Biomedical subjects

S M Meireles

Publications and source records attributed to S M Meireles.

9 recordsLinked to original sources

PubMed2002

Isokinetic evaluation of the knee in patients with rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis is a chronic inflammatory disease with a clinical picture of arthritis, muscle hypotrophy, loss of range of motion, loss of strength and disability. The objective of this study was to evaluate knees of patients with rheumatoid arthritis using an isokinetic dynamometer. METHODS: Fifty patients with a diagnosis of rheumatoid arthritis and 50 control subjects were evaluated using an isokinetic dynamometer (Cybex 6.000) regarding the following parameters: peak torque, peak torque angle, power, total work, peak torque acceleration time, set total work, torque acceleration energy ('explosion') and endurance. Comparisons between rheumatoid arthritis and the control group, left side versus right side, flexors and extensors and the proportion between flexors and extensors were made. The subjects were also evaluated through the Health Assessment Questionnaire, visual and analogical scale of pain, EPM-ROM and goniometry of the knee. RESULTS: The results showed that patients with rheumatoid arthritis have less strength than the control group (P < 0.05); the extensors are stronger than the flexors (P < 0.05); no significant differences between the right and the left knee for rheumatoid arthritis and the control group were observed and the proportion between flexors and extensors is the same in both groups. We concluded that regarding the isokinetic parameters, the knees of subjects with rheumatoid arthritis are different from normal knees with decreased strength parameters, maintaining the proportion between flexors and extensors with a global loss of strength in the segment, excluding the high speeds in some of the parameters.

Adult↗

PubMed2001

Ca2+ sensitivity of synaptic vesicle dopamine, gamma-aminobutyric acid, and glutamate transport systems.

The effect of Ca2+ on the uptake of neurotransmitters by synaptic vesicles was investigated in a synaptic vesicle enriched fraction isolated from sheep brain cortex. We observed that dopamine uptake, which is driven at expenses of the proton concentration gradient generated across the membrane by the H+-ATPase activity, is strongly inhibited (70%) by 500 microM Ca2+. Conversely, glutamate uptake, which essentially requires the electrical potential in the presence of low Cl- concentrations, is not affected by Ca2+, even when the proton concentration gradient greatly contributes for the proton electrochemical gradient. These observations were checked by adding Ca2+ to dopamine or glutamate loaded vesicles, which promoted dopamine release, whereas glutamate remained inside the vesicles. Furthermore, similar effects were obtained by adding 150 microM Zn2+ that, like Ca2+, dissipates the proton concentration gradient by exchanging with H+. With respect to gamma-aminobutyric acid transport, which utilizes either the proton concentration gradient or the electrical potential as energy sources, we observed that Ca2+ or Zn2+ do not induce great alterations in the gamma-aminobutyric acid accumulation by synaptic vesicles. These results clarify the nature of the energy source for accumulation of main neurotransmitters and suggest that stressing concentrations of Ca2+ or Zn2+ inhibit the proton concentration gradient-dependent neurotransmitter accumulation by inducing H+ pump uncoupling rather than by interacting with the neurotransmitter transporter molecules.

ATP-Binding Cassette Transporters↗

PubMed2000

Ca(2+) regulation of the carrier-mediated gamma-aminobutyric acid release from isolated synaptic plasma membrane vesicles.

The regulation of the carrier-mediated gamma-aminobutyric acid (GABA) efflux was studied in isolated synaptic plasma membrane (SPM) vesicles, which are particularly useful to study neurotransmitter release without interference of the exocytotic machinery. We investigated the effect of micromolar intravesicular Ca(2+) on the GABA release from SPM vesicles under conditions of basal release (superfusion with 150 mM NaCl), homoexchange (superfusion with 500 microM GABA) and K(+) depolarization-induced release (superfusion with 150 mM KCl). We observed that, in the presence of intravesicular Ca(2+) (10 microM), the maximal velocity (J(max)) of K(+) depolarization-induced GABA release is decreased by about 64%, and this effect was abolished in the presence of the channel blocker, La(3+). In contrast, the other mechanisms were not significantly altered by these cations. In agreement with our earlier results, inhibition of GABA uptake by intravesicular Ca(2+) was also observed by determining the kinetic parameters (K(0.5) and J(max)) of influx into the SPM vesicles. Under these conditions, the J(max) of GABA uptake was 17.4 pmol/s per mg protein, whereas in control experiments (absence of Ca(2+)), this value achieved 25.5 pmol/s per mg protein. The inhibitory effect of Ca(2+) on translocation of GABA across SPM appears to be mediated by calcium/calmodulin activation of protein phosphatase 2B (calcineurin), since it was completely relieved by W7 (calmodulin antagonist) and by cyclosporin A (calcineurin inhibitor). These results show that the GABA transport system, operating either in forward or backward directions, requires phosphorylation of internally localized calcineurin-sensitive sites to achieve maximal net translocation velocity.

Animals↗

PubMed2000

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex.

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.

Adenosine Triphosphate↗

PubMed2000

Methods for analysis of Ca(2+)/H(+) antiport activity in synaptic vesicles isolated from sheep brain cortex.

The involvement of Ca(2+)-storage organelles in the modulation of synaptic transmission is well-established [M.K. Bennett, Ca(2+) and the regulation of neurotransmitter secretion, Curr. Opin. Neurobiol. 7 (1997) 316-322 [1]; M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1998) 13-26 [2]; Ph. Fossier, L. Tauc, G. Baux, Calcium transients and neurotransmitter release at an identified synapse, Trends Neurosci. 22 (1999) 161-166 [7] ]. Various Ca(2+) sequestering reservoirs (mitochondria, endoplasmic reticulum and synaptic vesicles) have been reported at the level of brain nerve terminals [P. Kostyuk, A. Verkhratsky, Calcium stores in neurons and glia, Neuroscience 63 (1994) 381-404 [18]; V. Mizuhira, H. Hasegawa, Microwave fixation and localization of calcium in synaptic terminals using X-ray microanalysis and electron energy loss spectroscopy imaging, Brain Res. Bull. 43 (1997) 53-58 [21]; A. Parducz, Y. Dunant, Transient increase of calcium in synaptic vesicles after stimulation, Neuroscience 52 (1993) 27-33 [23]; O.H. Petersen, Can Ca(2+) be released from secretory granules or synaptic vesicles?, Trends Neurosci. 19 (1996) 411-413 [24] ]. However, the knowledge of the specific contribution of each compartment for spatial and temporal control of the cytoplasmic Ca(2+) concentration requires detailed characterization of the Ca(2+) uptake and Ca(2+) release mechanisms by the distinct intracellular stores. In this work, we described rapid and simple experimental procedures for analysis of a Ca(2+)/H(+) antiport system that transport Ca(2+) into synaptic vesicles at expenses of the energy of a DeltapH generated either by activity of the proton pump or by a pH jumping of the vesicles passively loaded with protons. This secondary active Ca(2+) transport system requires high Ca(2+)100 microM) for activation, it is dependent on the chemical component (DeltapH) of the proton electrochemical gradient across the synaptic vesicle membrane and its selectivity is essentially determined by the size of the transported cation [P.P. Gonçalves, S.M. Meireles, C. Gravato, M.G. P. Vale, Ca(2+)-H(+)-Antiport activity in synaptic vesicles isolated from sheep brain cortex, Neurosci. Lett. 247 (1998) 87-90 [10]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Ionic selectivity of the Ca(2+)/H(+) antiport in synaptic vesicles of sheep brain cortex, Mol. Brain Res. 67 (1999) 283-291 [11]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Synaptic vesicle Ca(2+)/H(+) antiport: dependence on the proton electrochemical gradient, Mol. Brain Res. 71 (1999) 178-184 [12] ]. The protocols described here allow to ascertain the characteristics of the Ca(2+)/H(+) antiport in synaptic vesicles and, therefore, may be useful for clarification of the physiological role of synaptic vesicles in fast buffering of Ca(2+) at various sites of the neurotransmission machinery.

Acridine Orange↗

PubMed1999

Synaptic vesicle Ca2+/H+ antiport: dependence on the proton electrochemical gradient.

Synaptic vesicles isolated from sheep brain cortex accumulate Ca2+ by a mechanism of secondary active transport associated to the H(+)-pump activity. The process can be visualized either by measuring Ca(2+)-induced H+ release or DeltapH-dependent Ca2+ accumulation. We observed that the amount of Ca2+ taken up by the vesicles increases with the magnitude of the DeltapH across the membrane, particularly at Ca2+ concentrations (approximately 500 microM) found optimal for the antiporter activity. Similarly, H+ release induced by Ca2+ increased with the magnitude of DeltapH. However, above 60% DeltapH (high H(+)-pump activity), the net H+ release from the vesicles decreased as the pump-mediated H+ influx exceeded the Ca(2+)-induced H+ efflux. We also observed that the Ca2+/H+ antiport activity depends, essentially, on the DeltapH component of the electrochemical gradient (approximately 3 nmol Ca2+ taken up/mg protein), although the Deltaphi component may also support some Ca2+ accumulation by the vesicles (approximately 1 nmol/mg protein) in the absence of DeltapH. Both Ca(2+)-induced H+ release and DeltapH-dependent Ca2+ uptake could be driven by an artificially imposed proton motive force. Under normal conditions (H+ pump-induced DeltapH), the electrochemical gradient dependence of Ca2+ uptake by the vesicles was checked by inhibition of the process with specific inhibitors (bafilomycin A(1), ergocryptin, folymicin, DCCD) of the H(+)-pump activity. These results indicate that synaptic vesicles Ca2+/H+ antiport is indirectly linked to ATP hydrolysis and it is essentially dependent on the chemical component (DeltapH) of the electrochemical gradient generated by the H(+)-pump activity.

Animals↗

PubMed1999

Ionic selectivity of the Ca2+/H+ antiport in synaptic vesicles of sheep brain cortex.

As we previously reported, synaptic vesicles isolated from sheep brain cortex contain a Ca2+/H+ antiport that permits Ca2+ accumulation inside the vesicles ( approximately 5 nmol/mg protein) at expenses of the pH gradient generated by the H+-pumping ATPase. We observed that the system associates Ca2+ influx to H+ release and operates with low affinity for Ca2+. In the present work, we found that Ca2+/H+ antiport mediates exchange of protons with other cations such as Zn2+ and Cd2+, suggesting that these cations and Ca2+ share the same transporter molecules to enter the intravesicular space. Zn2+ and Cd2+ induce H+ release in a concentration-dependent manner (fluorimetrically evaluated) and they inhibit the antiport-mediated Ca2+ uptake by the vesicles (isotopically measured). In contrast, large cations such as Ba2+ and Cs+ do not alter Ca2+ influx and they are unable to induce proton release from the vesicles. With respect to Sr2+, which has an intermediary size relatively to the other groups of cations, we found that it does not induce H+ liberation from the vesicles, but it has a concentration-dependent inhibitory effect on the Ca2+-induced H+ release and Ca2+ uptake by the vesicles. These results indicate that the cation selectivity of the synaptic vesicles Ca2+/H+ antiport is essentially determined by the size of the cation transported into the vesicles.

Adenosine Triphosphate↗

PubMed1999

Regulation of the gamma-aminobutyric acid transporter activity by protein phosphatases in synaptic plasma membranes.

The influence of the phosphorylation dephosphorylation states on the gamma-aminobutyric acid (GABA) transporter activity of synaptic plasma membranes (SPM) was studied by using either specific phosphatase inhibitors or activators. Calyculin A and okadaic acid (phosphatase 1 and phosphatase 2A inhibitors) inhibited the GABA uptake by isolated SPM vesicles, whereas cyclosporin A (phosphatase 2B inhibitor) had a stimulatory effect (approximately 10%) which was higher (approximately 38%) when all these drugs were present in the reaction medium. On the other hand, intravesicular Ca2+, up to about 10 microM, inhibited the GABA uptake (approximately 50%) in a manner which appeared to be facilitated in the presence of PP1 and PP2A inhibitors and this inhibition was relieved by the calmodulin antagonist W-7. We also observed that isolated SPM vesicles contain both Ca(2+)-independent phosphatase activity that is significantly inhibited by PP1 and PP2A inhibitors, and Ca(2+)-dependent phosphatase activity that is abolished in the presence of the PP2B inhibitor, cyclosporin A. These results indicate that regulation of the SPM GABA transporter is determined by the internally localized Ca-calmodulin-dependent phosphatase activity (calcineurin), and that other phosphorylated sites, sensitive to PP1 and PP2A inhibitors, potentiate either the positive or negative effects exerted by those internal sites when they are in their phosphorylated or dephosphorylated states, respectively.

Animals↗

PubMed1998

Ca2+-H+ antiport activity in synaptic vesicles isolated from sheep brain cortex.

Synaptic vesicles isolated from sheep brain cortex exhibit an ATP-dependent Ca2+ accumulation that is inhibited by the protonophore uncoupler carbonyl cyanide m-chorophenylhydrazone (CCCP) and completely released by the Ca2+ ionophore ionomycin. This transport activity was sensitive to the V-type ATPase inhibitor, bafilomycin, but not to the P-type ATPase inhibitor, vanadate. We also observed that the proton gradient, established across the synaptic vesicle membranes in the presence of ATP, is partially dissipated by the addition of Ca2+ (100-860 microM) in correlation to an increase of ATP hydrolysis by the H+-pumping ATPase. In contrast, the activity of the H+-ATPase, measured under uncoupling conditions (presence of CCCP), appears to be unaltered by the calcium ion. The Ca2+-induced H+ release visualized by fluorescence quenching of acridine orange correlates well with the Ca2+ uptake determined isotopically. These results indicate that synaptic vesicles accumulate Ca2+, via a low affinity Ca2+-H+ antiport system energized by the protonmotive force originated from the H+-pumping ATPase activity.

Adenosine Triphosphate↗