How to make and use calcium-specific mini- and microelectrodes.
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Animals↗
Biomedical subjects
S Baudet
Publications and source records attributed to S Baudet.
Paradoxical twitch potentiation after rest in cardiac muscle: increased fractional release of SR calcium.
Rest interval dependent changes in contractile force (rest decay and rest potentiation) were studied in rabbit, rat and ferret ventricular muscle and myocytes. The SR Ca content was assessed by rapid cooling contractures or caffeine induced contractures. Intracellular Ca transients, action potentials and Ca current were also recorded. Rest decay of twitches in rabbit ventricle are roughly paralleled by a decline in SR Ca content. Rat ventricle exhibits primarily rest potentiation, which is not necessarily paralleled by an increased SR Ca content. Ferret ventricle exhibits both rest potentiation and rest decay. However, the SR Ca content in ferret appears to decline monotonically throughout the rest. It is demonstrated that the rest potentiation is not due to an increase in Ca current or in action potential duration. We conclude that there is an increase in the fraction of SR Ca content which is released during the time that rest potentiation develops. The differences in post-rest contractile function among different cardiac preparations can be described by a simple unifying mechanistic model. In this model there is an exponential time dependent recovery of the ability of the SR to release Ca (e.g. recovery from inactivation) which can be considered to increase the fraction of SR Ca release in response to activation. This fractional SR Ca release is multiplied by the SR Ca content (which may decline exponentially) to provide a measure of the Ca available for activation of contractile force.
Animals↗
Effects of thapsigargin and cyclopiazonic acid on twitch force and sarcoplasmic reticulum Ca2+ content of rabbit ventricular muscle.
Thapsigargin (TG) and cyclopiazonic acid (CPA) are reported to be specific high-affinity inhibitors of the sarcoplasmic reticulum (SR) Ca2+ pump in isolated membranes and cells, with TG causing complete pump inhibition at nanomolar concentrations. To evaluate the effectiveness of TG and CPA in small multicellular cardiac preparations, we used rapid cooling contractures (RCCs) to assess the SR Ca2+ load. In contrast to observations in single myocytes, TG caused remarkably slow and incomplete SR Ca2+ depletion in multicellular preparations. A 45-minute exposure to 500 microM TG at 30 degrees C and 0.5-Hz stimulation only decreased RCCs by 76 +/- 5% (and 100 microM CPA reduced RCCs by 59 +/- 10% [mean +/- SEM]). In contrast, 10 minutes with 20 mM caffeine completely abolished RCCs. This confirms that there was still a caffeine-sensitive pool of Ca2+ in the TG-treated muscle. The time constant of rest decay of RCCs was accelerated by both TG (from 83 +/- 18 to 26 +/- 6 seconds) and CPA (from 68 +/- 11 to 10 +/- 5 seconds). This might be expected since Ca2+ leaking from the SR during rest cannot be taken back up as efficiently, favoring Ca2+ extrusion by the sarcolemmal Na(+)-Ca2+ exchanger. TG and CPA decreased twitch force (by 44 +/- 7% and 40 +/- 11%, respectively) and increased twitch duration, presumably because of the SR effects. We conclude that complete blockade of SR Ca2+ uptake by TG or CPA in multicellular preparations cannot be assumed, even at high [TG] or [CPA], unless evaluated (eg, by RCC).
Animals↗
Basket cells or shadow cells of Gumprecht: a scanning electron microscope study, and the correlation between percentages of basket cells, and cells with altered chromatin structure (dense cells), in chronic lymphocytic leukemia.
For over 50 years the received wisdom has been that the shadow cells of Gumprecht otherwise known as basket cells (BC) are in artefact, produced during preparation of films when a drop of blood is spread on a slide. The assumption has been that they are therefore of no significance. They are commonly seen in blood films from patients with lymphoproliferative syndromes and particularly in chronic lymphocytic leukemia (CLL). In 96 patients with CLL a statistically significant correlation existed between the basket cells observed in films and the lymphocytes with dense chromatin (DC) determined by flow-cytometry. There was no statistically significant correlation between the number of BC and DC, and the anatomic-clinical stage of the disease.
Chromatin↗
[Automation in hematologic cytology].
Automation in hematological cytology can identify blood cells in suspension by new parameters: electrical field variations, light intensity modifications absorbtion or diffraction by one or two lasers, fluorescence intensity, cytochemical reactions or specific lysis. The new technology change the strategy of hematological laboratories. Automation realizes perfectly the white blood cell differential and modifies hematological language with respect to anemia and white blood cells diseases.
Automation↗
Effect of haemodynamic pressure overload of the adult ferret right ventricle on inotropic responsiveness to external calcium and rest periods.
The inotropic effects of external calcium concentration ([Ca2+]o] and rest periods have been compared in papillary muscles isolated from control (n = 4) and pressure-overloaded right (n = 5) ventricles of adult ferrets. Hypertrophy was induced by pulmonary artery clipping for 30-45 days. Under control conditions (3 mM [Ca2+]o, 0.1 Hz), the isometric twitch force of hypertrophied muscles was decreased by 75%, time to peak was increased by 30% and time to half-relaxation was increased by 50% compared with non-hypertrophied preparations. The sensitivity of contraction to [Ca2+]o was decreased in hypertrophied muscles compared with control ([Ca2+] required for half-maximal contraction: 4.1 mM vs 1.7 mM) and the maximal contraction reached at high [Ca2+]o was smaller in pressure-overloaded muscles compared with control (8.3 +/- 2.0 mN mm-2 vs 19.0 +/- 2.1 mN mm-2 respectively). In both groups, rest periods longer than the steady-state interval were initially accompanied by a potentiation of the first post-rest contraction compared with steady-state. Peak potentiation occurred after a rest of 120 s in hypertrophied muscles and after a rest of 60 s in control. The maximal relative potentiation, i.e. compared with the steady-state twitch, was higher in hypertrophied muscles (+75%) than in control (+20%). After peak potentiation, the amplitude of the first post-rest contraction progressively decreased with increasing periods of rest, although at a slower rate in hypertrophy compared with control. The time constants of post-rest decay were 1203 +/- 99 s and 528 +/- 24 s respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
External calcium sensitivity of low sodium contractures in the control and hypertrophied right ventricle of the ferret.
The existence of possible differences of calcium (Ca2+) fluxes through the sarcolemmal sodium-calcium (Na+/Ca2+) exchanger during hypertrophy has been tested by comparing the characteristics of the contracture--as an indicator of the intracellular Ca2+ concentration--induced by partial or total withdrawal of external sodium (Na+), in the absence of external potassium, in the right ventricular trabeculae of adult ferret hearts. Pressure-overload was induced by pulmonary artery clipping and led to an increase of the right ventricular weight of 60%. At an external Ca2+ concentration ([Ca2+]o) of 3 mM, the dependence of the contractures on extracellular sodium concentration ([Na+]o), the rate of tension development, the time course of spontaneous relaxation and the time course for the repriming of the contracture were unchanged by hypertrophy. However, the relationship between [Ca2+]o and contracture amplitude at various [Na+]o showed that the apparent affinity of the contracture for [Ca2+]o was decreased in hypertrophied preparations. Thus, in 0 mM [Na+]o, half-maximal contracture was induced at a [Ca2+]o of 0.012 +/- 0.016 mM and 0.171 +/- 0.021 mM in control (n = 11) and hypertrophy (n = 12) respectively (P less than 0.001). Although these data may be indicative of a decreased Ca2+ influx through the Na+/Ca2+ exchanger, it cannot be excluded that intracellular buffering mechanism may also be involved in this differential response to [Na+]o withdrawal.
Animals↗
Effects of reduced external sodium concentration and multivalent cations on caffeine contractures in young ferret atrial trabeculae.
The characteristics of caffeine (1.25-80 mM) transient contractures have been examined in small atrial trabeculae (diameters 50-250 microns) isolated from young (1-1.5 months) ferret hearts. In the control medium, the half-saturation constant and the maximum contracture strength (at infinite caffeine concentration) were 37.8 +/- 10.2 mM and 0.9 +/- 0.2 kN.kg-1 (n = 11), respectively. The contractile response to caffeine was markedly enhanced following reduction of external sodium (70-0 mM). The perfusion of young ferret trabeculae with the sodium-free medium (up to 3 min) decreased the half-saturation constant by a factor of three (12.4 +/- 1.6 mM, n = 8) with an increase in maximum contracture strength (1.09 +/- 0.3 kN.kg-1, n = 8). The effects of various divalent and trivalent cations have been tested on the 10 mM caffeine contracture in trabeculae perfused with Na-containing (140 mM) solution. The order of cation effectiveness is Gd3+ (half effect 0.04-0.07 mM) greater than Cd2+ (0.15-0.25 mM) greater than Ni2+ (2-2.5 mM) greater than Co2+ (7-7.5 mM) much greater than Mn2+. In conclusion, the present work has shown that in atrial trabeculae isolated from young ferret hearts, the strength of the caffeine contracture was markedly affected by the activity of the sarcolemmal Na-Ca exchange.
Animals↗
Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution.
The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was co-crystallized with the deoxy-dinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 A resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulfate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root-mean-square C alpha movement being 0.45 A. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighboring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so-called recognition loop as it does in other complexes of guanine nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position where the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be non-productive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolyzed bond.
Amino Acid Sequence↗
Intracellular Na activity measurements in the control and hypertrophied heart of the ferret: an ion-sensitive micro-electrode study.
Because of the role of intracellular Na on cardiac contractility and of the depressed isometric contractile response of the hypertrophied myocardium, the effects of pressure overload on the intracellular Na activity (aiNa) have been investigated in papillary muscles isolated from the ferret right ventricle. In animals subjected to pulmonary artery clipped for 1-2 months, right ventricle-to-body weight ratio was increased by about 39% in comparison with the control group. aiNa was measured in quiescent papillary muscles, by means of Na-sensitive micro-electrodes, at room temperature (19-22 degrees C). aiNa values were, in the control ventricular cells, 7.8 +/- 1.1 mM (mean +/- SD; n = 20) and in the hypertrophied ones, 8.0 +/- 1.2 mM (n = 49). During superfusion by medium with a reduced extracellular Na concentration ([Na]0), aiNa declined in control and pressure-overloaded muscles to similar steady-state levels at a given [Na]0. aiNa fall was mono-exponential and was characterized by a smaller time constant in the hypertrophied group upon total withdrawal of Na0 (control 209 +/- 19 s, n = 4; hypertrophied 128 +/- 42 s, n = 6). In the absence of external K, aiNa increased to levels that were not significantly different between both groups. It was concluded that, in quiescent preparations, steady-state aiNa was not modified by the hypertrophic process. However, pressure overload induced a modification of aiNa regulation by a possible alteration of the sarcolemmal Na/Ca exchange, although other mechanisms, such as mitochondrial Ca transport, could be involved in the differential response to Na0 removal.
Animals↗
Triggered activity as a possible mechanism for arrhythmias in ventricular hypertrophy.
To study the cellular mechanisms of arrhythmias occurring in cardiac hypertrophy, we performed standard microelectrode studies on papillary muscles isolated from control (group N) and hypertrophied ferrets right ventricles. Different stages of hypertrophy, induced by pulmonary banding, were studied: 10-22 days (group H1), 4-6 weeks (H2), and 5 1/2-6 months (H3). During the development of hypertrophy, under beta-adrenergic stimulation, triggered activity (TA) induced by delayed afterdepolarizations appeared in 2 of 5 muscles in group H1 and 8 of 8 in group H2. This arrhythmia was absent in N muscles, as well as in H3, despite a pronounced prolongation of the action potentials at 50% (100 +/- 9.3 msec in group H3 vs 67 +/- 5.7 msec in H2; P less than 0.01) and 90% of repolarization (225 +/- 8.7 in H3 vs 185 +/- 7.4 msec in H2; P less than 0.02). The presence of TA was associated with an increase in the intracellular calcium activity (144 +/- 60 nM in H2 vs 47 +/- 9 nM in N; P less than 0.05). TA properties were as follows. Triggering frequency increased as beta-adrenergic stimulation increased, as pacing cycle length (PCL) decreased, and as duration of the prestimulative pause increased. The duration of salvos of TA increased as duration of the prestimulative pauses increased (NS). The coupling interval of the first triggered beat decreased as PCL decreased (P less than 0.001). The minimal cycle length of salvos of TA was not modified by these parameters. It is concluded that delayed afterdepolarizations-induced TA may occur under beta-adrenergic stimulation during the first stages of ventricular hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)
Action Potentials↗
Differential effects of caffeine on skinned fibers from control and hypertrophied ferret hearts.
The effects of caffeine on the mechanical properties of detergent-skinned fibers from control and pressure-overloaded ferret hearts have been compared. Right ventricle hypertrophy was studied 6 wk after pulmonary artery ligation, an intervention that increased the right ventricular weight-body weight ratio by 60%. Although no changes were observed in the Ca sensitivity of contraction between control [pCa for one-half-maximal activation (pCa50) = 5.80 +/- 0.05] and hypertrophied (pCa50 = 5.85 +/- 0.02) muscles, the leftward shift of the force-pCa relationship induced by caffeine was more pronounced in hypertrophied fibers than in control; pCa50, was 5.86 +/- 0.02, 5.94 +/- 0.01, 6.03 +/- 0.01 in control and 5.97 +/- 0.03, 6.10 +/- 0.03, 6.24 +/- 0.01 in control and 5.97 +/- 0.03, 6.10 +/- 0.03, 6.24 +/- 0.03 in hypertrophied fibers for 2, 5, 10 mM caffeine, respectively. This was accompanied in the hypertrophied muscles by a decrease in the Hill coefficient. On the other hand, maximal force was not affected by 10 mM caffeine. From these results it is possible that the site of action of caffeine in myofibrillar proteins is the thin filament. The increased responsiveness of hypertrophied fibers to caffeine may be mediated by a pressure overload-induced change in the thin filament regulatory proteins. Moreover, inotropic agents may act differently in hypertrophied than in control myocardium.
Animals↗
Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence.
The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.
Acrylamide↗
Changes in resting membrane potential induced by active sensitization in the guinea-pig vas deferens.
Smooth muscles hyperresponsiveness is a common feature in anaphylaxis and allergic diseases. The aim of the present work was to investigate whether the enhanced reactivity of sensitized guinea-pig vas deferens was associated with changes in the resting membrane potential (Er) of the smooth muscle cells. Active sensitization was performed by subcutaneous injection of egg albumen. Er was measured in vitro in isolated vas deferens with conventional KCl-filled microelectrodes. Quantification of [3H]ouabain binding sites, measurements of 86Rb efflux, and measurements of Na and K contents were also performed. In normal physiological solution, at 35 degrees C, Er was a mean of -54.1+/-0.3 mV (mean +/- SEM) in control vas deferens. Sensitization resulted in depolarizing Er by about 7 mV. In control and sensitized preparations, the 3H-ouabain binding site concentration, the efflux of 86Rb, and the K content were similar. In guinea-pig vas deferens, active sensitization induced a partial depolarization of the resting membrane potential of the smooth muscle cells, which did not result from a downregulation of Na+ -K+ pump sites.
Animals↗