Componente lento da cinética do VO2: determinantes fisiológicos e implicações para o desempenho em exercícios aeróbios

A cinética do consumo de oxigênio (VO2) e a resposta do lactato sanguíneo durante o exercício de carga constante em diferentes intensidades permitem caracterizar os domínios moderado, pesado e severo do exercício. Em exercício de intensidade constante, o perfil da resposta do VO2, analisada por ajustes exponenciais, apresenta as fases cardiodinâmica, fundamental e lenta. A ocorrência do componente lento (CL) tem sido associada a fatores como recrutamento de fibras do tipo II e acúmulo de metabólitos, como lactato, íons H+, fosfato inorgânico e ADP. O CL expressa uma redução da eficiência muscular e tem sido associado à menor tolerância de exercícios aeróbios de intensidade alta. O presente estudo teve por objetivo detalhar a fundamentação teórica sobre sua ocorrência, a influência na tolerância ao exercício, bem como prover os diferentes procedimentos adotados em sua quantificação.

Antiplatelet and Antithrombotic Activities of Non-Steroidal Anti-Inflammatory Drugs Containing an N-Acyl Hydrazone Subunit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Behavior of respiratory metabolism and pyridine nucleotide levels in Oxyrrhis marina during starvation

Máster en Oceanografía

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.

Effect of atorvastatin and pravastatin on platelet inhibition by aspirin and clopidogrel treatment in patients with coronary stent thrombosis

We sought to determine a potential interaction between statins and antiplatelet therapy with aspirin and clopidogrel. Previous laboratory studies have shown a possible drug-drug interaction of statins metabolized by cytochrome P450 3A4 and clopidogrel (prodrug metabolized by cytochrome P450 3A4), resulting in an impaired inhibitory effect of clopidogrel on platelet aggregation. However, conclusive prospective data assessing this potentially relevant interaction are lacking. In 73 patients, 23 with previous coronary stent thrombosis (ST) (ST group) and 50 without coronary ST (control group), platelet aggregation was measured 3 times in monthly intervals using light transmission aggregometry (adenosine diphosphate [ADP] and arachidonic acid induction). Measurements were carried out with aspirin monotherapy (100 mg/day), dual antiplatelet therapy with aspirin plus clopidogrel (75 mg/day), and additional treatment of 20 mg/day of atorvastatin or 40 mg/day of pravastatin. ADP (5 and 20 micromol)-induced platelet aggregation was significantly decreased with clopidogrel (p <0.001) but remained stable under additional treatment with atorvastatin or pravastatin in the 2 groups. Patients with previous ST showed a higher ADP-induced aggregation level than control subjects. This difference was not influenced by clopidogrel or statin treatment. In conclusion, patients with previous ST show a higher aggregation level than control subjects independent of statin treatment. Atorvastatin and pravastatin do not interfere with the antiaggregatory effect of aspirin and clopidogrel. In conclusion, drug-drug interaction between dual antiplatelet therapy and atorvastatin or pravastatin seems not to be associated with ST.

Preemptive analgesia by lornoxicam--an NSAID--significantly inhibits perioperative platelet aggregation

BACKGROUND AND OBJECTIVE: To investigate whether preemptive administered lornoxicam changes perioperative platelet function during thoracic surgery. METHODS: A total of 20 patients scheduled for elective thoracic surgery were randomly assigned to receive either lornoxicam (16 mg, i.v.; n = 10) or placebo (n = 10) preoperatively. All patients underwent treatment of solitary lung metastasis and denied any antiplatelet medication within the past 2 weeks. Blood samples were drawn via an arterial catheter directly into silicone-coated Vacutainer tubes containing 0.5 mL of 0.129 M buffered sodium citrate 3.8% before, 15 min, 4 h and 8 h after the study medication was administered. Platelet aggregation curves were obtained by whole blood electrical impedance aggregometry (Chrono Log). RESULTS: Platelet aggregation was significantly reduced 15 min, 4 h and 8 h after lornoxicam administration compared to placebo (P < 0.05) for collagen, adenosine diphosphate and arachidonic acid as trigger substances. Adenosine diphosphate-induced platelet aggregation decreased by 85% 15 min after lornoxicam administration, and remained impaired for 8 h. CONCLUSION: Platelet aggregation assays are impaired for at least 8 h after lornoxicam application. Therefore perioperative analgesia by use of lornoxicam should be carefully administered under consideration of subsequent platelet dysfunction.

Profile and Prevalence of Aspirin Resistance in Indian Patients with Coronary Artery Disease

Background: Aspirin resistance is considered to be an enigma and the data available on aspirin resistance is scarce. This study was initiated to prospectively evaluate the prevalence of aspirin resistance in patients with stable coronary artery disease by using an established method of optical platelet aggregation. Methods and Results: We studied 50 patients who were on 150 mg of aspirin for the previous 7 days. Fasting blood samples were assessed using optical platelet aggregation (Chronolog Corp, USA). The mean platelet aggregation with 10 μm of adenosine diphosphate in our patient group was 49.42 ± 23.29% and with 0.5 mg/ ml of arachidonic acid it was 13.58 ± 21.40%. Aspirin resistance was defined as a mean aggregation of ≥70% with 10 μm of adenosine diphosphate and a mean aggregation of ≥ 20% with 0.5 mg/ml of arachidonic acid. Aspirin semi responders were defined as those meeting only one of the criteria. Based on these criteria, 2.08% patients were found to be aspirin-resistant, 39.58% were aspirin semi responders and 58.33% were aspirin responders. Females tended to be more aspirin semi responsive (p = 0.08). All other parameters tested, namely, age, smoking, diabetes mellitus, hypertension, obesity, lipids, hemoglobin, platelet count, ejection fraction and drug intake did not show any statistically significant difference among the groups. Thus, in our group 41.66% patients showed inadequate response to aspirin. Conclusions: This study shows that aspirin resistance and aspirin semi responsiveness do occur in the Indian patients and there are no reliable clinical predictors for this condition. The diagnosis therefore relies primarily on laboratory tests.

Prolonged platelet activation in individuals with elevated blood pressure in response to a moderate exercise challenge

We examined the magnitude of 20-min moderate exercise-induced platelet activation in 50 volunteers with normal (n=31) or elevated blood pressure (EBP; n=19). Blood was drawn before, immediately after, and 25 min after exercise. Antibody-staining for platelet activation markers, P-selectin, and fibrinogen receptors was done with and without adenosine diphosphate (ADP) stimulation in whole blood for flow cytometric analyses. Exercise led to increases in percent aggregated platelets and percent platelets expressing P-selectin or PAC-1 binding (ps< or =.001). This increase in percent platelets expressing P-selectin continued even after a 25-min rest only in the EBP group (p< or =.01) accompanied by an increase in percent of aggregated platelets (p< or =.05). Although ADP stimulation led to increased platelet activation at rest, it was attenuated following exercise, even among EBP individuals. A moderate exercise challenge induced prolonged platelet activation in individuals with EBP but attenuation in activation to further stimulation by an agonist. Findings suggest that a recovery period after physical stress appears critical in individuals with high BP regarding platelet activation and aggregation, which can lead to an acute coronary syndrome in vulnerable individuals.

Seawater carbonate chemistry and biological processes of oysters Crassostrea virginica during experiments, 2010

Estuarine organisms are exposed to periodic strong fluctuations in seawater pH driven by biological carbon dioxide (CO2) production, which may in the future be further exacerbated by the ocean acidification associated with the global rise in CO2. Calcium carbonate-producing marine species such as mollusks are expected to be vulnerable to acidification of estuarine waters, since elevated CO2 concentration and lower pH lead to a decrease in the degree of saturation of water with respect to calcium carbonate, potentially affecting biomineralization. Our study demonstrates that the increase in CO2 partial pressure (pCO2) in seawater and associated decrease in pH within the environmentally relevant range for estuaries have negative effects on physiology, rates of shell deposition and mechanical properties of the shells of eastern oysters Crassostrea virginica (Gmelin). High CO2 levels (pH ~7.5, pCO2 ~3500 µatm) caused significant increases in juvenile mortality rates and inhibited both shell and soft-body growth compared to the control conditions (pH ~8.2, pCO2 ~380 µatm). Furthermore, elevated CO2 concentrations resulted in higher standard metabolic rates in oyster juveniles, likely due to the higher energy cost of homeostasis. The high CO2 conditions also led to changes in the ultrastructure and mechanical properties of shells, including increased thickness of the calcite laths within the hypostracum and reduced hardness and fracture toughness of the shells, indicating that elevated CO2 levels have negative effects on the biomineralization process. These data strongly suggest that the rise in CO2 can impact physiology and biomineralization in marine calcifiers such as eastern oysters, threatening their survival and potentially leading to profound ecological and economic impacts in estuarine ecosystems.

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Molecular Analysis of (R)-(+)-Mandelonitrile Lyase Microheterogeneity in Black Cherry1

The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10), which plays a key role in cyanogenesis in rosaceous stone fruits, occurs in black cherry (Prunus serotina Ehrh.) homogenates as several closely related isoforms. Biochemical and molecular biological methods were used to investigate MDL microheterogeneity and function in this species. Three novel MDL cDNAs of high sequence identity (designated MDL2, MDL4, and MDL5) were isolated. Like MDL1 and MDL3 cDNAs (Z. Hu, J.E. Poulton [1997] Plant Physiol 115: 1359–1369), they had open reading frames that predicted a flavin adenine dinucleotide-binding site, multiple N-glycosylation sites, and an N-terminal signal sequence. The N terminus of an MDL isoform purified from seedlings matched the derived amino acid sequence of the MDL4 cDNA. Genomic sequences corresponding to the MDL1, MDL2, and MDL4 cDNAs were obtained by polymerase chain reaction amplification of genomic DNA. Like the previously reported mdl3 gene, these genes are interrupted at identical positions by three short, conserved introns. Given their overall similarity, we conclude that the genes mdl1, mdl2, mdl3, mdl4, and mdl5 are derived from a common ancestral gene and constitute members of a gene family. Genomic Southern-blot analysis showed that this family has approximately eight members. Northern-blot analysis using gene-specific probes revealed differential expression of the genes mdl1, mdl2, mdl3, mdl4, and mdl5.

Heteromeric association creates a P2Y-like adenosine receptor

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that Gi/o protein-coupled A1 adenosine receptor (A1R) and Gq protein-coupled P2Y1 receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A1R and P2Y2 receptor, but not A1R and dopamine D2 receptor, also were found to coimmunoprecipitate in cotransfected cells. A1R agonist and antagonist binding to cell membranes were reduced by coexpression of A1R and P2Y1R, whereas a potent P2Y1R agonist adenosine 5′-O-(2-thiotriphosphate) (ADPβS) revealed a significant potency to A1R binding only in the cotransfected cell membranes. Moreover, the A1R/P2Y1R coexpressed cells showed an ADPβS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1R antagonist, indicating that ADPβS binds A1R and inhibits adenylyl cyclase activity via Gi/o proteins. Also, a high degree of A1R and P2Y1R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A1R with P2Y1R generates A1R with P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.

Promotion of sleep mediated by the A2a-adenosine receptor and possible involvement of this receptor in the sleep induced by prostaglandin D2 in rats.

A 6-hr continuous infusion of 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine (CGS21680), a selective A2a-adenosine agonist, into the subarachnoid space underlying the ventral surface region of the rostral basal forebrain, which has been defined as the prostaglandin (PG) D2-sensitive sleep-promoting zone, at rates of 0.02, 0.2, 2.0, and 12 pmol/min increased slow-wave sleep (SWS) and paradoxical sleep (PS) in a dose-dependent manner up to 183% and 202% of their respective baseline levels. The increments produced by the infusion of CGS21680 at 0.2 and 2.0 pmol/min were totally diminished when the rats had been pretreated with an i.p. injection of (E)-1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF17837; 30 mg/kg of body weight), a selective A2-adenosine antagonist. In contrast, the infusion of N6-cyclohexyladenosine (CHA), a selective A1-adenosine agonist, at 2 pmol/min significantly suppressed SWS before causing an increase in SWS, and a decrease in PS was also markedly visible. Essentially the same effects of CGS21680 and CHA were observed when these compounds were administered to the parenchymal region of the rostral basal forebrain through chronically implanted microdialysis probes. Thus, we clearly showed that stimulation of A2a-adenosine receptors in the rostral basal forebrain promotes SWS and PS. Furthermore, i.p. injections of KF17837 at 30 and 100 mg/kg of body weight dose-dependently attenuated the magnitude of the SWS increase produced by the infusion of PGD2 into the subarachnoid space of the sleep-promoting zone, thus indicating that the A2a-adenosine receptors are crucial in the sleep-promoting process triggered by PGD2.