Received November 21, 2006; Revised December 15, 2006; Accepted December 21, 2006. Author to whom all correspondence and reprint requests should be addressed: Prof. W. J. Malaisse, Laboratory of Experimental Hormonology, Brussels Free University, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: malaisse@ulb.ac.be Stimulus-Secretion Coupling of Hypotonicity-Induced Insulin Release in BRIN–BD11 Cells Renaud Beauwens, 1 Len Best, 2 Nicolas Markadieu, 1 Raphael Crutzen, 1 Karim Louchami, 3 Peter Brown, 2 Allen P. Yates, 2 Willy J. Malaisse, 3 and Abdullah Sener 3 Laboratories of 1 Cell and Molecular Physiology and 3 Experimental Hormonology, Brussels Free University, Brussels, Belgium; and 2 Department of Medicine, Manchester University, Manchester, UK Endocrine, vol. 30, no. 3, 353–363, December 2006 0969–711X/06/30:353–363/$30.00 ENDO (Online) ISSN 1559-0100 © 2006 by Humana Press Inc. All rights of any nature whatsoever reserved. 353 The stimulus-secretion coupling for hypotonicity-in- duced insulin release was investigated in BRIN–BD11 cells. A 50 mM decrease in extracellular NaCl caused a twofold increase in insulin release. The release of in- sulin evoked by hypotonicity progressively decreased in an exponential manner. The response to extracellu- lar hypotonicity displayed a threshold value close to 20 mOsmol/L and a maximal response at about 70 mOsmol/ L. Hypotonicity also caused a rapid increase in cell vol- ume followed by a regulatory volume decrease (RVD), cell membrane depolarization with induction of spike activity, and a rise in cytosolic Ca 2+ concentration. 5- Nitro-2-(3-phenylpropylamino)benzoate inhibited the secretory response to hypoosmolarity, failed to affect the early increase in cell volume but prevented the RVD, and suppressed the hypotonicity-induced plasma membrane depolarization. Insulin release provoked by hypotonicity was inhibited by verapamil, absence of Ca 2+ , thapsigargin, furosemide, tributyltin, and diazox- ide. On the contrary, tolbutamide augmented modestly insulin release recorded in the hypoosmolar medium. Last, a rise in extracellular K + concentration, while aug- menting basal insulin output, failed to affect insulin release in the hypoosmolar medium. Thus, the insulin secretory response to hypotonicity apparently represents a Ca 2+ -dependent process triggered by the gating of vol- ume-sensitive anion channels with subsequent depolar- ization and gating of voltage-sensitive Ca 2+ channels. Key Words: Hypotonicity; insulin release; BRIN–BD11 cells. Introduction Exposure of insulin-producing cells to a medium of low osmolarity provokes insulin release (1–6). The mechan- ism(s) involved in such a process are not well understood. To cite only one example, it remains a matter of debate whether the secretory response to hypotonicity is causally linked to the activation of chloride channels (6). The major aim of the present study was to gain further insight in the stimulus-secretion coupling for hypotonicity-induced in- sulin secretion. In order to explore all variables specifically in insulin-producing cells, the experiments were conducted in BRIN–BD11 cells, an insulin-secreting cell line estab- lished by electrofusion of normal pancreatic B-cells from New England Deaconess Hospital rats with immortalized RINm5F cells (7). Results Insulin Release Comparison Between Three Insulin-Producing Cell Lines In a first set of experiments, the secretory response to hypo- tonicity, as provoked by a decrease in NaCl concentration by 50 mM, was examined in three lines of insulin-produc- ing cells. The results of these experiments are summarized in Table 1. In MIN-6 cells exposed to the hypotonic medium, the release of insulin over 30 min incubation averaged 118.3 ± 1.9% (n = 12; p < 0.001) of the mean corresponding value found within the same experiment(s) under isotonic condi- tions (100.0 ± 1.4%; n = 12). The former percentage was lower ( p < 0.001) than that found in either INS-1 cells (174.4 ± 10.8%; n = 12; p < 0.001 vs isotonic control val- ues) or BRIN–BD11 cells (265.5 ± 26.9%; n = 18; p < 0.001 vs isotonic control values). The latter two mean per- centages were also significantly different from one another ( p < 0.02). Despite lower absolute values for insulin output (expressed per cell), the BRIN–BD11 cells thus offered the advantage of a relatively greater secretory responsiveness to hypotonicity (Table 1; upper panel). In some of these pilot experiments, the ratio between in- sulin output under hypotonic/isotonic conditions was also measured at increasing concentrations of D-glucose (1.1,