RESUMO
Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.
Assuntos
Cálcio , alfa-Sinucleína , Cálcio/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de LigaçãoRESUMO
The primary function of angiotensin II, the main peptide of central renin-angiotensin system (RAS), is regulation of blood pressure. Recently, new functions of so-called local (or tissue) RAS have been discovered in brain. AT1 and AT2 angiotensin receptors, found in many parts of central nervous system (CNS), stimulate various signalling pathways. Gamma-amniobutyric acid (GABA), which acts by three types of receptors, is the crucial inhibitory neurotransmitter. GABA and angiotensins are found in brain regions like paraventricular nucleus of hypothalamus, nucleus tractus solitari and rostral ventrolateral medulla, all involved in blood pressure regulation. The influence of angiotensin II on GABA action is different in various CNS regions, but mainly it is associated with cardiovascular neurons activity. There are other neurotransmitters which may interact with angiotensins action. Adenosine has inhibitory effect and play important role in epilepsy. Its beneficial influence may be stronger in presence of angiotensin. Angiotensins also interact with dopamine (DA) activity by stimulation of DA-synthesizing nerves.
Assuntos
Angiotensinas/metabolismo , Encéfalo/metabolismo , Neurotransmissores/metabolismo , Adenosina/metabolismo , Animais , Epilepsia/metabolismo , Humanos , Neurônios/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Calmodulin (CaM) is a sensor protein, which takes part in calcium-dependent signaling, regulating processes like growth, differentiation, proliferation and motility. Calmodulin binds calcium ions during induction of intracellular signaling. It is also involved in silencing of calcium signal through activation of plasma membrane Ca(2+)-ATPase (directly) or SERCA pump (indirectly). Calmodulin may affect various channels, e.g. voltage gated calcium channels (VGCCs), transient receptor potential channels (TRPCs), NMDA receptors, calcium channels dependent on cyclic nucleotides or these located in endoplasmic reticulum (ryanodine receptors and all isoforms of IP3-dependent receptors).
Assuntos
Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , Animais , Canais de Cálcio/metabolismo , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
GABA (γ-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.
Assuntos
ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Ácido gama-Aminobutírico/biossíntese , Animais , Linhagem Celular , Glutamato Descarboxilase/biossíntese , Glutamato Descarboxilase/genética , Isoenzimas/genética , Isoenzimas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , RatosRESUMO
Gamma-aminobutyric acid is a major inhibitory neurotransmitter in the central nervous system. GABA metabolism is dependent on the activity of three enzymes: glutamic acid decarboxylase, GABA-transaminase and succinic semialdehyde dehydrogenase. Decreased activity of these enzymes may cause many neurological syndromes, such as stiff-person syndrome, chronic fatigue syndrome, anxiety disorders and seizures. This article is a review of most important problems related to an impairment of GABA metabolism.
Assuntos
Doenças do Sistema Nervoso Central/enzimologia , Ácido gama-Aminobutírico/metabolismo , Transtornos de Ansiedade/enzimologia , Síndrome de Fadiga Crônica/enzimologia , Humanos , Convulsões/enzimologia , Rigidez Muscular Espasmódica/enzimologiaRESUMO
Angiotensin II (AngII), the main peptide of the reninangiotensin system (RAS), is involved in the proliferation of different types of cells, normal and pathological as well. The protein tyrosine kinases (PTKs) play an important role in the growth, differentiation and apoptosis of cells. AngII action depends on the hormonal milieu of the cell, and on sex steroid influence. Angiotensin 17 (Ang17), metabolite of AngII, shows opposite action to AngII in cells. The present study aimed to examine the influence of 17ßestradiol and testosterone on AngII and Ang17 action on PTK activity in androgenindependent humane prostate cancer cell line DU145. Cell cultures of human prostate cancer DU145 cells were used as a source of PTKs. Cultures were exposed to different concentrations of AngII (5x1011 to 5x109 M). The incubation with hormones lasted 15 min to limit the genomic effects of steroids. In the phosphorylation reaction, we used γ32PATP as a donor of phosphate and a synthetic peptide, Poly(Glu, Tyr) (4:1), as a substrate. The specific activities of PTKs were defined as pmol of 32P incorporated into 1 mg of exogenous Poly(Glu, Tyr) per minute (pmol/mg/min). Our findings suggest that testosterone and 17ßestradiol may change the effects of angiotensins in a rapid nongenomic way, probably via membranelocated receptors. The most significant change was caused by testosterone, whose effect was most significant on changes caused by Ang17. AngIIinduced changes in phosphorylation appeared to be insensitive to the presence of testosterone, but were modified by 17ßestradiol.
Assuntos
Androgênios/metabolismo , Angiotensinas/farmacologia , Estradiol/farmacologia , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Quinases/metabolismo , Testosterona/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Esteroides/metabolismo , Esteroides/farmacologiaRESUMO
A close link between Ca(2+), ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca(2+) may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca(2+) in cytosol. In differentiation process plasma membrane Ca(2+) ATPase (PMCA) is considered as one of the major players for Ca(2+) homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2 consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher [Ca(2+)]c resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.
Assuntos
Diferenciação Celular , Metabolismo Energético , Inativação Gênica , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Morte Celular , Respiração Celular , Citosol/metabolismo , Regulação para Baixo , Citometria de Fluxo , Glucose/metabolismo , Glicólise , Isoenzimas/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Fosforilação Oxidativa , Células PC12 , Ratos , Tubulina (Proteína)/metabolismoRESUMO
Plasma membrane Ca(2+)-ATPase (PMCA) by extruding Ca(2+) outside the cell, actively participates in the regulation of intracellular Ca(2+) concentration. Acting as Ca(2+)/H(+) counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca(2+) overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca(2+) clearance and partially attenuated cellular acidification during KCl-stimulated Ca(2+) influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca(2+) overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca(2+)-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Mitocôndrias/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Ácido Bongcréquico/farmacologia , Diferenciação Celular , Membrana Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Homeostase/fisiologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Células PC12 , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Cloreto de Potássio/farmacologia , Ratos , Transdução de SinaisRESUMO
Microsomal glutathione-S-transferase 1 (Mgst1) plays a specific role in protection of cells against oxidative stress. In this study, we assayed the effect of Mgst1 downregulation on cells behavior using differentiated PC12 line, a widely accepted neuronal model system. We have developed stable transfected cells with downregulated Mgst1 (PC12_M), which were differentiated with 1 mM dibutyryl-cAMP (db-cAMP). Mgst1 reduction induced necrosis, decreased ATP amount, and increased thiobarbituric acid reacting substances (TBARS) content. However, in PC12_M cell population, we detected more intensive neuritogenesis than that in mock-transfected cells. Interestingly, total glutathione as well as GSH level were significantly higher than those in control PC12 line. Real-time PCR and Western blot analyses showed elevated expression of enzymes involved in glutathione metabolism-a rate-limiting γ-glutamylcysteine ligase and glutathione reductase. The present study shows for the first time that under stress conditions induced by Mgst1 downregulation, a rescue pathway can be activated and thereby enables differentiated PC12 cells to survive. Since Mgst1expression was reported to decline with age, our results could represent a putative adaptive process during aging. It could also be an early mechanism protecting neuronal cells against some neurodegenerative insults.
Assuntos
Diferenciação Celular , Regulação para Baixo , Glutationa Transferase/metabolismo , Microssomos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Primers do DNA , Citometria de Fluxo , Células PC12 , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Changes in PMCA2 and PMCA3 expression during neuronal development are tightly linked to structural and functional modifications in Ca(2+) handling machinery. Using antisense strategy we obtained stably transfected PC12 lines with reduced level of PMCA2 or PMCA3, which were then subjected to dibutyryl-cAMP differentiation. Reduced level of neuron-specific PMCAs led to acceleration of differentiation and formation of longer neurites than in control PC12 line. Treatment with dibutyryl-cAMP was associated with retraction of growth cones and intensified formation of varicosities. In PMCA2-reduced cells development of apoptosis and DNA laddering were detected. Higher amounts of constitutive isoforms PMCA1 and PMCA4, their putative extended location to gaps left after partial removal of PMCA2 or PMCA3, together with increased SERCA may indicate the induction of compensatory mechanism in modified cells. Functional studies showed altered expression of certain types of VDCCs in PMCA-reduced cells, which correlated with their higher contribution to Ca(2+) influx. The cell response to PMCAs suppression suggests the interplay between transcription level of two opposite calcium-transporting systems i.e. voltage- and store depletion-activated channels facilitating Ca(2+) influx and calcium pumps responsible for Ca(2+) clearance, as well highlights the role of both neuron-specific PMCA isoforms in the control of PC12 cells differentiation.