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J Satrústegui

Publications and source records attributed to J Satrústegui.

10 recordsLinked to original sources
PubMed1992

Pyruvate dehydrogenase dephosphorylation in rat brain synaptosomes and mitochondria: evidence for a calcium-mediated effect in response to depolarization, and variations due to ageing.

The phosphorylation state of P42, the phosphorylated, catalytically inactive, alpha-subunit of pyruvate dehydrogenase (PDH), decreased markedly (42.4%) in response to K(+)-depolarization of synaptosomes. The dephosphorylation was rapid (5-15 s), calcium-dependent and could also be observed in isolated mitochondria exposed to a rise in extramitochondrial calcium, suggesting that P42 dephosphorylation may act as a calcium sensor in the mitochondrial matrix. The depolarization-dependent dephosphorylation rate of P42 was decreased in synaptosomes derived from 24-month-old animals with respect to 3-month-old adults. The relevance of these results in terms of PDH activation during ageing is discussed.

Aging↗
PubMed1992

Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.

Aging↗
PubMed1992

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.

Adenosine Triphosphate↗
PubMed1992

Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipocytes.

The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been investigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal 32P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3-month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the Km for ATP was not modified. In contrast, the insulin-stimulated Vmax value was decreased by two-fold in 24-month-old rats. The decrease in Vmax does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor beta-subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor.

Adenosine Triphosphate↗
PubMed1992

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.

Adenosine Diphosphate↗
PubMed1991

Conditions restricting depolarization-dependent calcium influx in synaptosomes reveal a graded response of P96 dephosphorylation and a transient dephosphorylation of P65.

Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High-K+ depolarization in media of low [Na+]o (32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K(+)-stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na(+)-based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 microM veratridine at 0.5 mM external calcium. Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) of the phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed.

Acetylcholine↗
PubMed1991

Prolactin increases cytosolic free calcium concentration in hepatocytes of lactating rats.

PRL at a physiological concentration (10(-8) M) produced a very rapid and transient increase in 45Ca efflux in freshly isolated hepatocytes, which reached the highest value within 5 min and returned to baseline level after 20 min. PRL-induced 45Ca2+ efflux resulted in a loss of 15% of total cell calcium, which was similar to that found in vasopressin-treated cells. However, in contrast with the PRL effect, 45Ca2+ efflux induced by vasopressin was sustained. We demonstrate by using two different approaches, glycogen phosphorylase-a activation and direct cytosolic calcium concentration [( Ca2+]i) measurements, that PRL elicits a [Ca2+]i increase. The treatment of hepatic cells with PRL caused a 4-fold stimulation in glycogen phosphorylase-alpha activity after 2 min of PRL addition. Direct [Ca2+]i determination in fluo-3-loaded hepatocytes showed a 11% increase after 5 min of PRL addition. Similar data were observed in hepatocytes stimulated either with vasopressin (10(-7) M) or calcium ionophore A23187 (200 nM). The increase in [Ca2+]i promoted by PRL was independent of extracellular calcium or voltage-operated calcium channels. The data demonstrate that calcium is involved in the intracellular signaling of PRL in liver cells and that PRL initiates its action by a Ca2+ mobilization from the intracellular stores.

Animals↗
PubMed1990

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i.

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.

Aminoquinolines↗
PubMed1990

Rapid postnatal developmental changes in the passive proton permeability of the inner membrane in rat liver mitochondria.

Titration of mitochondrial respiration against the membrane potential with the inhibitor malonate has been carried out during the perinatal period in isolated rat liver mitochondria. Neonatal and adult mitochondria exhibited the characteristic "nonohmic" behavior for the proton conductance (CmH+). In contrast, fetal mitochondria exhibited an "anomalous" "ohmic" behavior for CmH+. The calculated passive proton permeability of the membrane undergoes a profound reduction during the first postnatal hour. The results reported demonstrate that the hypothesis [Pollak, J.K. & Sutton, R. (1980) Trends Biochem. Sci. 5, 23-27] of the existence of a "leaky" mitochondria in the fetal rat liver, and of its sudden neonatal change towards a state of higher energy conservation of the proton electrochemical gradient, is correct.

Animals↗