thapsigargin and Pancreatic-Neoplasms

Topics: Adenocarcinoma; Biomarkers, Tumor; Case-Control Studies; Cell Line, Tumor; Drug Screening Assays, Antitumor; Gene Regulatory Networks; Humans; Integrin beta Chains; Integrin beta1; MicroRNAs; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Thapsigargin

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

Topics: 14-3-3 Proteins; Animals; Cell Death; Cell Line; Endoplasmic Reticulum Stress; Exenatide; Glucagon-Like Peptide-1 Receptor; Insulinoma; Pancreatic Neoplasms; Peptides; Phosphorylation; Protein Interaction Maps; Protein Processing, Post-Translational; Proteome; Proteomics; Rats; Thapsigargin; Venoms

The present study examined the expression of glucose‑regulated protein 78 (GRP78/Bip) in human pancreatic cancer cell lines and the effect of knockdown of GRP78 on the cleavage of poly(ADP-ribose) polymerase (PARP). Human pancreatic cancer cell lines (KP-2, MIAPaCa-2, Panc-1 and SUIT-2), constitutively expressed GRP78. We also demonstrated that ER stress induced by thapsigargin upregulated protein levels of GRP78. In the presence of thapsigargin, knockdown of GRP78 enhanced the PARP cleavage in the human pancreatic cancer cells. These results provide evidence that GRP78 is a potential therapeutic target for 'difficult-to-treat' pancreatic cancer, in which ER stress signaling in part falls into disorder.

Topics: Activating Transcription Factor 4; Activating Transcription Factor 6; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Gene Expression; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Pancreatic Neoplasms; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Phosphatase 1; Protein Processing, Post-Translational; Proteolysis; Regulatory Factor X Transcription Factors; Thapsigargin; Transcription Factors

Diabetes manifests from a loss in functional β-cell mass, which is regulated by a dynamic balance of various cellular processes, including β-cell growth, proliferation, and death as well as secretory function. The cell cycle machinery comprised of cyclins, kinases, and inhibitors regulates proliferation. However, their involvement during β-cell stress during the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two pharmacological inducers of β-cell stress, dexamethasone and thapsigargin. We hypothesized that β-cell stress upregulates p21 to activate the apoptotic pathway and suppress cell survival signaling. To this end, p21 was adenovirally overexpressed in pancreatic rat islets and 832/13 cells. As expected, p21 overexpression resulted in decreased [(3)H]thymidine incorporation. Flow cytometry analysis in p21-transduced 832/13 cells verified lower replication, as indicated by a decreased cell population in the S phase and a block in G2/M transition. The sub-G0 cell population was higher with p21 overexpression and was attributable to apoptosis, as demonstrated by increased annexin-positive stained cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the antiapoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the proapoptotic proteins Bax and Bak. Therefore, an intact intrinsic apoptotic pathway is central for p21-mediated cell death. In summary, our findings indicate that β-cell apoptosis can be triggered by p21 during stress and is thus a potential target to inhibit for protection of functional β-cell mass.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dexamethasone; Diabetes Mellitus, Type 2; Enzyme Inhibitors; Gene Expression; Glucocorticoids; Insulin-Secreting Cells; Insulinoma; Mitochondria; Oncogene Protein p21(ras); Pancreatic Neoplasms; Rats; Signal Transduction; Thapsigargin; Up-Regulation

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin naturally found in grapes and red wine, is a redox-active compound endowed with significant positive activities. In this study, the effects of resveratrol on intracellular free Ca(2+) concentration ([Ca(2+)](c)) and on cell viability in tumoral AR42J pancreatic cells are examined. The results show that resveratrol (100 μM and 1 mM) induced changes in [Ca(2+)](c), that consisted of single or short lasting spikes followed by a slow reduction toward a value close to the resting level. Lower concentrations of resveratrol (1 and 10 μM) did not show detectable effects on [Ca(2+)](c). Depletion of intracellular Ca(2+) stores by stimulation of cells with 1 nM CCK-8, 20 pM CCK-8 or 1 μM thapsigargin, blocked Ca(2+) responses evoked by resveratrol. Conversely, prior stimulation of cells with resveratrol inhibited Ca(2+) mobilization in response to a secondary application of CCK-8 or thapsigargin. In addition, resveratrol inhibited oscillations in [Ca(2+)](c) evoked by a physiological concentration of CCK-8 (20 pM). On the other hand, incubation of cells in the presence of resveratrol induced a reduction of cell viability. Finally, incubation of AR42J cells in the presence of resveratrol led to activation of c-Jun N-terminal kinase (JNK), a mitogen-activated protein kinase responsive to stress stimuli. Activation of JNK was reduced in the absence of extracellular Ca(2+). In summary, the results show that resveratrol releases Ca(2+) from intracellular stores, most probably from the endoplasmic reticulum, and reduces AR42J cells viability. Reorganization of cell's survival/death processes in the presence of resveratrol may involve Ca(2+)-mediated JNK activation.

Topics: Animals; Antioxidants; Calcium; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Pancreatic Neoplasms; Rats; Resveratrol; Sincalide; Stilbenes; Thapsigargin

We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.

Topics: Alanine; Asparagine; Cell Membrane; Curcumin; Cyclic AMP; Drug Design; Glucagon; Green Fluorescent Proteins; HEK293 Cells; Humans; Molecular Chaperones; Mutation; Pancreatic Neoplasms; Protein Transport; Pyridines; Pyrroles; Receptors, Glucagon; Serine; Thapsigargin

Pancreatic beta cell destruction in type 1 diabetes may be mediated by cytokines such as IL-1β, IFN-γ and TNF-α. Endoplasmic reticulum (ER) stress and nuclear factor-κB (NFκB) signalling are activated by cytokines, but their significance in beta cells remains unclear. Here, we investigated the role of cytokine-induced ER stress and NFκB signalling in beta cell destruction.. Isolated mouse islets and MIN6 beta cells were incubated with IL-1β, IFN-γ and TNF-α. The chemical chaperone 4-phenylbutyric acid (PBA) was used to inhibit ER stress. Protein production and gene expression were assessed by western blot and real-time RT-PCR.. We found in beta cells that inhibition of cytokine-induced ER stress with PBA unexpectedly potentiated cell death and NFκB-regulated gene expression. These responses were dependent on NFκB activation and were associated with a prolonged decrease in the inhibitor of κB-α (IκBα) protein, resulting from increased IκBα protein degradation. Cytokine-mediated NFκB-regulated gene expression was also potentiated after pre-induction of ER stress with thapsigargin, but not tunicamycin. Both PBA and thapsigargin treatments led to preferential upregulation of ER degradation genes over ER-resident chaperones as part of the adaptive unfolded protein response (UPR). In contrast, tunicamycin activated a balanced adaptive UPR in association with the maintenance of Xbp1 splicing.. These data suggest a novel mechanism by which cytokine-mediated ER stress interacts with NFκB signalling in beta cells, by regulating IκBα degradation. The cross-talk between the UPR and NFκB signalling pathways may be important in the regulation of cytokine-mediated beta cell death.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Survival; Cytokines; Diabetes Mellitus, Type 1; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Insulin-Secreting Cells; Interferon-gamma; Interleukin-1beta; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreatic Neoplasms; Phenylbutyrates; RNA, Small Interfering; Signal Transduction; Thapsigargin; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Tunicamycin; Unfolded Protein Response

Pancreatic cancer is a highly lethal disease and gemcitabine is considered to be the standard of care for the treatment of advanced pancreatic cancer. However, the outcome of the patients treated with gemcitabine is still unstatisfactory and further development of new treatments is required. We recently found that short wavelength ultra-violet (UV-C) suppresses cell proliferation with downregulation of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells, but not in normal pancreatic epithelial (PE) cells. In this study, we investigated the effect of UV-C on apoptosis in several cell lines derived from the pancreas. UV-C induced poly(ADP-ribose) polymerase (PARP) cleavage, which is a marker of cells undergoing apoptosis, in Panc1, MiaPaca2, KP3 and BxPC3 pancreatic cancer cells, but not in PE cells. We also observed similar effects in Hoechst 33258 staining, which shows DNA fragmentation. While p53, a tumor suppressor protein, plays a critical role in UV-C-induced cell damage, we did not observe the correlation between the sensitivity to UV-C and p53 status. Thapsigargin, an agent that promotes endoplasmic reticulum (ER) stress by depletion of lumenal calcium stores, as well as cis-diamineplatinum (II) dichloride, a classical anti-cancer drug that causes DNA damage, induced PARP cleavage even in PE cells. Moreover, UV-C-induced apoptosis in Panc1 and KP3 cells was associated with the release of cytochrome c, indicating that it was mediated via mitochondrial pathway. Taken together, UV-C has a potent anti-cancer effect on pancreatic cancer cells without adverse effect on normal cells and it could be useful for the treatment of human pancreatic cancers.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA Fragmentation; Enzyme Inhibitors; Epithelial Cells; Humans; Mitochondria; Pancreas; Pancreatic Neoplasms; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Signal Transduction; Thapsigargin; Tumor Suppressor Protein p53; Ultraviolet Rays

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.

Topics: Apoptosis; Cell Line, Tumor; Ceramides; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Enzymologic; Group VI Phospholipases A2; Humans; Hydrolysis; Insulinoma; Pancreatic Neoplasms; Phosphodiesterase Inhibitors; Retroviridae; RNA, Small Interfering; Spectrometry, Mass, Electrospray Ionization; Sphingomyelin Phosphodiesterase; Sphingomyelins; Thapsigargin; Transfection

The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic beta-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses.. Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic beta-cell-derived MIN6 cells, and Akita mouse-derived Ins2 (96Y/Y) insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted.. Islet beta-cells were the major site of Wfs1 expression. This expression was also found in delta-cells, but not in alpha-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 (96Y/Y) insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 (96Y/Y) cells than Ins2 (wild/wild) cells.. Wfs1 expression, which is localized to beta- and delta-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.

Topics: Animals; Anti-Bacterial Agents; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Gene Expression; Humans; Insulinoma; Ionophores; Islets of Langerhans; Membrane Proteins; Mice; Pancreatic Neoplasms; Promoter Regions, Genetic; Stimulation, Chemical; Thapsigargin; Transcriptional Activation; Tunicamycin; Up-Regulation

Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative that is a selective and potent inhibitor of the proteasome. We hypothesized that proteasome inhibition would lead to an accumulation of misfolded proteins in the cell resulting in endoplasmic reticulum (ER) stress. The ability of bortezomib to induce ER stress and the unfolded protein response was investigated in a human pancreatic cancer cell line, L3.6pl. Bortezomib increased expression of ER stress markers, CHOP and BiP, but inhibited PKR-like ER kinase and subsequent phosphorylation of eukaryotic initiation factor 2alpha (eif2alpha), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2alpha phosphorylation, ubiquitylated protein accumulation, and proteotoxicity.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Calcium; Caspase Inhibitors; Caspases; Caspases, Initiator; Cycloheximide; eIF-2 Kinase; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Molecular Chaperones; Oxidative Stress; Pancreas; Pancreatic Neoplasms; Phosphorylation; Protein Biosynthesis; Protein Processing, Post-Translational; Pyrazines; RNA, Small Interfering; Thapsigargin; Transcription Factor CHOP; Ubiquitin

Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH(2)-terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca(2+) levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin.

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Calcium; Carcinogens; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cisplatin; Cytochromes c; Drug Interactions; Endoplasmic Reticulum; Enzyme Activation; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Pyrazines; RNA, Small Interfering; Thapsigargin; Tumor Stem Cell Assay; Tunicamycin

Apoptosis is probably the main form of beta-cell death in both type 1 diabetes mellitus (T1DM) and T2DM. In T1DM, cytokines contribute to beta-cell destruction through nuclear factor-kappaB (NF-kappaB) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are beta-cell toxic also via NF-kappaB activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced beta-cell apoptosis and determine whether TNFalpha, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-kappaB activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified beta-cells by oleate, palmitate, and/or cytokines (IL-1beta, interferon-gamma, TNFalpha). Palmitate and IL-1beta induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNFalpha did not potentiate FFA toxicity in primary beta-cells. The NF-kappaB-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1beta but not by FFAs. Cytokines activated NF-kappaB in INS-1E and beta-cells, but FFAs did not. Moreover, FFAs did not enhance NF-kappaB activation by TNFalpha. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA- and cytokine-induced beta-cell death but the mechanisms involved are different. Whereas cytokines induce NF-kappaB activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-kappaB- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of beta-cell death in T1DM and T2DM.

Topics: Animals; Apoptosis; Carcinogens; Cell Line, Tumor; Cytokines; Drug Synergism; Endoplasmic Reticulum; Fatty Acids, Nonesterified; Flow Cytometry; Insulinoma; Interferon-gamma; Interleukin-1; Islets of Langerhans; Male; NF-kappa B; Oleic Acid; Palmitates; Pancreatic Neoplasms; Rats; Rats, Wistar; Thapsigargin; Tumor Necrosis Factor-alpha

The mechanisms by which bradykinin (BK) increases glucagon release were investigated. BK (0.1-10 microM) increased [Ca(2+)](i) and glucagon release in clonal alpha-cells In-R1-G9. BK-induced glucagon release was lower in the absence than in the presence of extracellular Ca(2+), but it still increased glucagon release while [Ca(2+)](i) was stringently deprived. Depletion of intracellular Ca(2+) store with thapsigargin abolished both the BK-induced Ca(2+) peak and sustained plateau. Microinjection of heparin abolished BK-induced Ca(2+) release. Pertussis toxin (PTX) did not block BK-induced [Ca(2+)](i) increase or glucagon release. U-73122 (8 microM), a phospholipase C (PLC) inhibitor, abolished BK-induced increases in [Ca(2+)](i), but only reduced BK-induced glucagon release by 40%. A phospholipase D (PLD) inhibitor zLYCK reduced BK-induced glucagon release by 60%. The combination of U-73122 and zLYCK abolished BK-induced glucagon release. Both SK&F 96365, a receptor-operated Ca(2+) channel (ROC) blocker and nimodipine, an L-type Ca(2+) channel blocker, reduced BK-induced [Ca(2+)](i) increase and glucagon release. These findings suggest that BK increase glucagon release through a PTX-insensitive G protein and both Ca(2+)-dependent and -independent pathways. The Ca(2+)-dependent pathway is attributable to PLC activation. PLC catalyzes IP(3) formation, inducing Ca(2+) release from the endoplasmic reticulum, which, in turn, triggers Ca(2+) influx via both ROCs and L-type channels. PLD activation may be involved in Ca(2+)-dependent and/or -independent pathway.

Topics: Animals; Bradykinin; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Chelating Agents; Clone Cells; Cricetinae; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Estrenes; Glucagon; Glucagonoma; GTP-Binding Proteins; Heparin; Imidazoles; Inositol 1,4,5-Trisphosphate Receptors; Mesocricetus; Microinjections; Pancreatic Neoplasms; Pertussis Toxin; Phospholipase D; Pyrrolidinones; Receptor, Bradykinin B2; Receptors, Bradykinin; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases

Sphingosine (10 microM) induced mobilization of intracellular Ca2+ stores in the pancreatic duct adenocarcinoma cell line CFPAC-1. The effect was specific for sphingosine, since the sphingosine analog C2-ceramide had no effect. Sphingosine did not cause Ca2+ entry from extracellular medium, as also shown by following Mn2+ quenching of Fura-2 fluorescence. Furthermore, sphingosine, similarly to the mitochondrial inhibitors rotenone and oligomycin, strongly inhibited the rate of Mn2+ entry triggered by both thapsigargin- and agonist-induced depletion of intracellular stores. The uptake of rhodamine 123, a lipophilic cation which estimates mitochondrial energy level, was reduced by sphingosine to an extent similar to that observed in the presence of mitochondrial inhibitors. It is suggested that impairment of mitochondrial function might be responsible for inhibition of capacitative Ca2+ entry caused by sphingosine.

Topics: Adenocarcinoma; Biological Transport; Calcium; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Manganese; Membrane Potentials; Mitochondria; Oligomycins; Pancreatic Ducts; Pancreatic Neoplasms; Propidium; Rhodamine 123; Rhodamines; Rotenone; Sphingosine; Thapsigargin; Tumor Cells, Cultured; Uncoupling Agents; Uridine Diphosphate

The importance of Ca2+ in the regulation of secretion is well-known. However, recent experiments suggest that a rise in intracellular Ca2+ (Ca2+i) does not necessarily trigger secretion in pancreatic acinar cells. In AR4-2J cells the role of the Ca2+ mobilization induced by cholecystokinin/gastrin (CCK/G), which is dependent of the intracellular calcium store and the calcium influx operating through voltage-dependent calcium channels, has never been directly demonstrated. Therefore, we attempted to determine whether Ca2+i and/or extracellular Ca2+ (Ca2+e) mobilized by CCK/G plays a role in the amylase secretion of these cells. We measured the [Ca2+]i by spectrofluorometry and amylase release in different experimental procedures modulating the two pools of calcium. Ionomycin increased both [Ca2+]i and amylase related. In Ca(2+)-depleted cells or in the presence of thapsigargin the transient rise in Ca2+i and the amylase secretion induced by CCK/G were suppressed. A 50 mM K+ solution or Bay K 8644, which activated the Ca2+ influx, did not induce any variation of the basal amylase secretion. Moreover, amylase secretion induced by CCK/G did not change significantly in Ca(2+)-free medium or in the presence of nifedipine. These results indicate that in AR4-2J cells, amylase secretion is dependent of the large increase in Ca2+i induced by CCK/G and independent of the Ca2+ influx through voltage-dependent calcium channels dihydropyridine sensitive.

Topics: Amylases; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cholecystokinin; Enzyme Inhibitors; Gastrins; Intracellular Fluid; Ion Channel Gating; Ionomycin; Ionophores; Nifedipine; Pancreatic Neoplasms; Rats; Spectrometry, Fluorescence; Terpenes; Thapsigargin; Tumor Cells, Cultured

To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar-type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP3) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 microM did not affect calcium sequestration by nonmitochondrial, InsP3-sensitive stores at all; 50 microM carbonylcyanide m-chlorophenylhydrazone, 50 microM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against signficant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3-sensitive stores in RINm5F cells. The proton-potassium-exchanging ionophore nigericin (20-100 microM), however, inhibited calcium sequestration by nonmitochondrial and InsP3-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3-sensitive calcium-storing organelles.

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Cell Membrane; Cell Membrane Permeability; Endoplasmic Reticulum; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Insulinoma; Ionophores; Islets of Langerhans; Liver; Macrolides; Nigericin; Pancreatic Neoplasms; Potassium; Proton Pump Inhibitors; Proton Pumps; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured; Vacuoles; Vanadates

This study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells.

Topics: Alkaloids; Animals; Biological Transport; Calcium; Cytosol; Insulin; Insulin Secretion; Insulinoma; Manganese; Pancreatic Neoplasms; Protein Kinase C; Rats; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.

Topics: Animals; Calcium; Cell Nucleus; Cytoplasm; Egtazic Acid; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Kinetics; Methacholine Chloride; Microscopy, Electron; Microscopy, Fluorescence; Mitochondria; Nuclear Envelope; Pancreatic Neoplasms; Rats; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured