RESUMO
In squid nerves, MgATP modulation of the Na(+)/Ca(2+) exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin superfamily of lipid-binding proteins (LBP or FABP) of 132 amino acids. We called it regulatory protein of squid nerve sodium/calcium exchanger (ReP1-NCXSQ, access to GenBank EU981897).ReP1-NCXSQ was cloned, expressed, and purified. Circular dichroism, far-UV, and infrared spectroscopy suggest a secondary structure, predominantly of beta-sheets. The tertiary structure prediction provides ten beta-sheets and two alpha-helices, characteristic of most of LPB. Functional experiments showed that, to be active, ReP1-NCXSQ must be phosphorylated by MgATP, through the action of a kinase present in the plasma membrane. Moreover, PO4-ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. An additional crucial observation was that, in proteoliposomes containing only the purified Na(+)/Ca(2+) exchanger, PO4-ReP1-NCXSQ promotes activation; therefore, this upregulation has no other requirement than a lipid membrane and the incorporated exchanger protein.Recently, we solved the crystal structure of ReP1-NCXSQ which was as predicted: a "barrel" consisting of ten beta-sheets and two alpha-helices. Inside the barrel is the fatty acid coordinated by hydrogen bonds with Arg126 and Tyr128. Point mutations showed that neither Tyr20Ala, Arg58Val, Ser99Ala, nor Arg126Val is necessary for protein phosphorylation or activity. On the other hand, Tyr128 is essential for activity but not for phosphorylation. We can conclude that (1) for the first time, a role of an LBP is demonstrated in the metabolic regulation of an ion exchanger; (2) phosphorylation of this LBP can be separated from the activation capacity; and (3) Tyr128, a candidate to coordinate lipid binding inside the barrel, is essential for activity.
Assuntos
Decapodiformes , Proteínas do Tecido Nervoso , Trocador de Sódio e Cálcio , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Clonagem Molecular , Decapodiformes/química , Decapodiformes/genética , Decapodiformes/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/isolamento & purificação , Trocador de Sódio e Cálcio/metabolismoRESUMO
The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP ß-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na(+)/Ca(2+) exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na(+)/Ca(2+) exchanger from squid nerve.
Assuntos
Decapodiformes/química , Trocador de Sódio e Cálcio/química , Animais , Decapodiformes/genética , Modelos Moleculares , Mutação , Filogenia , Estrutura Terciária de Proteína , Trocador de Sódio e Cálcio/genética , Homologia Estrutural de ProteínaRESUMO
PURPOSE: In search for new drugs derived from natural products for the possible treatment of cancer, we studied the action of agelasine B, a compound purified from a marine sponge Agelas clathrodes. METHODS: Agelasine B was purified from a marine sponge Agelas clathrodes and assayed for cytotoxicity by MTT on two human breast cancer cells (MCF-7 and SKBr3), on a prostate cancer cells (PC-3) and on human fibroblasts. Changes in the intracellular Ca(2+) concentrations were assessed with FURA 2 and by confocal microscopy. Determination of Ca(2+)-ATPase activity was followed by Pi measurements. Changes in the mitochondria electrochemical potential was followed with Rhodamine 123. Apoptosis and DNA fragmentation were determined by TUNEL experiments. RESULTS: Upon agelasine B treatment, cell viability of both human breast cancer cell lines was one order of magnitude lower as compared with fibroblasts (IC(50) for MCF-7 = 2.99 µM; SKBr3: IC(50) = 3.22 µM vs. fibroblasts: IC(50) = 32.91 µM), while the IC(50) for PC-3 IC(50) = 6.86 µM. Agelasine B induced a large increase in the intracellular Ca(2+) concentration in MCF-7, SKBr3, and PC-3 cells. By the use of confocal microscopy coupled to a perfusion system, we could observe that this toxin releases Ca(2+) from the endoplasmic reticulum (ER). We also demonstrated that agelasine B produces a potent inhibition of the ER Ca(2+)-ATPase (SERCA), and that this compound induced the fragmentation of DNA. Accordingly, agelasine B reduced the expression of the anti-apoptotic protein Bcl-2 and was able to activate caspase 8, without affecting the activity of caspase 7. CONCLUSIONS: Agelasine B in MCF-7 cells induce the activation of apoptosis in response to a sustained increase in the [Ca(2+)]( i ) after blocking the SERCA activity. The reproduction of the effects of agelasine B on cell viability and on the [Ca(2+)]( I ) obtained on SKBr3 and PC-3 cancer cells strongly suggests the generality of the mechanism of action of this toxin.
Assuntos
Agelas/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Naftalenos/farmacologia , Purinas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/isolamento & purificação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Concentração Inibidora 50 , Masculino , Microscopia Confocal , Naftalenos/administração & dosagem , Naftalenos/isolamento & purificação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Purinas/administração & dosagem , Purinas/isolamento & purificaçãoRESUMO
The Naâº/Ca²âº exchangers are structural membrane proteins, essential for the extrusion of Ca²âº from most animal cells. Apart from the transport sites, they have several interacting ionic and metabolic sites located at the intracellular loop of the exchanger protein. One of these, the intracellular Ca²âº regulatory sites, are essential and must be occupied by Ca²âº to allow any type of ion (Na⺠or Ca²âº) translocation. Intracellular protons and Na⺠are inhibitory by reducing the affinity of the regulatory sites for Ca²âº; MgATP stimulates by antagonizing H⺠and Naâº. We have proposed a kinetic scheme to explain all ionic and metabolic regulation of the squid nerve Naâº/Ca²âº exchanger. This model uniquely accounts for most of the new kinetic data provided here; however, none of the existing models can explain the trans effects of the Ca(i)²âº-regulatory sites on external cation transport sites; i.e. all models are incomplete. MgATP up-regulation of the squid Naâº/Ca²âº exchanger requires a cytosolic protein, which has been recently identified as a member of the lipocalin super family of Lipid Binding Proteins (LBP or FABP) of 132 amino acids (ReP1-NCXSQ, access to GenBank EU981897). This protein was cloned, expressed and purified. To be active, ReP1-NCXSQ must be phosphorylated from MgATP by a kinase present in the plasma membrane. Phosphorylated ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. Experiments with proteoliposomes proved that this up-regulation can take place just with the lipid membrane and the exchanger protein. The structure of ReP1-NCXSQ predicted from the amino acid sequence has been confirmed by X-ray crystal analysis; it has a "barrel" formed by ten beta sheets and two alpha helices, with a lipid coordinated by hydrogen bonds with Arg 126 and Tyr 128.
Assuntos
Sistema Nervoso/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Decapodiformes , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Sistema Nervoso/citologia , Trocador de Sódio e Cálcio/genéticaRESUMO
The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect H(i)(+) and (H(i)(+) + Na(i)(+)) inhibition of Ca(i)(2+)-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na(+)/Ca(2+) exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca(2+) concentration that seems to be the main factor responsible for cell damage.
Assuntos
Axônios/metabolismo , Cálcio/metabolismo , Decapodiformes , Estresse Oxidativo/fisiologia , Trocador de Sódio e Cálcio/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Axônios/efeitos dos fármacos , Cálcio/antagonistas & inibidores , Cálcio/farmacologia , Quelantes/farmacologia , Diálise , Ácido Egtázico/farmacologia , Peróxido de Hidrogênio/farmacocinética , Concentração de Íons de Hidrogênio , Loligo , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Ácido Peroxinitroso/farmacologia , Fenantrolinas/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/efeitos dos fármacosRESUMO
Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca(2+) entry, SOCE. Sarcoplasmic reticulum (SR) Ca(2+) stores were depleted by repetitive exposures to high K(+) or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca(2+). SR Ca(2+) store replenishment was substantially reduced using 5 microM cyclopiazonic acid (CPA). Readmission of external Ca(2+) (5 mM) increased basal [Ca(2+)](i) under two modalities. In mode 1 [Ca(2+)](i) initially increased at a rate of 0.8 +/- 0.1 nM/s and later at a rate of 12.3 +/- 2.6 nM/s, reaching a final value of 477.8 +/- 36.8 nM in 215.7 +/- 25.9 s. In mode 2, [Ca(2+)](i) increased at a rate of 0.8 +/- 0.1 nM/s to a value of 204.9 +/- 20.6 nM in 185.4 +/- 21.1 s. FCCP, 2 microM, reduced this Ca(2+) entry. In nine FCCP-poisoned fibers, the initial rate of Ca(2+) increase was 0.34 +/- 0.1 nM/s (mean +/- SEM), reaching a plateau of 149.2 +/- 14.1 nM in 217 +/- 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca(2+), we show that during depletion [Ca(2+)](mito) initially increases and later diminishes. Finally, we show that the increase in basal [Ca(2+)](i), associated with SOCE activation, diminishes upon external Na(+) withdrawal. Na(+) entry through the SOCE pathway and activation of the reversal of Na(+)/Ca(2+) exchanger could explain this SOCE modulation by Na(+).
Assuntos
Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/toxicidade , Técnicas In Vitro , Indóis/farmacologia , Camundongos , Microscopia Confocal , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismoRESUMO
In a previous work we demonstrated that, in dialyzed squid axons, an impairment of the Ca2+(i)-regulatory site affected the apparent affinities for external Na+ and Ca2+ in a way opposite to that predicted by the exiting (ping-pong) models for the exchangers. In the present work, we used model simulations and actual experiments where the Ca2+(i)-regulatory remained always saturated while [Ca2+](i) was either limiting or near saturating for the internal Ca2+ transport sites. Under these conditions, both the theoretical and experimental transport activation curves for external Na+ and Ca2+ were those expected from the current kinetic schemes. These observations have two important implications: on the one hand, they confirm the ping-pong translocation schemes for Na+/Ca2+ exchange. On the other, they call for caution in interpreting kinetic data in membrane transport systems possessing intracellular ionic and/or metabolic regulation.
Assuntos
Axônios/metabolismo , Decapodiformes/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Cinética , Modelos Biológicos , Sódio/metabolismoRESUMO
Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of beta-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten beta-sheets and two short alpha-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.
Assuntos
Trifosfato de Adenosina/farmacologia , Decapodiformes/fisiologia , Gânglios/fisiologia , Neurônios/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Regiões 5' não Traduzidas/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Cinética , Dados de Sequência Molecular , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Espectrofotometria Infravermelho , Raios UltravioletaRESUMO
We recently demonstrated that rat cerebellar Type-1 astrocytes express a very active Na(+)/Ca(2+) exchanger highly colocalized with ryanodine receptors (RyRs), which in turn play a key role in glutamate-induced Ca(2+) signaling through a calcium-induced calcium release (CICR) mechanism. In this work we have explored whether the Na(+)/Ca(2+) exchanger has any role in the Ca(2+)(i) signal induced by hypo-osmotic stress in these cells, using microspectrofluorometric measurements with Fura-2, pharmacological tools, and confocal microscopy image analysis. We present evidence for the first time that the increase in [Ca(2+)](i) in rat cerebellar Type-1 astrocytes, resulting from moderate hypotonic shock, is mediated by Ca(2+) release from ryanodine-operated Ca(2+)(i) stores, and that the magnitude of the intracellular Ca(2+) signal induced by hypotonicity in the short term (up to 240 s) is small and controlled by the activity of the Na(+)/Ca(2+) exchanger operating in its extrusion mode. With longer times in the hypotonic medium, intracellular Ca(2+) store depletion leads to Ca(2+) entry through store-operated Ca(2+) channels. We found it interesting that the activity of the Na(+)/Ca(2+) exchanger measured during this reverse mode operation (Ca(2+) entry in exchange for internal Na(+)) was found to be greatly increased in hypotonic solutions and decreased in hypertonic ones. The buffering of the [Ca(2+)](i) rise induced by hypo-osmotic stress may prevent excessive increases in [Ca(2+)](i), which otherwise might impair the normal function of this glial cell.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio , Cerebelo/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Tamanho Celular , Células Cultivadas , Cerebelo/citologia , Soluções Hipotônicas , Microscopia Confocal , Microespectrofotometria , Pressão Osmótica , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sódio/metabolismo , Espectrometria de Fluorescência , Fatores de TempoRESUMO
We propose a steady-state kinetic model for the squid Na(+)/Ca(2+) exchanger that differs from other current models of regulation in that it takes into account, within a single kinetic scheme, all ionic [intracellular Ca(2+) (Ca(i)(2+))-intracellular Na(+) (Na(i)(+))-intracellular H(i)(+)] and metabolic (ATP) regulations of the exchanger in which the Ca(i)(2+)-regulatory pathway plays the central role in regulation. Although the integrated ionic-metabolic model predicts all squid steady-state experimental data on exchange regulation, a critical test for the validity of it is the predicted dual effect of Na(i)(+) on steady-state Ca(2+) influx through the exchanger. To test this prediction, an improved technique for the estimation of isotope fluxes in squid axons was developed, which allows sequential measurements of ion influx and effluxes. With this method, we report here two novel observations of the squid axon Na(+)/Ca(2+) exchanger. First, at intracellular pH (7.0) and in the absence of MgATP, Na(i)(+) has a dual effect on Ca(2+) influx: inhibition at low concentrations followed by stimulation at high Na(i)(+) concentrations, reaching levels higher than those seen without Na(i)(+). Second, in the presence of MgATP, the biphasic response to Na(i)(+) disappears and is replaced by a sigmoid activation. Furthermore, the model predicts that Ca(2+) efflux is monotonically inhibited by Na(i)(+), more pronouncedly without than with MgATP. These results are predicted by the proposed kinetic model. Although not fully applicable to all exchangers, this scheme might provide some insights on expected net Ca(2+) movements in other tissues under a variety of intracellular ionic and metabolic conditions.
Assuntos
Axônios/metabolismo , Cálcio/metabolismo , Condução Nervosa , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Diálise , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Loligo , Potenciais da Membrana , Modelos Neurológicos , Reprodutibilidade dos TestesRESUMO
In squid axons, intracellular Mg2+ reduces the activity of the Na+/Ca2+ exchanger by competing with Ca2+ i for its regulatory site. The state of the Ca i-regulatory site (active-inactive) also alters the apparent affinity of intra- and extracellular transport sites. Conditions that hinder the binding of Ca2+ i (low pH i, low [Ca2+]i, high [Mg2+]i) diminish the apparent affinity of intracellular transport sites, in particular for Na i due to its synergism with H+ inhibition, but less noticeably for Ca2+ i because of its antagonism towards (Ha i + Na+ i) and Mg2+ i inhibitions. These are kinetic effects unrelated to the true affinity of the sites. With the Ca i-regulatory site saturated, the intracellular transporting sites are insensitive to [H+]i and to ATP. Likewise, the state of the Ca i-regulatory site (activated or inactivated) influences the affinity of the extracellular Ca o and Na o-transport sites (trans effects). In this case, the effects are opposite to those predicted by any of the transport schemes proposed for the Na+/Ca2+exchanger; i.e. its mechanism remains unexplained. In addition to their intrinsic importance for a full understanding of the properties of the Na+/Ca2+ exchanger, these findings show a new way by which the state of the Ca i-regulatory site may determine net movements of Ca2+ through this system.
Assuntos
Axônios/metabolismo , Cálcio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ligação Competitiva , Concentração de Íons de Hidrogênio , Cinética , Loligo , Magnésio/metabolismo , Ligação ProteicaRESUMO
In squid nerve MgATP upregulation of Na+/Ca2+ exchange requires a soluble cytosolic regulatory protein (SCRP) of about 13 kDa; phosphoarginine (PA) stimulation does not. MgATP-gamma-S mimics MgATP. When a 30-10-kDa cytosolic fraction is exposed to 0.5 mM [32P]ATP in the same solution used for transport assays, and in the presence of native membrane vesicles, a 13-kDa and a 25-kDa band become phosphorylated. Membrane vesicles alone do not show these phosphorylated bands and heat denaturation of the cytosolic fraction prevents phosphorylation. Moreover, staurosporine, a general inhibitor of kinases, does not affect MgATP + SCRP stimulation of the exchanger or the phosphorylation of the 13 kDa but prevents phosphorylation of the 25-kDa cytosolic band. The 30-10-kDa fraction phosphorylated in the presence of staurosporine stimulates Na+/Ca2+ exchange in vesicles in the absence of ATP but with Mg2+ in the medium. The 30-10-kDa fraction is not phosphorylated by PA. In membrane vesicles two protein bands, at about 60 kDa and 70 kDa identified as the low molecular weight neurofilament (NF), are phosphorylated by PA, but not by MgATP. This phosphorylation is specific for PA, insensitive to staurosporine (similar to the PA-stimulated fluxes), and labile. In addition, co-immunoprecipitation was observed between the NF and the exchanger protein. Under the conditions of these experiments no phosphorylation of the exchanger is detected, either with MgATP or PA.
Assuntos
Trifosfato de Adenosina/farmacologia , Arginina/análogos & derivados , Sistema Nervoso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Arginina/farmacologia , Decapodiformes , Compostos Organofosforados/farmacologia , Fosforilação , Estaurosporina/farmacologiaRESUMO
We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/fisiologia , Astrócitos/metabolismo , Cálcio/fisiologia , Cerebelo/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Cátions , Imunofluorescência , Técnicas In Vitro , Microscopia Confocal , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Espectrometria de FluorescênciaRESUMO
The Na(+)/Ca(2+) exchanger's family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding Ca(2+) from the cell. Two basic properties characterize them. 1) Their activity is not predicted by thermodynamic parameters of classical electrogenic countertransporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na(+) and Ca(2+)) and nontransported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra-, intra-, and transmembrane protein domains. 2) Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This review emphasizes the interrelationships between ionic and metabolic modulations of Na(+)/Ca(2+) exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP-related pathways in these two systems is that although they are different (phosphatidylinositol bisphosphate in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: Na(+)-Ca(2+)-H(+) interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger is discussed in detail as well as its relevance in cellular Ca(i)(2+) homeostasis.
Assuntos
Trocador de Sódio e Cálcio/metabolismo , Trocador de Sódio e Cálcio/fisiologia , Animais , Axônios/metabolismo , Evolução Biológica , Transporte Biológico Ativo , Decapodiformes , Humanos , Íons/metabolismo , Trocador de Sódio e Cálcio/químicaRESUMO
Sphingolipids comprise a very important class of second messengers involved in cell growth, differentiation, and apoptosis, among other different functions. Recently, these lipids have been implicated in calcium mobilization in different cell lines, including Jurkat T-lymphocytes. However, the effect of each particular sphingolipid appears to be cell-line specific. Among them, the least studied is ceramide-1-P (Cer-1-P). Here, we show that Cer-1-P increased the intracellular Ca(2+) concentration in Jurkat T-cells. Furthermore, laser-scanning confocal microscopy indicated that Ca(2+) is released from the endoplasmic reticulum. An effect on store-operated Ca(2+) channels was evidenced by whole-cell "patch clamp" measurements after Cer-1-P induced Ca(2+) store depletion. The mechanism of action of Cer-1-P resembles that of the Jurkat anti-TCR antibody, but differs from that of ceramide, since Cer-1-P induced an increase in Ins(1,4,5)-P(3).
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Ceramidas/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Linfócitos T/efeitos dos fármacos , Humanos , Células Jurkat , Técnicas de Patch-Clamp , Linfócitos T/metabolismoRESUMO
The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/química , Ceramidas/metabolismo , Citoplasma/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Ceramidas/química , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/química , Células Jurkat , Linfócitos/metabolismo , Magnésio/química , Manganês/química , Microscopia Confocal , Técnicas de Patch-Clamp , Transdução de Sinais , Esfingolipídeos/química , Esfingosina/química , Tapsigargina/farmacologia , Fatores de TempoRESUMO
The effects of a new, potent, and selective inhibitor of the Na(+)/Ca(2+) exchange, SEA-0400 (SEA), on steady-state outward (forward exchange), inward (reverse exchange), and Ca(2+)/Ca(2+) transport exchange modes were studied in internally dialyzed squid giant axons from both the extra- and intracellular sides. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: it increases synergic intracellular Na(+) (Na(i)(+)) + intracellular H(+) (H(i)(+)) inactivation, is antagonized by ATP and intracellular alkalinization, and is enhanced by intracellular acidification even in the absence of Na(+). Inhibition by SEA is still present even after 1 h of its removal from the experimental solutions, whereas removal of the cointeracting agents of inhibition, Na(i)(+) and H(i)(+), even in the continuous presence of SEA, releases inhibition, indicating that SEA facilitates the reversible attachment of the natural H(i)(+) and Na(i)(+) synergic inhibitors. On the basis of a recent model of squid Na(+)/Ca(2+) exchange regulation (DiPolo R and Beauge L. J Physiol 539: 791-803, 2002), we suggest that SEA acts on the H(i)(+) + Na(i)(+) inactivation process and can interact with the Na(+)-free and Na(+)-bound protonized carrier. Protection by ATP concurs with the antagonism of the nucleotide by H(i)(+) + Na(i)(+) synergic inhibition.
Assuntos
Compostos de Anilina/farmacologia , Neurônios/fisiologia , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Decapodiformes , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Trocador de Sódio e Cálcio/antagonistas & inibidoresRESUMO
In squid nerves the Na(+)-Ca(2+) exchanger is up-regulated by ATP and phosphoarginine (PA). ATP regulation involves drastic alterations in the Na(+)(i), H(+)(i) and Ca(2+)(i) interactions with the large intracellular cytoplasmic loop of the exchanger protein. In this work we explored the mechanisms associated with PA regulation in intracellular dialysed squid axons and squid optic nerve membrane vesicles. Dialysed axons were used to measure the four modes of exchange fluxes (Na(+)(o)-Ca(2+)(i) or forward exchange, Ca(2+)(o)-Na(+)(i) or reverse exchange, Ca(2+)(o)-Ca(2+)(i) exchange and Na(+)(o)-Na(+)(i) exchange) under controlled intra- and extracellular conditions. Inside-out membrane vesicles allowed measurement of the Na(+)-gradient-dependent (45)Ca(2+) uptake (forward mode) as influenced by ligands and digestion with chymotrypsin from the intracellular side. The results show that, unlike ATP, PA regulation does not affect the H(+)(i), Na(+)(i) and Ca(2+)(i) interactions with the intracellular 'regulatory' loop, but increases the affinity of the intracellular transport sites, preferentially for Ca(2+)(i) (about 20-fold) over Na(+)(i) (50%); i.e. PA favours the forward mode over the other exchange modes. Intracellular chymotrypsin digestion removed ATP regulation while leaving modulation by PA unmodified. Western blot analysis suggested that chymotrypsin disrupts the large intracellular loop. Together these results indicate that ATP and PA regulations are associated with different structures inside and outside the exchanger protein. Based on these observations we expanded our previous model for metabolic regulation of the Na(+)-Ca(2+) exchanger by adding to the original 'ATP region' a new zone, the 'PA region', related to the intracellular transport sites for Na(+)(i) and Ca(2+)(i). This new model is able to explain most previous and present results.
Assuntos
Trifosfato de Adenosina/farmacologia , Arginina/análogos & derivados , Arginina/farmacologia , Nervo Óptico/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Trocador de Sódio e Cálcio/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Decapodiformes , Relação Dose-Resposta a Droga , Técnicas In Vitro , Ligantes , Nervo Óptico/fisiologia , Trocador de Sódio e Cálcio/agonistasRESUMO
Previous studies have shown that in contrast to other neuronal cells, Na(+)/Ca(2+) exchange contributes little to Ca(i)(2+) homeostasis in rat cerebellar Purkinje neurons under intracellular perfused conditions and at room temperature [Fierro et al.: J Physiol (Lond) 510: 499-512, 1998]. The purpose of this study was to clarify the role of this transporter in cerebellar Purkinje neurons by using intact cells at nearly physiological body temperature. Using Fluo-3 microfluorometry, we have examined the role of the Na(+)/Ca(2+) exchange in the buffering of calcium loads in cultured rat Purkinje neurons at two temperatures: 20 and 34 degrees C. At 20 degrees C, the recovery of the K(+)-induced [Ca(2+)](i) signal was little affected by the presence of external Na(+) (tau(e) = 35.5 +/- 1.2 s [n = 49]), or by its absence (tau(e) = 36.6 +/- 2.2 s [n = 29]), i.e. in a Li(+)-containing medium. In contrast, at 34 degrees C, the recovery of the [Ca(2+)](i) signal was highly dependent on external Na, i.e. tau(e) = 19.9 +/- 1.2 s (n = 119) and tau(e) = 41.7 +/- 2.6 s (n = 39), in Li(+)-containing media, respectively. A comparison of the rate of clearance of [Ca(2+)](i) in Na(+) or Li(+) media, shows that at a room temperature of 20 degrees C, the Na(+)/Ca(2+) exchange contributes at most to 15-20% of the total [Ca(2+)](i) clearance, compared to 55-65% at 34-36 degrees C. We also demonstrate that under normal physiological conditions forward and reverse Na(+)/Ca(2+) exchanges operate in the same neuron. We conclude that the Na(+)/Ca(2+) exchange is strongly suppressed at room temperature and therefore its role should be reevaluated among different neuronal preparations.
Assuntos
Cálcio/metabolismo , Células de Purkinje/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Animais , Técnicas de Cultura de Células , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , TemperaturaRESUMO
Intracellular Na(+) and H(+) synergistically inhibit the squid Na(+)/Ca(2+) exchanger by reducing the affinity for Ca(2+) of its regulatory site. MgATP antagonizes H(+)(i) and Na(+)(i) inhibition; this effect must occur through a phosphorylation-dephosphorylation process, because exogenous protein phosphatases prevent MgATP activation of the exchanger. Protection by ATP against H(+)(i) and Na(+)(i) inhibition happens by decreasing the apparent affinity for the synergistic binding of these cations to the carrier. In this way ATP modifies the apparent affinity for Ca(2+) of its regulatory site. Mg(2+) ions play an important role in the process because they are essential for ATP activation of Na(+)/Ca(2+) exchange but can also promote deactivation of the ATP upregulated exchanger. At constant [ATP], activation at low [Mg(2+)](i) is followed by deactivation as [Mg(2+)](i) is increased. The most likely explanation for deactivation is stimulation of endogenous phosphatases. We developed a kinetic model that predicts all H(+)(i), Na(+)(i), and MgATP described above. This scheme includes the following conditions: (i) The binding of Ca(2+) to the regulatory site is essential for the binding of Na(+)(i) or Ca(2+)(i) to the transporting sites. (ii) The binding of a first H(+)(i) to the carrier displaces Ca(2+)(i) from its regulatory site and allows binding of one Na(+) forming a H.E(1).Na complex. The H.E(1).Na complex can bind a second H(+)(i) forming a dead-end inhibitory H(2).E(1).Na complex. (iii) MgATP, through an unspecified phosphorylation process, decreases the apparent affinity for the synergistic H(+)(i) and Na(+)(i) binding to the carrier.