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Biomedical subjects

F Orrego

Publications and source records attributed to F Orrego.

At least 37 records · Page 2Linked to original sources
PubMed1985

Methylphenidate, but not other CNS stimulants, inhibits red blood cell calcium-activated potassium efflux.

The first-order rate constant of net potassium efflux, measured in human red blood cell (RBC) suspensions by means of a K+-sensitive electrode, was increased 26 fold (from 0.0025 min-1 to 0.0656 min-1) by 0.5 microM of the calcium ionophore A 23187. Both the basal or the calcium-stimulated potassium efflux remained unchanged following the addition of different CNS stimulants (nikethamide (1 mM), pentylenetetrazol (1mM), doxapram (1 mM), strychnine (0.1 mM), picrotoxin (0.1 mM), or nomifensine (0.1 mM). Methylphenidate (10-100 microM), however, inhibited in a concentration-dependent manner the calcium-stimulated, but not the basal potassium efflux. An IC50 of 190 microM was estimated for this effect.

Calcium↗
PubMed1984

A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations.

The possible excitatory effect of N-acetyl-alpha- aspartylglutamate ( NAAG ) was studied in 3 different systems. First on the increase in 45Ca2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM L-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of L-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 micrograms) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.

Animals↗
PubMed1984

Calcium and the mechanism of action of digitalis.

A critical review is made of the mechanism by which digitalis increases the force of contraction of heart muscle. First, it is concluded that the initial step is always an inhibition of the sodium pump, and that the postulated stimulation of the pump by low digitalis concentrations is, possibly, not a real phenomenon. Secondly, the major theories that try to explain the inotropic effect of digitalis are analyzed, and it is tentatively concluded that the effect occurs because Na increases close to the inner side of the plasma membrane, and this decreases Ca efflux through the Na-Ca "exchange" mechanism. An internal Ca store, probably the sarcoplasmic reticulum, that competes with the plasma membrane for Ca, is then able to capture and, subsequently release, a larger fraction of the Ca mobilized during each transient. It is also concluded that the digitalis-induced larger Ca transients can be entirely explained because of a greater Ca "injection" into the cytoplasm during each beat, and not because of changes in resting pCa. A comprehensive model is presented that seems to explain in some detail both the inotropic and the toxic effect of digitalis.

Animals↗
PubMed1983

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro.

Kainate (0.62-5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl-DL-aspartate (50-1000 microM), L-glutamate (0.62-5 mM), DL-homocysteate (0.62-2.5 mM), and ibotenate (6-170 microM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino acids N-acetyl-L-aspartate, and alpha-methyl-DL-aspartate (both 1.25-5 mM), did not increase such influx. We suggest that enhanced calcium uptake may represent the basis for the neurotoxic effects of these compounds.

Absorption↗
PubMed1983

Guanethidine effects on the guinea pig vas deferens are antagonized by the blockers of calcium-activated potassium conductance, apamin, methylene blue, and quinine.

The blocking effects of guanethidine on electrically induced, neurally mediated, contractions of the guinea pig vas deferens in vitro could be markedly antagonized by the bee venom polypeptide apamin (20-60 nM), by 0.1 mM methylene blue, and (less regularly) by 0.1-0.15 mM quinine, three substances known to inhibit calcium-activated potassium conductance in a variety of cells. Guanethidine (20 microM) was also found to inhibit (by 88%) the release of [3H]norepinephrine induced by electrical stimulation (20-Hz, 2-msec, biphasic pulses of supramaximal voltage). Such inhibition was decreased to 39% when 20 nM apamin was present together with guanethidine, thus showing that the effect of this polypeptide is presynaptic. On the basis of these findings, we suggest that guanethidine may block adrenergic neurons by activating their calcium-activated potassium conductance, presumably by releasing intracellular calcium.

Animals↗
PubMed1982

A search in rat brain cortex synaptic vesicles for endogenous ligands for kainic acid receptors.

Conditions were found for stabilizing rat brain cortex kainic acid (KA) receptors. Such receptors had the same Hill number (about 0.6) for KA and for glutamate. The receptors were then used as detectors for endogenous ligands present in brain cortex synaptic vesicle (SV) soluble extracts. When these SV extracts were fractionated by gel filtration on Sephadex G-10, by thin-layer chromatography, or by high voltage electrophoresis, a single endogenous component, that in all cases comigrated with glutamic acid, was found.

Animals↗
PubMed1982

A study of calcium compartments in rat brain cortex thin slices: effects of veratridine, lithium and of a mitochondrial uncoupler.

The efflux kinetics of 45Ca from rat brain cortex thin slices previously equilibrated with it, was studied in a superfusion system. Two first order kinetic components of efflux from the tissue were found: k2 = 0.0667 min-1, that was unchanged by lowering the temperature from 37 degrees C to 15 degrees, and k3 = 0.0167 min-1 at 37 degrees C, that was reduced to 0.0897 min-1 at 15 degrees C. This suggests that k2 represents efflux from the extracellular space, and k3 that from the cellular compartment. Addition of the mitochondrial uncoupler carbonyl cyanide, m-chlorophenylhydrazone (CCCP) (10(-5)M) increased the efflux fractional rate constant of 45Ca by 35%, while no change in efflux was induced by 10 mM caffeine. Veratridine (10(-5)M) drastically reduced 45Ca efflux if superfusion was with physiological salt solution (150 mM sodium present), but not if 50 mM lithium replaced an equivalent amount of sodium in the superfusion fluid. This lithium-containing solution did not affect 45Ca efflux in the absence of veratridine. These results indicate that mitochondria accumulate only a minor fraction of intracellular 45Ca; that 45Ca possibly turns over very rapidly in the endoplasmic reticulum, and that most of 45Ca is present in a different, non-mitochondrial, non endoplasmic reticular compartment, the nature of which can be only conjectured.

Animals↗
PubMed1981

Differential effects of ouabain and 2,4-dinitrophenol on contractile tension of and on sodium and calcium efflux from frog heart ventricular strips.

1 The efflux, from heart ventricular strips of Rana pipiens, of sodium ((22)Na) and calcium ((45)Ca) was measured simultaneously.2(22)Na efflux could be resolved into two first order kinetic components: k(I) = 0.105 min(-1), thought to represent efflux from the extracellular space, and k(II) = 0.0182 min(-1) representing efflux from the cells.3(45)Ca efflux was also resolved into an extracellular component, k(I) = 0.1216 min(-1); and an intracellular one, k(II) = 0.0102 min(-1). (45)Ca k(II) was greatly increased by 2,4-dinitrophenol (DNP), but unchanged by caffeine. This suggests that it represents a mitochondrial calcium compartment.4(22)Na k(II) was not changed by DNP. This indicates that, at the time of DNP addition, (22)Na was passively bound to undefined intracellular components.5 Ouabain (10(-6) M) decreased (45)Ca efflux (k(II)) initially but at later periods slightly increased it. The former effect is thought to be due to an action at the plasma membrane level, while the latter probably represents an increased exchangeability of mitochondrial calcium. The same effects were always found when ouabain was applied at different times of strip superfusion.6 Ouabain (0.25 to 4 muM) did not decrease the k(II) of (22)Na efflux. Kinetic reasons are presented which indicate that, in this preparation, the activity of the sodium pump may be too fast to be measured by means of (22)Na efflux, therefore these findings do not necessarily mean that ouabain does not inhibit active sodium transport.7 The time course of the inotropic effect of ouabain was also studied in ventricular strips of Rana pipiens heart that were stimulated at 0.2 Hz with biphasic, 2 ms pulses of supramaximal intensity, and incubated in Ringer solution containing 1.1 mM calcium, or in ;calcium-free' Ringer (residual calcium: 5.2 muM), or in ;calcium-free' Ringer with 0.1 mM of the calcium chelator ethyleneglycol bis (beta-aminoethylether) N,N'-tetraacetic acid (EGTA).8 In Ringer, the inotropic effect of ouabain was already observed at 5-10 s after steroid addition, even with the lowest concentration tested (0.25 muM), while signs of toxicity appeared only after 15 min in 4 muM ouabain, the highest concentration used.9 When the strips were incubated in ;calcium-free' Ringer solution, force of contraction decayed to 1-2% of that in 1.1 mM calcium. Addition of 4 muM ouabain to these hypodynamic strips led to a progressive increase in contractile force of up to 300%, that started after a 50 s latency period. No signs of toxicity were observed.10 Incubation of the strips in EGTA-Ringer also reduced contractile force to about 2% of that in Ringer, and 4 muM ouabain also increased force of contraction by approximately the same amount as seen in ;calcium-free' Ringer, but the effect began after a 10 min latency period. The concentration of calcium ion (Ca(2+)) in the extracellular space of strips incubated in EGTA-Ringer, was approximately 800 fold lower than in Ringer, and 60 fold lower than in ;calcium-free' Ringer solution.11 Caffeine (20 mM) induced, in strips previously incubated for 1 h in 4.4 mM calcium Ringer solution plus 10(-6) M ouabain, a marked initial contracture, that relaxed spontaneously, and was followed by slow waves of contracture. This was not observed if the strips were incubated, prior to caffeine, in 4.4 mM calcium Ringer without ouabain, or in 1.1 mM calcium Ringer solution that contained 10(-6) M ouabain.12 Based on these findings, a hypothesis that can explain the inotropic effect of cardioactive steroids is presented.

2,4-Dinitrophenol↗
PubMed1980

Electrically induced release of amino acids formed from (U14C) glucose in rat brain cortex slices, studied by a simplified dansylation procedure.

The nonessential amino acids glutamate, aspartate, glutamine, gamma-aminobutyrate (GABA), alanine, glycine, and proline present in rat thin brain cortex slices were labeled by in vitro incubation of these with [U-14C]glucose, and the efflux of such endogenous radioactive amino acids and of lactate was studied in a superfused system, under control conditions or when the slices were depolarized by varous procedures. When electrical stimuli known to induce selective neurotransmitter release (1 or 1.5 volt, sine wave 60 Hz) were applied for 10 sec to the slices, no significant increase in amino acid efflux was found. When more intense stimuli (4 volt, 60 Hz) were applied for 60 sec, or extracellular potassium was raised to 56 mM, both conditions being known to induce nonselective substance release, the efflux of essentially all amino acids and of lactate was markedly increased. Increases in efflux were proportionately larger for glutamate, aspartate, and gamma-aminobutyrate, and this could be accounted for by their greater intracellular chemical (or electrochemical) potentials, but not because of a selective release mechanism for them. Amino acids were analyzed as their 1-dimethylaminoaphthalene-5-sulfonyl (dansyl) derivatives, by a modification of existing procedures in which the dansyl (DNS) derivatives were efficiently extracted from acidified incubation fluid into an organic phase. This rapidly desalted the derivatives and allowed their concentration and chromatographic separation on thin-layer silica gel sheets with little loss.

Amino Acids↗
PubMed1977

Effects of tetrodotoxin, elevated calcium and calcium antagonists on electrically induced 3H-noradrenaline release from brain slices.

Electrically induced release of 3H-noradrenaline from superfused rat brain cortex slices was completely inhibited by tetrodotoxin (1 microgram/ml) if 0.5 or 1 V stimuli were used, while inhibition was 97% with 2 V, and nil with 12 V stimuli. 20 mM calcium depressed noradrenaline release at all applied potentials, and shifted the release versus voltage curve to higher potentials. Manganese (5 mM) and D-600 (10-100 microgram/ml) also considerably inhibited release. This suggests that stimuli of up to 2 V induce transmitter release by exciting intracortical noradrenergic axons in their non-terminal regions.

Animals↗
PubMed1977

Electrically induced release of [3H]dopamine from slices obtained from different rat brain cortex regions. Evidence for a widespread dopaminergic innervation of the neocortex.

Slices obtained from the deeper layers of the rat dorsal frontal, parietal and occipital brain cortex were incubated in vitro with 6.25 X 10-7 M [3H]dopamine (DA), and subsequently superfused and electrically stimulated, while held on quick transfer electrodes, and changes in the efflux of 3H and of the individual amines measured. The separation of the amines, with quantitative recoveries, was performed by chromatography on cation-exchange resins eluted sequentially with water, 1 N HCL AND 6 M urea i 1 N HCl. When no drugs were used, the prestimulation efflux was entirely formed by deaminated metabolites, while following stimulation there was an increase in the efflux of deaminated metabolites, and considerable amounts of [3H]-noradrenaline (NA) now appeared. No DA was present in the pre- or poststimulation medium. Similar results were obtained in all the regions studied. When the slices were incubated with 10-5 M desmethylimipramine (DMI), 10-4 Mnialamide and 10-4 M tropolone, before and during incubation with [3H]DA, it was observed that, prior to stimulation, the efflux was composed of deaminated metabolites, DA and 3-methoxytyramine (MTA), and following the electrical stimulus there was an increased release of DA, NA and deaminated compounds (in order of decreasing release), while no change in that of MTA was evident. The stimulus-induced release of DA was greatest from frontal slices, intermediate from parietal, and lowest from occipital ones. DMI-resistant uptake of [3H]DA also diminished when passing from frontal to occipital. These findings are interpreted as due to the presence of dopaminergic axon terminals in all the regions studied, but with a density that diminishes in a rostrocaudal direction.

Adrenergic Fibers↗
PubMed1977

Subcellular distribution of rat brain cortex high-affinity, sodium-dependent, glycine transport sites.

The subcellular distribution of the membrane components, present in rat brain cortex homogenates, that interact with glycine in the presence of sodium ions was studied. The distribution in the primary fractions, as per cent of total binding in the homogenate, was: P1 ('nuclear'), 58%; P2 (large granule), 39%; P3 (microsomal), 2%9 Of the subfractions obtained by centrifuging P1 in a linear 0.32--1.5 M sucrose gradient, only the lighter fraction (P1-III) formed by large myelin fragments was enriched in specific binding activity with respect to P1. The pellet formed by purified nuclei had negligible binding, and fractions of intermediate density had a lower activity than P1. Transient exposure of P1-III to 1.5 M sucrose did not diminish its binding ability. Similarly, in the subfractions obtained by centrifuging P1 in a discontinuous sucrose gradient, only the least dense one, P1-A, that is formed exclusively by large myelin fragments, was enriched with respect to P1. The electron microscopy of these fractions is presented. The P2 subfractions, obtained in a linear 2--18% Ficoll gradient, had the following sodium-dependent activity (counts/min/mg protein, fractions being in the order of decreasing density): pellet, 0; P2-I, O; P2-II, 450; P2-III, 1770; P2-IV, 4130; unfractionated P2, 880; P2-IV, the least dense fraction being composed mainly of myelin. With P2 subfractions obtained in a discontinuous sucrose gradient (0.32, 0.8 and 1.2 M sucrose layers), it was also found that sodium-dependent glycine binding was only enriched, with respect to P2, in the myelin fraction P2-A. Glycine binding to purified brain cortex myelin was also found to be very high, while binding to non-myelin membranes, obtained during the purification procedure, was only 0--7% of that seen with myelin. These results suggest that high-affinity glycine binding is located in myelin proper, and possibly also in some other glial plasma membranes, but not in nuclei, mitochondria, endoplasmic reticulum or synaptosomes. The relevance of these findings for interpreting previous reports on high-affinity glycine transport in the central nervous system is analyzed.

Animals↗