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Biomedical subjects

I Mocchetti

Publications and source records attributed to I Mocchetti.

At least 55 records · Page 3Linked to original sources
PubMed1991

Stimulation of nerve growth factor biosynthesis in developing rat brain by reserpine: steroids as potential mediators.

The stimulation of beta-adrenergic receptors by isoproterenol increases nerve growth factor (NGF) biosynthesis in C6 rat glioma cells, suggesting that norepinephrine may regulate NGF biosynthesis in vivo. We have tested this hypothesis in 21-day-old rats by depleting catecholamine stores with reserpine. Northern blot analysis of NGF mRNA, in combination with a two-site enzyme immunoassay for NGF, showed that depletion of catecholamines was associated with a 3-fold increase in NGF mRNA, which was followed by a significant increase in the NGF content of cerebral cortex. The increase in NGF mRNA was most marked 9 hr after reserpine administration (2 mg/kg, subcutaneously) and was no longer apparent 24 hr after drug administration, when brain monoamine stores were still depleted. Moreover, the lowest dose of reserpine that significantly increased NGF mRNA levels induced only a small change in the content of cortical catecholamines. These results suggest that reserpine mediates the increase in NGF production by a mechanism other than monoamine depletion. Because reserpine increases plasma glucocorticoid concentrations through the pituitary-adrenal axis, we investigated whether adrenal steroids could be responsible for the induction of NGF biosynthesis. The effect of reserpine on NGF biosynthesis was abolished in adrenalectomized rats. Moreover, dexamethasone, a synthetic glucocorticoid, given at a dose of 0.5 mg/kg, subcutaneously, increased the amount of NGF mRNA and NGF in cerebral cortex. NGF biosynthesis in the central nervous system may, thus, be regulated by adrenocortical hormonal secretion.

Adrenal Cortex Hormones↗
PubMed1990

Increased nerve growth factor receptor mRNA in contused rat spinal cord.

Peripheral nerve injury induces the expression of nerve growth factor receptor (NGFR). To determine whether a similar induction results from injury of the spinal cord, NGFR mRNA content was determined using Northern blot hybridization analysis of total RNA from spinal cords of rats contused in the mid-thoracic region. By four days after contusion NGFR mRNA was significantly increased in the thoracic segments that included the injury site. The induction was maximal at 7 days, about 5- to 7-fold the level of uninjured controls, and remained 4 times higher than controls at 14 and 28 days after injury. These results suggest that axotomy in the CNS may also trigger the molecular mechanism(s) leading to up-regulation of NGFR expression.

Animals↗
PubMed1990

Regulation of nerve growth factor receptor mRNA content by dexamethasone: in vitro and in vivo studies.

Northern blot hybridization analysis was used to study regulation of nerve growth factor receptor (NGFR) mRNA content by glucocorticoids. Treatment for 6 h with dexamethasone (1 microM) caused a 40% decrease of NGFR mRNA content in PC12 cells and a 60% decrease in C6-2B glioma cells which was time and dose dependent. Dexamethasone (1 microM/kg) administered s.c. for two days to 21-day-old rats, elicits a 60% decrease in NGFR mRNA content in septum. These results suggest that the expression of NGFR gene in the brain could be inhibited by endogenous glucocorticoids. Whether dexamethasone inhibits NGFR gene expression by directly affecting cis-regulatory elements in the promoter regions of the gene remains to be elucidated.

Adrenal Gland Neoplasms↗
PubMed1990

Molecular biology of diazepam binding inhibitor peptide.

Complementary DNA (cDNA) clones containing the entire coding sequence for Diazepam Binding Inhibitor (DBI) peptide, a 10-kDa precursor of putative natural ligands of benzodiazepine recognition sites, were isolated from rat, human and cow libraries. The sequence of all these clones is highly conserved; however, the N-terminal sequence predicted by the human DBI clone differed from that of the other two clones. DBI cDNA, utilized as hybridization probe in Southern blot analysis, revealed that DBI of both human and rat might be encoded by a multiple family of 4-6 genes. Furthermore, we have used in situ chromosomes hybridization to map human DBI genes. The results indicate that a human DBI gene is localized on chromosome 2 and that three of the four hybridization signals detected by the human DBI probe are located on three other chromosomes. These findings raise a question as whether multiple DBI genes encode for different molecular forms of DBI. In the attempt to test this hypothesis, cow cDNA and human genomic libraries were screened with DBI cDNA. In this paper I report the isolation of clones from these libraries which, although hybridizing well to DBI cDNA, possess a low percentage of homology (46.7%), randomly distributed within the coding region of DBI cDNA. Whether or not these clones encode for peptides sharing the same physiological role as DBI is under investigation.

Amino Acid Sequence↗
PubMed1989

Down-regulation of proopiomelanocortin synthesis and beta-endorphin utilization in hypothalamus of morphine-tolerant rats.

We investigated regulation of the dynamic state of enkephalin and endorphin brain stores during morphine tolerance and dependence using cDNA hybridization and radioimmunoassay of the biologically active peptide(s) and their respective peptide precursors. Rats were made tolerant to morphine with the subcutaneous implantation of three morphine pellets (75 mg each) for a period of five days. Hypothalamic proopiomelanocortin (POMC) mRNA, POMC, and corticotropin-like intermediate lobe peptide content were decreased by 50% in morphine-dependent rats. However, beta-endorphin content remained unchanged. Enkephalin and proenkephalin mRNA content in various brain structures failed to change. A single injection of naltrexone (2 mg/kg) 1 hour before decapitation did not reverse the decrease in POMC mRNA and POMC content elicited by morphine. However, a slower, spontaneous withdrawal caused by removal of the pellets did reverse (after two days) the down-regulation of the hypothalamic POMC system. A single injection of morphine (10 mg/kg) failed to affect any parameter used to assess the dynamic state of opioid peptides.

Animals↗
PubMed1989

Regulation of nerve growth factor biosynthesis by beta-adrenergic receptor activation in astrocytoma cells: a potential role of c-Fos protein.

The chain of events that results in increased production of nerve growth factor (NGF) following beta-adrenergic receptor (BAR) stimulation has been investigated in the C6-2B rat astrocytoma cell line. Exposure of these cells to the BAR agonist isoproterenol elicits the following cascade of events: (i) increase of cAMP content; (ii) increase of c-Fos mRNA content; (iii) accumulation of c-Fos protein immunoreactivity in the nucleus; (iv) increase of NGF mRNA content. The increase in c-Fos mRNA and its translation product are early events (15 and 40 min, respectively) and precede the accumulation of NGF mRNA, which peaks at 3 hr. The increase in the two mRNAs appears interrelated because cycloheximide inhibits the accumulation of c-Fos protein and NGF mRNA elicited by isoproterenol. Moreover, the accumulation of nuclear c-Fos protein and NGF mRNA induced by BAR stimulation is reduced by 2-aminopurine, an inhibitor of c-Fos mRNA induction. These data suggest that, in C6-2B astrocytoma cells, the nuclear accumulation of c-Fos protein is required for the induction of NGF mRNA expression by BAR stimulation.

2-Aminopurine↗
PubMed1988

Chromosomal localization of the human diazepam binding inhibitor gene.

We have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the gamma-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes.

Animals↗
PubMed1988

Beta adrenergic and prostaglandin receptor activation increases nerve growth factor mRNA content in C6-2B rat astrocytoma cells.

C6-2B rat astrocytoma cells were used to test whether the increase of cellular nerve growth factor (NGF) content and secretion induced by isoproterenol treatment are associated with an increase in the content of mRNA that encodes NGF (NGF mRNA). Incubation of cells with isoproterenol (10 microM) for different time periods produced an increase of NGF mRNA content (3- to 5-fold over control) reaching maximum levels at 3 hr and lasting up to 24 hr. The isoproterenol effect on NGF mRNA was antagonized by the beta adrenergic receptor antagonist I-propranolol (10 microM) but not by phentolamine (10 microM), an alpha adrenergic receptor antagonist. When C6-2B astrocytoma cells were exposed for a short time to isoproterenol (10 microM; 10 or 20 min) followed by a washout period of 3 hr, the NGF mRNA content was increased by about 2-fold. The increase of NGF mRNA was obtained also with 10 microM prostaglandin E1 and this effect was potentiated by 100 microM of 3-isobutyl-1-methylxanthine. Inasmuch as both isoproterenol and prostaglandin E1 increase cyclic AMP content, one can surmise that cyclic AMP is involved in the stimulation of NGF mRNA accumulation. Whether cyclic AMP directly activates NGF gene transcription or activates an intermediate step, however, cannot be assessed by the present experiments.

1-Methyl-3-isobutylxanthine↗
PubMed1987

In vivo studies of the regulation of neuropeptide stores in structures of the rat brain.

In order to evaluate the regulation of neuropeptide stores in structures of the rat brain, the content of the specific messenger ribonucleic acid for the peptide precursor, of the precursor and of its biologically active peptide fragment, resulting from the precursor processing was measured. Enkephalin stores of the striatum, but not that of other structures, were upregulated by repeated daily doses of dopaminergic receptor antagonists of the D-1 type. Morphine tolerance failed to change stores of enkephalin in brain but produced a down-regulation of the synthesis of pro-opiomelanocortin in the hypothalamus. The regulation of diazepam binding inhibitor (DBI), the precursor of a family of putative endogenous ligands of the benzodiazepine recognition site, was studied during tolerance to benzodiazepines. Tolerance to diazepam increased the dynamic state of an octadecaneuropeptide (ODN), derived from DBI, in the cerebellum and cortex. The steady state or dynamic state of ODN were not changed in other structures of the brain of the rat, tolerant to diazepam.

Animals↗
PubMed1987

Beta-adrenergic receptor regulation of NGF-mRNA content in rat C6-2B glioma cells.

In C6-2B astrocytoma cells the Beta NGF content and secretion rate are increased by isoproterenol activation of beta-adrenergic receptors (Schwartz and Costa, 1977). Utilizing poly (A+) RNA hybridization analysis with a cRNA probe for mouse Beta NGF it was found that isoproterenol activation of C6-2B cells produces also a 4 fold increase of the content of messenger RNA encoding Beta NGF. This increase is specifically antagonized by 1-propanolol, but not by phentolamine. Furthermore, addition of dibutyryl-cAMP induces an increase of Beta NGF mRNA content similar to that obtained with isoproterenol. These results are consistent with the hypothesis that regulation of Beta NGF synthesis in neuroglial cells may be modulated by beta-adrenergic receptor activation.

Animals↗
PubMed1987

Protracted treatment with diazepam increases the turnover of putative endogenous ligands for the benzodiazepine/beta-carboline recognition site.

DBI (diazepam-binding inhibitor) is a putative neuromodulatory peptide isolated from rat brain that acts on gamma-aminobutyric acid-benzodiazepine-Cl- ionophore receptor complex inducing beta-carboline-like effects. We used a cDNA probe complementary to DBI mRNA and a specific antibody for rat DBI to study in rat brain how the dynamic state of DBI can be affected after protracted (three times a day for 10 days) treatment with diazepam and chlordiazepoxide by oral gavage. Both the content of DBI and DBI mRNA increased in the cerebellum and cerebral cortex but failed to change in the hippocampus and striatum of rats receiving this protracted benzodiazepine treatment. Acute treatment with diazepam did not affect the dynamic state of brain DBI. An antibody was raised against a biologically active octadecaneuropeptide (Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys ) derived from the tryptic digestion of DBI. The combined HPLC/RIA analysis of rat cerebellar extracts carried out with this antibody showed that multiple molecular forms of the octadecaneuropeptide-like reactivity are present and all of them are increased in rats receiving repeated daily injections of diazepam. It is inferred that tolerance to benzodiazepines is associated with an increase in the turnover rate of DBI, which may be responsible for the gamma-aminobutyric acid receptor desensitization that occurs after protracted benzodiazepine administration.

Animals↗
PubMed1987

Opioid peptide biosynthesis: enzymatic selectivity and regulatory mechanisms.

Certain general principles determine the biosynthesis of most biologically active peptides, including the opioid peptides, from large protein precursors. In almost all instances, the active peptide is embedded in the precursor flanked on both sides by pairs of basic amino acids. The first step in processing involves a trypsinlike enzyme, cleaving to the carboxyl terminus of basic amino acids, and leaving the active peptide with a basic amino acid on the carboxyl terminus. A carboxy-peptidase peptidase B-like enzyme then removes the remaining basic amino acid. It has been unclear whether any endopeptidases with trypsinlike activity are selective for one or another basic amino acid. Recently a soluble endopeptidase has been identified that can cleave to both the carboxyl and amino termini of basic amino acids. Enkephalin convertase (carboxypeptidase E, H) (EC 3.4.17.10) has considerable selectivity, and appears to be physiologically associated with the biosynthesis of enkephalin as well as a limited number of other neuropeptides. The turnover of opioid peptides and other neuropeptides is most effectively ascertained by measuring levels of mRNA either biochemically or by in situ hybridization. Striking dynamic alterations include a pronounced increase in levels of proenkephalin mRNA in the corpus striatum after blockade of dopamine receptors, but changes in opioid peptide mRNA after opiate addiction are less clear.

Animals↗
PubMed1987

Regulation of striatal enkephalin turnover in rats receiving antagonists of specific dopamine receptor subtypes.

The hypothesis that striatal dopamine regulates enkephalin (ENK) synthesis is supported by the increase of striatal proenkephalin mRNA and ENK after intranigral injection of 6-hydroxydopamine. In order to elucidate which dopamine receptor subtype is operative in the regulation of the dynamic state of ENK, the effect of drugs that block D-1 or D-2 receptor selectively was studied. Daily administration of 140 mumol/kg s.c. of the D-2 antagonist I-sulpiride twice daily for 2 weeks produces a 30% decrease in the content of striatal proenkephalin mRNA and ENK. In contrast, a 50% increase was observed after 2 weeks of treatment with the D-1 antagonist SCH 23390 at 74 nmol/kg s.c. three times a day. Hence, it can be inferred that the endogenous activation of D-1 tonically decreases striatal ENK synthesis. Removal of this neurally mediated regulation either by a specific pharmacologic blockage of D-1 or by lesioning with 6-hydroxydopamine increases the biosynthesis of ENK. The increase of ENK biosynthesis elicited by denervation with 6-hydroxydopamine cannot be due to the endogenous activation of D-2 receptors and must be due to the inactivation of the tonic inhibition exerted by D-1 receptors.

Animals↗
PubMed1986

Putative diazepam binding inhibitor peptide: cDNA clones from rat.

cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes.

Amino Acid Sequence↗
PubMed1986

Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells.

In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone (1 microM) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 microM veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 microM dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 microM) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.

Adrenal Medulla↗
PubMed1985

Studies on the up regulation of alpha-adrenoceptors on rat hippocampal perikarya by chemical lesion of the median raphe nucleus.

Neurotoxin-induced lesion of the serotonergic raphe-hippocampal pathway produced about a 50% increase in the density of a nM affinity alpha-adrenergic binding site for (3H)WB-4101 in rat hippocampus 18 days postlesion without altering the specific binding of (3H)5-HT to serotonergic receptors. The chronic i.c.v. infusion of serotonin by minipump started at the appropriate time averted or reverted the effect. The dynamics of noradrenergic neurotransmission in the hippocampus was not impaired by lesion of the median raphe nucleus as determined by the uptake and turnover of noradrenaline as well as its release - as reflected by the normetanephrine concentration. In addition, neurotoxin-induced lesion of the dorsal noradrenergic bundle failed to alter either the Bmax or the Kd of (3H)WB-4101 binding to the nM site. Kainic acid-induced destruction of perikarya depressed the nM (3H)WB-4101 binding sites by 60% and completely prevented the up regulation caused by lesion of the median raphe nucleus. Thus, the supersensitivity-like response of the adrenoceptors to the lack of serotonin appears to be localized on kainate-sensitive cells within the hippocampus.

Animals↗
PubMed1985

Presynaptic and transynaptic mechanisms involved in the subsensitivity of rat cortical noradrenergic system after long-term antidepressant treatment.

Chronic treatment with antidepressants has been shown to produce a subsensitivity of noradrenergic neurons, both at presynaptic and postsynaptic sites. Important mechanisms, whereby the activity of noradrenergic neurons is regulated, could be the sensitivity of presynaptic alpha 2-adrenoceptors and the participation of transynaptic mechanisms involving other neurons. In this report we demonstrate that transynaptic factors involving the serotonergic system may be relevant to the regulation of the function of alpha 2-receptors in antidepressant chronically treated animals. In fact, we provide evidence of a markedly deminished responsiveness of noradrenergic neurons to an alpha 2-agonist (clonidine) or antagonist (mianserin) in biochemical and behavioral studies following serotonergic denervation with 5,7-dihydroxytryptamine. These results indicate that a functional interrelationship between serotonergic and noradrenergic systems might play an important role in the adaptive changes which bring the noradrenergic neurons to a lower level of activity after chronic antidepressant administration.

5,7-Dihydroxytryptamine↗