RESUMEN
Acute pancreatitis (AP) is serious inflammatory disease of the pancreas. Accumulating evidence links diabetes with severity of AP, suggesting that endogenous insulin may be protective. We investigated this putative protective effect of insulin during cellular and in vivo models of AP in diabetic mice (Ins2Akita) and Pancreatic Acinar cell-specific Conditional Insulin Receptor Knock Out mice (PACIRKO). Caerulein and palmitoleic acid (POA)/ethanol-induced pancreatitis was more severe in both Ins2Akita and PACIRKO vs control mice, suggesting that endogenous insulin directly protects acinar cells in vivo. In isolated pancreatic acinar cells, insulin induced Akt-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) which upregulated glycolysis thereby preventing POA-induced ATP depletion, inhibition of the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) and cytotoxic Ca2+ overload. These data provide the first mechanistic link between diabetes and severity of AP and suggest that phosphorylation of PFKFB2 may represent a potential therapeutic strategy for treatment of AP.
Asunto(s)
Células Acinares/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Glucólisis/efectos de los fármacos , Insulina/metabolismo , Insulina/farmacología , Pancreatitis/metabolismo , Sustancias Protectoras/farmacología , Células Acinares/efectos de los fármacos , Enfermedad Aguda , Animales , ATPasas Transportadoras de Calcio/metabolismo , Ceruletida , Diabetes Mellitus Experimental/metabolismo , Ácidos Grasos Monoinsaturados , Masculino , Ratones , Ratones Noqueados , Páncreas/metabolismo , Pancreatitis/tratamiento farmacológico , Pancreatitis/patologíaRESUMEN
Cancer is caused by excessive cell proliferation and a propensity to avoid cell death, while the spread of cancer is facilitated by enhanced cellular migration, invasion, and vascularization. Cytosolic Ca2+ is central to each of these important processes, yet to date, there are no cancer drugs currently being used clinically, and very few undergoing clinical trials, that target the Ca2+ signalling machinery. The aim of this review is to highlight some of the emerging evidence that targeting key components of the Ca2+ signalling machinery represents a novel and relatively untapped therapeutic strategy for the treatment of cancer.
Asunto(s)
Pancreatitis , Canales Catiónicos TRPM , Células Acinares , Enfermedad Aguda , Calcio , HumanosRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is largely resistant to standard treatments leading to poor patient survival. The expression of plasma membrane calcium ATPase-4 (PMCA4) is reported to modulate key cancer hallmarks including cell migration, growth, and apoptotic resistance. Data-mining revealed that PMCA4 was over-expressed in pancreatic ductal adenocarcinoma (PDAC) tumors which correlated with poor patient survival. Western blot and RT-qPCR revealed that MIA PaCa-2 cells almost exclusively express PMCA4 making these a suitable cellular model of PDAC with poor patient survival. Knockdown of PMCA4 in MIA PaCa-2 cells (using siRNA) reduced cytosolic Ca2+ ([Ca2+]i) clearance, cell migration, and sensitized cells to apoptosis, without affecting cell growth. Knocking down PMCA4 had minimal effects on numerous metabolic parameters (as assessed using the Seahorse XF analyzer). In summary, this study provides the first evidence that PMCA4 is over-expressed in PDAC and plays a role in cell migration and apoptotic resistance in MIA PaCa-2 cells. This suggests that PMCA4 may offer an attractive novel therapeutic target in PDAC.
RESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus. METHODS: PDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca2+ overload and in situ Ca2+ clearance. PKM2 knockdown was achieved using siRNA. RESULTS: The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death. CONCLUSIONS: Cutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/tratamiento farmacológico , Proteínas Portadoras/genética , Proliferación Celular/genética , Proteínas de la Membrana/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Hormonas Tiroideas/genética , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas Portadoras/antagonistas & inhibidores , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Naftoquinonas/farmacología , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas de Unión a Hormona TiroideRESUMEN
The plasma membrane Ca2+-ATPase (PMCA) is a ubiquitously expressed, ATP-driven Ca2+ pump that is critical for maintaining low resting cytosolic Ca2+ ([Ca2+]i) in all eukaryotic cells. Since cytotoxic Ca2+ overload has such a central role in cell death, the PMCA represents an essential "linchpin" for the delicate balance between cell survival and cell death. In general, impaired PMCA activity and reduced PMCA expression leads to cytotoxic Ca2+ overload and Ca2+ dependent cell death, both apoptosis and necrosis, whereas maintenance of PMCA activity or PMCA overexpression is generally accepted as being cytoprotective. However, the PMCA has a paradoxical role in cell death depending on the cell type and cellular context. The PMCA can be differentially regulated by Ca2+-dependent proteolysis, can be maintained by a localised glycolytic ATP supply, even in the face of global ATP depletion, and can be profoundly affected by the specific phospholipid environment that it sits within the membrane. The major focus of this review is to highlight some of the controversies surrounding the paradoxical role of the PMCA in cell death and survival, challenging the conventional view of ATP-dependent regulation of the PMCA and how this might influence cell fate.
Asunto(s)
ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Supervivencia Celular , Humanos , Mitocondrias/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
AIM: To construct a global "metabolic phenotype" of pancreatic ductal adenocarcinoma (PDAC) reflecting tumour-related metabolic enzyme expression. METHODS: A systematic review of the literature was performed using OvidSP and PubMed databases using keywords "pancreatic cancer" and individual glycolytic and mitochondrial oxidative phosphorylation (MOP) enzymes. Both human and animal studies investigating the oncological effect of enzyme expression changes and inhibitors in both an in vitro and in vivo setting were included in the review. Data reporting changes in enzyme expression and the effects on PDAC cells, such as survival and metastatic potential, were extracted to construct a metabolic phenotype. RESULTS: Seven hundred and ten papers were initially retrieved, and were screened to meet the review inclusion criteria. 107 unique articles were identified as reporting data involving glycolytic enzymes, and 28 articles involving MOP enzymes in PDAC. Data extraction followed a pre-defined protocol. There is consistent over-expression of glycolytic enzymes and lactate dehydrogenase in keeping with the Warburg effect to facilitate rapid adenosine-triphosphate production from glycolysis. Certain isoforms of these enzymes were over-expressed specifically in PDAC. Altering expression levels of HK, PGI, FBA, enolase, PK-M2 and LDA-A with metabolic inhibitors have shown a favourable effect on PDAC, thus identifying these as potential therapeutic targets. However, the Warburg effect on MOP enzymes is less clear, with different expression levels at different points in the Krebs cycle resulting in a fundamental change of metabolite levels, suggesting that other essential anabolic pathways are being stimulated. CONCLUSION: Further characterisation of the PDAC metabolic phenotype is necessary as currently there are few clinical studies and no successful clinical trials targeting metabolic enzymes.
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Carcinoma Ductal Pancreático/enzimología , Metabolismo Energético , Glucosa/metabolismo , Neoplasias Pancreáticas/enzimología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Metabolismo Energético/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , FenotipoRESUMEN
Evidence suggests that the plasma membrane Ca(2+)-ATPase (PMCA), which is critical for maintaining a low intracellular Ca(2+) concentration ([Ca(2+)]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca(2+)]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca(2+)]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca(2+)]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.
Asunto(s)
Adenosina Trifosfato/metabolismo , Membrana Celular/enzimología , Glucólisis , Neoplasias Pancreáticas/patología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Adenocarcinoma/patología , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Galactosa/farmacología , Glucólisis/efectos de los fármacos , Humanos , Ácido Yodoacético/farmacología , Cetoácidos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidoresRESUMEN
Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca(2+) ([Ca(2+)]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca(2+)]i are linked critically by the ATP-driven plasma membrane Ca(2+)-ATPase (PMCA) important for maintaining low resting [Ca(2+)]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca(2+)]i was measured by fura-2 imaging. An in situ [Ca(2+)]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mM) and POAEE (100 µM) induced a small but irreversible Ca(2+) overload response but had no significant effect on PMCA activity. POA (50-100 µM) induced a robust Ca(2+) overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca(2+) overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca(2+) overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacología , Insulina/fisiología , Páncreas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Muerte Celular , Cromonas/farmacología , Etanol/administración & dosificación , Etanol/metabolismo , Ácidos Grasos/metabolismo , Fluorescencia , Morfolinas/farmacología , Páncreas/citología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca(2+) ([Ca(2+)]i), as the ATP-dependent plasma membrane Ca(2+) ATPase (PMCA) is critical for maintaining low [Ca(2+)]i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca(2+)]i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca(2+)]i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Glucólisis , Neoplasias Pancreáticas/patología , Adenocarcinoma/patología , Adenosina Trifosfato/deficiencia , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Citosol/metabolismo , Humanos , Mitocondrias/metabolismoRESUMEN
Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca(2+) ([Ca(2+)](i)) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca(2+) overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca(2+)-ATPase (PMCA) provides a final common path for cells to "defend" [Ca(2+)](i) during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca(2+) overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca(2+) overload, even in the face of impaired mitochondrial function.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Insulina/metabolismo , Páncreas Exocrino/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Células Cultivadas , Glucólisis/fisiología , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Estrés Oxidativo/fisiología , Páncreas Exocrino/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.
Asunto(s)
Proliferación Celular , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Potasio/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp , Pirazoles/metabolismo , Transducción de Señal/fisiología , Canales de Sodio/genética , Canales de Sodio/metabolismoRESUMEN
Calcium (Ca(2+))-activated K(+) (K(Ca)) channels regulate membrane excitability and are activated by an increase in cytosolic Ca(2+) concentration ([Ca(2+)](i)), leading to membrane hyperpolarization. Most patch clamp experiments that measure K(Ca) currents use steady-state [Ca(2+)] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca(2+)](i) changes dynamically, for example during [Ca(2+)](i) oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca(2+)](i) on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K(Ca) subtype in isolation were used to simultaneously measure agonist-evoked [Ca(2+)](i) signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca(2+)](i) oscillations induced a corresponding K(Ca) current that faithfully followed the [Ca(2+)](i) in 13-50% of cells, suggesting a good synchronization. However, [Ca(2+)](i) and K(Ca) current was much less synchronized in 50-76% of cells that exhibited Ca(2+)-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca(2+)](i) events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca(2+)](i) and K(Ca) current was least synchronized, 36% of total [Ca(2+)](i) spikes occurred without activating a corresponding K(Ca) current spike, suggesting that BK(Ca) channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca(2+)](i) and K(Ca) current suggests that this relationship is more complex than could be predicted from steady-state [Ca(2+)](i) and K(Ca) current. These phenomena may be important for encoding stimulus-response coupling in various cell types.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Carbacol/farmacología , Células Cultivadas , Fura-2 , Humanos , Indoles/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio Calcio-Activados/agonistasRESUMEN
Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.
Asunto(s)
Señalización del Calcio , Mitocondrias/metabolismo , Estrés Oxidativo , Páncreas Exocrino/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antimicina A/farmacología , Bioensayo , Ácido Bongcréquico/farmacología , Señalización del Calcio/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Ciclosporina/farmacología , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Oligomicinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Páncreas Exocrino/efectos de los fármacos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Compuestos de Rutenio/farmacología , Factores de Tiempo , Desacopladores/farmacologíaRESUMEN
Cross-talk between intracellular calcium ([Ca(2+)](i)) signaling and cAMP defines the specificity of stimulus-response coupling in a variety of cells. Previous studies showed that protein kinase A (PKA) potentiates and phosphorylates the plasma membrane Ca(2+)-ATPase (PMCA) in a Ca(2+)-dependent manner in parotid acinar cells (Bruce, J. I. E., Yule, D. I., and Shuttleworth, T. J. (2002) J. Biol. Chem. 277, 48172-48181). The aim of this study was to further investigate the spatial regulation of [Ca(2+)](i) clearance in parotid acinar cells. Par-C10 cells were used to functionally isolate the apical and basolateral PMCA activity by applying La(3+) to the opposite side to inhibit the PMCA. Activation of PKA (using forskolin) differentially potentiated apical [Ca(2+)](i) clearance in mouse parotid acinar cells and apical PMCA activity in Par-C10 cells. Immunofluorescence of parotid tissue slices revealed that PMCA1 was distributed throughout the plasma membrane, PMCA2 was localized to the basolateral membrane, and PMCA4 was localized to the apical membrane of parotid acinar cells. However, in situ phosphorylation assays demonstrated that PMCA1 was the only isoform phosphorylated by PKA following stimulation. Similarly, immunofluorescence of acutely isolated parotid acinar cells showed that the regulatory subunit of PKA (RIIbeta) translocated to the apical region following stimulation. These data suggest that PKA-mediated phosphorylation of PMCA1 differentially regulates [Ca(2+)](i) clearance in the apical region of parotid acinar cells because of a dynamic translocation of PKA. Such tight spatial regulation of Ca(2+) efflux is likely important for the fine-tuning of Ca(2+)-dependent effectors close to the apical membrane important for the regulation of fluid secretion and exocytosis.
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ATPasas Transportadoras de Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Glándula Parótida/citología , Glándula Parótida/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Colforsina/metabolismo , Cinética , Ratones , Modelos Biológicos , Fosforilación , Transporte de Proteínas , Ratas , Transducción de SeñalRESUMEN
Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca(2+)](i)) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca(2+)](i) signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca(2+)](i), and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca(2+)](i), which was sensitive to antioxidants and removal of external Ca(2+), and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca(2+)-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca(2+) uptake. This suggests that hydrogen peroxide promotes Ca(2+) release from the endoplasmic reticulum and the mitochondria and that it promotes Ca(2+) influx. Lower concentrations of hydrogen peroxide (10-100 muM) increased [Ca(2+)](i) and altered cholecystokinin-evoked [Ca(2+)](i) oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca(2+)](i) also upregulated the activity of the plasma membrane Ca(2+)-ATPase in a Ca(2+)-dependent manner, whereas higher concentrations (0.1-1 mM) inactivated the plasma membrane Ca(2+)-ATPase. This may be important in facilitating "Ca(2+) overload," resulting in cell injury associated with pancreatitis.
Asunto(s)
Señalización del Calcio/fisiología , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Páncreas Exocrino/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Membrana Celular/enzimología , Colagogos y Coleréticos/farmacología , Colecistoquinina/farmacología , Fluoresceínas , Técnicas In Vitro , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Páncreas Exocrino/citología , Pancreatitis/metabolismo , RatasRESUMEN
In salivary acinar cells, intracellular calcium ([Ca(2+)](i)) signaling plays an important role in eliciting fluid secretion through the activation of Ca(2+)-activated ionic conductances. Ca(2+) and cAMP have synergistic effects on fluid secretion such that peak secretion is elicited following activation of both parasympathetic and sympathetic pathways. We have recently demonstrated that cAMP exerts effects on Ca(2+) release, through protein kinase A (PKA)-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)R) in mouse parotid acinar cells. To extend these findings, in the present study cross-talk between Ca(2+) signaling and cAMP pathways in human parotid acinar cells was investigated. In human parotid acinar cells, carbachol stimulation evoked increases in the [Ca(2+)](i) and the initial peak amplitude was enhanced following PKA activation, consistent with reports from mouse parotid. Stimulation with ATP also evoked an increase in [Ca(2+)](i). The ATP-evoked Ca(2+) elevation was largely dependent on extracellular Ca(2+), suggesting the involvement of the P2X family of purinergic receptors. Pharmacological elevation of cAMP resulted in a approximately 5-fold increase in the peak [Ca(2+)](i) change evoked by ATP stimulation. This enhanced [Ca(2+)](i) increase was not dependent on intracellular release from InsP(3)R or ryanodine receptors, suggesting a direct effect on P2XR. Reverse transcription-polymerase chain reaction and Western blot analysis confirmed the presence of P2X(4)R and P2X(7)R mRNA and protein in human parotid acinar cells. ATP-activated cation currents were studied using whole cell patch clamp techniques in HEK-293 cells, a null background for P2XR. Raising cAMP resulted in a approximately 4.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X(4)R DNA but had no effects on currents in cells expressing P2X(7)R. These data indicate that in human parotid acinar cells, in addition to modulation of Ca(2+) release, Ca(2+) influx through P2X(4)R may constitute a further locus for the synergistic effects of Ca(2+) and PKA activation.
Asunto(s)
Adenosina Trifosfato/farmacología , Señalización del Calcio/fisiología , AMP Cíclico/metabolismo , Glándula Parótida/metabolismo , Receptores Purinérgicos P2/metabolismo , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Colforsina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Espacio Extracelular/metabolismo , Femenino , Humanos , Técnicas In Vitro , Masculino , Glándula Parótida/citología , Técnicas de Placa-Clamp , Receptores Purinérgicos P2X4RESUMEN
Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.
Asunto(s)
Calcio/metabolismo , Núcleo Celular/metabolismo , Glándula Parótida/citología , Adenosina Trifosfato/química , Animales , Calcio/química , Carbacol/farmacología , Citosol/metabolismo , Colorantes Fluorescentes/farmacología , Procesamiento de Imagen Asistido por Computador , Inositol 1,4,5-Trifosfato/metabolismo , Iones , Cinética , Luz , Ratones , Microscopía Confocal , Mitocondrias/metabolismo , NAD/química , Oscilometría , Compuestos de Rutenio/farmacología , Rojo de Rutenio/farmacología , Transducción de Señal , Factores de TiempoRESUMEN
Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.