J.M. de Gandarias

Lithium alters mu-opioid receptor expression in the rat brain.

Lithium can potentiate the effects of antidepressant drugs and alters morphine analgesia and phosphoinositide turnover. Analysis of mu-opioid receptor immunostaining after chronic lithium administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic lithium treatment. This could be a compensatory mechanism, induced in part by the effects of lithium on mu-opioid receptor transduction mechanism.

GENESIS AND EVOLUTION OF HIGH-PLOIDY TUMOUR CELLS EVALUATED BY MEANS OF THE PROLIFERATION MARKERS P34CDC2, CYCLIN B1, PCNA AND 3H-THYMIDINE

Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34cdc2, cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal‐ and in high‐ploidy subpopulations, almost all cells were p34cdc2‐positive. In the modal‐ploidy subpopulation only 17.1% cells were cyclin B1‐positive and 85.6% PCNA‐positive; in contrast, in the high‐ploidy subpopulation up to 91.8% cells were cyclin B1‐positive and 97.3% cells were PCNA‐positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34cdc2, both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24‐h incubation with 3[H]‐thymidine followed by withdrawal of the isotope, high‐ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modalploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal‐ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.

Developmental changes of pyroglutamate-peptidase I activity in several regions of the female and the male rat brain

In this work, we have described decreases of pGlu-peptidase I activity, detected using pGlu-beta-naphtylamide as substrate, from 9 to the 20 days after birth in five brain areas. In addition, we have found sexual differences in the enzyme activity in the hypothalamic-pituitary axis in all ages investigated. pGlu-peptidase cleaves the pGlu-aminoacid bond of several neuropeptides such as thyroliberin, luliberin and neurotensin. The decrease in the activity of pGlu-peptidase coincidents with the increases observed in brain thyroliberin concentration and the decreases in His-Pro diketopiperazine. It is suggested that this enzyme could play a part in the normal development of the rat brain.

Interaction mechanisms of imipramine and desipramine with enkephalin-degrading aminopeptidases in vitro.

In the last few years, considerable evidence has appeared concerning the importance of the opioid systems in the action mechanism of some antidepressant drugs. This action mechanism could be mediated through the inhibition of the enzymes responsible for enkephalin degradation. In this sense, imipramine treatment in vivo increases the enkephalin levels, and this effect is enhanced by inhibitors of enkephalin-degrading enzymes. The present work shows the effects in vitro of imipramine and its active metabolite desipramine on the activities of two membrane-bound enkephalin-degrading aminopeptidases present in rat brain. Imipramine and desipramine in vitro do not affect the aminopeptidase M activity, but they reversibly inhibits the aminoeptidase MII. The enzyme kinetic analysis shows that this enzyme molecule has two different binding sites for each drug, which exert a mixed type enzyme inhibition.

Effect of subacute benzene exposure on the activity of two neuropeptide-degrading enzymes in the rat brain.

Benzene (Bz) is an important industrial chemical, a petroleum by-product, a component of unleaded gas, and thus a ubiquitous environmental pollutant. It is well established that this organic solvent possesses neurotoxic and behavioral effects. However, the neurochemical mechanism of the solvent action remains obscure. The aminopeptidases (AP) are proteolytic enzymes that have been proposed as a candidate regulator of the degradation of several neuropeptides. In this work, changes in Lys- and Leu-aminopeptidase activities in several rat brain regions after benzene administration are described. The AP activity was determined by measuring the rate of hydrolysis of the artificial substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities decrease in the thalamus, hypothalamus, hippocampus, and amygdala after Bz treatment. It is suggested that these aminopeptidase activities play a part in the benzene action mechanism, possibly by regulating the activity of several neuroactive peptides.

Study of Binding of Benzyl-Thiouracil to Human Serum Albumin by Gel Filtration Chromatography

Abstract The binding of benzyl-thiouracil to human serum albumin was studied at 37°C and pH 7.4 by Sephadex filtration chromatography based upon Hummel and Dreyers method. As the benzyl-thiouracil (ligand) was adsorbed on to the gel matrix, the free ligand concentrations had to be corrected through the ligand distribution between the stationary and mobile phases. A good agreement was found between binding parameters—calculated by this method and by the classical method (equilibrium dialysis). Binding is characterized by one binding site with a moderate association constant (K1 = 5.7 × 104 M-1) and two binding sites with a low association constant (K2 = 7.8 × 103 M-1).

Membrane-bound tyrosine aminopeptidase activities in the rat brain throughout the estrous cycle

Puromycin sensitive and insensitive membrane-bound aminopeptidase activity levels during the estrous cycle in several brain areas have been described in this research. We have found the highest aminopeptidase M activity levels during the proetrous stage in the hypothalamus, the amygdala and the pituitary gland. Since this enzyme has been involved in opioid peptide metabolism, it is suggested that aminopeptidase M could play a part in the decrease in the inhibitory influence of the endogenous opioids peptides that participate in the LH surge.

Brain aminopeptidase activity after subacute xylene exposure

Xylene is a neurotoxic aromatic hydrocarbon widely used in industry. In this article, the effect of subacute xylene exposure on neutral and basic aminopeptidase activities in several regions of the rat brain is described. Neutral aminopeptidase activity only decreased significantly in the thalamus. There were no changes in basic aminopeptidase activity after the solvent administration. Thus, these brain aminopeptidase activities are largely unaffected by subacute exposure to xylene, which is not the case with benzene or carbon disulfide. This could be in line with its recognized lesser toxicity at the central nervous system level.

Neutral aminopeptidase activity levels during the estrous cycle and the pregnancy in the hypothalamus and the pituitary of the rat

Neutral aminopeptidase activity levels during the estrous cycle and pregnancy, in the hypothalamus and the pituitary of experimental animals, was studied in this research. The cyclic stages were the estrous, diestrous and proestrous (at morning and afternoon). The pregnancy phases were the 2nd, 7th, 14th and 20th day postinsemination. The parturition day was also studied. The higher cyclic enzyme levels were found in the proestrous stage (afternoon). No differences between estrous and diestrous were observed. During pregnancy, there was a rise at 7th and 14th day, with a decrease in the 20th day. In the hypothalamus, significant increases after parturition were shown, the latter not being observed in the pituitary. In general, the changes seem to be parallel to those of the luteinizing hormone.

H1‐histamine receptors may mediate the contractile response of guinea‐pig ileum to ‘histamine‐free’ splenic extracts

1 A water‐soluble splenic factor, which produces a contractile response of the guinea‐pig ileum, that is resistant to cholinoceptor and adrenoceptor antagonists is described. 2 The ileal contractions elicited by the splenic extract showed some significant differences from those elicited by 5‐hydroxytryptamine. The responses to splenic extract were not affected by the D‐tryptamine‐receptor antagonist, methysergide. 3 The effect of the splenic extract on the guinea‐pig ileum was similar to that of histamine. The H1‐histamine antagonists, (+)‐chloropheniramine and diphenhydramine, caused a parallel shift to the right of the splenic extract dose‐response curve without suppression of the maximum response. A pA2 value of 8.97 ± 0.03 for (+)‐chlorpheniramine and 7.55 ± 0.1 for diphenhydramine was calculated. 4 Significant histamine levels, as determined by fluorometric methods, could not be detected in the splenic extract. Likewise, the splenic factor did not release histamine from the intestinal preparation. 5 These results support the view that: (i) the splenic factor acts through H1‐histamine receptors; (ii) it is not histamine; (iii) it does not have any histamine releasing effect on the ileal smooth muscle.