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Under pathophysiological conditions, infiltration of leukocyte plays a key role in the progression of the neuroinflammatory reaction in the CNS. Prostaglandin E2 (PGE2) is known to accumulate at lesion sites of the post-ischemic brain. Although post-ischemic treatments with cyclooxygenase-2 inhibitors reduce blood-brain barrier (BBB) leukocyte infiltration, the direct effect of PGE2 on BBB has not been fully implemented. Therefore, the direct effect of increasing PGE2 infusion on translocation of labeled albumin into the brain was assessed. Under anesthesia rats were drilled stereo-taxicaly a burr hole in the right forebrain and PGE2 was infused into the forebrain and the hole was occluded. The animals were then injected with fluorescent labeled albumin (FA), via internal right jugular vein and decapitated at different infusion time points. The forebrain was removed and each forebrain hemisphere was homogenized and fluorescence intensities were measured in the supernatant. The fluorescence intensities measured in the right and left forebrain hemispheres of the control group (0.0 µg PGE2) were almost identical. Four hours after infusion of PGE2 at doses higher than 250 µg, fluorescence intensity increased in the right forebrain supernatant, even if it was not statistically significant. The fluorescence intensity was detectable in the brain supernatant 4 h after infusion of PGE2 in doses higher than 250 µg PGE2. The highest fluorescence intensity was 16 h after infusion of 500 µg PGE2, which returned to near control values after 48 h. Increased fluorescence intensity in the brain following PGE2 infusion is concluded to be associated with disruption of the BBB.

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Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinson's disease with L-DOPA.

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Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinsons disease with L-DOPA.(AU)

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A fluororeceptor assay (FRA) has been developed for the determination of antibodies against acetylcholine receptor (AChR), employing an antifluorescein serum and fluorescein-labeled AChR. Antiserum raised against rat muscle AChR in rabbit and the labeled AChR are incubated with antifluorescein serum at room temperature. At high levels of anti-AChR, binding of the labeled AChR prevented subsequent binding of the fluorescein groups by antifluorescein, resulting in little change in the signals of the label. Conversely, at low levels of anti-AChR, the free fraction of the labeled AChR is available to be bound by antifluorescein, which markedly reduced fluorescence intensity of the label. Thus, the fluorescence intensity of the assay mixture directly reflects the amount of anti-AChR antibodies in the serum. It is concluded that the availability of fluorescein-labeled AChR and the antibody directed against it permit measurement of anti-AChR antibodies in human myasthenia. The quality of the assay and its preliminary clinical application have been evaluated.

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The uptake and release of dopamine (DA) by rat brain striatal synaptosomes were studied in presence of glycine or glutamate (0.5-20 mM) in incubation media containing 5 or 55 mM KCl. In low K+ medium glycine, up to 5 mM had little effect on DA uptake, but higher concentrations of the amino acid inhibited the uptake. High K+ medium resulted in a decrease in DA uptake as compared to that of basal K+ medium. Glycine at 1 microM, but not at higher concentrations, prevented the inhibition induced by high K+ depolarization. However, glutamate in the low K+ medium and at a 0.5 mM concentration, stimulated DA uptake, but at higher concentrations it inhibited the uptake. In the high K+ medium, glutamate in all concentrations potentiated the inhibition of DA uptake induced by K+ depolarization. The DA release response of the synaptosomes to glycine concentrations (1-20 mM) in low K+ medium was a biphasic pattern, with a stimulation at 1 mM and an inhibition at higher concentrations. This pattern was reversed when DA release was measured in the high K+ medium. The pattern of DA release in the presence of glutamate concentrations (1-20 mM) was a triphasic one, with an inhibition at 1 mM, stimulation at 5 mM, and a less effective inhibition at higher concentrations. In the high K+ medium, glutamate at 1 mM concentration prevented the stimulation induced by K+ depolarization, but at 5 mM reversed the rate of release to the depolarization state. Results of this study suggest that glycine and glutamate have more than a simple inhibitory or excitatory transmitter role in the striatum, respectively. The identical effects of glycine with that of glutamate in certain concentrations is consistent with previous reports that glycine and N-methyl-D-aspartate (NMDA) act as coagonists of a common excitatory amino acid receptor.

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Nuclear poly(ADP-ribose) polymerase levels as well as the DNA strand break levels of whole-brain neuronal and astroglial cells were investigated. Three- and 30-month-old rats were used. Low-molecular-weight neurofilaments and glutamine synthetase served as neuronal and astroglial markers, respectively. A large increase in the poly(ADP-ribose) polymerase activity was observed in the neurons (threefold) and astrocytes (3.7-fold) derived from 30-month-old rats. Similarly, the amount of poly(ADP-ribose) polymerase, evaluated per milligram of DNA, increased approximately 3.5-fold in neurons and 3.9-fold in astrocytes prepared from 30-month-old rats. Whether the increase in the poly(ADP-ribose) polymerase activity was due to an enhanced rate of DNA strand break was investigated by determining the rate of DNA unwinding. A significant increase in DNA unwinding rate was detected in the neurons (2.7-fold), although a lower increase was observed in the astroglia (1.3-fold) of aged animals.

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The effect of castration on the levels of brain monoamines and their metabolites has been investigated in rats which became or did not become muricidal following long-term isolation. Fourteen brain areas were explored: olfactory bulbs (OB), olfactory tubercles (OT), septum (Se), striatum (Sr), amygdala (A), thalamus (Th), hypothalamus (Hy), hippocampus (Hi), superior colliculus (SC), inferior colliculus (IC), raphe (Ra), pons-medulla (PM), frontal cortex (FC), temporal cortex (TC) and parietal cortex (PC). Except in the raphe of non muricidal rats and in the striatum of muricidal animals, all other areas examined demonstrate some changes of monoamines neurotransmitter or their metabolites after castration. The strongest changes, always increases, were found in the thalamus. In several brain areas, the changes occurring after castration, differ quantitatively and qualitatively in muricidal and non-muricidal rats.

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Exogenously applied dopamine may interfere with cholinergic activity in the brain. The aim of the present work was to study the effect of L-dopa administration on acetylcholinesterase (EC 3.1.1.7) activity in rat (Wistar) brain striatum. Short-term administration of a mixture of L-dopa (10 mg/kg) and carbidopa (1 mg/kg) resulted in an increase in dopamine content and a decrease in acetylcholinesterase activity of the tissue. The greatest changes were found 30 min postinjection, and activity returned to near-control values after 2 h. When the drug mixture was injected for a period of 30 d and the animals were killed 24 h after the last injection, a lower dopamine content and higher enzyme activity were seen, compared to control values. It would appear that chronic administration of L-dopa gradually reduced the dopamine-storage capacity of the striatum and that the activity of acetylcholinesterase might be controlled by the levels of dopamine in the brain striatum.

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The concentration of copper in the rat brain hypothalamus showed a dose-dependent increase with the administration of copper ions. With doses larger than 3 mg/kg the copper content was higher when measured 30 min after administration of the metal and was depleted to near control values after 6 h. Copper ions in doses of 3, 5 and 10 mg/kg increased dopamine and decreased noradrenaline contents of the hypothalamus in a non-linear fashion. Peak hypothalamic dopamine content was found 30 min after injection of copper (5 mg/kg) which returned to normal levels after 6 h. Ascorbic acid (500 mg/kg) administration prevented the copper-induced dopamine increase in the brain. Ascorbic acid also caused the copper content of the tissue to decrease in both normal and copper-receiving rats. However, the effect of the vitamin on catecholamine content of the hypothalamus was opposite to that of copper ions, i.e. it caused noradrenaline to increase and dopamine to decrease in comparison to control values. The results suggest that ascorbic acid may reduce the effects of excessive copper deposition in the brain hypothalamus.

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The synthesis of dopamine and serotonin in rat brain striatal synaptosomes has been studied by direct measurement of dopamine and serotonin by HPLC employing electrochemical detection. In both cases the presence of dibutyryl cyclic AMP stimulated the rate of synthesis and this effect was prolonged in the presence of theophylline. Theophylline alone had no effect. The stimulating effect of 1 mM dibutyryl cyclic AMP on dopamine synthesis was prevented by 1 mM EDTA but not by NaF. EDTA alone did not effect dopamine synthesis but 20 mM NaF alone caused a marked stimulation (increase of 33%) comparable to that produced by 1 mM dibutyryl cyclic AMP. In contrast 1 mM EGTA caused a marked inhibition (30% decrease) of the synthesis of dopamine in this system. The stimulation of serotonin synthesis by 1 mM dibutyryl cyclic AMP was inhibited by 1 mM EDTA and 20 mM NaF. In contrast to dopamine synthesis, serotonin synthesis was markedly inhibited (40%) by 1 mM EDTA on its own, but not affected by 20 mM NaF. However, 1 mM EGTA caused a similar inhibition of serotonin synthesis to that of dopamine synthesis. Exogenously added noradrenaline, adrenaline and serotonin (1-100 ?M) markedly inhibited dopamine synthesis in a non linear fashion as did noradrenaline and adrenaline on serotonin synthesis. The addition of imipramine together with noradrenaline rendered the noradrenaline inhibition of both dopamine and serotonin synthesis less effective. The results are discussed with respect to the mechanisms of feed back inhibition of dopamine and serotonin synthesis by products and the stimulation of their synthesis by cyclic AMP linked systems. It is concluded that, whilst the cyclic AMP stimulation of dopamine synthesis in striatal synaptosomes is consistent with activation of tyrosine hydroxylation by phosphorylation, the cyclic AMP stimulation of serotonin synthesis occurs by an alternative mechanism, possibly at the level of the synaptosomal membrane.

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By use of high performance liquid chromatography with electrochemical detection to measure dopamine production, tyrosine hydroxylase (EC 1.14.16.2) activity has been measured in rat brain synaptosomes from striatum and forebrain. Normal specific activities three- to fivefold higher than previously reported in the literature for radiochemical methods of assay were found. It is suggested that synaptosomes contain a significant amount of endogenous substrate for tyrosine hydroxylase, which causes dilution of the added labelled tyrosine and hence underestimation of the activity of this enzyme when radiochemical methods are used.

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1. Male Sprague--Dawley rats were given 630 g/kg sucrose or starch with 2 mg/kg aflatoxin b1 for periods of 75, 145 and 200 d, and the 24 h urinary excretion of aflatoxin M1 was measured. 2. Less aflatoxin M1 was excreted by the rats fed on the sucrose-rich diet compared to those fed on the starch-rich diet. This difference was especially marked when expressed per g metabolizing tissue. 3. It is concluded that sucrose probably decreases the activity of aflatoxin B1 metabolism in a similar way to its previously found effect on the drug-metabolizing enzyme.

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