RESUMO
Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Bradicinina/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Calreticulina , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Citometria de Fluxo , Homeostase , Immunoblotting , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores da Bradicinina/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Tapsigargina/farmacologia , TransfecçãoRESUMO
Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Lisofosfolipídeos , Mitocôndrias/patologia , Transdução de Sinais , Esfingosina/análogos & derivados , Receptores de Esfingosina-1-FosfatoRESUMO
Antiapoptotic Bcl-2-family members are well known for their 'mitochondrial' functions as critical neutralizers of proapoptotic Bcl-2-family members, including the executioner multidomain proteins Bax and Bak and the BH3-only proteins. It has been clear for more than 20 years that Bcl-2 proteins can impact intracellular Ca(2+) homeostasis and dynamics. Moreover, altered Ca(2+) signaling is increasingly linked to oncogenic behavior. Specifically targeting the Ca(2+)-signaling machinery may thus prove to be a valuable strategy for cancer treatment. Over 10 years ago a major controversy was recognized concerning whether or not Bcl-2 proteins exerted their antiapoptotic functions via Ca(2+) signaling through lowering the filling state of the endoplasmic reticulum (ER) Ca(2+) stores or by suppressing Ca(2+) release from the ER without affecting the filling state of this Ca(2+) store. Further research from different laboratories indicated a wide variety of mechanisms by which Bcl-2-family members can impact Ca(2+) signaling. In this review, we propose that antiapoptotic Bcl-2-family members are multimodal regulators of intracellular Ca(2+)-signaling events in cell survival and cell death. We will discuss how different Bcl-2-family members impact cell survival and cell death by regulating Ca(2+) transport systems at the ER, mitochondria and plasma membrane and by impacting the organization of organelles and how these insights can be exploited for causing cell death in cancer cells. Finally, we propose that the existing controversy reflects the diversity of links between Bcl-2 proteins and Ca(2+) signaling, as certainly not all targets or mechanisms will be operative in every cell type and every condition.
Assuntos
Sinalização do Cálcio/genética , Retículo Endoplasmático/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Apoptose/genética , Cálcio/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.
Assuntos
Linfócitos B/imunologia , Proteínas de Membrana/fisiologia , Linfócitos T/imunologia , Transporte Ativo do Núcleo Celular , Animais , Apoptose , Linfócitos B/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Caspases/metabolismo , Sobrevivência Celular , Citoplasma/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Feminino , Leucopenia/genética , Leucopenia/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Obesidade/genética , Obesidade/imunologia , Baço/imunologia , Baço/patologia , Linfócitos T/metabolismoRESUMO
The Golgi apparatus is, like the endoplasmic reticulum, an inositol-1,4,5-trisphosphate-sensitive Ca2+ store, but its role in setting up Ca2+ signals is not well understood. We have now measured histamine-induced Ca2+ signals in HeLa cells pretreated with brefeldin A, a fungal metabolite that leads to the fragmentation and subsequent disappearance of the Golgi apparatus by its reabsorption within the endoplasmic reticulum. Ca2+ responses in which the free cytoplasmic Ca2+ concentration returned to resting levels during the histamine stimulation (mainly baseline Ca2+ oscillations or a single Ca2+ peak) occurred more often in brefeldin A pretreated cells, resulting in a lower Ca2+ plateau in population measurements. The latencies before the onset of the Ca2+ signals were longer after brefeldin A pretreatment. These results suggest that the integrity of the Golgi apparatus contributes to the shaping of intracellular Ca2+ signals.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Citosol/fisiologia , Complexo de Golgi/fisiologia , Brefeldina A/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Imunofluorescência , Complexo de Golgi/efeitos dos fármacos , Células HeLa/ultraestrutura , Histamina/farmacologia , Humanos , Tempo de Reação/efeitos dos fármacosRESUMO
ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.
Assuntos
Cálcio/metabolismo , Fosfatos de Inositol/metabolismo , Rim/metabolismo , Fosfatos Açúcares/metabolismo , Animais , Transporte Biológico , Proteínas de Ligação a Calmodulina/metabolismo , Compartimento Celular , Fracionamento Celular/métodos , Linhagem Celular , Sistema Livre de Células , Epitélio/metabolismo , Glucose-6-Fosfatase/metabolismo , Rim/citologia , Membranas/metabolismo , Mitocôndrias/enzimologia , Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , gama-Glutamiltransferase/metabolismoRESUMO
The calmodulin content of renal brush-border membrane vesicles, prepared by Mg2+-precipitation in EGTA-containing solutions, amounts to 1.8 micrograms per mg protein. The amount and the distribution of this EGTA-insensitive calmodulin was determined in membrane and cytoskeletal fractions prepared from the brush-border membrane vesicles by extraction with Triton X-100. The Triton X-100 insoluble pellet contains 21.2% of the protein and 52.2% of the EGTA-insensitive calmodulin, which amounts in this fraction to 4.4 micrograms per mg protein. Treatment of the Triton X-100 insoluble pellet, consisting of the microvillar core residue, with ATP and Mg2+ results in the solubilization of a relatively small number of proteins among which are actin, myosin, calmodulin and several calmodulin-binding proteins. The solubilization is partially reversible and a fraction of the proteins can be precipitated by centrifugation after the enzymatic hydrolysis of ATP. Readdition of ATP to the pellet results in the resolubilization of myosin, part of the actin, an 115-kDa calmodulin-binding protein and calmodulin. The calmodulin content of the final extract was 61.8 micrograms per mg protein. We have found roughly the same distribution pattern of calmodulin and ATP-solubilized, calmodulin-binding proteins in renal and intestinal brush-border preparations. The calmodulin content, however, as well as the relative amount of the calmodulin-binding proteins versus actin are about 4 to 5-times higher in intestinal than in renal microvillar core residues.
Assuntos
Proteínas de Ligação a Calmodulina/isolamento & purificação , Calmodulina/análise , Proteínas do Citoesqueleto/isolamento & purificação , Túbulos Renais Proximais/análise , Microvilosidades/análise , Trifosfato de Adenosina/farmacologia , Animais , Membrana Celular/análise , Galinhas , Citoesqueleto/análise , Ácido Egtázico , Intestino Delgado/análise , Magnésio/farmacologia , Octoxinol , Polietilenoglicóis/farmacologia , Solubilidade , SuínosRESUMO
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.
Assuntos
Fosfatase Alcalina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Córtex Renal/enzimologia , Microvilosidades/enzimologia , Fosfatase Alcalina/isolamento & purificação , Animais , Transporte Biológico Ativo , ATPase de Ca(2+) e Mg(2+) , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/isolamento & purificação , Cinética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Peso Molecular , Fosforilação , SuínosRESUMO
Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from the renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [125I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of 20 microM La2+ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.
Assuntos
Cálcio/farmacologia , Calmodulina/metabolismo , Córtex Renal/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Lantânio/metabolismo , Membranas/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Suínos , Tripsina/metabolismoRESUMO
Xenopus laevis oocytes (stages V and VI) are a widely used model system for the study of Ca2+ signaling. The properties of the Xenopus oocyte InsP3 receptor (InsP3R) are of paramount importance for our thinking about this system and for our efforts to model Ca2+ dynamics in the oocyte cytosol. The recent data regarding the molecular structure, the regulation and the functional properties of the Xenopus oocyte InsP3R are summarized in this review. The main properties of the Xenopus oocyte InsP3R are compared with the properties of the cerebellar InsP3R and are shown to be remarkably similar. The density of the InsP3R in Xenopus oocyte cytoplasm is estimated to a value between 1.1-4.1 x 10(14) tetrameric InsP3R/l. The use of these numbers in a quantitative model of Ca2+ wave propagation leads to values of Ca2+ wave amplitude (0.8-1.5 microM Ca2+) and velocity of the wave propagation (12-24 microns/s) that are in excellent agreement with the values observed experimentally. The density of InsP3Rs in Purkinje cells of the cerebellum is estimated to be about 20,000-fold higher, but in other types of neurons and in peripheral tissues the InsP3R density is estimated to be of the same order of magnitude as, or up to 20-fold higher than, in Xenopus oocytes. The implications of differences in InsP3R density for Ca2+ signaling are discussed.
Assuntos
Canais de Cálcio/metabolismo , Oócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Xenopus laevis/metabolismo , Animais , Feminino , Receptores de Inositol 1,4,5-Trifosfato , Transdução de SinaisRESUMO
2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release in bi-directional Ca2+ -flux conditions. We have now studied the effects of 2APB on the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ efflux from the non-mitochondrial Ca2+ stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3 -induced Ca2+ release, with a half maximal inhibition at 36 microM 2APB, without affecting [3H]IP3 binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+ concentration. The Ca2+ pumps of the non-mitochondrial Ca2+ stores were half-maximally inhibited at 91microM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+ from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3 -induced Ca2+ release. Our results can explain the various effects of 2APB observed in intact cells.
Assuntos
Compostos de Boro/farmacologia , Canais de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Músculo Liso Vascular/citologia , Ratos , Tapsigargina/farmacologiaRESUMO
ATP-driven calcium uptake was studied in basal-lateral membranes and in microsomal fractions, isolated from pig kidney cortex. The uptake is strongly enhanced in conditions where calcium inside the vesicles is precipitated by oxalate (5 mM) or phosphate (40 mM). Both anions were equally effective for the stimulation of calcium uptake in the microsomes but oxalate was less effective than phosphate in the basal-lateral membrane fraction. The active calcium pumps in the renal basal-lateral and microsomal fractions are different transport ATPases characterized by phosphorylated intermediates of 135 kDa and 115 kDa respectively. The subcellular distribution of the 135 kDa and 115 kDa phosphointermediates, reflects the distribution of typical marker enzymes for the basal-lateral membrane and for the endoplasmic reticulum. The calmodulin binding to the 135 kDa polypeptide as estimated by 125I-labelled calmodulin overlay, can be used as a specific marker for the basal-lateral plasma membrane calcium pump.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Córtex Renal/metabolismo , Trifosfato de Adenosina/farmacologia , Albuminas/farmacologia , Animais , Calmodulina/metabolismo , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Canais Iônicos/metabolismo , Córtex Renal/citologia , Cinética , Microssomos/metabolismo , Oxalatos/farmacologia , Ácido Oxálico , Fosfatos/farmacologia , Fosforilação , SuínosRESUMO
We have investigated how the immunosuppressant drug FK506 affected the basal Ca(2+) leak in permeabilized A7r5 cells. Non-mitochondrial Ca(2+) stores loaded to steady state with Ca(2+) slowly lost their accumulated Ca(2+) during incubation in a Ca(2+)-free efflux medium. FK506 up to 100 microM had no effect on the basal Ca(2+) leak. In contrast, the rate of Ca(2+) release proceeded much faster immediately after washing out FK506. The increase in rate of Ca(2+) release after washing out of this compound depended on both its initial concentration and on the time of pre-incubation. A similar effect was also observed after removing another immunosuppressant drug (rapamycin) and after removing the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin C. Since all these substances have a high octanol/H(2)O partition coefficient and accumulate in the endoplasmic reticulum membrane, we suggest that the transient increase in the basal Ca(2+) leak is due to the sudden removal of these lipophilic substances from the membrane.
Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Imunossupressores/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Tacrolimo/farmacologia , Animais , Aorta , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Compostos Macrocíclicos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Oxazóis/farmacologia , Ratos , Sirolimo/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Carcinoma/metabolismo , Neoplasias da Próstata/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma/fisiopatologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , Neoplasias da Próstata/fisiopatologia , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Tapsigargina/farmacologia , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP3R-IV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691-21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP3 binding affinity (Kd 3.8 +/- 0.8 nM, Bmax 0.40 +/- 0.08 pmol/mg protein) than microsomes from A7r5 cells (Kd 65 +/- 7 nM, Bmax 0.65 +/- 0.08 pmol/mg protein) or from cerebellum (Kd 135 +/- 14 nM, Bmax 7.35 +/- 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP3Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C3H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP3R-I was used to determine the presence of the various InsP3R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD (Xenopus oocytes) isoforms were recognized. Expression of InsP3R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP3R isoforms may differ to a large extent in their affinity for InsP3 and suggest that RBL-2H3 cells are a useful model for the study of InsP3R-IV.
Assuntos
Canais de Cálcio/fisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Leucemia Basofílica Aguda/patologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio/análise , Canais de Cálcio/classificação , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/imunologia , Receptores de Inositol 1,4,5-Trifosfato , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos C3H , Microssomos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Ratos , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/classificação , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/imunologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca2+ binding proteins in fraction F3 and the sucrose density gradient fractions. InsP3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCAs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.
Assuntos
Basófilos/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Fracionamento Celular , Membrana Celular , Centrifugação , Centrifugação com Gradiente de Concentração , Receptores de Inositol 1,4,5-Trifosfato , Leucemia , Coelhos , Ratos , Sacarose , Células Tumorais CultivadasRESUMO
Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT-PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+ pools may play distinct roles in cell proliferation and differentiation.
Assuntos
Canais de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Endotélio Vascular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Retículo Sarcoplasmático/enzimologia , Medula Suprarrenal/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Colágeno/metabolismo , Enzimas de Restrição do DNA/metabolismo , Combinação de Medicamentos , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Laminina/metabolismo , Masculino , Isoformas de Proteínas/metabolismo , Proteoglicanas/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Veias Umbilicais/metabolismoRESUMO
Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.
Assuntos
Equorina/biossíntese , Equorina/genética , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Equorina/metabolismo , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/farmacologiaRESUMO
Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.
Assuntos
Sinalização do Cálcio , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Queratinócitos/metabolismo , Animais , Células COS , ATPases Transportadoras de Cálcio/genética , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , TransfecçãoRESUMO
The non-mitochondrial Ca2+ stores in permeabilized A7r5 cells responded to a decrease in Mg-ATP concentration with a pronounced Ca2+ release if 20 microM CoA was present. This release was rather specific for the preincubation or removal of ATP. ATP gamma S was much less effective and AMP-PNP, GTP, ITP, CTP, UTP, ADP, AMP, adenosine and adenine had no effect. CoA activated with an EC50 of 6 microM. Dephospho-CoA was a less effective cofactor and desulfo-CoA was ineffective. The release induced by Mg-ATP removal did not occur in the presence of 2% fatty acid-free bovine serum albumin and did not develop at 4 degrees C. All these findings suggest that CoA had to be acylated by endogenous fatty-acyl-CoA synthetase to become effective. Myristoyl- and palmitoyl-CoA esters were identified as the most effective cofactors for the release. Ca2+ release induced by removing Mg-ATP did not occur if the osmolality of the medium was kept constant by addition of mannitol, sucrose, KCl, MgCl2 or Mg-GTP, indicating that the decrease in tonicity was the trigger for the release. Mg-ATP plus CoA also synergized with Ca2+ release induced by a hypotonic shock imposed by diluting the medium with H2O. Osmolality changes induced by decreasing the Mg-ATP concentration were more effective in releasing Ca2+ than equal decreases in concentration of all solutes. We conclude that fatty acyl-CoA esters sensitize the hypotonically induced Ca2+ release from the non-mitochondrial Ca2+ stores.