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Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporters and efflux by Na+ /Ca2+ exchangers (NCLX). Here, we describe a novel relationship among NCLX, intracellular Ca2+ , and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.
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Señalización del Calcio , Calcio , Clonazepam/análogos & derivados , Tiazepinas , Señalización del Calcio/fisiología , Calcio/metabolismo , Intercambiador de Sodio-Calcio , Mitocondrias/metabolismo , Autofagia , Sodio/metabolismoRESUMEN
BACKGROUND: The high similarity in anatomical and neurophysiological processes between pigs and humans make pigs an excellent model for metabolic diseases and neurological disorders. Lipids are essential for brain structure and function, and the polyunsaturated fatty acids (PUFA) have anti-inflammatory and positive effects against cognitive dysfunction in neurodegenerative diseases. Nutrigenomics studies involving pigs and fatty acids (FA) may help us in better understanding important biological processes. In this study, the main goal was to evaluate the effect of different levels of dietary soybean oil on the lipid profile and transcriptome in pigs' brain tissue. RESULTS: Thirty-six male Large White pigs were used in a 98-day study using two experimental diets corn-soybean meal diet containing 1.5% soybean oil (SOY1.5) and corn-soybean meal diet containing 3.0% soybean oil (SOY3.0). No differences were found for the brain total lipid content and FA profile between the different levels of soybean oil. For differential expression analysis, using the DESeq2 statistical package, a total of 34 differentially expressed genes (DEG, FDR-corrected p-value < 0.05) were identified. Of these 34 DEG, 25 are known-genes, of which 11 were up-regulated (log2 fold change ranging from + 0.25 to + 2.93) and 14 were down-regulated (log2 fold change ranging from - 3.43 to -0.36) for the SOY1.5 group compared to SOY3.0. For the functional enrichment analysis performed using MetaCore with the 34 DEG, four pathway maps were identified (p-value < 0.05), related to the ALOX15B (log2 fold change - 1.489), CALB1 (log2 fold change - 3.431) and CAST (log2 fold change + 0.421) genes. A "calcium transport" network (p-value = 2.303e-2), related to the CAST and CALB1 genes, was also identified. CONCLUSION: The results found in this study contribute to understanding the pathways and networks associated with processes involved in intracellular calcium, lipid metabolism, and oxidative processes in the brain tissue. Moreover, these results may help a better comprehension of the modulating effects of soybean oil and its FA composition on processes and diseases affecting the brain tissue.
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Aceite de Soja , Transcriptoma , Animales , Masculino , Encéfalo , Calcio , Dieta/veterinaria , Ácidos Grasos , Aceite de Soja/farmacología , PorcinosRESUMEN
Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+ /Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+ -dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.
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Astrocitos , Calcio , Ratones , Animales , Astrocitos/metabolismo , Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Mitocondrias/metabolismo , Glucólisis , Cognición , Sodio/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
RESUMEN El objetivo de este estudio fue evaluar los efectos de diferentes niveles de dureza total del agua en la muda, calcificación del exoesqueleto, crecimiento y supervivencia de Cryphiops caementarius. Los camarones machos fueron colectados del río Pativilca (Lima, Perú) y cultivados en recipientes individuales dentro de acuarios (55 L). Se emplearon cuatro niveles de dureza total del agua (100, 200, 300 y 400 mg CaCO3 L-1), con tres repeticiones, respectivamente. Los camarones cultivados en agua con dureza de 300 mg L-1 tuvieron menor periodo de muda (26,7 días) y mayor frecuencia de mudas (tres mudas). El contenido de calcio del exoesqueleto del camarón incrementó (p < 0,05) de 25 a 31 % en agua con dureza de 100 y 400 mg L-1, respectivamente. El mayor grosor del exoesqueleto (144 -jm en cefalotórax y 131 μm en abdomen) fue obtenido en agua con dureza de 400 mg L-1 y el menor grosor (93 -jm en cefalotórax y abdomen) en 100 mg L-1. El crecimiento en longitud fue mayor (p < 0,05) en agua con durezas de 200 y 300 mg L-1. El crecimiento en peso fue similar (p > 0,05) entre tratamientos. La mayor supervivencia (> 94,4 %) se mantuvo en agua con durezas de hasta 300 mg L-1 y la menor supervivencia (77,8 %) fue con 400 mg L-1. La dureza total del agua de 200 y 300 mg L-1 es conveniente para el cultivo del camarón, pero dureza del agua mayor o menor a este rango afectan la muda, el crecimiento y la supervivencia por deficiencia o exceso de calcio acumulado, respectivamente en el exoesqueleto del camarón.
ABSTRACT The aim of this work was to evaluate the effects of different levels of total hardness on the molting, calcification of exoskeleton, growth and survival of Cryphiops caementarius prawn. Male prawns were collected from Pativilca river (Lima, Peru) and cultivated in individual containers inside aquariums (55 L). Four levels of the total hardness of water (100, 200, 300 and 400 mg CaCO3 L-1) were used, with three repetitions, respectively. Prawns cultured in water with hardness of 300 mg L-1 has a shorter molting period (26.7 days) and a higher frequency of molts (three molts). The calcium content of the prawn exoskeleton increased (p < 0.05) from 25 to 31 % in water with hardness of 100 and 400 mg L-1, respectively. The greatest thickness of the exoskeleton (144 -jm cephalothorax and 131 -jm abdomen) was obtained in water with a hardness of400 mg L-1 and the smallest thickness (93 -jm in the cephalothorax and abdomen) in 100 mg L-1. The growth in length was greater (p < 0.05) in water with hardness of 200 and 300 mg L-1. The weight growth was similar (p > 0.05) between treatments. The highest survival (> 94.4 %) was maintained in water with hardness up to 300 mg L-1 and the lowest survival (77.8 %) was at 400 mg L-1. The total hardness of 200 and 300 mg L-1 is suitable for prawn farming, but hardness high or lower than this range affects the molting, growth and survival due to deficiency or excess of accumulated calcium, respectively, in the prawn exoskeleton.
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The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms "x" and "z". While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants "x" and "z", maintaining constant the most frequent splice variant "b" at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant "z" of the splicing site A.
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Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the assessment of such mitochondrial activities in several working models. Pollen tube mitochondrial studies represent one example of this, where tests have been often restricted due the physical impossibility of performing experiments with isolated mitochondria in enough quantities. Here we detail a method to measure in situ mitochondrial respiratory chain activity and calcium transport in tobacco pollen tubes. â¢Digitonin-mediated plasmalemma permeabilization allows efficient assessment of mitochondrial respiration and calcium uptake.â¢This method allows quick, reliable and portable measurements from low to high cellular densities, versus methods requiring intracellular calcium reporters.
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Calcium oxalate (CaOx) crystals occur as intravacuolar deposits in most angiosperm species. Different functions have been attributed to these crystals, some of which are very speculative, until now. Calcium regulation and homeostasis seem to be the most widespread function of CaOx crystals. Being rich in calcium, these crystals constitute a reserve of calcium for plants. However, despite being bioavailable, this reserve is functional in just a few situations due to the low mobility of calcium for phloem translocation. Therefore, CaOx crystals as a calcium reserve is a paradox because in most cases the reserve cannot be used. However, in most plants, these crystals occur in organs or tissues that will be discarded, which allows the elimination of excess calcium. This suggests that CaOx crystals have a functional role in excess calcium excretion. There is some evidence that, for calcium, this excretory function is relevant for plants since they lack an excretory system dedicated to discarding solid wastes, such as calcium salts.
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Oxalato de Calcio/metabolismo , Calcio/metabolismo , Plantas/metabolismo , Transporte Biológico , CristalizaciónRESUMEN
In addition to their role in providing cellular energy, mitochondria fulfill a key function in cellular calcium management. The present review provides an integrative view of cellular and mitochondrial calcium homeostasis, and discusses how calcium regulates mitochondrial dynamics and functionality, thus affecting various cellular processes. Calcium crosstalk exists in the domain created between the endoplasmic reticulum and mitochondria, which is known as the mitochondriaassociated membrane (MAM), and controls cellular homeostasis. Calcium signaling participates in numerous biochemical and cellular processes, where calcium concentration, temporality and durability are part of a regulated, finely tuned interplay in nontransformed cells. In addition, cancer cells modify their MAMs, which consequently affects calcium homeostasis to support mesenchymal transformation, migration, invasiveness, metastasis and autophagy. Alterations in calcium homeostasis may also support resistance to apoptosis, which is a serious problem facing current chemotherapeutic treatments. Notably, mitochondrial dynamics are also affected by mitochondrial calcium concentration to promote cancer survival responses. Dysregulated levels of mitochondrial calcium, alongside other signals, promote mitoflash generation in tumor cells, and an increased frequency of mitoflashes may induce epithelialtomesenchymal transition. Therefore, cancer cells remodel their calcium balance through numerous mechanisms that support their survival and growth.
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Calcio/metabolismo , Mitocondrias/metabolismo , Neoplasias/metabolismo , Animales , Transporte Biológico , Señalización del Calcio , Homeostasis , Humanos , Dinámicas MitocondrialesRESUMEN
Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration.
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Sistema Nervioso Central/microbiología , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/microbiología , Encéfalo/metabolismo , Encéfalo/microbiología , Calcineurina/metabolismo , Calcio/metabolismo , Línea Celular , Criptococosis/metabolismo , Criptococosis/microbiología , Modelos Animales de Enfermedad , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Meningoencefalitis/metabolismo , Meningoencefalitis/microbiología , Ratones , Ratones Endogámicos BALB C , Vacuolas/metabolismo , Vacuolas/microbiología , Virulencia/fisiología , Factores de Virulencia/metabolismoRESUMEN
We have previously demonstrated that melatonin (MEL) blocks the inhibition of the intestinal Ca(2+) absorption caused by menadione (MEN). The purpose of this study were to determine whether MEL not only restores but also prevents the intestinal Ca(2+) absorption inhibited either by MEN or BSO, two drugs that deplete glutathione (GSH) in different ways, and to analyze the mechanisms by which MEN and MEL alter the movement of Ca(2+) across the duodenum. To know this, chicks were divided into four groups: 1) controls, 2) MEN treated, 3) MEL treated, and 4) treated sequentially with MEN and MEL or with MEN and MEL at the same time. In a set of experiments, chicks treated with BSO or sequentially with BSO and MEL or with BSO and MEL at the same time were used. MEL not only restored but also prevented the inhibition of the chick intestinal Ca(2+) absorption produced by either MEN or BSO. MEN altered the protein expression of molecules involved in the transcellular as well as in the paracellular pathway of the intestinal Ca(2+) absorption. MEL restored partially both pathways through normalization of the O2(-) levels. The nitrergic system was not altered by any treatment. In conclusion, MEL prevents or restores the inhibition of the intestinal Ca(2+) absorption caused by different GSH depleting drugs. It might become one drug for the treatment of intestinal Ca(2+) absorption under oxidant conditions having the advantage of low or null side effects.
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Calcio/metabolismo , Glutatión/metabolismo , Absorción Intestinal/efectos de los fármacos , Melatonina/farmacología , Animales , Pollos , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Vitamina K 3/farmacologíaRESUMEN
The transport of calcium to the extracellular space carried out by plasma membrane Ca(2+) pumps (PMCAs) is essential for maintaining low Ca(2+) concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca(2+)-calmodulin to the calmodulin-binding autoinhibitory sequence, which in the human PMCA is located in the C-terminal segment and results in a PMCA of high maximal velocity of transport and high affinity for Ca(2+). Autoinhibition involves the intramolecular interaction between the autoinhibitory domain and a not well defined region of the molecule near the catalytic site. Here we show that the fusion of GFP to the C terminus of the h4xb PMCA causes partial loss of autoinhibition by specifically increasing the Vmax. Mutation of residue Glu(99) to Lys in the cytosolic portion of the M1 transmembrane helix at the other end of the molecule brought the Vmax of the h4xb PMCA to near that of the calmodulin-activated enzyme without increasing the apparent affinity for Ca(2+). Altogether, the results suggest that the autoinhibitory interaction of the extreme C-terminal segment of the h4 PMCA is disturbed by changes of negatively charged residues of the N-terminal region. This would be consistent with a recently proposed model of an autoinhibited form of the plant ACA8 pump, although some differences are noted.
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Calcio/metabolismo , Membrana Celular/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Calmodulina/metabolismo , Catálisis , Citosol/metabolismo , Eliminación de Gen , Glutamina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisina/metabolismo , Mutación , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Unión Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismoRESUMEN
The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.
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Adenosina Trifosfato/química , Membrana Eritrocítica/enzimología , Modelos Químicos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , Adenosina Trifosfato/metabolismo , Membrana Eritrocítica/química , Humanos , Transporte Iónico/fisiología , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The Ca(2+)-calcineurin signaling pathway in the human fungal pathogen Cryptococcus neoformans is essential for adaptation to the host environment during infection. Calcium transporters regulate cytosolic calcium concentrations, providing Ca(2+) loading into storage organelles. The three calcium transporters that have been characterized in C. neoformans, Cch1, Eca1 and Vcx1, are required for fungal virulence, supporting a role for calcium-mediated signaling in cryptococcal pathogenesis. In the present study, we report the functional characterization of the putative vacuolar calcium ATPase Pmc1 in C. neoformans. Our results demonstrate that Pmc1 provides tolerance to high Ca(2+) concentrations. The double knockout of C. neoformans PMC1 and VCX1 genes impaired the intracellular calcium transport, resulting in a significant increase in cytosolic calcium levels. Furthermore, Pmc1 was essential for both the progression of pulmonary infection and brain colonization in mice, emphasizing the crucial role of calcium signaling and transport for cryptococcal pathogenesis.
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ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Cryptococcus neoformans/enzimología , Proteínas Fúngicas/metabolismo , Animales , ATPasas Transportadoras de Calcio/clasificación , ATPasas Transportadoras de Calcio/genética , Criptococosis/metabolismo , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Ratones , Filogenia , Vacuolas/enzimología , VirulenciaRESUMEN
Crustaceans show discontinuous growth and have been used as a model system for studying cellular mechanisms of calcium transport, which is the main mineral found in their exoskeleton. Ucides cordatus, a mangrove crab, is naturally exposed to fluctuations in calcium and salinity. To study calcium transport in this species during isosmotic conditions, dissociated gill cells were marked with fluo-3 and intracellular Ca(2+) change was followed by adding extracellular Ca(2+) as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5mM), together with different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with Vmax=0.137±0.001 ∆Ca(2+)i (µM×22.10(4)cells(-1)×180s(-1); N=4; r(2)=0.99); Km=0.989±0.027mM. The use of different inhibitors for gill cells showed that amiloride (Na(+)/Ca(2+) exchange inhibitor) inhibited 80% of Ca(2+) transport in gill cells (Vmax). KB-R, an inhibitor of Ca influx in vertebrates, similarly caused a decrease in Ca(2+) transport and verapamil (Ca(2+) channel inhibitor) had no effect on Ca(2+) transport, while nifedipine (another Ca(2+) channel inhibitor) caused a 20% decrease in Ca(2+) affinity compared to control values. Ouabain, on the other hand, caused no change in Ca(2+) transport, while vanadate increased the concentration of intracellular calcium through inhibition of Ca(2+) efflux probably through the plasma membrane Ca(2+)-ATPase. Results show that transport kinetics for Ca(2+) in these crabs under isosmotic conditions is lower compared to a hyper-regulator freshwater crab Dilocarcinus pagei studied earlier using fluorescent Ca(2+) probes. These kinds of studies will help understanding the comparative mechanisms underlying the evolution of Ca transport in crabs living in different environments.
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Braquiuros/metabolismo , Calcio/metabolismo , Branquias/metabolismo , Tolerancia a la Sal , Animales , Proteínas de Artrópodos/metabolismo , Braquiuros/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Branquias/citología , Cinética , Salinidad , Vanadatos/farmacología , Verapamilo/farmacología , HumedalesRESUMEN
Calcium signaling through calmodulin and the phosphatase calcineurin are required for key events of the biology of the human pathogen Cryptococcus neoformans, including mating, morphogenesis, growth at 37°C and virulence. In a recent work we described the functional characterization of a new component of this calcium signaling network: the vacuolar calcium exchanger Vcx1. This transporter is involved in calcium tolerance and virulence in C. neoformans. Two other uncharacterized calcium transporters which are putative orthologs of Saccharomyces cerevisiae PMC1 (a vacuolar calcium ATPase) and PMR1 (a Golgi calcium ATPase) are also functional in C. neoformans. No ortholog of CRZ1, the target of calcineurin in other fungi, has been identified in C. neoformans, indicating a high complexity in cryptococcal calcium-related pathways. Future studies are necessary for the complete understanding of calcium signaling regulation in C. neoformans.
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Myotoxic effects of local anesthetics on skeletal muscle fibers involve the inhibition of sarcoplasmic reticulum Ca2+-dependent ATPase activity and Ca2+ transport. Lidocaine is a local anesthetic frequently used to relieve the symptoms of trigeminal neuralgia. The aim of this work was to test the inhibitory and/or stimulatory effect of lidocaine on sarcoplasmic reticulum Ca2+-dependent ATPase isolated from rabbit temporalis muscle. Ca2+-dependent ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca2+- dependent ATPase, Ca2+ transport, and phosphorylation of the enzyme by ATP were determined with radioisotopic techniques. Lidocaine inhibited the Ca2+-dependent ATPase activity in a concentration- dependent manner. The preincubation of the sarcoplasmic reticulum membranes with lidocaine enhanced the Ca2+- dependent ATPase activity in the absence of calcium ionophore. Lidocaine also inhibited both Ca2+ uptake and enzyme phosphorylation by ATP but had no effect on Ca2+-binding to the enzyme. We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent ATPase from temporalis muscle is due to the drug's direct interaction with the enzyme and the increased permeability of the sarcoplasmic reticulum membrane to Ca.
La toxicidad de los anestesicos locales sobre las fibras musculares esqueleticas involucra a la inhibicion de la actividad de la calcio ATPasa del reticulo sarcoplasmico y a la inhibicion del transporte del calcio. Tales efectos inhibitorios no han sido aun descriptos en el musculo temporal. La lidocaina es un anestesico local habitualmente usado para aliviar los sintomas de la neuralgia del trigemino por medio de la anestesia infiltrativa de la region temporal. El objetivo del trabajo fue demostrar el efecto inhibitorio y/o activador de la lidocaina sobre la calcio ATPasa del reticulo sarcoplasmico del musculo temporal del conejo. La actividad de la calcio ATPasa se determino empleando un metodo colorimetrico. La union del calcio a la enzima, el transporte del calcio y la fosforilacion de la ATPasa por ATP se determinaron mediante el empleo de tecnicas radioisotopicas. La lidocaina inhibio a la actividad de la calcio ATPasa. El efecto inhibitorio incremento en funcion de la concentracion del anestesico. La preincubacion de las membranas del reticulo sarcoplasmico en lidocaina incremento la actividad de la calcio ATPasa en ausencia de un ionoforo de calcio. Tal resultado avala el efecto permeabilizante del anestesico local sobre las membranas del reticulo sarcoplasmico del musculo temporal. La lidocaina inhibio la captacion del calcio y la fosforilacion de la calcio ATPasa por ATP, pero no evidencio efecto sobre la union del calcio a la enzima. Concluimos que el efecto de la lidocaina sobre la calcio ATPasa del reticulo sarcoplasmico del musculo temporal se debe a la accion directa de la droga sobre la enzima y al incremento inducido de la permeabilidad de la membrana del reticulo sarcoplasmico al Ca.