Urszula Rafałowska

Astroglial reaction during the early phase of acute lead toxicity in the adult rat brain

The developing nervous system is susceptible to lead (Pb) exposure but less is known about the effect of this toxic agent in adult rat brain. Since astrocytes serve as a cellular Pb deposition site, it is of importance to investigate the response of astroglial cells in the adult rat brain in a model of acute lead exposure (25 mg/kg b.w. of lead acetate, i.p. for 3 days). An increased immunoreactivity of glial fibrillary acidic protein (GFAP) on Western blots was noticeable in fractions of astroglial origin-glial plasmalemmal vesicles (GPV) and in homogenates from the hippocampus and cerebral cortex but not in the cerebellum. The features of enhanced astrocytic reactivity (i.e. large accumulation of mitochondria, activated Golgi apparatus and increment of gliofilaments) were observed in electron microscopy studies in the same tissues. Total glutathione levels increased both in GPV fractions and in brain homogenates-in the cerebellum (120% above control) and in hippocampus (30% above control). The results of current studies indicate that acute lead exposure is accompanied by astrocyte activation connected with the presence of the enhanced expression of GFAP. It may indicate lead-induced neuronal injury. At the same time, a regional enhancement of detoxicative mechanisms (GSH) was noticed, suggesting activation of astrocyte-mediated neuroprotection against toxic Pb action.

Is neurotransmitter histamine predominantly inactivated in astrocytes

Rat synaptosomes and astroglia cell-enriched fraction were tested for the uptake of histamine (HA) and its precursor histidine, and the activities of the HA-synthesizing enzyme, histidine decarboxylase (HD) and HA-metabolizing enzyme, histamine methyltransferase (HMT). While histidine uptake was more active into synaptosomes than into astrocytes, only astrocytes were capable of a significant HA uptake. Kinetic analysis of the astrocytic HA uptake revealed a high affinity-low capacity system (Km = 5 X 10(-7) M, Vmax = 1.6 X 10(-12) mol.min-1 X mg-1) similar to the astroglial transport systems for other neurotransmitters. HMT was 70% more active in astrocytes than in synaptosomes, whereas HD activity was not different in these two preparations. The results indicate that astrocytes could be the major site of neurotransmitter HA inactivation.

DOES LEAD PROVOKE THE PEROXIDATION PROCESS IN RAT BRAIN SYNAPTOSOMES

Up to now there has been no information concerning the effect of lead on the peroxidation process in brain nerve endings. We have examined whether lead acetate (in chronic and acute models of toxicity in vivo and in vitro) affected the level of free radicals in synaptosomes obtained from rat brain. Simultaneously, we have checked the effect of peroxidation of Pb2+ on brain homogenates and microsomal fraction. Our results indicated that the lead level in synaptosomal fraction obtained from lead-treated rats was much higher than in controls. We did not observe induction of spontaneous and Fe(3+)-dependent peroxidation either in synaptosomes or in homogenates and brain microsomes after chronic and acute lead administration to the rats. Lead itself also did not enhance both processes when added in vitro to the control brain synaptosomes in micromolar concentrations. The lack of the lead effect on the peroxidation process in subcellular fractions of brain was rather surprising, because lead is known to be the accelerator of Fe(3+)-dependent peroxidation processes in liver. Additionally, livers from rats under the same toxicity conditions were examined. We have found that lead did not provoke spontaneous peroxidation in liver, but contrary to brain fractions, it drastically increased iron-dependent peroxidation in liver homogenates and microsomes. The lack of the effect of lead on inducing peroxidation processes in brain is probably the consequence of the brain having stronger protective mechanisms against its toxicity than the liver.

LEAD AS AN INDUCTOR OF SOME MORPHOLOGICAL AND FUNCTIONAL CHANGES IN SYNAPTOSOMES FROM RAT BRAIN

Summary1. The effect of lead (in vivo) on the uptake of GABA, dopamine, and histidine as a precursor of histamine in synaptosomes obtained from chronically lead-treated rats was studied.2. Lead decreased the uptake of GABA, increased the uptake of dopamine, and did not change the uptake of histidine. These effects were independent of calcium concentration.3. Lead administration to the rat changed the morphology of the synaptosomes, as manifested in the decreased number of synaptic vesicles and disturbed mitochondrial structure.4. The results suggest the existence of several mechanisms of lead toxicity on uptake, related to individual neurotransmitters, which are not necessarily connected with a Pb2+/Ca2+ interaction.

Astrocytic response in the rodent model of global cerebral ischemia and during reperfusion

The present study investigated alterations in astrocytic cells after global cerebral ischemia resulting from cardiac arrest immediately and at several intervals after reperfusion when excessive formation of highly cytotoxic free radicals is known to occur. The cellular fraction of astrocytic origin (glial plasmalemmal vesicles - GPV) was examined by biochemical and immunochemical procedures. A tendency towards an elevation in immunocontent of glial fibrillary acidic protein (GFAP) was noticed after 24 hours whereas a significant increase was observed 7 days post ischemic event. The features of astrocytic stimulation were also observed in electron microscopy studies. An enhanced amount of gliofilaments was noticed in brain sections obtained from rats after 7 days of recovery. Simultaneously, a gradual decrease of total glutathione level, depending on the duration of reperfusion, was observed in brain homogenates and in fractions of astroglial origin. The most considerable reduction was observed on day 1 (52%) and day 7 (65%) after reperfusion in brain homogenates and on day 7 (47%) in GPV fraction. The results indicate an enhanced reactivity of astrocytic cells in ischemic conditions concomitantly with a long lasting decrease of total glutathione. Obviously, the inability of astrocytic glutathione system to detoxify free radicals formed during ischemic/reoxidation conditions may lead to damage to cerebral neurons by oxidative stress.

Alteration of dopamine transport and dopamine D2 receptor binding in the brain induced by early and late consequences of global ischaemia caused by cardiac arrest in the rat

This study was designed to determine the effects of global cerebral ischaemia caused by temporary cardiac arrest and the early and late consequences of this ischaemia on dopamine transport and dopamine D(2) receptor binding in rat brain. The effects of 10 min of global ischaemia were measured immediately and after 1 h and 7 days post-resuscitation. A decrease of dopamine uptake in the rats by synaptosomes was noted immediately following global ischaemia and 1 h after resuscitation. However, at 7 days post-resuscitation, the dopamine uptake returned to control values. Reversibility of the changes in the synaptosomal dopamine uptake is undoubtedly a favourable sign. Global ischaemia and reperfusion after 1 h or 7 days did not show altered rates of dopamine release but did affect the dopamine D(2) receptor. An observed increase of receptor affinity may be an adaptive response to the reduction in binding capacity. The reduction of visible D(2) receptor binding sites in the early post-resuscitation phase, which was extended to the period of 7 days after resuscitation without recovery, is probably associated with neuronal necrotic damage.

Metabolic changes in rat brain synaptosomes after ex- posure to sulfide in vivo

The effects of exposure of rats to sulfide and the action of exogenous heme were studied in rat-brain synaptosomes. Exposure to sulfide impaired the respiration of synaptosomes which was reversed by heme (4 mg/kg body weight). Sodium sulfide caused partial inhibition of gamma-aminobutyric acid (GABA) and dopamine uptake, strongly inhibited veratridine-dependent release of these neurotransmitters and reduced veratridine-dependent changes in transmembrane electrical potential. Heme treatment did not reverse these changes.

Transport of malate and citrate into rat brain mitochondria under hypoxia and anesthesia

Hypoxia and anesthesia inhibited penetration of malate and citrate into the brain mitochondria by 60% and 40%, respectively. Anesthetized animals exposed to low oxygen tension showed similar changes as those subjected to hypoxia without anesthesia. Recovery from anesthesia was rapid and the rates of citrate and malate uptake returned to the control values in approximately 60 min. In the case of hypoxia, days were required to restore the control values of citrate and malate uptake. Free fatty acids had no effect on the entry of malate and citrate into the mitochondria. However, changes in the levels of protein—SH groups were observed which may be responsible for the impaired transport of citrate and malate under hypoxic condition.

Hypoxia and ischemia modifies histamine metabolism and transport in brain synaptosomes.

Histamine level (HA), the activities of the HA synthetizing enzyme--histidine decarboxylase (HD) and HA metabolizing enzyme--histamine methyltransferase (HMT) and the uptake and release of histidine and histamine were analyzed in synaptosomal preparations obtained from rats with brain hypoxia and ischemia. Hypoxia produced only non-significant changes in all the parameters studied, whereas ischemia induced increase of both enzyme activities and histidine release, with simultaneous decreased of histidine uptake and HA level. The effect of ischemia appeared to be reversible; the changes retreated within 1 h of resuscitation together with the vital functions of rats.

Synaptosomal susceptibility on global ischaemia caused by cardiac arrest correlated with early and late times after recirculation in rats.

The aim of the study was to assess the sensitivity of brain synaptosomes and their mitochondria to the effects of global cerebral ischaemia caused by temporary cardiac arrest and the early and late consequences. The effects of 10 min of global ischaemia were measured immediately and after 1 h, 24 h and 7 days post-resuscitation. Ischaemia caused a reduction in oxygen consumption by synaptosomes of about 20%, a drop in ATP/ADP ratio of about 40%, a decrease in CrP/Cr ratio at about 45% and a reduction of synaptic vesicles and disturbances in the mitochondrial structure in isolated synaptosomes and in nerve endings of brain specimens. Morphometric analysis showed that ischaemic conditions caused a decrease in synaptic vesicles by about 61% and an increase of mitochondrial damage to 58 and 50% after 1 and 24 h postreperfusion time, respectively. Seven days postresuscitation, all the observed changes returned to normal but small numbers (about 2%) of neurones which were destroyed neurons appeared at that time. It is concluded that global ischaemia with early resuscitation after cardiac arrest may lead to damage of synaptosomes and synaptic mitochondria. This, in turn, modifies substrate oxidation, synthesis of energy variables and affects neurotransmitter function. The observed disturbances return to normal later after resuscitation but the ischaemic events and reoxygenation caused selective morphological injury of certain neurones and this may form the basis for irreversible brain damage.