b-Cell toxicity of ATP-sensitive K þ channel-blocking insulin secretagogues Ingo Rustenbeck a,* , Andrea Krautheim b,1 , Anne Jo¨rns c , Hans Ju¨rgen Steinfelder b a Institute of Pharmacology and Toxicology, Technical University of Braunschweig, D-38106 Braunschweig, Germany b Institute of Pharmacology and Toxicology, University of Go¨ttingen, D-37075 Go¨ttingen, Germany c Institute of Anatomy, Hannover Medical School, D-30623 Hannover, Germany Received 18 August 2003; accepted 16 January 2004 Abstract A prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100 mM each), the sulfonylurea tolbutamide (500 mM), or the alkaloid quinine (100 mM) resulted in morphological damage of 4–18% of b-cells compared to less than 2% in controls. Thus, the question arose whether K ATP channel-blocking insulin secretagogues are b-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24 h with idazoxan reduced MTT conversion by 50% at 100 mM and by 98% at 1000 mM. Phentolamine and quinine reduced viability comparably at 1000 mM, but were less toxic at 100 mM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000 mM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100 mM of idazoxan and phentolamine, whereas at 1000 mM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca 2þ influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for b-cells in intact mouse islets. In summary, b-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca 2þ influx via L-type Ca 2þ channels. Thus, secretagogue-mediated closure of K ATP channels is apparently not per se b-cell toxic. # 2004 Elsevier Inc. All rights reserved. Keywords: Pancreatic islets; HIT-cells; Imidazolines; Tolbutamide; Apoptosis; Necrosis 1. Introduction In a recent investigation, the desensitization of insulin secretion by a panel of structurally diverse inhibitors of ATP-dependent K þ channels (K ATP channels) was studied. Isolated incubated mouse islets were examined by electron microscopy in order to determine the content of secretory granules. Unexpectedly, islets cultured for 18 h in the presence of the secretagogues did not only show varying degrees of degranulation but also contained a higher number of ultrastructurally damaged b-cells than con- trol-cultured islets [1]. The b-cell damage was most pro- minent after exposure to the imidazoline idazoxan but there was also an increased number of damaged b-cells follow- ing exposure to the sulfonylurea tolbutamide [1]. This observation was reminiscent of a recent report that several imidazolines, a group of investigational antidia- betic drugs, negatively affected viability of insulin-secret- ing cell lines [2]. In this investigation some imidazolines like idazoxan were found to be markedly toxic, while others like efaroxan were apparently completely devoid of such an effect. Thus, the authors concluded that the toxicity was due to individual properties of the imidazo- lines and was not the consequence of the common K ATP channel-blocking and insulin-releasing effect. On the other hand, Berggren and co-workers had shown earlier that tolbutamide as well as high glucose concentrations induced apoptosis in isolated pancreatic b-cells and islets: this effect appeared to be dependent on Ca 2þ influx through Biochemical Pharmacology 67 (2004) 1733–1741 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.01.016 Abbreviations: K ATP channels, ATP-dependent K þ channels; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide * Corresponding author. Tel.: þ49-531-391-5670; fax: þ49-531-391-8287. E-mail address: i.rustenbeck@tu-bs.de (I. Rustenbeck). 1 Present address: Clinic of Dermatology and Venerology, University of Magdeburg, D-39106 Magdeburg, Germany.