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P Donoso

Publications and source records attributed to P Donoso.

At least 19 recordsLinked to original sources
PubMed2000

Sulfhydryl oxidation overrides Mg(2+) inhibition of calcium-induced calcium release in skeletal muscle triads.

We studied the effect of oxidation of sulfhydryl (SH) residues on the inhibition by Mg(2+) of calcium-induced calcium release (CICR) in triad-enriched sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. Vesicles were either passively or actively loaded with calcium before eliciting CICR by dilution at pCa 4.6-4.4 in the presence of 1.2 mM free [ATP] and variable free [Mg(2+)]. Native triads exhibited a significant inhibition of CICR by Mg(2+), with a K(0.5) approximately 50 microM. Partial oxidation of vesicles with thimerosal produced a significant increase of release rate constants and initial release rates at all [Mg(2+)] tested (up to 1 mM), and shifted the K(0.5) value for Mg(2+) inhibition to 101 or 137 microM in triads actively or passively loaded with calcium, respectively. Further oxidation of vesicles with thimerosal completely suppressed the inhibitory effect of [Mg(2+)] on CICR, yielding initial rates of CICR of 2 micromol/(mg x s) in the presence of 1 mM free [Mg(2+)]. These effects of oxidation on CICR were fully reversed by SH reducing agents. We propose that oxidation of calcium release channels, by decreasing markedly the affinity of the channel inhibitory site for Mg(2+), makes CICR possible in skeletal muscle.

Adenosine Triphosphate↗
PubMed1998

Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles.

Fast (milliseconds) Ca2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (approximately 0.2 pH units) of the lumen occurred within the same time scale (t(1/2) = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5'-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5-10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.

Adenosine Triphosphate↗
PubMed1997

Rapid kinetic studies of SH oxidation-induced calcium release from sarcoplasmic reticulum vesicles.

We studied the kinetics of calcium release induced by SH oxidation in triads isolated from frog and rabbit skeletal muscle by measuring calcium fluxes by a fast filtration method. In both species SH oxidation induced release of 70-80% of the passively loaded calcium with a rate constant of 1 s-1. This rate constant is 3 times higher than the rate constant of calcium-induced calcium release and 15 times lower than the rate constant of ATP-induced release. Calcium release induced by SH oxidation exhibited the same calcium dependence of calcium-induced calcium release and was also inhibited by physiological [MG2+]. Neither SH oxidation-induced calcium release nor calcium-induced calcium release were regulated by luminal calcium. The redox state of thiol groups does not seem to control ATP-induced calcium release since the rate constant of calcium release after SH oxidation was not different from the rate constant measured in the presence of the reducing agent dithiothreitol. Our results do not support a role for SH oxidation, per se, as an essential step for the release of calcium from sarcoplasmic reticulum.

2,2'-Dipyridyl↗
PubMed1996

Luminal pH regulated calcium release kinetics in sarcoplasmic reticulum vesicles.

Calcium binding to triads isolated from rabbit skeletal muscle followed a single hyperbolic function in the pH range 5.5-8.0. Maximal binding was obtained at pH 8.0; decreasing the pH decreased the binding capacity and, at pH < or = 6.0, increased Kd 2-fold. These results indicate that lowering the pH diminished calcium binding to calsequestrin, since this protein is the primary source of calcium binding sites in triads. Luminal pH had a marked effect on calcium release induced by 2 mM ATP, at pCa 5.0, pH 6.8. At a constant luminal [Ca2+] of 0.1 mM, release rate constants (k) and initial rates of release increased steadily as a function of decreasing luminal pH; at luminal pH 7.5, values of k < 0.4 s-1 were found, whereas at pH 5.5 values of k approximately 10 S-1 were obtained. Increasing luminal [Ca2+] from 0.05 mM to 0.7 mM had no effect on the k values measured at luminal pH 5.5. In contrast, at pH 6.8, increasing luminal [Ca2+] produced a marked increase in k values, that reached maximal values of k approximately 10 S-1 at 0.7 mM luminal [Ca2+]. Control experiments using fluorescent pH indicators showed that luminal pH did not change significantly during calcium release. It is proposed that luminal protons or calcium induces conformational changes in calsequestrin that in turn promote activation of the calcium release channels.

Animals↗
PubMed1996

Protons induce calsequestrin conformational changes.

Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes extensive calcium-induced conformational changes at neutral pH that cause distinct intrinsic fluorescence changes. The results reported in this work indicate that pH has a marked effect on these calcium-induced intrinsic fluorescence changes, as well as on calorimetric changes produced by the addition of Ca(2+) to calsequestrin. The addition of Ca(2+) at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrinsic fluorescence was not affected by the addition of Ca(2+), and the same intrinsic fluorescence as that measured in millimolar calcium at neutral pH was obtained. The magnitude and the cooperativity of the calcium-induced intrinsic fluorescence changes decreased as either [H+] or [K+] increased. The evolution of heat production, determined by microcalorimetry, observed upon increasing the molar ratio of Ca(2+) to calsequestrin in 0.15 M KCl, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content changes produced by Ca(2+). We propose that protons bind to calsequestrin and induce protein conformational changes that are responsible for the observed proton-induced intrinsic fluorescence and calorimetric changes.

Animals↗
PubMed1996

Ca(2+)- and pH-dependent halothane stimulation of Ca2+ release in sarcoplasmic reticulum from frog muscle.

The effect of halothane on calcium release kinetics was studied in triad-enriched sarcoplasmic reticulum vesicles from frog skeletal muscle. Release from vesicles passively equilibrated with 3 mM 45CaCl2 was measured in the millisecond time range by use of a fast-filtration system. Halothane (400 microM) increased release rate constants at pH 7.1 and 7.4 as a function of extravesicular pCa. In contrast, halothane at pH 6.8 produced the same stimulation of release from pCa 7.0 to 3.0; no release took place in these conditions in the absence of halothane. Halothane shifted the calcium activation curve at pH 7.1, but not at pH 7.4, to the left and increased channel open probability at pH 7.1 in the cis pCa range of 7.0 to 5.0. These results indicate that cytosolic pCa and pH modulate the stimulatory effects of halothane on calcium release. Furthermore, halothane stimulated release in frog skeletal muscle at low pH and resting calcium concentration, indicating that in frog muscle halothane can override the closing of the release channels produced by these conditions, as it does in malignant hyperthermia-susceptible porcine muscle.

Animals↗
PubMed1995

Luminal calcium regulation of calcium release from sarcoplasmic reticulum.

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.

Animals↗
PubMed1995

Luminal calcium regulates calcium release in triads isolated from frog and rabbit skeletal muscle.

Triads isolated from frog and rabbit skeletal muscle were equilibrated with different external [Ca2+], ranging from 0.025 to 10 mM. Vesicular calcium increased with external [Ca2+] as the sum of a linear plus a saturable component; the latter, which vanished after calsequestrin removal, displayed Bmax values of 182 and 132 nmol of calcium/mg of protein, with Kd values of 1.21 and 1.14 mM in frog and rabbit vesicles, respectively. The effect of luminal [Ca2+] on release kinetics in triads from frog and rabbit skeletal muscle was investigated, triggering release with 2 mM ATP, pCa 5, pH 6.8. In triads from frog, release rate constant (k) values increased sixfold after increasing luminal [Ca2+] from 0.025 to 3 mM. In triads from rabbit, k values increased 20-fold when luminal [Ca2+] increased from 0.05 to 0.7 mM. In both preparations, k values remained relatively constant (10-12 s-1) at higher luminal [Ca2+], with a small decrease at 10 mM. Initial release rates increased with luminal [Ca2+] in both preparations; in triads from rabbit the increase was hyperbolic, and in triads from frogs the increase was sigmoidal. These results indicate that, although triads from frog and rabbit respond differently, in both preparations luminal [Ca2+] has a distinctive effect on release, presumably by regulating sarcoplasmic reticulum calcium channels.

Animals↗
PubMed1994

Comparison of the effects of caffeine and other methylxanthines on [Ca2+]i in rat ventricular myocytes.

1. The effects of caffeine and other methylxanthines were investigated on intracellular calcium concentration ([Ca2+]i) and contraction in rat isolated ventricular myocytes. The use of the fluorescent indicator, Indo-1, allowed simultaneous measurement of [Ca2+]i and the intracellular concentration of the methylxanthines. 2. Rapid application of caffeine (10 mM) produced a transient rise of [Ca2+]i which decayed to resting levels. This was accompanied by a transient contraction which decayed to a level above baseline. The addition of theophylline also produced a transient increase of [Ca2+]i. However, following the initial transient, contraction decayed before redeveloping to a maintained level. 3. Direct measurements showed that [caffeine]i rose more quickly than did [theophylline]i. The slower rise of [theophylline]i was associated with a delay in the increase of [Ca2+]i. At lower concentrations of the methylxanthines, theophylline was less effective than caffeine at initiating Ca release. The rate of entry of theobromine was similar to that of theophylline. 4. Isocaffeine did not produce a rise of [Ca2+]i. The rate of rise of [isocaffeine]i was much slower than that of either caffeine or theophylline. 5. Measurements of the oil:water partition coefficient showed that the order of relative partitioning into oil was: caffeine > theophylline > theobromine > isocaffeine. This is similar to the order of rate of entry into the cell. 6. We conclude that many of the differences in the effects of these methylxanthines can be attributed to differences in membrane permeability due to differences in oil:water partition.

Animals↗
PubMed1994

[Anemia associated with acute post streptococcal glomerulonephritis].

Thirty-three children with post-streptococcal acute glomerulonephritis, age mean: 8.3 years (range: 6 - 12) were studied prospectively. Mean initial hematocrit (Hct) was 31.6% with 90% showing Hct under the normal lower limit for this age group. Reticulocyte index (RI) was < 0.5 in half of the cases. Serum iron concentration, total iron binding capacity (TIBC) and percentage of transferrin saturation were normal for this age group although 75% of the children had increased serum ferritin levels. At the time of discharge, Hct increased to 35.1% but 44% still had anemia. Hct increased spontaneously for 105 days stabilizing at 38%. Based on Hct changes, 3 groups were defined: Group I (3 individuals): normal upon discharge; Group II (19): partial recovery at discharge, slow recovery stabilizing after 105 days; Group III (11): lower Hct, slower recovery but with RI significantly higher than group II (0.96 vs 0.45 p < 0.01). Our data suggest that although hemodilution is present in all, it may be considered the solely factor only in 3 cases (Group I). In group II, evidence of bone marrow depression was indicated by the low RI. On the other hand, the intense anemia that could not be justified only by hemodilution and marrow depression in group III, suggests other pathogenic factors.

Acute Disease↗
PubMed1993

pH-sensitive calcium release in triads from frog skeletal muscle. Rapid filtration studies.

Triad vesicles from frog skeletal muscle exhibited calcium-induced calcium release highly sensitive to extravesicular pH; calcium induced release at pH > 7.4 but not at pH 6.8. In contrast, triads isolated from rabbit skeletal muscle exhibited significant calcium-induced calcium release at pH 6.8. At pH 7.4, there was no stimulation of calcium release in triads from frog at pCa 7, maximum stimulation at pCa 5, and complete inhibition at pCa 3. Addition of ATP at pCa 5, pH 6.8, induced calcium release with the same high rate constants in both preparations. In triads from frog, ATP-induced calcium release at pCa 5 had the same kinetics at pH 6.8 and 7.4, whereas ATP-induced calcium release at pCa > 8, pH 6.8, was partial, with a decrease in the amounts released but not in rate constants. In contrast, triads from rabbit displayed the opposite behavior, with a decrease in rate constants but not in the amounts of calcium released at pCa > 8, pH 6.8. In triads from frog ATP-induced calcium release decreased at pCa < 4, to reach total inhibition at pCa 2; addition of magnesium (free > 0.5 mM) completely abolished ATP-induced calcium release at pH 6.8, pCa 7 or 5. The potential physiological relevance of these results is discussed.

Adenosine Triphosphate↗
PubMed1993

Triads and transverse tubules isolated from skeletal muscle contain high levels of inositol 1,4,5-trisphosphate.

We measured the content of inositol 1,4,5-trisphosphate in sarcoplasmic reticulum, transverse tubules, and triads isolated from frog skeletal muscle, as well as in triads isolated from rabbit skeletal muscle. We found that acid extracts of both transverse tubules and triads contained significant amounts of inositol 1,4,5-trisphosphate, in the range of 300-400 pmol/mg of protein as determined by a radioreceptor assay, whereas no detectable amounts were found in sarcoplasmic reticulum vesicles. The identity of inositol 1,4,5-trisphosphate in the extracts was confirmed by comigration with [3H]inositol 1,4,5-trisphosphate on polyethyleneimine-cellulose plates, and by phosphorylation to inositol 1,3,4,5-tetrakisphosphate using the inositol 1,4,5-trisphosphate 3-kinase present in muscle extracts. These findings may have important physiological implications. First, the results indicate that the muscle plasma membrane regions differentiated as transverse tubules contain high amounts of inositol 1,4,5-trisphosphate, suggesting that they might possess a high density of binding sites for this compound. Second, since inositol 1,4,5-trisphosphate has been proposed as a chemical transmitter in excitation-contraction coupling in skeletal muscle, our finding that this second messenger is present in high density at the site of coupling may contribute to the understanding of its role in this process.

Animals↗
PubMed1993

Relationship between intracellular pH and metabolite concentrations during metabolic inhibition in isolated ferret heart.

1. Intracellular pH (pHi) and phosphorus metabolites were measured in isolated ferret hearts with 31P nuclear magnetic resonance (NMR). 2. The application of cyanide (to mimic hypoxia) produced a fall of the concentration of phosphocreatine ([PCr]) and a rise of those of inorganic phosphate ([Pi]) and sugar phosphates. These were accompanied by an intracellular acidosis. 3. If glycolysis was partly inhibited by prior exposure to a glucose-free solution then the application of cyanide also produced a fall of [ATP]. The acidosis was similar to that observed in the presence of glucose. 4. If glycolysis was completely inhibited by iodoacetate then the acidosis produced by subsequent addition of cyanide developed more quickly. 5. The results are reproduced by a model which incorporates lactic acid production as well as the effects of protons released and absorbed by the changes in metabolite concentrations. The results suggest that the acidosis produced by cyanide (without inhibition of glycolysis) is largely due to lactic acid production. When glycolysis is partly inhibited (glucose-free solution) the acidosis produced by cyanide is partly due to protons released by ATP breakdown. Finally, when glycolysis is entirely inhibited the acidosis is completely due to ATP breakdown. There is no need to postulate a contribution on this time scale from inhibition of pH regulating mechanisms.

Acidosis↗
PubMed1992

Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes.

1. The fluorescent Na+ indicator SBFI was incorporated into isolated ventricular myocytes using the acetoxymethyl (AM) ester. 2. The excitation spectrum was found to be shifted about 20 nm in the cell compared to in vitro. In the cell, an increase of [Na+] decreased fluorescence at 380 nm (F380) and had no effect at 340 nm (F340). The ratio (R = F340/F380) was used as a measure of [Na+]i. 3. In vivo calibration of SBFI for [Na+]i was obtained by equilibrating [Na+] across the plasma membrane with a divalent-free solution in the presence of gramicidin D. 4. Selective removal of the surface membrane with saponin or digitonin released only about 50% of the indicator. Following saponin treatment, cyanide or carbonylcyanide m-chlorphenylhydrazone (CCCP) increased the apparent [Na+] measured by the remaining (presumably mitochondrial) SBFI. It is suggested that mitochondrial [Na+] is normally less than cytoplasmic. 5. Attempts to examine the effects of metabolic inhibition on [Na+]i were hampered by changes of autofluorescence due to changes of [NADH]. It is shown that this effect can be corrected for using the isosbestic signal (excited at 340 nm). 6. Inhibition of both aerobic metabolism (with CN-) and glycolysis (glucose removal or iodoacetate) produced a gradual increase of [Na+]i. This began before the resting contracture developed and may (via Na(+)-Ca2+ exchange) account for some of the rise of diastolic [Ca2+]i seen in previous work. The rise of [Na+]i began at about the same time as the decrease of systolic contraction and therefore at a time when [ATP]i had begun to fall.

Animals↗
PubMed1991

Factors affecting distribution of alveolar surfactant during resting ventilation.

Few studies have been done to establish the ventilatory factors affecting alveolar surfactant under resting conditions. Experiments in which ventilatory variables were recorded for 4 h were performed in 12 adult cats breathing spontaneously under pentobarbital sodium anesthesia. After the animals were killed and bronchoalveolar lavages (BALs) were performed, the resulting fluid was subjected to differential centrifugation and determinations of proteins, phospholipids (PL), and disaturated phosphatidylcholine (DSPC). Pellet P1+2 was obtained by two centrifugations at 140 g; the supernatant was centrifuged at 1,000 g to obtain pellet P3 and subsequently at 60,000 g to obtain P4. Pellets P3 and P4 had the higher contents of PL and DSPC. Bivariate and multivariate correlational analyses indicate that 1) total PL in BAL was not related to any of the ventilatory variables studied, 2) PL in P3 and P4 fractions was directly correlated to the physiological range of variations in the frequency of large spontaneous gasps (fL), and 3) PL in P1+2 fraction was inversely related to fL. Cats subjected to bilateral section of carotid nerves, although presenting reduced chemosensory drive and ventilatory chemoreflexes, did not exhibit significant differences in resting ventilatory variables nor in alveolar surfactant components. Present results indicate that the total content of alveolar surfactant is not modified by ventilatory variations within physiological range but that the spontaneous occurrence of large gasps increases the proportion of more active forms of alveolar surfactant. This may mediate the role of augmented breaths in keeping lung compliance and preventing atelectasis under resting ventilatory conditions.

Animals↗