Biomedical subjects

Adalberto Vieyra

Publications and source records attributed to Adalberto Vieyra.

10 recordsLinked to original sources

PubMed2006

Phosphate sorption and desorption on pyrite in primitive aqueous scenarios: relevance of acidic --> alkaline transitions.

Phosphate (P(i)) sorption assays onto pyrite in media simulating primeval aquatic scenarios affected by hydrothermal emissions, reveal that acidic conditions favour P(i) sorption whereas mild alkaline media--as well as those simulating sulfur oxidation to SO(2-) (4)--revert this capture process. Several mechanisms relevant to P(i) availability in prebiotic eras are implicated in the modulation of these processes. Those favouring sorption are: (a) hydrophobic coating of molecules, such as acetate that could be formed in the vicinity of hydrothermal vents; (b) water and Mg(2+) bridging in the interface mineral-aqueous media; (c) surface charge neutralization by monovalent cations (Na+ and K+). The increase of both the medium pH and the SO(2-) (4) trapping by the mineral interface would provoke the release of sorbed P(i) due to charge polarization. Moreover it is shown that P(i) self-modulates its sorption, a mechanism that depends on the abundance of SO(2-) (4) in the interface. The relevance of the proposed mechanisms of P(i) capture, release and trapping arises from the need of abundant presence of this molecule for primitive phosphorylations, since--similarly to contemporary aqueous media--inorganic phosphate concentrations in primitive seas should have been low. It is proposed that the presence of sulphide minerals with high affinity to P(i) could have trapped this molecule in an efficient manner, allowing its concentration in specific niches. In these niches, the conditions studied in the present work would have been relevant for its availability in soluble form, specially in primitive insulated systems with pH gradients across the wall.

Adsorption↗

PubMed2005

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A.

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.

Adenosine Triphosphatases↗

PubMed2005

Protein kinase C-mediated inhibition of renal Ca2+ ATPase by physiological concentrations of angiotensin II is reversed by AT1- and AT2-receptor antagonists.

Angiotensin II (Ang II) increases the cytosolic Ca2+ concentration in different cell types. In this study, we investigate the effect of Ang II on the Ca2+ ATPase of purified basolateral membranes of kidney proximal tubules. This enzyme pumps Ca2+ out of the cytosol in a reaction coupled to ATP hydrolysis, and it is responsible for the fine-tuned regulation of cytosolic Ca2+ activity. Ca2+-ATPase activity is inhibited by picomolar concentrations of Ang II, with maximal inhibition being attained at approximately 50% of the control values. The presence of raising concentrations (10(-11) to 10(-7) M) of losartan (an AT1-receptor antagonist) or PD123319 (an AT2-receptor antagonist) gradually reverts inhibition by Ang II. Both the phospholipase C (PLC) inhibitor U-73122 (10(-6) M) and the inhibitor of protein kinase C (PKC) staurosporine (10(-7) M) prevent inhibition of the Ca2+ pump by Ang II. Incubation of the previously isolated membranes with a PKC activator-the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (10(-8) M)-mimics the inhibition found with Ang II, and the effects of the compounds are not additive. Taken as a whole, these results indicate the Ang II inhibits Ca2+-ATPase by activation of a PKC system present in primed state in these membranes after binding of the hormone to losartan- and PD123319-sensitive receptors coupled to a PLC. Therefore, inhibition of the basolateral membrane Ca2+-ATPase by kinase-mediated phosphorylation appears to be one of the pathways by which Ang II promotes an increase in the cytosolic Ca2+ concentration of proximal tubule cells.

Angiotensin II↗

PubMed2005

Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C.

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.

Adenosine Triphosphate↗

PubMed2004

Ca2+ binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca2+ sites in the presence of ADP.

Sarcoplasmic reticulum (SR) Ca2+-ATPase was phosphorylated by Pi at pH 8.0 in the presence of dimethyl sulfoxide (Me2SO). Under these conditions, it was possible to measure transient 45Ca2+ binding to the phosphoenzyme. Binding reached 1.2 Ca2+ per phosphoenzyme (E-PCax) within 10 min in 30% Me2SO, 20 mM MgCl2 and 0.1 mM Pi and the phosphoenzyme only decreased by 23% during this period. This Ca2+ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca2+-ATPase. At 40% Me2SO, simultaneous addition of Ca2+ and ADP increased Ca2+ binding up to almost four Ca2+ per phosphoenzyme (ADPE-PCay), revealing a species bearing simultaneously four Ca2+ sites. Both E-PCax and ADPE-PCay were further identified as distinct species by (2',3'-O-2(2,4,6-trinitrophenyl)adenosine 5'-triphosphate) fluorescence, which revealed long-range modifications in the Ca2+-transport sites induced by ADP binding to E-P. In addition, E-PCax was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me2SO was diluted. These findings indicate that more than two functional Ca2+-sites exist on the functional Ca2+-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca2+-ATPase allowing simultaneous binding of Ca2+ at lumenal and cytosolic sites. The stoichiometries for Ca2+ binding found here could either be interpreted as binding of four Ca2+ on a Ca2+-ATPase monomer considered as the functional unit or as binding of two Ca2+ per monomer of a functional dimer.

Adenosine Diphosphate↗

PubMed2004

Cholesterol depletion by methyl-beta-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei.

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

Actinin↗

PubMed2004

The plasma membrane Ca2+ pump from proximal kidney tubules is exclusively localized and active in caveolae.

Plasma membrane Ca2+-ATPase is involved in the fine-tuned regulation of intracellular Ca2+. In this study, the presence of Ca2+-ATPase in caveolae from kidney basolateral membranes was investigated. With the use of a discontinuous sucrose gradient, we show that Ca2+-ATPase is exclusively located and fully active in caveolin-containing microdomains. Treatment with methyl-beta-cyclodextrin--a cholesterol chelator--leads to a spreading of both caveolin and completely inactive Ca2+-ATPase toward high-density fractions. These data support the view that Ca2+ fluxes mediated by Ca2+-ATPase in kidney epithelial cells occur only in caveolae, being strictly dependent on the integrity of these microdomains.

Animals↗

PubMed2004

A mutational study in the transmembrane domain of Ccc2p, the yeast Cu(I)-ATPase, shows different roles for each Cys-Pro-Cys cysteine.

Ccc2p is homologous to the human Menkes and Wilson copper ATPases and is herein studied as a model for human copper transport. Most studies to date have sought to understand how mutations in the human Menkes or Wilson genes impair copper homeostasis and induce disease. Here we analyze whether eight conserved amino acids of the transmembrane domain are important for copper transport. Wild-type Ccc2p and variants were expressed in a ccc2-Delta yeast strain to check whether they were able to restore copper transport by complementation. Wild-type Ccc2p and variants were also expressed in Sf9 cells using baculovirus to study their enzymatic properties on membrane preparations. The latter system allowed us to measure a copper-activated ATPase activity of about 20 nmol/mg/min for the wild-type Ccc2p at 37 degrees C. None of the variants was as efficient as the wild type in restoring copper homeostasis. The mutation of each cysteine of the (583)CPC(585) motif into a serine resulted in nonfunctional proteins that could not restore copper homeostasis in yeast and had no ATPase activity. Phosphorylation by ATP was still possible with the C583S variant, although it was not possible with the C585S variant, suggesting that the cysteines of the CPC motif have a different role in copper transport. Cys(583) would be necessary for copper dissociation and/or enzyme dephosphorylation and Cys(585) would be necessary for ATP phosphorylation, suggesting a role in copper binding.

Adenosine Triphosphatases↗

PubMed2003

Phosphate immobilization by oxide precursors: implications on phosphate availability before life on earth.

The present study is based on the proposal that if the aqueous phosphorus-capture mechanism by iron oxide precursors was inhibited in prebiotic anoxic scenarios then soluble phosphates could have been more available than what is observed now. Supporting this conjecture, we examine prevailing contemporary trapping mechanisms of orthophosphate (Pi) and pyrophosphate (PPi). To illustrate its efficiency, the attachment of (Pi) onto aggregates of iron-3 oxyhydroxide is compared with the one reported for the product of its condensation, PPi. The electrophoretic profiles of the Pi- and PPi-aggregate complexes reveal different pH-modulated interactions of the phosphorylated compounds with both the aggregate and its aqueous surrounding layers. The observed differences of Pi/PPi sorption and desorption mechanisms are discussed in terms of their consequences to the prebiotic availability of soluble orthophosphate and of a phosphorylated compound having the high-energy phosphoanhydride linkage and a molecule representative of condensed oligophosphates.

Diphosphates↗

PubMed2003

Sphingosine-1-phosphate formation activates phosphatidylinositol-4 kinase in basolateral membranes from kidney cells: crosstalk in cell signaling through sphingolipids and phospholipids.

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.

1-Phosphatidylinositol 4-Kinase↗