R1 RAPID COMMUNICATION

C Involvement of and synaptotagmin Ca2 sensors in 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 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 . 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 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 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 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 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. Cells unit (Amersham Pharmacia), blocked with 5% milk powder, were then incubated in G200 mM E64 for 24 h. Total protein and probed with antibodies as follows: m- and m-calpain content was measured using BCA kit (Pierce Biotechnology antibody (1:5000 incubated overnight at 4 8C), calpain-10

Journal of Endocrinology (2006) 190, R1–R7 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access Calpain, synaptotagmin and secretion $ E AGANNA and others R3 antibody (1:3000), and synaptotagmin antibody (1:1000). (a) INS-1 cells

Immunoreactivity was detected using HRP-conjugated goat P< 0·005 anti-rabbit (1:3000) or goat anti-mouse (1:1000) and visualized 500 using ECL plus chemiluminescent detection and exposed to ECL Hyperfilm (Amersham Pharmacia). 400 300

Statistical analysis 200 n.s. (% of control) All graphical data were prepared using GraphPad Prism 3.0 Insulin secretion 100 (GraphPad, San Diego, CA, USA) and analyzed using pre- 0 programmed analysis equations within Prism. Secretion data E64: − + − + were presented as normalized data pooled from multiple Basal Stim experiments. Where appropriate, an ANOVA was performed on data followed by Tukey’s multiple comparison test, accepting P!0$05 as significant. (b) GH3 cells 200 P< 0·001

n.s. Results 100 Sensitivity of hormone secretion to cysteine protease inhibition GH secretion We and other researchers have previously shown that (% of control) pharmacological intervention with calpain inhibitors 0 abolishes secretagog-stimulated insulin secretion from pan- E64: − + − + creatic b-cells (Ort et al. 2001, Zhou et al. 2003, Marshall et al. Basal Stim 2005, Parnaud et al. 2005). However, an understanding of the role of the calpain family in hormone secretion from other endocrine cells is less clear. In order to determine whether (c) AtT20 cells hormone secretion per se is either calpain-mediated or calpain 200 independent, we pre-incubated GH3 and AtT20 pituitary n.s. cells with E64 for 48 h, an inhibitor and incubation time that n.s. have been previously shown to be highly effective in 100 inhibiting regulated insulin secretion from both pancreatic islets (Zhou et al. 2003) and INS-1 b-cells (Marshall et al. (% of control) ACTH secretion 2005). E64 effects on secretion were determined relative to those observed in INS-1 cells, and as can be seen in Fig. 1, 0 both GH3 and AtT20 cells also secrete hormone via a basal E64: − + − + pathway that does not require secretagog to induce secretion. Basal Stim This is completely insensitive to pre-incubation with E64 Figure 1 Sensitivity of hormone secretion to calpain inhibition. (Fig. 1b and c; left panels). In marked contrast, stimulated (a) INS-1 cells were incubated for 3 h in Krebs–Ringer solution alone (Basal) or Krebs–Ringer solution containing 15 mM glucose secretion of GH from GH3 cells was completely abolished (Stim) in the presence or absence of 24 h pre-incubation with following pre-incubation with E64 (Fig. 1b; right panels). 200 mM E64. (b) GH3 cells were incubated for 24 h in either media However, this was not the case with ACTH secretion from alone (Basal) or media containing 100 mM FSK (Stim) in the AtT20 cells, where stimulated secretion was completely E64 presence or absence of 48 h pre-incubation with 200 mM E64. insensitive (Fig. 1c; right panels). Therefore, whilst GH3 cells (c) AtT20 cells were incubated for 24 h in either media alone (Basal) or media containing 1 mM CRF (Stim) in the presence or absence of display similar sensitivity to calpain inhibition as INS-1 b-cells 48 h pre-incubation with 200 mM E64. For each treatment, hormone (Fig. 1a), this is not the case in AtT20 cells. Our results suggest secretion was normalized and expressed as percentage of control that stimulated ACTH secretion from AtT20 cells occurs via a (nZ6). mechanism that is independent of the calpain family, whilst stimulated secretion from GH3 cells is calpain dependent. As INS-1 cells displayed the greatest secretagog-stimulated increase in secretion relative to basal, we used these cells for this E64 has no effect on cellular hormone content or apoptotic status series of experiments. As stimulated secretion from INS-1 cells is In order to determine whether E64 action was specific to the also extremely sensitive to E64 inhibition (Fig. 1), they should be regulated secretory pathway, we analyzed cellular hormone similarly sensitive to any E64-mediated changes in insulin content and apoptotic status of cells treated with or without E64. biosynthesis, granule storage, or apoptosis. However, as can be www.endocrinology-journals.org Journal of Endocrinology (2006) 190, R1–R7

Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access R4 E AGANNA and others $ Calpain, synaptotagmin and secretion

seen in Fig. 2a, there is no effect of E64 upon insulin content in m-Calpain expression INS-1 cells, despite treating cells with the same concentration of We sought to address whether the differences in secretory E64 and for the same period of time as in the secretion pathway sensitivity of different endocrine cells to E64 might experiments. Therefore, E64 inhibition of secretion is not a consequence of any action upon either the insulin biosynthetic arise from differences in expression of individual calpains m machinery or upon insulin storage. between the different cell types. -Calpain has previously In order to determine whether E64 might instead inhibit been shown to facilitate insulin secretion through its action on insulin secretion by inducing apoptosis, we again incubated the cytoskeleton (Ort et al. 2001). Therefore, in order to cells with or without E64, then measured their apoptotic address whether m-calpain-mediated changes in cytoskeletal status using FACS analysis of cells stained with propidium dynamics might reflect the differential sensitivity of hormone iodide and annexin V. Incubation with staurosporine positive secretion to E64, we examined m-calpain expression and control (Fig. 2b), resulted in a 79$2G1$5% reduction in cell localization. As can be seen in Fig. 3, we observed no major viability. By contrast, we observed no reduction whatsoever differences between the three cell lines with regard to in cell viability of cells incubated for 24 h with E64. m-calpain expression. Three times more membranes were Therefore, the inhibitory action of E64 upon insulin loaded onto gels relative to cytosol, but even after taking this secretion is not a consequence of any reduction in cell into account there was still a surprisingly high affinity of viability arising from a pro-apoptotic action of this cysteine m-calpain for microsomal membranes, densitometric analysis protease inhibitor. revealing that there was between 57 and 60% present on microsomal membranes in all the three cell types. However, as (a) 2500 both the expression level and the degree of partitioning between membrane and cytosol were not significantly different among the three cell types, the differences in E64 2000 sensitivity observed in Fig. 1 were not a reflection of any differences in either m-calpain expression or localization. 1500

m-Calpain expression 1000 g cellular protein) µ Insulin content As can be seen in Fig. 4, there is an abundant expression of (pg/ 500 m-calpain in INS-1 and GH3 cells. Moreover, densitometric analysis revealed that w8% is associated with INS-1 w 0 microsomal membranes and 15% is found associated with GH3 microsomal membranes. However (despite equal E64: − + loading of protein between all cell types), there is a noticeably low-relative abundance of m-calpain in AtT20 cells, with (b) 100 little detected on either microsomal membranes or in cytosol. This raises the possibility that m-calpain might be a key protein-mediating hormone secretion from INS-1 and GH3 80 cells, but not AtT20 cells. If so, its presence in INS-1 and GH3 cells could reflect the fact that these cells are susceptible 60 to inhibition by E64, whereas AtT20 cells which possess relatively little m-calpain are not. 40 % Viable cells µ-Calpain 20

0 Positive E64: − + control GH3 GH3 INS-1 AtT20 INS-1 AtT20 Figure 2 Calpain inhibition has no effect on insulin content or Membrane Cytosol apoptotic status. (a) INS-1 cells were incubated in the presence or absence of 24 h pre-incubation with 200 mM E64 and total insulin Figure 3 Immunoblot analysis of m-calpain. Subcellular membrane cellular content determined. (b) INS-1 cells were incubated for 24 h and cytosol fractions from INS-1, GH3, and AtT20 cells were in the presence or absence of 200 mM E64. Cells were stained with normalized for protein level, then subjected to 8% SDS-PAGE, annexin V-FITC and propidium iodide and apoptosis determined electrotransferred to PVDF membrane, and western blotted with using FACS analysis. Staurosporine (1 mM) was used as a positive antibody raised specifically against m-calpain. This figure is the control for the presence of apoptosis. representative of three independent experiments.

Journal of Endocrinology (2006) 190, R1–R7 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access Calpain, synaptotagmin and secretion $ E AGANNA and others R5

m-Calpain a similar role in the secretion of GH from GH3 cells. If so, then the absence of appreciable levels of this isoform in AtT20 cells would render these cells unable to utilize this protein to mediate stimulus–secretion coupling.

GH3 GH3 INS-1 AtT20 INS-1 AtT20 Synaptotagmin expression Membrane Cytosol There are currently known to be at least 15 isoforms of Figure 4 Immunoblot analysis of m-calpain. Subcellular membrane synaptotagmin in vertebrates (Sudhof 2002), and previous and cytosol fractions from INS-1, GH3, and AtT20 cells were studies have carefully documented expression levels of these normalized for protein level, then subjected to 8% SDS-PAGE, individual synaptotagmin isoforms in INS-1, GH3, and AtT20 electrotransferred to PVDF membrane, and western blotted with antibody raised specifically against m-calpain. This figure is the cells respectively. Therefore, rather than blotting for each representative of four independent experiments. synaptotagmin isoform independently we instead chose to use a polyclonal anti-synaptotagmin antibody raised against peptide 2C Calpain-10 expression sequence that incorporates the highly conserved C2ACa - binding domain. In this way,we sought to address whether there Calpain-10-mediated insulin secretion from INS-1 pancreatic were substantial differences in global synaptotagmin expression b-cells has previously been documented (Marshall et al. 2005) between the different endocrine cells. As can be seen in Fig. 6, and this is clearly an attractive candidate to similarly regulate INS-1 cells have very little expression of synaptotagmin relative hormone secretion from other endocrine cells. In order to to GH3 and AtT20 cells, where the chemiluminescent signal is address this possibility, we immunoblotted membrane and extremely strong. In particular, AtT20 cells display intense bands cytosol fractions from INS-1, GH3, and AtT20 cells with a which, based upon both the extent of C2A domain homology highly specific and well-characterized anti-calpain-10 and the migrating protein molecular weights, are assigned as antibody (Ma et al. 2001, Marshall et al. 2005). As can be synaptotagmins I, III, and V. GH3 cells also display a similar seen in Fig. 5, there was little difference in the total amount of banding pattern, albeit at a lesser intensity.While sample loading calpain-10 expression between the three cell types. However, was normalized to protein, in INS-1 there is only one the membrane association pattern is markedly different moderately expressed band that is readily visible. As previously between cell types for some of the isoforms. In particular, studies have shown that synaptotagmin III is the major there is a pronounced lack of the 54 kDa isoform in AtT20 synaptotagmin implicated in insulin secretion from b-cells w cells, and there is also the presence of a 60 kDa band on (Mizuta et al. 1994, 1997, Brown et al. 2000), we suggest that this GH3 cell membranes. While there is at present no proposed is the isoform we observe in INS-1 cells. function for the novel w60 kDa isoform, the mere fact that it is absent in both INS-1 and AtT20 cells seemingly rules this isoform out as a candidate mediator of differential calpain Discussion inhibitor sensitivity. However, given that the 54 kDa isoform is the very isoform proposed to trigger exocytosis in It has previously been reported that m-calpain mediates pancreatic b-cells (Marshall et al. 2005), it probably performs secretion through proteolysis of ICA512, a secretory granule

Calpain-10 KDa

75

50

GH3 GH3 INS-1 AtT20 INS-1 AtT20

Membrane Cytosol Figure 5 Immunoblot analysis of calpain-10. Normalized subcellular membrane and cytosolic fractions from INS-1, GH3, and AtT20 cells were separated on 10% SDS-PAGE and electrotransferred to PVDF. Specific isoforms of calpain-10 were detected by western blotting with anti-calpain-10 antibody. This figure is the representative of three independent experiments. www.endocrinology-journals.org Journal of Endocrinology (2006) 190, R1–R7

Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access R6 E AGANNA and others $ Calpain, synaptotagmin and secretion

KDa Synaptotagmin Syt 75

I / III

50

V

35

MMMCC C INS-1 GH3 AtT20 Figure 6 Immunoblot analysis of synaptotagmin. Western blot analysis of membrane (M) and cytosol (C) fractions from INS-1, GH3, and AtT20 cells using anti-synaptotagmin antibody. The fractions were normalized for protein level before being subjected to 8% SDS-PAGE and transferred to PVDF. Migration positions of synaptotagmin (Syt) isoforms are indicated on the right. This figure is the representative of three independent experiments.

protein linked to the cytoskeletal remodeling, which is protease inhibition. Future studies utilizing antisense oligo- associated with second-phase insulin secretion (Ort et al. nucleotide or siRNA technology might enable us to determine 2001). However, as the endocrine cells we studied in this the precise roles of each of the calpains in stimulated secretion. report all expressed similar amounts of m-calpain, this is However, given that the calpain-10 gene is subject to differential unlikely to account for the differences we observed in splicing, which generates as many as eight isoforms in humans sensitivity to calpain inhibition. The involvement of (Horikawa et al. 2000), the situation is complex. m-calpain in hormone secretion is not as well documented, Neurotransmission and endocrine secretion both utilize although specific knockdown of m-calpain mRNA has been soluble N-ethyl maleimide sensitive fusion protein attachment reported to result in a significant reduction of regulated receptor molecules, or their associated homologues, during secretion from alveolar epithelial cells (Li et al. 1998). The exocytotic membrane fusion. Furthermore, both processes C report of secretagog-induced proteolysis of spectrin in share a similar Ca2 requirement (Burgoyne & Clague 2003, alveolar epithelial cells (Zimmerman et al. 1992)offersa Stojilkovic 2005), although the kinetics of neurotransmission clue to possible action, as spectrin has been shown to be are much more rapid than those of endocrine secretion. This C involved in the cytoskeletal rearrangement that accompanies gives rise to the possibility that different classes of Ca2 sensor stimulated secretion (Mercier et al. 1989). Another possible might therefore trigger the respective exocytotic fusion events, function of m-calpain might be in secretory granule their differing molecular modes of action being reflected in the maturation, as in vitro proteolysis of and adaptins differing kinetics. Our results in pituitary cells are in agreement has been observed in response to addition of exogenous with previous studies (Jacobsson & Meister 1996, Xi et al. m-calpain to pituitary cell lysates (Ohkawa et al. 2000). Were 1999), which showed that synaptotagmins I and III were the either of these mechanisms the source of the observed major species of synaptotagmin in the pituitary. The function differential E64 sensitivity in this report then, as with of synaptotagmins in pancreatic b-cell secretion however is far m-calpain, this would represent a role for m-calpain in the less clear, although a role for synaptotagmins Vand VII–IX has slower but more sustained phase of secretion that follows the been suggested, based upon either molecular- or antibody- initial rapid burst of exocytotic granule fusion. based techniques (Gut et al. 2001, Iezzi et al. 2004). However, C Calpain-10 has recently emerged as a Ca2 sensor in in these studies, both peptide competition and RNA stimulus–secretion coupling, the 54 kDa isoform being shown interference yielded only partial inhibition of secretion. As to regulate first-phase insulin secretion (Marshall et al. 2005). such, it is highly likely that these isoforms of synaptotagmin are C Interestingly, we saw an abundance of this particular isoform in not the sole Ca2 sensors in stimulus–secretion coupling in INS-1 and GH3 cells, but severely diminished expression in the these cells. Instead, perhaps they work either in combination cysteine protease-insensitive AtT20 cells. These findings are with other synaptotagmins, or alternatively in concert with C consistent with this isoform performing a similar function in one or more additional Ca2 sensor, such as calpain-10. GH3 cells to that previously reported in INS-1 cells. As such, the Additionally, a number of publications have shown that AtT20 C low abundance of this isoform in AtT20 cells would also explain and GH3 pituitary cells both utilize the Ca2 sensor why secretion from these cells was insensitive to cysteine synaptotagmin to secrete hormone in response to secretory

Journal of Endocrinology (2006) 190, R1–R7 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access Calpain, synaptotagmin and secretion $ E AGANNA and others R7 stimulation by CRF or FSK respectively. Therefore, it is Jacobsson G & Meister B 1996 Molecular components of the exocytotic C extremely likely that both cAMP and Ca2 have active roles to machinery in the rat pituitary gland. Endocrinology 137 5344–5356. Jia L,Patwari Y,Srinivasula SM, Newland AC, Fernandes-Alnemri T,Alnemri ES play during the secretory process in these cells, and there is & Kelsey SM 2001 Bax translocation is crucial for the sensitivity of leukaemic likely to be cross-talk between these signaling pathways. cells to etoposide-induced apoptosis. Oncogene 20 4817–4826. In conclusion, stimulated secretion from INS-1 and GH3 Li HL, Feinstein SI, Liu L & Zimmerman UJP 1998 An antisense cells is calpain mediated, in contrast to stimulated secretion from oligodeoxyribonucleotide to m-calpain mRNA inhibits secretion from AtT20 cells which is calpain independent. Calpain-10, in alveolar epithelial type II cells. Cellular Signalling 10 137–142. Ma H, Fukiage C, Kim YH, Duncan MK, Reed NA, Shih M, Azuma M & particular, might determine sensitivity of secretory cells to E64 Shearer TR 2001 Characterization and expression of calpain-10. Journal of sensitivity, as like synaptotagmin (Schiavo et al. 1997, Gerona Biological Chemistry 276 28525–28531. et al. 2000, Earles et al. 2001, Zhang et al. 2002) it also has been Marshall C, Hitman GA, Partridge CJ, Clark A, Ma H, Shearer TR & reported to interact with synaptosomal-associated protein Turner MD 2005 Evidence that an isoform of calpain-10 is a regulator of 2C exocytosis in pancreatic b-cells. Molecular Endocrinology 19 213–224. (SNAP)-25 during Ca -triggered exocytosis (Marshall et al. Mercier F, Reggio H, Devilliers G, Bataille D & Mangeat P 1989 Membrane- 2005). Furthermore, the proposed conformational change in cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin SNARE protein complex caused by calpain-10 action (Marshall upon stimulation of gastric acid secretion. Journal of Cell Biology 108 441–453. et al. 2005) resonateswith the direct action of synaptotagmin that Mizuta M, Inagaki N, Nemoto Y, Matsukura S, Takahashi M & Seino S 1994 has been observed on neuronal t-SNAREs (Bhalla et al. 2006). Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells. Journal of Cell Biology 269 11675–11678. However, there is no direct evidence at this time to conclusively Mizuta M, Kurose T, Miki T, Shoji-Kasai Y, Takahashi M, Seino S & prove or disprove whether calpain-10 and synaptotagmin Matsukura S 1997 Localization and functional role of synaptotagmin III in perform discreet or complementary actions, but it is nonetheless insulin secretory vesicles in pancreatic beta-cells. Diabetes 46 2002–2006. tempting to speculate that the relative abundance of both calpain Ohkawa K, Takada K, Asakura T, Hashizume Y, Okawa Y, Tashiro K, Ueda J, and synaptotagmin isoforms decrees precisely how cells perform Itoh Y & Hibi N 2000 Calpain inhibitor inhibits secretory granule maturation and secretion of GH. Neuroreport 11 4007–4011. stimulus–secretion coupling. Ort T,Voronov S, Guo J, Zawalich K, Froehner SC, Zawalich W & Solimena M C 2001 Dephosphorylation of b2-syntrophin and Ca2 /m-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion. EMBO Journal 20 Acknowledgements 4013–4023. Parnaud G, Hammar E, Rouiller DG & Bosco D 2005 Inhibition of calpain M D T and G A H would like to thank Diabetes UK for blocks pancreatic beta-cell spreading and insulin secretion. American project grant support (BDA:RD03/0002689). The authors Journal of Physiology. Endocrinology and Metabolism 289 E313–E321. Sabatini BL & Regehr WG 1999 Timing of synaptic transmission. Annual declare that there is no conflict of interest that would Review of Physiology 61 521–542. prejudice the impartiality of this scientific work. Schiavo G, Stenbeck G, Rothman JE & Sollner TH 1997 Binding of the SV v-SNARE, synaptotagmin, to the t-SNARE, SNAP-25 can explain docked vesicles at neurotoxin-treated . PNAS 94 997–1001. C References Stojilkovic SS 2005 Ca2 -regulated exocytosis and SNARE function. Trends in Endocrinology and Metabolism 16 81–83. C Bhalla A, Chicka MC, Tucker WC & Chapman ER 2006 Ca2 - Sudhof TC 2002 Synaptotagmins: why so many? Journal of Biological Chemistry synaptotagmin directly regulates t-SNARE function during reconstituted 277 7629–7632. membrane fusion. Nature Structural and Molecular Biology 13 323–329. Sudhof TC 2004 The cycle. Annual Review of Neuroscience 27 Brown H, Meister B, Deeney J, Corkey BE, Yang SN, Larsson O, Rhodes CJ, 509–547. 2C Seino S, Berggren PO & Fried G 2000 Synaptotagmin III isoform is Tokuoka H & Goda Y 2003 Synaptotagmin in Ca -dependent exocytosis: compartmentalized in pancreatic beta-cells and has a functional role in dynamic action in a flash. Neuron 38 521–524. exocytosis. Diabetes 49 383–391. Xi D, Chin H & Gainer H 1999 Analysis of synaptotagmin I–IV messenger Burgoyne RD & Clague MJ 2003 Calcium and in membrane RNA expression and developmental regulation in the rat hypothalamus and fusion. Biochimica et Biophysica Acta 1641 137–143. pituitary. Neuroscience 88 425–435. Earles CA, Bai J, Wang P & Chapman ER 2001 The tandem C2 domains of Yoshihara M, Adolfsen B & Littleton JT 2003 Is synaptotagmin the calcium C synaptotagmin contain redundant Ca2 binding sites that cooperate to engage sensor? Current Opinion in Neurobiology 13 315–323. t-SNAREs and trigger exocytosis. Journal of Cell Biology 154 1117–1123. Zhang X, Kim-Miller MJ, Fukuda M, Kowalchyk JA & Martin TF 2002 2C Gerona RR, Larsen EC, Kowalchyk JA & Martin TF 2000 The C terminus of Ca -dependent synaptotagmin binding to SNAP-25 is essential for C 2C SNAP25 is essential for Ca2 -dependent binding of synaptotagmin to Ca -triggered exocytosis. Neuron 34 599–611. SNARE complexes. Journal of Biological Chemistry 275 15458–15465. Zhou Y-P,Sreenan S, Pan C-Y,Currie KPM, Bindokas VP,Horikawa Y,Lee J-P, Gut A, Kiraly CE, Fukuda M, Mikoshiba K, Wollheim CB & Lang J 2001 Ostrega DM, AhmedN, Baldwin AC et al.2003 A 48-h exposure ofpancreatic Expression and localisation of synaptotagmin isoforms in endocrine beta-cells: islets to calpain inhibitors impairs mitochondrial fuel metabolism and the their function in insulin exocytosis. Journal of Cell Science 114 1709–1716. exocytosis of insulin. Metabolism 52 528–534. Iezzi M, Kouri G, Fukuda M & Wollheim CB 2004 Synaptotagmin Vand IX Zimmerman UJ, Speicher DW & Fisher AB 1992 Secretagogue-induced C isoforms control Ca2 -dependent insulin exocytosis. Journal of Cell Science proteolysis of lung spectrin in alveolar epithelial type II cells. Biochimica et 117 3119–3127. Biophysica Acta 1137 127–134. Henkel AW & Almers W 1996 Fast steps in exocytosis and endocytosis studied by capacitance measurements in endocrine cells. Current Opinion in Received 4 January 2006 Neurobiology 6 350–357. Received in final form 21 June 2006 Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE et al. 2000 Genetic variation in the Accepted 29 June 2006 gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nature Made available online as an Accepted Preprint Genetics 26 163–175. 24 July 2006 www.endocrinology-journals.org Journal of Endocrinology (2006) 190, R1–R7

Downloaded from Bioscientifica.com at 10/01/2021 05:24:29PM via free access