Glutamatergic system: another target for the action of porphyrinogenic agents.

The N-methyl-diethyl-aspartate (NMDA) receptor has been reported to play an important role in several acute and chronic neuropathologic syndromes. 5-aminolevulinic acid (ALA) accumulates in acute porphyrias due to a deficiency in the heme biosynthetic pathway. Considering that glutamate uptake inhibition caused by ALA could be one of the reasons conducing to porphyric neuropathy, it was of interest to evaluate the effect of porphyrinogenic agents on NMDA glutamatergic system. To this end receptor levels and apparent affinity (Kd) were analyzed in mice brain cortex and cerebellum. NMDA levels were diminished after chronic Isoflurane anaesthesia in brain cortex. In cerebellum, a diminution was observed after acute Enflurane and Isoflurane and allylisopropylacetamide, while ethanol administration showed a significant increase. ALA administration diminished NMDA levels only in cerebellum. Affinity constant was only reduced in brain cortex after chronic Isoflurane treatment. In conclusion, glutamatergic system appears to be involved in the action of some of the porphyrinogenic drugs studied mainly in cerebellum. Receptors regulation should therefore be considered an important mechanism in the cellular response to specific drugs, with the aim of designing new therapies and elucidating the mechanisms leading to porphyric neuropathy and acute attack triggering.

Exogenous NGF alters a critical motor period in rat striatum.

In previous studies we found that there is a critical period during rat postnatal development when motor training starting at age 30 days (P30) but not before or after this age, induces a bilateral lifetime drop in Bmax of the muscarinic radioligand [3H]QNB in striatum. We examined the possibility that striatal NGF level would be a determining factor for the normal occurrence of this synaptic plasticity. With this aim, rats underwent training at P30-37 with or without simultaneous NGF perfusion into the left striatum. At P70, we found the expected bilateral enduring fall of striatal [3H]QNB sites in trained controls. While the non-cannulated side of NGF-treated trained rats showed a similar drop in [3H]QNB binding, the perfused striata from these animals were not affected by training. Thus, the findings add new evidence in favour of a major role of NGF in this critical period of activity-dependent permanent adjustment in the striatal muscarinic system.

Nerve growth factor preserves a critical motor period in rat striatum.

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.

Decreased GAP-43/B-50 phosphorylation in striatal synaptic plasma membranes after circling motor behavior during development.

We evaluated the in vitro phosphorylation of the presynaptic substrate of protein kinase C (PKC), GAP-43/B-50 and the PKC activity in the striatum of rats submitted to a circling training (CT) test during postnatal development. Motor activity at 30 days of age, but not at other ages, produced a unilateral reduction (-29.5%; p<0.001) in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum with respect to the ipsilateral side, while non-trained control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of trained animals also showed a significant reduction (-29.3%; p<0. 001) in the incorporation of 32P-phosphate into GAP-43. This decreased in vitro GAP-43 phosphorylation was seen at 30 min, but not immediately after circling motor behavior. This contralateral change in GAP-43 phosphorylation correlated with the running speed developed by the animals [(r=0.9443, p=0.0046, n=6, relative to control group) and (r=0.8813, p=0.0203, n=6, with respect to the ipsilateral side of the exercised animals)]. On the contrary, GAP-43/B-50 immunoblots did not show changes in the amount of this phosphoprotein among the different experimental groups. Back phosphorylation assays, performed in the presence of bovine purified PKC, increased the level of GAP-43/B-50 phosphorylation in the striatum contralateral to the sense of turning [(+22%; p<0.05, with respect to ipsilateral side of the same trained group) and (+21%; p<0.05, relative to control group)]. Taken together, these results demonstrate that the activity developed in the CT test induces a reduction in the phosphorylation state of GAP-43/B-50 in the specific site for PKC. We conclude that general markers of activity-dependent neuronal plasticity are also altered in the same period that long-lasting changes in striatal neuroreceptors are triggered by circling motor behavior.

Decreased phosphorylation of GAP-43/B-50 in striatal synaptic plasma membranes after circling motor activity.

The effects of spontaneous circling motor activity on the in vitro phosphorylation of the protein kinase C substrate GAP-43/B-50 was studied on striatal membranes of developing rats (30 days of age). At this time of postnatal development, permanent plastic changes in cholinergic and dopaminergic systems are produced by physiological motor activity. Exercised animals showed a significant reduction of 31% in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum respect to the ipsilateral side (P < 0.01), while control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of exercised animals showed a 33% reduction in the incorporation of 32P-phosphate into GAP-43/B-50 30 minutes post-exercise (P < 0.01). This change in GAP-43/B-50 phosphorylation was correlated with the running speed developed by the animals (r:0.8986, P = 0.015). GAP-43/B-50 immunoblots revealed no changes in the amount of this protein in any group. Moreover, a significant variation of 25% (P < 0.05) in the PKC activity was seen between both exercised striata. Interhemispheric differences were not found in control animals. We conclude that endogenous phosphorylation of this protein is also altered by motor activity in the same period that permanent changes in striatal neuroreceptors are triggered after motor training.

Cold stress related alteration of RNA biosynthesis in brain cortex of mother-deprived newborn rats.

We studied the influence of maternal deprivation on the RNA biosynthesis in the brain cortex of 10 day-old rats. Mother-deprived pups, placed at 25 degrees C showed a reduction in body temperature of 6 +/- 1 degree C. After mother retrieval, RNA biosynthesis decreased 27% and 34% in total brain cortex and in isolated neurons, respectively. This fall is proportional to the body temperature reduction and can be avoided placing the pups at 37 degrees C immediately after the separation. Rethermostatization of offsprings, after one hour at 25 degrees C, showed an overshoot of RNA biosynthesis (145%) with further stabilization of synthesis rates to normal levels after 100 min. This classical physiological mechanism was further studied in vitro. Comparing in vivo and in vitro experiments, it is concluded that overshooting can not be observed in vitro if temperature reduction was not previously performed in vivo. Thus, this phenomenon seems to respond to humoral factors in order to be triggered. Afterwards, in vitro overshooting following cold stress in vivo, demonstrates that the depressed tissue by itself has the capability to turn back to normal RNA levels in the same way as observed in vivo.

Co-alteration of dopamine D2 receptor and muscarinic acetylcholine receptor binding in rat striatum after circling training.

CIRCLING training (CT) decreases muscarinic acetyl-choline receptor (mAchR) binding in rat striatum. As cholinergic and dopaminergic systems interact strongly we evaluated the expression of D2-subtype dopamine receptor (DA D2) and mAchR together after CT. Animals trained from 30 to 37 days of age and sacrificed 2 months later showed an enduring drop in Bmax of 40% in DA D2 and 34% in mAchR. Plotting the percentage of binding drop of both receptors for each animal showed that the reduction of one system correlates with the other (r2 = 0.71, p < 0.01; n = 8). Neither mAchR nor DA D2 were affected when training started at 20 or 60 days. We conclude that the presence of a period where CT exerts long term alterations during development involves both cholinergic and dopaminergic systems.

Permanent alteration of muscarinic acetylcholine receptor binding in rat striatum after circling training during development.

We evaluated the effect of circling training (CT) in the expression of muscarinic acetylcholine receptor (mAchR) in developing rat striatum. For this, male and female rats were subjected to CT at 20, 30, 40 and 60 days of age during 7 days. Animals trained at 30 days but not at other ages showed an average decreased binding to mAchR of 33% in males and 24% in females, representing a significant difference with respect to control non-trained animals (males P < 0.001, females P < 0.005), and showing also a differential response between sex (P < 0.01). mAchR drop was found invariably either 2 months or 1 year after training indicating a long term plastic change due to circling training. Scatchard analysis showed that altered binding represents a variation of the total receptor number instead of its binding affinity, with no significant differences found among Kd (P > 0.1). mAchR variation was correlated with the motor performance accomplished in the test. Regarding total distance run, male rats trained for 3 days (300 meters. run), for 5 days (600 meters) and for 7 days (900 meters) showed a drop of 19, 28 and 33% respectively (r2 = 0.91, P < 0.001), while female changes were of 21, 23 and 24% (r2 = 0.78, P < 0.001). Nevertheless, no correlation with running speed was found (r2 = 0.13 male, r2 = 0.02 female; P > 0.1). In summary, these results demonstrate the presence of a limited sensitivity period during striatum development where mAchR expression may be affected by the activity performed during CT, representing a permanent alteration of the receptor levels.

The molecular basis of the self/nonself selectivity of a coelenterate toxin.

Coelenterates produce potent hemolysins inhibited by sphingomyelin (SM). Remarkably, instead of this lipid, their membranes contain a phosphono analogue of it. Using coelenterolysin (CL), a toxin produced by the sea anemone Phymactis clematis, we have examined a possible connection between these two peculiar traits. Our experiments showed that, while SM binds this lysin and inhibits its hemolytic activity, the endogenous PnSL do neither. In addition, liposomes made of bovine erythrocyte lipids are rapidly disrupted by CL, while those made of P. clematis lipids are completely resistant to it. However, if small amounts of SM are added to the P. clematis lipids, the resulting liposomes become sensitive to CL. Taken together, our results show for the first time that substitution of SM by its phosphono analogue is the molecular basis for the selectivity of an anthozoan toxin. We therefore propose that exotoxin production and membrane composition are coadapted traits that confer on the coelenterates a significant evolutionary advantage.

The circling training rat model as a behavioral teratology test.

The properties of circling training (CT) for detecting behavioral teratologic drug-induced effects was evaluated by prenatal administration of two behavioral teratogenic drugs: vitamin A (80,000 IU/kg/day) and haloperidol (2.5 mg/kg/day). The circling training was started at 30 days of age and performed for 8 days in an automated apparatus. Statistically significant differences between drug-treated and control animals regarding the measured response (turns per minute) were found. Two components may affect the response measured by the CT: associative learning and motor performance. The incidence of these components was discriminated with behavioral and mathematical approaches. In the experimental conditions used the most affected parameter was motor performance. The results indicate that CT can be used as an instrumental conditioning test where the quantifiable endpoint is the on-going motor performance. Further applications of the CT for neurochemical evaluation of drug induced effects are also discussed.

Kinetics of carboxylmethylation of the charge isoforms of myelin basic protein by protein methyltransferase II.

The charge isoforms (C1-C5) of bovine myelin basic protein (MBP) were used as substrates for the rat brain enzyme protein carboxylmethyltransferase (PM II). The objective of these experiments was to ascertain whether the kinetic behavior of the MBP isoforms reflected differences in the structures of this molecular family. Initial velocity plots as a function of the MBP-isoform concentration showed significant differences (p less than 0.05) among the assayed isoforms except for isoforms C2 and C4. Under the conditions of our experiment all the curves exhibited a consistent sigmoidicity. The kinetic data were best fitted by a model, previously described for the enzyme D-beta-hydroxybutyrate dehydrogenase, in which two independent sites must be randomly occupied before any catalytic activity can occur. This mechanism is substantially different from that proposed by other investigators for similar PM II enzymes and other substrates. The differences in the rates of isoform carboxylmethylation are largely accounted for by the different apparent dissociation constants Ks and is explained on the basis of inherent structural differences among the charge isoforms.

Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties.

Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.

Detection of drug effects on neurons: protein synthesis inhibition by low doses of amphetamine.

The effects of in vivo administered amphetamine (4 and 15 mg/kg) on neuronal and unfractionated brain cortex protein synthesis were evaluated in albino rats. In vitro incubation of brain cortex prisms with 14C-leucine and bulk isolation of neuronal perikarya were performed 1 h after drug administration, and 14C was measured by liquid scintillation counting in hot TCA-insoluble fraction. Amphetamine 15 mg/kg significantly decreased protein synthesis in both fractions, neuronal protein synthesis being more inhibited than total cortex. A lower dose (4 mg/kg) significantly inhibited neuronal protein synthesis, even when it failed to affect total cortex protein synthesis. Results presented here show that neurons may be more sensitive than other brain cells to changes in body temperature. The usefulness of the procedure in the evaluation of drug effects is also considered.

Identification of polydisperse RNA in the cytoplasm of neurons isolated from the immature brain cortex.

The cytoplasmic RNA of 10-day-old rats was studied in bulk-isolated, cortical neurons, obtained under conditions which minimize nuclear contamination. Two RNA fractions, one enriched rRNA and the other in "polydisperse" RNA, were obtained by differential extraction with phenol. Gel electrophoresis and pulse labelling with 5-[3H]uridine were used to confirm the delayed appearance of newly synthesized rRNA in the cytoplasm and to demonstrate its stability. "Polydisperse" RNA appeared in the cytoplasm earlier and had a shorter half-life than rRNA. A wide range of molecular weights for this RNA was found with no predominant individual species. A set of cytoplasmic RNA components of molecular size between 28S and 18S was also present probably reflecting the in vivo degradation of rRNA. The significance of the unexpectedly high amounts of neuronal polydisperse RNA is discussed.

Nucleic acids quantitation in polyacrylamide disc gels with a modified double beam spectrophotometer.

Quantitation of RNA and protein bands separated in polyacrylamide disc gel electrophoresis is time consuming and not reliable when performed after staining processes. The best theoretical approach is the use of absorption spectra of the samples. The present paper reports the construction of an efficient and inexpensive adapter to perform direct scanning of 100 mm gels in a Unicam SP-800 double beam spectrophotometer. Conditions for gel casting and electrophoresis were established for RNA. A linear response between 0.25 micrograms and 15 micrograms per individual peak was found with a quantitative limit of detection of 0.25 micrograms. Spectral analysis of each band can be performed allowing identification of samples as nucleotides and detection of contaminants.