R1 RAPID COMMUNICATION C Involvement of calpain and synaptotagmin Ca2 sensors in hormone secretion from excitable endocrine cells Ebun Aganna, Jacky M Burrin1, Graham A Hitman and Mark D Turner Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, Barts and The London, Queen Mary’s School of Medicine and Dentistry, University of London, Whitechapel, London E1 2AT, UK and 1Centre for Molecular Endocrinology, William Harvey Research Institute, Barts and The London, Queen Mary’s School of Medicine and Dentistry, University of London, Charterhouse Square, London EC1M 6BQ, UK (Requests for offprints should be addressed to M D Turner; Email: [email protected]) Abstract C The requirement for Ca2 to regulate hormone secretion from secretagog-stimulated secretion from both INS-1 and GH3 cells endocrine cells is long established, but the precise function of was completely abolished following pre-incubation with C Ca2 sensors in stimulus–secretion coupling remains unclear. the cysteine protease inhibitor E64, whereas stimulated In the current study, we examined the expression of calpain and secretion from AtT20 cells was modest and completely synaptotagmin in INS-1 pancreatic and GH3 and AtT20 insensitive to E64 inhibition. These results are in stark contrast pituitary cells, and investigated the sensitivity of hormone to synaptotagmin data. Synaptotagmin expression in AtT20 cells secretion from these cells to inhibition of the calpain family of is abundant, whereas INS-1 cells express extremely low C cysteine proteases. Little difference in expression of m-calpain levels of this Ca2 sensor, relative to the pituitary cells. We was observed between the different endocrine cells. However, hypothesize that the expression pattern of calpain and AtT20 cells did exhibit an extremely low abundance of synaptotagmin isoforms may reflect alternative mechanisms of both m-calpain and the 54 kDa isoform of calpain-10 relative stimulus–secretion coupling in excitable endocrine cells. to their expression in INS-1 and GH3 cells. Interestingly, Journal of Endocrinology (2006) 190, R1–R7 Introduction the fine detail of synaptotagmin I action, nonetheless after years of debate there finally appears to be a consensus that this is the C C The Ca2 requirement for stimulated secretion is well primary mediator of Ca2 sensing in neuronal stimulus– C documented, although the contribution of individual Ca2 secretion coupling. However, there are a number of differences sensors and their precise function in stimulus–secretion coupling between the secretory dynamics of hormone secretion and those remains poorly understood. Recently, the calpain family has of neurotransmission. In particular, whilst neurotransmission is been shown to be an important class of molecule mediating an extremely rapid process (Sabatini & Regehr 1999)endocrine insulin secretion from pancreatic b-cells (Ort et al. 2001, Zhou secretion is multiphasic, with rapid exocytosis often only a et al. 2003, Marshall et al. 2005, Parnaud et al. 2005). Moreover, minor component of total regulated secretion (Henkel & C evidence has emerged for a role for calpain-10 as a Ca2 sensor Almers 1996). Therefore, whilst the core fusion machinery mediating the actual exocytotic fusion event itself (Marshall et al. operating in all neuroendocrine cells is very similar, there are 2005). However, despite m-calpain (calpain-1; Ort et al. 2001) likely to be different regulatory molecules operating between and calpain-10 (Marshall et al. 2005) being specifically shown to the systems. The current study examines the contribution of be regulators of insulin secretion, little is known as to whether calpain and synaptotagmin to stimulus–secretion coupling in individual calpain family members play cell-specific or more three different endocrine cell types. general roles in endocrine secretion per se. This also raises the question, are calpains capable of interchanging function, or do they perform isoform-specific roles based upon their respective C Materials and Methods Ca2 requirements for activation? C The major focus of neuronal Ca2 -sensing studies has largely Materials focused around the synaptotagmin family, and in particular synaptotagmin I (Tokuoka & Goda 2003, Yoshihara et al. 2003, Antibodies used were obtained as follows: rabbit anti-m-calpain Sudhof 2004). Whilst numerous questions still remain regarding (Calbiochem, San Diego, CA, USA), rabbit anti-m-calpain Journal of Endocrinology (2006) 190, R1–R7 DOI: 10.1677/joe.1.06737 0022–0795/06/0190–R1 q 2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access R2 E AGANNA and others $ Calpain, synaptotagmin and secretion (Calbiochem), mouse anti-synaptotagmin (BD Biosciences, Inc.), and used to normalize insulin values obtained. Media San Diego, CA, USA), rabbit anti-calpain-10 was as described were removed from wells from each plate, and 500 ml previously (Ma et al. 2002, Marshall et al. 2005), goat anti- extraction solution containing 1.5% hydrochloric acid rabbit-HRP conjugate (Bio-Rad), and goat anti-mouse-HRP (37%), 18.5% distilled water, 80% ethanol (95%) added to conjugate (Dako, Ely, UK). E64 (Roche Diagnostics) were each well. After 24 h, at 4 8C, 500 mlof0$1 M sodium K dissolved in methanol to make a stock of 2!10 5 M and hydroxide was added to neutralize the samples, which were stored at K20 8C. Corticotrophin-releasing factor (CRF) then assayed for insulin content using standard ELISA was diluted in sterile distilled water to form a stock solution of protocols (Mercodia, Uppsala, Sweden). K 10 4 M and stored at K20 8C until diluted in culture medium. Forskolin (FSK) was dissolved in sterile dimethylsulf- K FACS analysis oxide to form a stock solution of 10 3 M and stored at 4 8C until diluted in culture medium. All other reagents were INS-1 cells were seeded at 1!106 cells/well in six-well tissue purchased from Sigma unless otherwise stated. culture plates and incubated in G200 mM E64 for 24 h. Cells were trypsinized, pelleted, and washed twice with cold PBS. From each dish, 1!106 cells were suspended in 1 ml binding Cell culture buffer, and 100 mlofthissolution(1!105 cells) transferred to INS-1 pancreatic b-cell line was cultured in RPMI-1640 separate tubes. Five microliters of annexin V-FITC and medium (Sigma) using standard protocols. Both AtT20 and propidium iodide (PI; Annexin V-FITC Apoptosis Detection GH3 cells were cultured in DMEM (high glucose; Gibco) Kit I; BD Pharmingen, San Diego, CA, USA) were added to containing 2 mM L-glutamine and 25 mM glucose, and each tube, after which they were vortexed and then incubated in supplemented with 10% (v/v) horse serum (HS; Gibco), the dark for 15 min at room temperature. To each tube, 400 ml 100 U/ml penicillin-G, and 10 mg/ml streptomycin sulfate at binding buffer was added and then annexin V and PI 37 8C, in a 5% CO2–95% air atmosphere. fluorescence measured by flow cytometry (Becton Dickinson FACScan; Cambridge, UK). Clumped cells and cell debris were excluded from analysis using the method of Jia et al. (2001). Secretion Staurosporine (1 mM) was used as a positive control for the For secretion studies, AtT20 cells were seeded at 5! presence of apoptosis (overnight stimulation). 103 cells/well and GH3 cells at 1!104 cells/well in six-well tissue culture plates 24 h prior to incubation GE64 (200 mM) Sub-cellular fractionation for 48 h. For the subsequent 24 h, AtT20 cells were incubated K GCRF (10 7 M) and GH3 cells were incubated GFSK Equal numbers of INS-1, GH3, and AtT20 cells were cultured K (10 5 M) in the continued presence or absence of E64. At the to 80% confluence in 6 cm diameter Petri dishes, then scraped end of this incubation period, media were collected and into 5 ml PBS and spun at 90 g for 5 min. Each pellet was analyzed using a two-site solid-phase IRMA for adreno- resuspended in 150 ml buffer A (10 mM MES-NaOH, pH 7$4, corticotrophic hormone (ACTH) (Euro-Diagnostica AB, 10 mM CaCl2,0$2 mM phenylmethylsulphonyl fluoride) and Malmo, Sweden) or a competitive RIA for growth hormone homogenized by passing through a 25-gauge syringe needle (GH) (Biocode-Hycel, Lie`ge, Belgium). Within- and eight times. Aliquots of homogenate were taken for total between-batch variation (%coefficient of variation) were cellular protein quantification using the BCA assay kit (Pierce). less than 10% for both assays. The homogenates were centrifuged at 500 g at 4 8C for 10 min INS-1 cells were seeded at 1!106 cells/well in six-well to produce post-nuclear supernatants (PNS). Each PNS was tissue culture plates and incubated in G200 mM E64 for 24 h. further centrifuged at 23 000 g at 4 8C for 30 min. The The cells were then washed with Krebs–Ringer solution and supernatant corresponding to cytosol fraction was transferred incubated for 3 h in Krebs–Ringer solution, G15 mM to a clean tube, while the pellet containing the membrane C glucose and 1 mM extracellular Ca2 . Supernatant was fraction was resuspended in distilled water. Equal sample collected and spun down to remove cell debris, complete volume of 2! Laemmli buffer was added to each fraction and protease cocktail was added, and insulin level measured by boiled for 5 min before storing at K20 8C. ELISA rat insulin assay kit (Mercodia, Uppsala, Sweden). Cellular protein content of lysed cells was assayed as per BCA Western blotting kit protocol (Pierce Biotechnology, Inc., Rockford, USA) and used to normalize secretion data. Membrane and cytosol fractions were normalized for cellular protein content and separated on appropriate percentage of SDS-PAGE gels (see figure legends). Protein was transferred Insulin content onto PVDF membrane using a Hoefer TE 70 semi-dry transfer INS-1 cells were seeded as described for secretion data.