoceptors of the -cell (Robertson and Porte, enbeck et al., 1997). 1973; Efendic et al., 1975). The observation that the insuli- The demonstration that phentolamine and other imidazo- notropic effect of this compound was not shared by other lines block ATP-sensitive Kϩ channels (K channels) in ␣ ATP antagonists at 2-adrenoceptors but was shared by other pancreatic ␤-cells offered an explanation for their insulino- compounds with imidazoline moieties (Ostenson et al., 1988; tropic property (Plant and Henquin, 1990; Chan and Morgan, Schulz and Hasselblatt, 1989) led to the hypothesis that the 1990). However, it remained unclear how the effect on the insulinotropic effect was mediated by a ␤-cell-specific sub- KATP channel was related to the nonadrenergic imidazoline type of the imidazoline receptor (Chan et al., 1994). In other binding sites on ␤-cells (Rustenbeck et al., 1997). The concept tissues, at least two nonadrenergic imidazoline binding sites of a ␤-cell imidazoline receptor mediating multiple effects by have been identified, and consequently the hypothetical signal transduction cascades was rendered less likely by the ␤-cell subtype was named I receptor (Eglen et al., 1998; 3 observation that the K channel block by imidazolines was Morgan, 1999). In contrast to the I and I sites where bind- ATP 1 2 exerted directly at Kir6.2, the pore-forming subunit of the channel (Proks and Ashcroft, 1997; Grosse-Lackmann et al., This work was supported by grants from the Deutsche Forschungsgemein- 2003). Kir6.2 was then suggested to be the correlate of the I3 schaft [Ru 368/4-1, Ru 368/5-1]; and by the German Diabetes Society. receptor (Monks et al., 1999; Morgan, 1999). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. The demonstration that the imidazoline RX871024 in- doi:10.1124/jpet.109.152751. creased insulin secretion not only by blocking KATP channels ϩ 2ϩ ABBREVIATIONS: KATP channel, ATP-sensitive K channel; RX871024, 2-(imidazolin-2-yl)-1-phenylindole; [Ca ]c, cytosolic free calcium concentration; BL11282, 5-chlor-3-(imidazolin-2-yl)-2-methylindole; NNC77-0074, (ϩ)-2-(2-(4,5-dihydro-1H-imidazol-2yl)-thiophene-2-yl-ethyl)- pyridine; SUR, sulfonylurea receptor; KO, knockout; KIC, ␣-ketoisocaproic acid; KU14R, 2-(2-ethyl-2,3-dihydrobenzo[b]furan-2-yl)-1H-imidazole. 1033 1034 Hatlapatka et al. ϩ but also by acting at a site distal to Ca2 influx led to the mixing for 1 min. Islets and single islet cells were cultured in cell alternative hypothesis that imidazolines exert effects at mul- culture medium RPMI 1640 with 10% fetal calf serum (5 mM glu- tiple independent sites (Zaitsev et al., 1996). The experimen- cose) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Electrophysiological Recordings. K channel activity was tal model to demonstrate the KATP channel-independent ef- ATP fect was the permeabilized cell where the cytosolic free measured by a standard patch-clamp technique using the cell-at- 2ϩ 2ϩ tached configuration (Hamill et al., 1981). Pipettes were pulled from calcium concentration ([Ca ]c) was clamped by use of Ca - buffered incubation media. RX871024 (Zaitsev et al., 1996) borosilicate glass (2 mm o.d. and 1.4 mm i.d.; Hilgenberg, Malsfeld, and later efaroxan (Chan et al., 2001) were shown to increase Germany) by a two-stage vertical puller (List Electronics, Darm- stadt, Germany) and had resistances between 3 and 6 M⍀ when insulin release from such permeabilized cell preparations. It filled with solution. Currents were recorded by an EPC 7 patch- was hypothesized that these imidazolines sensitize the exo- 2ϩ clamp amplifier (List Electronics) and low pass-filtered by a four-pole cytotic machinery to [Ca ]c, which in turn would be in- Bessel filter at 2 kHz and stored on a videotape. The pipette holding creased by the KATP channel-blocking effect of these com- potential was 0 mV in cell-attached recordings. The membrane po- pounds. A similar combination of effects, comprising a tential of ␤-cells was determined using the whole-cell and perforated protein kinase C-mediated sensitization of the exocytotic ma- patch modes under current clamp condition (Smith et al., 1990). chinery in addition to a KATP channel block, has been sug- Exposure to the test compounds and washout was done by changing gested to explain the insulinotropic characteristics of sulfo- the bath medium with a slow bath perfusion system. The composi- nylureas (Eliasson et al., 1996). The hypothesis that the tions of the bath and pipette media were as given by Zu¨nkler et al. dependence on stimulatory glucose concentrations is due to (1988). All experiments were performed at room temperature (20– Downloaded from an effect at a site distal to Ca2ϩ influx seemed to be con- 23°C). Data were analyzed off-line using pClamp 6.03 software (Axon firmed by the development of imidazolines (e.g., BL11282 or Instruments, Foster City, CA). Microfluorimetric Measurements of the [Ca2؉] . Islets were NNC77-0074), which do not block KATP channels, but stim- c ulate insulin secretion nevertheless (Efanov et al., 2001b; cultured on collagen-coated glass cover slips in Petri dishes and were Hoy et al., 2003). These imidazolines were then termed “sec- used from days 2 to 4 after isolation. Fura-PE3/AM was loaded at a concentration of 2 ␮M (dissolved in Krebs-Ringer medium with 5 mM ond-generation imidazolines” to distinguish them from the jpet.aspetjournals.org glucose) for 45 min at 37°C. The coverslip with the attached cells or K channel-blocking imidazolines, which were termed ATP islets was inserted in a purpose-made perifusion chamber on the “first-generation imidazolines” (Efendic et al., 2002). stage of an epifluorescence microscope fitted with a Zeiss Fluar (40ϫ) However, a sensitization of the exocytotic machinery to 2ϩ objective. The fluorescence (excitation at 340 or 380 nm; emission [Ca ]c should also amplify subthreshold signals of non-nu- Ͼ470 nm) was recorded by a slow-scan charge-coupled device camera trient secretagogues and thus seems an unlikely explanation (TILL Photonics, Gra¨felfing, Germany). All perifusions were per- for the selective enhancement by efaroxan and RX871024 of formed at 35°C using a HEPES-buffered Krebs-Ringer-bicarbonate the effect of suprathreshold glucose concentrations (Zaitsev medium. Image pairs were taken at intervals as indicated in the at ASPET Journals on March 10, 2015 et al., 1996; Efendic et al., 2002; Bleck et al., 2004). In this figures; illumination time for each image was 800 ms. RX871024, but study, we present evidence that the selective enhancement not efaroxan, proved to be fluorescent with an excitation maximum 2ϩ does not consist of a sensitization of exocytosis to [Ca ]c at 331 nm and a broad emission peak with a maximum at 460 nm. increases but of an enhanced efficacy of amplifying signals This necessitated performing the experiments in duplicate using derived from the metabolism of suprathreshold concentra- sham-loaded islets to subtract the RX871024 fluorescence from the tions of nutrient secretagogues (Henquin, 2000). Despite this Fura fluorescence emission.

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