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/metabolismo

RESUMO

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 & purificação

RESUMO

The Na⁺/Ca²âº exchangers are structural membrane proteins, essential for the extrusion of Ca²âº 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 Ca²âº regulatory sites, are essential and must be occupied by Ca²âº to allow any type of ion (Na⁺ or Ca²âº) translocation. Intracellular protons and Na⁺ are inhibitory by reducing the affinity of the regulatory sites for Ca²âº; MgATP stimulates by antagonizing H⁺ and Na⁺. We have proposed a kinetic scheme to explain all ionic and metabolic regulation of the squid nerve Na⁺/Ca²âº 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)²âº-regulatory sites on external cation transport sites; i.e. all models are incomplete. MgATP up-regulation of the squid Na⁺/Ca²âº 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/genética

RESUMO

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/metabolismo

RESUMO

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/metabolismo

RESUMO

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 Ultravioleta

RESUMO

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 Proteica

RESUMO

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 Testes

RESUMO

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/farmacologia

RESUMO

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 Fluorescência

RESUMO

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/química

RESUMO

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/metabolismo

RESUMO

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 Tempo

RESUMO

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 & inibidores

RESUMO

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/agonistas

RESUMO

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 , Temperatura

RESUMO

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.

Assuntos

Axônios/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/farmacologia , Decapodiformes , Homeostase , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Modelos Biológicos , Nervo Óptico/fisiologia , Sódio/farmacologia

RESUMO

In the last decade, there has been a large increase in the study of the Na(+)/Ca(2+) exchanger due to its implications in physiological and pathophysiological processes at the cell and organ levels. Key areas of these studies have been molecular biology, regulation and physiology-pathophysiology of the exchanger. There are three main types of regulation that take place at the large intracellular loop of the Na(+)/Ca(2+) exchanger: (i) ionic (sodium inactivation, calcium regulation and proton inhibition), (ii) metabolic (ATP as phosphoryl group donor), and (iii) genetic (alternative splicing). This review analyzes the most recent data on the mutual interactions of regulatory ionic ligands (Ca(2+), Na(+), H(+)) and how they are secondarily modulated by MgATP, emphasizing the importance of the binding of Ca(2+) to its regulatory site as an essential requirement for the exchange function. Intracellular protons and sodium inhibit the Na(+)/Ca(2+) exchanger by reducing the apparent affinity of the Ca(i)-regulatory site for Ca(2+). Although the metabolic pathways are different in the mammalian heart (membrane lipids) and squid nerve cells (soluble cytosolic regulatory protein), the final mechanism for the protective effect of MgATP is the same: a reduction of Na(i)(+)-H(i)(+) binding affinities facilitating the attachment of Ca(2+) to its regulatory site. Kinetic models, which partially analyzed some of these ionic and metabolic interactions, can be integrated into a single scheme where the Ca(i)-regulatory site plays a central role.

Assuntos

Trifosfato de Adenosina/metabolismo , Trocador de Sódio e Cálcio/química , Animais , Sítios de Ligação , Cátions , Cinética , Ligantes , Modelos Biológicos , Fosforilação , Prótons , Trocador de Sódio e Cálcio/metabolismo

RESUMO

Intracellular Na(+) and H(+) inhibit Na(+)-Ca(2+) exchange. ATP regulates exchange activity by altering kinetic parameters for Ca(2+)(i), Na(+)(i) and Na(+)(o). The role of the Ca(2+)(i)regulatory site on Na(+)(i)-H(+)(i)-ATP interactions was explored by measuring the Na(+)(o)-dependent (45)Ca(2+) efflux (Na(+)(o)-Ca(2+)(i) exchange) and Ca(2+)(i)-dependent (22)Na(+) efflux (Na(+)(o)-Na(+)(i) exchange) in intracellular-dialysed squid axons. Our results show that: (1) without ATP, inhibition by Na(+)(i) is strongly dependent on H(+)(i). Lowering the pH(i) by 0.4 units from its physiological value of 7.3 causes 80 % inhibition of Na(+)(o)-Ca(2+)(i) exchange; (2) in the presence of MgATP, H(+)(i) and Na(+)(i) inhibition is markedly diminished; and (3) experiments on Na(+)(o)-Na(+)(i) exchange indicate that the drastic changes in the Na(+)(i)-H(+)(i)-ATP interactions take place at the Ca(2+)(i) regulatory site. The increase in Ca(2+)(i) affinity induced by ATP at acid pH (6.9) can be mimicked by a rise in pH(i) from 6.9 to 7.3 in the absence of the nucleotide. We conclude that ATP modulation of the Na(+)-Ca(2+) exchange occurs by protection from intracellular proton and sodium inhibition. These findings are predicted by a model where: (i) the binding of Ca(2+) to the regulatory site is essential for translocation but not for the binding of Na(+)(i) or Ca(2+)(i) to the transporting site; (ii) H(+)(i) competes with Ca(2+)(i) for the same form of the exchanger without an effect on the Ca(2+)(i) transporting site; (iii) protonation of the carrier increases the apparent affinity and changes the cooperativity for Na(+)(i) binding; and (iv) ATP prevents both H(+)(i) and Na(+)(i)-effects. The relief of H(+) and Na(+) inhibition induced by ATP could be important in cardiac ischaemia, in which a combination of acidosis and rise in [Na(+)](i) occurs.

Assuntos

Trifosfato de Adenosina/farmacologia , Axônios/metabolismo , Membranas Intracelulares/metabolismo , Prótons , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trifosfato de Adenosina/deficiência , Animais , Cálcio/metabolismo , Decapodiformes , Diálise , Sódio/farmacologia

RESUMO

El uso de la preparación de axón gigante de calamar para realizar estudios de homeostasis de calcio ha sentado las bases para el entendimiento de los procesos que regulan el calcio iónico intracelular ([Ca2+]). En particular, el transportador Na/Ca ha podido ser estudiado en términos de aquellos procesos que modulan su actividad. Este sistema de contra-transporte juega un papel crítico en fenómenos fisiológico y fisiopatológicos como son la contractilidad cardíaca, la foto-recepción, la hipertensión arterial esencial y las arritmias cardíacas. Bajo condiciones fisiológicas la habilidad del intercambiador Na/Ca para regular la [Ca2+] depende tanto de la dirección (salida o entrada de calcio), como de la magnitud del transporte Na/Ca. La dirección de Ca depende del gradiente electroquímico para sodio y calcio (factores termodinámicos); la magnitud del transporte va a estar regulada por factores de tipo cinéticos. Recientemente se ha realizado un importante esfuerzo en elucidar mecanismos que regulan la actividad del intercambiador Na/Ca. En particular la modulación por nucleóticos y por [Ca2+]. En la hora nerviosa hemos encontrado que MgTP estimula la actividad del intercambiador Na/Ca a través de un proceso de fosforilación-desfosforilación probablemente mediada por un sistema de quinasa/fosfatasa. Hemos demostrado la presencia de un sitio regulatorio para iones calcio de alta afinidad, localizado en la cara citoplasmática del intercambiador cuya función sería la de controlar su actividad. En este artículo describimos los hallazgos más relevantes referentes a la regulación metabólica o iónica del intercambiador Na/Ca

Assuntos

Axônios , Cálcio , Decapodiformes , Homeostase , Troca Iônica , Íons , Nucleotídeos