RESUMO
OBJECTIVE: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. METHODS: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. RESULTS: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 µM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. CONCLUSION: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.
Assuntos
Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Metilaminas/farmacologia , Propionatos/farmacologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.
Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células , Compostos de Silício/química , Animais , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Adesões Focais , Fator de Crescimento Neural/farmacologia , Neurônios , Células PC12 , Polilisina/química , Ratos , Propriedades de SuperfícieRESUMO
The sweet taste receptor is expressed in the taste bud and is activated by numerous sweet molecules with diverse chemical structures. It is, however, not known whether these sweet agonists induce a similar cellular response in target cells. Using MIN6 cells, a pancreatic ß-cell line expressing endogenous sweet taste receptor, we addressed this question by monitoring changes in cytoplasmic Ca2+ ([Ca2+]i) and cAMP ([cAMP]i) induced by four sweet taste receptor agonists. Glycyrrhizin evoked sustained elevation of [Ca2+]i but [cAMP]i was not affected. Conversely, an artificial sweetener saccharin induced sustained elevation of [cAMP]i but did not increase [Ca2+]i. In contrast, sucralose and acesulfame K induced rapid and sustained increases in both [Ca2+]i and [cAMP]i. Although the latter two sweeteners increased [Ca2+]i and [cAMP]i, their actions were not identical: [Ca2+]i response to sucralose but not acesulfame K was inhibited by gurmarin, an antagonist of the sweet taste receptor which blocks the gustducin-dependent pathway. In addition, [Ca2+]i response to acesulfame K but not to sucralose was resistant to a Gq inhibitor. These results indicate that four types of sweeteners activate the sweet taste receptor differently and generate distinct patterns of intracellular signals. The sweet taste receptor has amazing multimodal functions producing multiple patterns of intracellular signals.
Assuntos
Transdução de Sinais/efeitos dos fármacos , Edulcorantes/farmacologia , Papilas Gustativas/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Ácido Glicirrízico/farmacologia , Células Secretoras de Insulina/metabolismo , Camundongos , Proteínas de Plantas/farmacologia , Sacarina/farmacologia , Sacarose/análogos & derivados , Paladar/efeitos dos fármacos , Papilas Gustativas/efeitos dos fármacos , Tiazinas/farmacologiaRESUMO
Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic ß-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.
Assuntos
Células Secretoras de Insulina/citologia , Insulina/metabolismo , Animais , Bioquímica/métodos , Linhagem Celular , Células Endócrinas/citologia , Corantes Fluorescentes/farmacologia , Genes Reporter , Glucose/metabolismo , Insulinoma/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Plasmídeos/metabolismo , Vesículas Secretórias/metabolismoRESUMO
Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC gamma, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells.
Assuntos
Microscopia de Fluorescência/métodos , Microscopia de Interferência/métodos , Transdução de Sinais/fisiologia , Animais , Células COS , Sinalização do Cálcio/fisiologia , Chlorocebus aethiops , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Diacilglicerol Quinase/fisiologia , Corantes Fluorescentes , Microscopia de Fluorescência/instrumentação , Microscopia de Interferência/instrumentação , Proteína Quinase C/fisiologiaRESUMO
BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+) ([Ca(2+)](c)) and cAMP ([cAMP](c)) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+)](c). The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+)](c) response. The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor. Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Paladar , Animais , Sequência de Bases , Linhagem Celular , Citoplasma/metabolismo , Primers do DNA , Ativação Enzimática , Secreção de Insulina , Camundongos , Proteína Quinase C/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sacarose/análogos & derivados , Sacarose/farmacologiaRESUMO
BACKGROUND: In endotoxemia, plasminogen activator inhibitor-1 (PAI-1) increases and develops clinical symptoms by suppressing fibrinolysis. We analyzed therapeutic advantage of nafamostat, a broad-range protease inhibitor, on fibrinolysis in an animal sepsis model. METHODS: Male Wister rats infused with lipopolysaccharide (LPS) (50 mg/kg) alone or together with nafamostat (0.1 mg/kg/hr) for 4 hours were analyzed. RESULTS: Plasma PAI-1 (4.2: 4.0-5.0 ng/mL, median and interquartile range) increased after LPS infusion (3700: 3400-4000), which was attenuated by nafamostat (2300: 2100-2600, p < 0.05). Fibrin(ogen) degradation products after LPS injection (173: 152-182 microg/mL) were further elevated by nafamostat (205: 205-228, p < 0.05), Nafamostat attenuated polymorphonuclear neutrophils infiltration in the liver, and tended to suppress plasma tumor necrosis factor-alpha levels. Nafamostat did not affect thrombin generation, platelet count, markers of liver and kidney function, and overall mortality. CONCLUSIONS: Nafamostat appeared to improve impaired fibrinolysis by suppressing the increase of PAI-1 in plasma, though it did not largely improve clinical parameters.
Assuntos
Fibrinogênio/metabolismo , Fibrinólise/efeitos dos fármacos , Guanidinas/farmacologia , Fígado/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidores de Proteases/farmacologia , Sepse/metabolismo , Inibidores de Serina Proteinase/metabolismo , Animais , Benzamidinas , Interações Medicamentosas , Interleucina-10/sangue , Lipopolissacarídeos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Both urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) are associated with a poor prognosis in cancer patients. We demonstrate that PAI-1 inhibits human fibrosarcoma cell (HT-1080) adhesion to vitronectin (Vn) via alpha (v)beta (5) integrin, and stimulates cell migration from Vn toward collagen type IV (Col). The cells attached more strongly to Vn and Col than to fibronectin (Fn), whereas PAI-1 interfered with cell attachment to Vn only. An integrin antagonist, RGD peptide, and anti-alpha (v)beta (5) integrin antibodies, which similarly inhibited cell attachment to Vn, also stimulated cell migration from Vn toward Col. u-PA did not modify cell attachment directly, but reversed the PAI-1-mediated inhibitory effect on cell adhesion to Vn, and its stimulatory effect on cell migration from Vn toward Col. Thus HT-1080 cell migration appears to be modified by u-PA and PAI-1, altering cell adhesion to Vn via alpha (v)beta (5) integrin. This may be related to their tumor-promoting effect.
Assuntos
Movimento Celular , Fibrossarcoma/patologia , Integrinas/metabolismo , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Receptores de Vitronectina/metabolismo , Vitronectina/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Humanos , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium-evoked insulin secretion. In rat beta cells transfected with green fluorescent protein-tagged PKC-alpha (PKC-alpha-EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca(2)(+)](c)), which was accompanied by transient translocation of PKC-alpha-EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC-theta-EGFP, the C1 domain of PKC-gamma and PKC-epsilon-GFP. A high concentration of glucose induced repetitive elevation of [Ca(2)(+)](c) and repetitive translocation of PKC-alpha-EGFP. Diazoxide completely blocked both elevation of [Ca(2)(+)](c) and translocation of PKC-alpha-EGFP. We then studied the role of conventional PKC in calcium-evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Go-6976, an inhibitor of conventional PKC, and PKC-alpha pseudosubstrate fused to antennapedia peptide (Antp-PKC(19-31)) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö-6976 and Antp-PKC(19-31). However, when islets were stimulated for 60 min with high glucose, both Gö-6976 and Antp-PKC(19-31) reduced glucose-induced insulin secretion. Similar results were obtained by transfection of dominant-negative PKC-alpha using adenovirus vector. Taken together, PKC-alpha is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization-induced insulin secretion per se, but it also augments glucose-induced secretion.