Pietro Fumagalli

Vulnerability of mitochondrial complex I in PC12 cells exposed to manganese.

The present findings provide experimental evidence for the hypothesis that an impairment of mitochondrial function may be involved in manganese neurotoxicity. Specifically, the treatment of dopaminergic neuronal-derived cell line (PC12) with MnCl2 produced a significant inhibition of some mitochondrial complexes of the respiratory chain, while in the glial-derived cell line (C6) this effect was not observed. In PC12 the decrease in complex I activity was more pronounce than in other mitochondrial complexes. However treatment of cells with ZnSO4 exerted no significant variations in enzymatic activities. A direct exposure of mitochondrial fraction to MnCl2 reduced enzymatic activities of mitochondria in both cell lines adding further support to the proposed theory that the different sensitivity of the cells to manganese may be explained by a difference in uptake or intracellular storage. These data indicate that manganese neurotoxicity could be the result of a direct effect just on complex I activity or due to a secondary effect of oxidative stress induced by an excess of this transition metal.

Characterization and Mapping of Melatonin Receptors in the Brain of Three Mammalian Species: Rabbit, Horse and Sheep

Melatonin receptors were characterized in the brains of three mammals (rabbit, horse and sheep) by an in vitro binding technique, using 2-[125I]iodomelatonin as labelled ligand. Although binding sites for melatonin have been described recently in several vertebrate species (including the sheep), the rabbit and the horse have not been the subject of investigation so far. Apart from characterization, the present report describes receptor distribution in a number of brain regions, thus allowing for direct interspecies comparison under the same methodological conditions. 2-[125I]iodomelatonin labelled high-affinity binding sites in crude membrane preparations from these species. A series of kinetic and saturation experiments revealed that the binding was rapid, stable, saturable, reversible, of high affinity (Kd in the low picomolar range) and low capacity (Bmax between 1 and 20 fmol/mg protein). The competition studies showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding was as follows: 2-iodomelatonin greater than 6-chloromelatonin greater than melatonin much much greater than 5-methoxytryptophol greater than 5-methoxytryptamine, and that it was similar in the different brain regions. Prazosin, which has been reported as an extremely potent melatonin analog in the hamster brain, possessed no potency in all preparations from different regions in the three species under investigation. The regional distribution of the receptor showed insignificant species differences. Highest density was always recorded in the median eminence/pars tuberalis (ME/PT) area. Other regions (SCN, POA and certain cortical areas), showed lower, but significant, receptor content. Saturation and competition studies revealed that these binding sites were also of high affinity, low capacity and high specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and characterization of melatonin binding sites in the brain of the rabbit (Oryctolagus cuniculus) by autoradiography and in vitro ligand-receptor binding

The distribution and the properties of the melatonin binding sites were characterized in the brain of the rabbit by combined use of autoradiography and in vitro ligand-receptor binding. Autoradiography revealed widespread specific binding in the brain. The pars tuberalis of the pituitary gland, suprachiasmatic nuclei, ventromedial hypothalamic nuclei, tapetum, hippocampus, indusium griseum, cingulate gyrus, cortex and the choroid plexus were intensely labelled. Diffuse specific binding was recorded in the olfactory bulb and the anterior hypothalamus. Series of in vitro ligand-receptor binding experiments, using the anterior hypothalamus, confirmed that the binding was of high affinity and specificity. Coincubation with a non-hydrolyzable GTP analogue provoked a shift in the binding affinity, the numerical values of the Kd increasing from 20-30 pM to 280-300 pM. Apparently the melatonin receptor in the rabbit brain is linked to its second messenger via a G protein, similarly to what has been described for the brain of other vertebrates.

Cold stress in the rat induces parallel changes in plasma and pituitary levels of endorphin and ACTH

Endorphin and ACTH-like materials levels in rat plasma and pituitary were measured by radioimmunoassay under baseline and cold stress conditions. Cold stress significantly increased plasma beta-endorphin and ACTH immunoreactivity. A rise in these two peptides was also found in the neurointermediate lobe of the pituitary, while in the anterior lobe their levels were unaffected. These findings suggest that the rise of beta-endorphin and ACTH content in the neurointermediate lobe occurs as a compensatory biosynthetic mechanism for the peptides released from the adenohypophysis.

Benzodiazepines and their antagonists interfere with opioid-dependent stress-induced analgesia

Timing or intensity of shocks significantly modify the characteristics of the analgesia induced by footshock, and conditioning to footshock induces analgesia, independently from the time and shock parameters used for conditioning. However, whatever the parameters of shock, and the presence of conditioning or not, the stress has to be inescapable in order to produce an increase in pain thresholds. This observation suggests that anxiety plays a major role in the development of stress-induced analgesia. In order to test this hypothesis we investigated the effects of the benzodiazepine agonists diazepam and clonazepam, the antagonists RO 15-1788, CGS 8216, CGS 9896, and the inverse agonists FG 7142 and FG 7041 on the development and maintenance of stress-induced analgesia. Benzodiazepine receptor agonists decreased the analgesic effect of inescapable footshock, benzodiazepine receptor antagonists increased the footshock induced analgesia, whereas inverse agonists did not modify the analgesia induced by the shock. All the benzodiazepine receptor ligands blocked the antagonism of the footshock analgesia induced by naloxone.

Isoelectric point determination of human and camel β-endorphin, α-endorphin and enkephalins

Abstract The isoelectric point of the camel and the human β-endorphin, of the α-endorphin and the enkephalins were determined by analytical isoelectric focusing on 1 mm thin polyacrylamide gel slab. The difficulty of staining peptides as short as β-endorphin or smaller was overcomed using a modification of Bibring and Baxandalls or Faupel and Von Arxs staining method. The camel β-endorphin gives two bands having isoelectric point of 10.3 and 10.4, the human β-endorphin focus at pH 9.9, while α-endorphin, leu and met-enkephalin at pH 5.9, 5.5 and 5.45 respectively. The staining method described coupled with the isoelectric focusing seems to be fit for discriminating β-endorphin in a crude rat pituitary extract.

Regulation of the androgen receptors in the Harderian gland of the male Syrian hamster : influence of photoperiod, castration, and chronic melatonin treatment

Abstract: Male Syrian hamsters that were exposed for 8 weeks to short photoperiod (LD 10:14) or treated with melatonin in the late afternoon under long photoperiod conditions (LD 14:10) had a significantly higher content of androgen receptors in the Lipidex‐purified soluble fractions isolated from the Harderian glands as compared to the long photoperiod (LD 14:10) exposed controls. Simultaneous computer‐assisted analyses of all series of saturation and competition experiments revealed that the numerical value of the apparent Kd, as determined by using the synthetic androgen R‐1881 (methyltrienolone), was not different between the experimental groups, and ranged from 0.050 to 0.067 nM. Of the principal natural androgens, testosterone (T) was most potent in inhibiting methyltrienolone binding to the receptor (Ki values from 0.33 to 0.55 nM), and 5α‐dihydrotestosterone (DHT) and α4‐androstenedione (AD) were less effective (Ki values between 1 and 1.9 nM). In the hypothalami and pituitaries of the same animals, used in parallel control assays, DHT was twice as potent as T. Short‐term castration (24 hr post‐orchidectomy) did not result in significant changes in the receptor binding characteristics. Following 8 weeks exposure to a long photoperiod (LD 14:10) the Bmax values demonstrated a four‐fold increase in castrated animals (179 fmoles/mg protein vs. 47 fmoles/mg protein) over intact controls. The relative binding affinity of the major androgens under these conditions remained unchanged, with the exception of AD, where a five‐fold increase in the numerical Ki values (decrease in the binding affinity) was recorded (Ki = 9.6 nM). Studies on in vitro metabolism of the natural androgens by the cytosol during incubation under conditions identical to those employed in the binding studies revealed that in the normal intact animal AD is metabolized to T in elevated amounts. The ability of the Harderian gland to make this conversion is dramatically diminished following long‐term castration. This could explain the apparent “shift” in the high affinity of AD toward a much lower one. Testosterone, however, was not further metabolized. No DHT was formed and small amounts of AD were recovered. Thus, in the androgen receptor system of the hamster Harderian gland testosterone appears to be a strong androgen per se, binding to the receptor with affinity higher than that of DHT. This receptor system is apparently flexible in its response to changes in the photoperiod and to removal of the natural steroids from the endogenous milieu following long‐term castration. Both situations are accompanied by a significant increase in receptor numbers, though castration seemingly results in concomitant changes in the androgen metabolism in the gland.

Isoelectrofocusing on flat bed for determining and separating β-endorphin

Camel synthetic beta-endorphin focused in three bands by isoelectrofocusing on 1 mm polyacrylamide thin gel. All the bands have opioid activity, measured on guinea pig ileum, and radioimmunologically react with beta-endorphin antiserum. Since beta-endorphin from rat pituitary gland, particularly from the neurointermediate lobe, also focused in several bands, we hypothesized that the camel peptide occurs in different conformations. A quick, simple technique based on histoelectrofocusing is proposed as a good approach to separating and measuring beta-endorphin from rat pituitary lobes. The method gives very high recovery.