RESUMO
KEY POINTS: Gain-of-function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM-associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK-7975A prevents the constitutive activity of TAM-associated channels and might be used in therapy for patients suffering from TAM. ABSTRACT: Skeletal muscle differentiation relies on store-operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+ -permeable ORAI channels. Gain-of-function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK-293T cells or human primary myoblasts, the mutated channels increased basal and store-operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK-7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1-V107M constitutive activity and revealed larger STIM1-gated V107M- and T184M-mediated currents with conserved fast and slow Ca2+ -dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1-/- /Stim2-/- mouse embryonic fibroblasts expressing the binding-deficient STIM1-F394H mutant, indicating a higher sensitivity for STIM1-mediated gating, with ORAI1-T184M gain-of-function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain-of-function mutations in ORAI1.
Assuntos
Ativação do Canal Iônico , Miopatias Congênitas Estruturais/genética , Proteína ORAI1/genética , Animais , Benzamidas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Fibroblastos/fisiologia , Mutação com Ganho de Função , Células HEK293 , Humanos , Camundongos Knockout , Mioblastos/fisiologia , Miopatias Congênitas Estruturais/fisiopatologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/química , Proteína ORAI1/fisiologia , Domínios Proteicos , Pirazóis/farmacologia , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
Major-histocompatibility-complex (MHC) proteins are used to display, on the surface of a cell, peptides derived from foreign material - such as a virus - that is infecting that cell. Cytotoxic T lymphocytes then recognize and kill the infected cell. The HIV-1 Nef protein downregulates the cell-surface expression of class I MHC proteins, and probably thereby promotes immune evasion by HIV-1. In the presence of Nef, class I MHC molecules are relocalized from the cell surface to the trans-Golgi network (TGN) through as-yet-unknown mechanisms. Here we show that Nef-induced downregulation of MHC-I expression and MHC-I targeting to the TGN require the binding of Nef to PACS-1, a molecule that controls the TGN localization of the cellular protein furin. This interaction is dependent on Nef's cluster of acidic amino acids. A chimaeric integral membrane protein containing Nef as its cytoplasmic domain localizes to the TGN after internalization, in an acidic-cluster- and PACS-1-dependent manner. These results support a model in which Nef relocalizes MHC-I by acting as a connector between MHC-I's cytoplasmic tail and the PACS-1-dependent protein-sorting pathway.
Assuntos
Capsídeo/metabolismo , Proteínas de Transporte , Produtos do Gene nef/metabolismo , HIV-1/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Antígenos CD4/metabolismo , Capsídeo/antagonistas & inibidores , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Furina , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Produtos do Gene nef/farmacologia , Complexo de Golgi/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Estrutura Terciária de Proteína/genética , Ratos , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Subtilisinas/metabolismo , Transfecção , Proteínas de Transporte Vesicular , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Efficient mechanisms of H(+) ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase-in analogy with mitochondrial cytochromes-has an inherent H(+) channel activity remains uncertain: electrophysiological studies did not find altered H(+) currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H(+) currents in human eosinophils. The "classical" H(+) current had properties similar to previously described H(+) conductances and was present in CGD cells. In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H(+) extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.
Assuntos
Eosinófilos/metabolismo , Doença Granulomatosa Crônica/metabolismo , Adulto , Criança , Transporte de Elétrons , Ativação Enzimática , Feminino , Doença Granulomatosa Crônica/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Potenciais da Membrana , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Explosão RespiratóriaRESUMO
The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.
Assuntos
Adesão Celular/fisiologia , Citosol/química , Neutrófilos/citologia , Citoesqueleto de Actina/efeitos dos fármacos , Antígenos CD18/fisiologia , Movimento Celular , Tamanho Celular , Citocalasina D/farmacologia , Inibidores Enzimáticos/farmacologia , Fluoresceínas , Corantes Fluorescentes , Doença Granulomatosa Crônica/imunologia , Humanos , Concentração de Íons de Hidrogênio , Ionóforos/farmacologia , Microscopia de Vídeo , NADH NADPH Oxirredutases/antagonistas & inibidores , NADPH Oxidases , Neutrófilos/metabolismo , Nigericina/farmacologia , Oniocompostos/farmacologia , Explosão Respiratória/fisiologia , Rodaminas/farmacologia , Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/fisiologia , Superóxidos/metabolismoRESUMO
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
Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).
Assuntos
Processamento Alternativo , Hidrogênio/metabolismo , Canais Iônicos/genética , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Sequência de Aminoácidos , Linhagem Celular , Citosol/metabolismo , Condutividade Elétrica , Transporte de Elétrons , Etiquetas de Sequências Expressas , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/química , Técnicas de Patch-Clamp , Prótons , Transfecção , Células Tumorais Cultivadas , Zinco/farmacologiaRESUMO
Ca(2+) entry in endothelial cells is a key signaling event as it prolongs the Ca(2+) signal activated by a receptor agonist, and thus allows an adequate production of a variety of compounds. The possible routes that lead to Ca(2+) entry in non-excitable cells include the receptor-activated Ca(2+) entry (RACE), which requires the presence of an agonist to be activated, and the store-operated Ca(2+) entry (SOCE) pathway, whose activation requires the depletion of the ER Ca(2+) store. However, the relative importance of these two influx pathways during physiological stimulation is not known. In the present study we experimentally differentiated these two types of influxes and determined under which circumstances they are activated. We show that La(3+) (at 10 microM) is a discriminating compound that efficiently blocks SOCE but is almost without effect on histamine-induced Ca(2+) entry (RACE). In line with this, histamine does not induce massive store depletion when performed in the presence of extracellular Ca(2+). In addition, inhibition of mitochondrial respiration significantly reduces SOCE but modestly affects RACE. Thus, agonist-induced Ca(2+) entry is insensitive to La(3+), and only modestly affected by mitochondrial depolarization. These data shows that agonist relies almost exclusively on RACE for sustained Ca(2+) signaling in endothelial cells.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Transporte Biológico/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Retículo Endoplasmático/metabolismo , Células Endoteliais/efeitos dos fármacos , Histamina/farmacologia , Humanos , Imidazóis/farmacologia , Lantânio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.
Assuntos
Cálcio/metabolismo , Fosfatos de Inositol/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Citosol/metabolismo , Humanos , Indóis/farmacologia , Potenciais da Membrana , Células Tumorais CultivadasRESUMO
We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.
Assuntos
Endossomos/metabolismo , Endossomos/ultraestrutura , Receptores da Transferrina/metabolismo , ATPases Vacuolares Próton-Translocadoras , Animais , Caveolina 1 , Caveolinas/metabolismo , Caveolinas/ultraestrutura , Linhagem Celular , Colesterol/metabolismo , Cães , Endocitose , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Concentração de Íons de Hidrogênio , Membranas Intracelulares/química , ATPases Translocadoras de Prótons/metabolismo , Receptores da Transferrina/genética , Esfingomielinas/metabolismo , Frações Subcelulares/química , Transfecção , Transferrina/metabolismoRESUMO
Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.
Assuntos
Potenciais da Membrana/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Sequência de Bases , Células CHO , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Cricetinae , DNA/análise , DNA/química , DNA/genética , Primers do DNA/análise , Primers do DNA/química , Primers do DNA/genética , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Isomerismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/fisiologia , Macrófagos Peritoneais/ultraestrutura , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , TransfecçãoRESUMO
We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.
Assuntos
Trifosfato de Adenosina/fisiologia , Nucleotídeos/metabolismo , Fosfolipídeos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , DNA/biossíntese , Eletrofisiologia , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência , Técnicas de Patch-Clamp , RatosRESUMO
Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Angiotensina II/metabolismo , Antígenos CD36/metabolismo , Ésteres do Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Zona Glomerulosa/metabolismo , Angiotensina II/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Bovinos , Células Cultivadas , Humanos , Receptores Depuradores , Receptores Depuradores Classe B , Transdução de Sinais/efeitos dos fármacosRESUMO
Cells of the pituitary tumour cell line GH(4) C(1) were exposed to epidermal growth factor, estradiol and insulin for 5 days, a treatment which resulted in 1) increased prolactin storage in secretory granules, 2) the loss of spontaneous [Ca(2+) ](1) oscillations, and 3) a selective reduction of the protein G(s) α, seen in immunoblots, cholera toxin labelling, and vasoactive intestinal peptide stimulation of adenylyl cyclase. In contrast, the glucocorticoid dexamethasone, which increases the expression of G(s) α, partially restored spontaneous [Ca(2+) ](1) oscillations and decreased prolactin storage. It is concluded that G(s) α levels in tumoral cells result in spontaneous electrical activity which may empty prolactin stores by the continuous activation of exocytosis.
Assuntos
Cálcio/análise , Cálcio/sangue , Concentração de Íons de Hidrogênio , Neutrófilos/fisiologia , Citosol/metabolismo , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Exocitose , Corantes Fluorescentes , Humanos , Indóis , Cinética , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Células Tumorais CultivadasRESUMO
BACKGROUND AND PURPOSE: Amiloride derivatives are blockers of the Na(+)/H(+) exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action. EXPERIMENTAL APPROACH: We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca(2+) concentration ([Ca(2+)](ER)) with a 'cameleon' indicator, D1ER. KEY RESULTS: Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca(2+)](ER), mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca(2+)](ER). The decrease in [Ca(2+)](ER) evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cytosolic Ca(2+) concentration. CONCLUSIONS AND IMPLICATIONS: Amiloride derivatives disrupt ER and cytosolic Ca(2+) homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca(2+) depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives.
Assuntos
Amilorida/toxicidade , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Diuréticos/toxicidade , Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Amilorida/análogos & derivados , Antígenos de Diferenciação/genética , Proteínas de Ciclo Celular/genética , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Cinética , Proteína Fosfatase 1 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia , Tapsigargina/farmacologia , Fator de Transcrição CHOP/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Na+/H+ antiport is a major determinant of intracellular pH (pHi) and also plays an important role in the maintenance of cellular volume. Na+/H+ exchange through NHE-1, the ubiquitous isoform of the antiporter, is accelerated by cytosolic acidification and also by osmotically induced cell shrinking, thereby promoting recovery of the physiological pHi and volume, respectively. Although hydrolysis of ATP is not required for transport of ions through the antiporter, metabolic depletion exerts a marked inhibitory effect. Depletion of ATP also prevents osmotic activation and volume regulation. Contrary to earlier suggestions, however, changes in the phosphorylation state of the antiporter itself are not involved in the effects of either metabolic depletion or osmotic stimulation. Nevertheless, the cytosolic carboxy-terminal segment of the antiporter, which contains the major phosphorylation sites, is essential for the ATP dependence as well as for osmotic activation. It is conceivable that this domain interacts with ancillary phosphorylated or nucleotide-binding proteins, with the cytoskeleton and/or with specific phospholipids, which modulate the rate of transport. Nucleotide dependence and osmotic sensitivity have been compared in three different isoforms of the antiporter, heterologously expressed in fibroblastic cells. Like NHE-1, NHE-2 and NHE-3 were severely inhibited by depletion of ATP. In contrast, whereas NHE-2 was stimulated by osmotic shrinkage, NHE-3 was inhibited. The possible physiological significance of the ATP-dependence and osmotic responsiveness of the antiporter isoforms is discussed.
Assuntos
Trifosfato de Adenosina/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Fenômenos Fisiológicos Celulares , Células/citologia , Cricetinae , Homeostase , Humanos , Modelos Biológicos , Ratos , Trocador 3 de Sódio-HidrogênioRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) increases cAMP production and stimulates hormone release from a variety of anterior pituitary cells. However, in anterior pituitary gonadotrophs PACAP stimulates oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) that appear to be independent of cAMP. To study the mechanisms involved in this response, we used the patch-clamp technique to microperfuse various agents into single rat gonadotrophs while monitoring [Ca2+]i with microfluorometry. Extracellular application of PACAP to single gonadotrophs stimulated high amplitude (> 1 microM) oscillations in [Ca2+]i, which were blocked by intracellular application of GDP beta S (guanosine 5'-O-2-thiodiphosphate), indicating the involvement of a G-protein. To identify the intracellular messenger(s) involved, we microperfused gonadotrophs with cAMP, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, an antagonist of the Ins(1,4,5)P3 receptor. A high concentration of cAMP (100 microM) had no significant effect on basal [Ca2+]i and did not alter the PACAP-stimulated Ca2+ response. Heparin, but not its inactive isoform, completely blocked the PACAP-stimulated increase in [Ca2+]i, while Ins(1,4,5)P3 stimulated oscillations in [Ca2+]i very similar to those observed in response to PACAP. These results strongly suggest that PACAP mobilizes Ca2+ through an Ins(1,4,5)P3-dependent mechanism. The fact that PACAP stimulates two signaling pathways in pituitary cells could substantially enhance the signaling potential of this hypothalamic peptide.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Neuropeptídeos/fisiologia , Neurotransmissores/fisiologia , Adeno-Hipófise/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Masculino , Neuropeptídeos/antagonistas & inibidores , Neurotransmissores/antagonistas & inibidores , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.
Assuntos
Cálcio/metabolismo , Indóis/farmacologia , Transporte Biológico , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Cátions Bivalentes/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Fura-2 , Hidroquinonas/farmacologia , Imidazóis/farmacologia , Micotoxinas/toxicidade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Terpenos/farmacologia , TapsigarginaRESUMO
Inorganic ions constitute the predominant osmolytes of blood cells and are therefore the main determinant of the cellular volume. In leukocytes, maintenance of constant cell size is accomplished by modulating the ionic permeability of the membrane. In addition, the plasmalemmal ionic permeability dictates the transmembrane potential, which influences a variety of cellular responses. Inorganic ions are also essential for the regulation of the intracellular pH and play an active role in signal transduction during phagocyte activation. This chapter reviews recent progress in the area of ion transport in phagocytic leukocytes, with special reference to the physiologic implications to the activation process.
Assuntos
Transporte de Íons , Fagócitos/fisiologia , Animais , Cloretos/farmacocinética , Humanos , Potássio/farmacocinética , Bombas de Próton , Sódio/farmacocinética , Canais de Sódio/fisiologia , Trocadores de Sódio-HidrogênioRESUMO
Na+/H+ exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibition of the antiport. No evidence has been found for direct binding of nucleotide to exchangers or alteration in their state of phosphorylation, suggesting ancillary factors may be involved. This possibility was tested by comparing the activity of dog red blood cells (RBC) and their resealed ghosts. Immunoblotting experiments using isoform-specific polyclonal and monoclonal antibodies indicated RBC membranes express Na+/H+ exchanger isoform 1 (NHE1). In intact RBC, uptake of Na+ was greatly stimulated when the cytosol was acidified. The stimulated uptake was largely eliminated by amiloride and by submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also be elicited by acidification in resealed ghosts containing ATP, the absolute rate of transport was markedly diminished at comparable pH. Dissipation of the pH gradient was ruled out as the cause of diminished transport rate in ghosts. This was accomplished by a "pH clamping" procedure based on continued export of base equivalents by the endogenous anion exchanger. These observations suggest a critical factor required to maintain optimal Na+/H+ exchange activity is lost or inactivated during preparation of ghosts. Depletion of ATP, achieved by incubation with 2-deoxy-D-glucose, inhibited Na+/H+ exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate of Na+/H+ exchange in ATP-depleted but otherwise intact cells was similar to the transport rate of ghosts. Therefore, we tentatively conclude that full activation of NHE1 requires both ATP and an additional regulatory factor, which may mediate the action of the nucleotide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) that is inactivated or missing in ghosts.