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Biomedical subjects

A Guidotti

Publications and source records attributed to A Guidotti.

At least 199 records · Page 11Linked to original sources
PubMed1984

Effect of alpha-bungarotoxin and etorphine on acetylcholine-evoked release of endogenous and radiolabeled catecholamines from primary culture of adrenal chromaffin cells.

Cell cultures of adrenal medulla have become an important research tool to study basic processes that regulate catecholamine storage, release and synthesis. Release has been studied either by labeling with [3H]norepinephrine and measuring release of radioactivity or by measuring the endogenous catecholamines released with HPLC. Acetylcholine (5 X 10(-6)-10(-4) M) appears to release preferentially norepinephrine, although the cells store more epinephrine than norepinephrine. Etorphine and alpha-bungarotoxin antagonize the release of catecholamines elicited by acetylcholine. This inhibitory action appears to be greater when the measurement of endogenous catecholamines rather than radioactive norepinephrine is used to monitor the action of acetylcholine. The data suggest that the measurement of endogenous catecholamines by HPLC is preferable to the [3H]NE loading and release technique, especially when analyzing the effects of low concentrations of drugs that are thought to affect nicotinic receptor function.

Acetylcholine↗
PubMed1984

Intrinsic GABAergic system of adrenal chromaffin cells.

Histochemical and biochemical studies demonstrate that gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (EC 4.1.1.15), and GABA aminotransferase (EC 2.6.1.19) are present in bovine adrenal chromaffin cells. Moreover, [3H]GABA can be taken up and stored by primary cultures of adrenal chromaffin cells. Nicotinic receptor stimulation or KCl depolarization releases the [3H]GABA taken up by these cell cultures. GABA and benzodiazepine recognition sites located in chromaffin cells interact with each other with modalities similar to those described for GABA and benzodiazepine recognition sites located in synaptic membranes prepared from brain tissue. Bicuculline facilitates the release of catecholamine from chromaffin cells induced by nicotinic receptor stimulation but it fails to influence the release of catecholamine evoked by K+ depolarization. Since the GABA-benzodiazepine receptor system appears to modulate nicotinic receptor function, it is suggested that GABA transmission might participate in modulating responsiveness of chromaffin cells to incoming cholinergic stimuli.

4-Aminobutyrate Transaminase↗
PubMed1984

Increase in the Bmax of gamma-aminobutyric acid-A recognition sites in brain regions of mice receiving diazepam.

gamma-Aminobutyric acid (GABA) receptors were characterized in vivo by studying ex vivo the binding of [3H]muscimol to cerebellum, cortex, hippocampus, and corpus striatum of mice receiving intravenous injections of tracer doses of high-specific-activity (approximately equal to 30 Ci/mmol) [3H]muscimol. This ligand binds with high affinity (apparent Kd, 2-3 X 10(-9) M) to a single population of binding sites (apparent Bmax, 250-180 fmol per 10 mg of protein). Pharmacological studies using drugs that selectively bind to GABAA or GABAB receptors suggest that [3H]muscimol specifically labels a GABAA recognition site. Moreover, diazepam (1.5 mumol/kg, i.p.) increases the Bmax but fails to change the affinity of [3H]muscimol binding to different brain areas. This diazepam-elicited increase in Bmax is blocked in mice receiving the diazepam antagonist Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4] benzodiazepine-3-carboxylate). Since the diazepam-induced increase of [3H]muscimol binding is paralleled by a significant potentiation of the inhibitory effect of muscimol on locomotor activity, it is proposed that the facilitatory action on GABAergic transmission elicited in vivo by diazepam is mediated by an increase in the Bmax of the binding sites of GABAA receptors.

Animals↗
PubMed1984

Regulation of the GABA receptor complex by a phosphorylation mechanism.

GABA- modulin , a regulatory component of the GABA/benzodiazepine receptor complex, is phosphorylated by cyclic-AMP-, Ca/calmodulin-, and Ca/phospholipid-dependent protein kinases at distinct sites in the molecule. Phosphorylation of GM by the cyclic-AMP-dependent process results in a complete loss of GM inhibitory activity on specific 3H-GABA binding to synaptic membrane recognition sites. The effect of the Ca2+-dependent phosphorylation, dependent on CaM or PS, of GM is presently unknown but may involve a synergistic or antagonistic action on the cyclic-AMP-dependent phosphorylation. Alternatively, the Ca2+-dependent protein kinases may regulate another function of GM, perhaps its postulated role as a coupler of the GABA/benzodiazepine recognition sites. The results of these experiments strongly implicate a role for protein phosphorylation in the regulation or modulation of GABA receptor function, and such a mechanism may be extrapolated to other neurotransmitter receptor complexes.

Animals↗
PubMed1984

Frequencies of cribra orbitalia in central Italy (19th century) under special consideration of their degrees of expression.

According to several hematological and anthropological studies the formation of hyperostosis spongiosa orbitae can be attributed to different types of anemias. Since especially in Central Italy the different forms of thalassemia as well as Plasmodium vivax, Plasmodium malariae and Plasmodium falciparum have been rather vastly distributed a study of this trait (differentiating between degrees of expression) on a population sample (125 males, 80 females) including adult, mature and senile skulls has been carried out (anatomical series of the medical faculty of the University of Siena ). However, the high incidence of this trait, though rather weakly expressed (male 56.8%, female 70.0%), and clearly classifiable in three degrees of expression also indicates the existence of other factors involved. Among these factors the nutritional ones could be excluded because this region was scarcely populated and rich in nutritional resources. On the other hand, the low content of Fe and Cl anions in the water could be made up with the assumption of foods abounding for these elements.

Adolescent↗
PubMed1984

Morphometrical considerations on occipital condyles.

From the study of the surface area of the occipital condyles and of skull volume resulted that the surface area is not proportional to skull volume. The measurements were conducted on a collection of 424 male and 317 female skulls. It was observed that the two condyles are rarely symmetrical and that also flat condyles appear. Bi-partitioned condyles can be divided into three types: roof-like bipartitions, simple bipartitions and total bipartitions.

Cephalometry↗
PubMed1984

Enkephalin biosynthesis in adrenal medulla. Modulation of proenkephalin mRNA content of cultured chromaffin cells by 8-bromo-adenosine 3',5'-monophosphate.

Incubation of primary cultures of chromaffin cells from bovine adrenal medulla with 8-bromo-adenosine 3',5'-monophosphate (8-Br-cyclic AMP) resulted in an increase in proenkephalin mRNA content. The mRNA that increased was detected by hybridization analysis using a cDNA probe and migrated with an apparent size of approximately 1400 bases. The increase in proenkephalin mRNA following 8-Br-cyclic AMP treatment was apparent in 12 hr and continued over 2 days. Corresponding changes were detected in enkephalin-like immunoreactivity but with a 24-hr lag: the cellular content increased significantly after 2 days of treatment and continued to rise over the next 2 days, whereas changes in the amount released to the medium followed the same time course. Dose-response curves for the increase in the content of proenkephalin mRNA and of enkephalin-containing peptides were essentially identical. Chromatographic characterization of the enkephalin-like peptides demonstrated that 8-Br-cyclic AMP increased both the high molecular weight fraction and the low molecular weight fraction, which was shown by high-pressure liquid chromatography to contain Met5-enkephalin, Leu5-enkephalin, and Met5-enkephalin-Arg6-Phe7. Previous results in chromaffin cells have demonstrated that the synthesis of tyrosine hydroxylase is also regulated by cyclic AMP, with a similar time course. These results therefore suggest the possibility of coordinate regulation by cyclic AMP of the expression of the cotransmitters, catecholamines and enkephalin peptides, in the adrenal medulla.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Purification and characterization of a bungarotoxin polypeptide which blocks nicotinic receptor function in primary culture of adrenal chromaffin cells.

In primary cultures of bovine chromaffin cells, commercially available preparations of alpha-bungarotoxin inhibit the acetylcholine (ACh)- or nicotine-evoked release of endogenous catecholamines. The potency of different lots of alpha-bungarotoxin is not related to the alpha-bungarotoxin peptide content but to that of another peptide (termed P-4 bungarotoxin) present as an impurity in the alpha-bungarotoxin preparations. P-4 Bungarotoxin was isolated and purified to homogeneity by high-pressure liquid chromatography (HPLC). Homogeneity was established by a variety of means, including polyacrylamide gel electrophoresis, HPLC, end carboxy group analysis and NH2-terminal amino acid sequence. Purified P-4 bungarotoxin contains approximately 121 amino acid residues, and it is different in its amino composition, molecular weight, and amino acid sequence from alpha-bungarotoxin and beta-bungarotoxin. P-4 Bungarotoxin (IC50 congruent to 1 nM) blocked the ACh-induced release of endogenous catecholamines but failed to block the KCl-induced catecholamine release. Although P-4 bungarotoxin is endowed with phospholipase A2 activity, its effect on ACh-evoked catecholamine release persists when the phospholipase activity is blocked (99.9%) by treatment of the toxin with p-bromophenacyl bromide. P-4 Bungarotoxin may represent a useful tool with which to study nicotinic receptor function in sympathetic and central nervous system neurons.

Acetylcholine↗
PubMed1983

In vivo modulation of brain dopamine recognition sites: a possible model for emission computed tomography studies.

The content of authentic 3H-spiroperidol and of its metabolites was measured in brain regions of rat, guinea pig and mouse receiving tracer doses of 3H-spiroperidol intravenously (0.2 to 0.5 micrograms/kg). The time course of the 3H-spiroperidol content of various brain regions shows that a steady state was maintained between 2 and 6 hrs; the lowest 3H-spiroperidol content was attained in cerebellum where the value approached that of blood plasma. Since the cerebellum contains an insignificant number of dopamine receptors but many serotonin receptors and other sites that bind 3H-spiroperidol, the 3H-spiroperidol contained in cerebellum was considered background binding. In the striatum and olfactory tubercle of rats receiving two daily doses for 3 weeks of haloperidol or amphetamine the amount of 3H-spiroperidol that binds in vivo is decreased or increased, respectively. If the kinetic characteristics of in vivo binding of 3H-spiroperidol observed in the rat, guinea pig and mouse can be replicated in man using spiroperidol containing a gamma- or a position-emitting label, one might have a probe to study dopamine receptors in vivo with emission computed tomography scanning.

Animals↗
PubMed1983

GABAergic synapses. Supramolecular organization and biochemical regulation.

Extraneurally released gamma-aminobutyric acid (GABA) interacts with specific recognition sites associated with proteins located in postsynaptic neuronal membranes that function as chloride (Cl-)ionophores. As a result of the interaction between GABA and the recognition sites, Cl- ionophores are opened causing an influx or an efflux of Cl-, depending on the values of the Cl- equilibrium potential and of the membrane potential. Hyperpolarization or depolarization will result from inward or outward Cl- fluxes, respectively. Independently of the change in conductivity elicited by GABA, this amino acid transmitter will reduce the effectiveness of the sodium ion (Na+) excitatory potential. In attempts to elucidate the molecular mechanism, whereby benzodiazepines facilitate the action of GABA on membrane conductance without changing the activity of Cl- or other ionophore, a basic protein (GABA-modulin, GM) has been isolated from rat brain which is similar in structure to the small molecular weight myelin basic protein, found in rodent brain. While GABA-modulin is located in synaptosomes, the small molecular weight myelin basic protein is located in the myelin fraction: more important, GABA-modulin inhibited the high affinity binding of GABA to crude synaptic membranes while the basic myelin protein did not. Also, amino acid composition and molecular weight differentiate the two proteins. The GABA-modulin can be phosphorylated with different stoichiometry by cyclic AMP-dependent protein kinase (4 mol PO4(-3)) or Ca2+-dependent protein kinase (1 mol PO4(-3)). Only cyclic AMP-dependent phosphorylation inhibited the action of GABA-modulin on GABA binding.

Animals↗
PubMed1983

On a brain polypeptide functioning as a putative effector for the recognition sites of benzodiazepine and beta-carboline derivatives.

Stimulation of benzodiazepine recognition sites by various ligands can elicit opposite types of responses such as proconvulsant and anticonvulsant or proconflict and anticonflict actions. The study of the pharmacological profile of various ligands makes it possible to distinguish three classes of compounds: (1) those that elicit anticonflict responses in the Vogel test, inhibit the convulsions due to an impairment of GABAergic transmission and increase the Bmax of the high affinity recognition site for GABA; (2) those that displace benzodiazepines from specific binding sites, facilitate convulsions due to impairment of GABAergic mechanisms, elicit proconflict responses in Vogel's test and inhibit the facilitation by benzodiazepines of GABA binding and (3) those that displace benzodiazepines and beta-carboline-derivatives from specific binding sites, antagonize the anticonflict, the proconflict, the anticonvulsant and the proconvulsant actions of the two preceding groups of substances and in very large doses elicit a small proconvulsant action. Examples of the latter are an imidazobenzodiazepine (RO 15-1788) and a pyrazolquinolinone derivative (CGS 8216). The nomenclature for these three classes of drugs should be kept flexible until the action of the endogenous ligand that functions as the physiological effector of the benzodiazepine/beta-carboline recognition site is known. A putative ligand for this site (DBI = diazepam binding inhibitor) has been isolated and purified to homogeneity. It includes 104 amino acid residues, the sequence of the last 45 amino acids has been determined. This compound elicits a proconflict action, displaces beta-carboline derivatives more than anxiolytic benzodiazepines, but its high molecular weight and relative low affinity for the binding sites in brain might suggest that it is a precursor, rather than the putative effector for benzodiazepine and/or beta-carboline recognition sites.

Animals↗
PubMed1983

Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors.

A brain polypeptide termed diazepam-binding inhibitor (DBI) and thought to be chemically and functionally related to the endogenous effector of the benzodiazepine recognition site was purified to homogeneity. This peptide gives a single band of protein on NaDodSO4 and acidic urea gel electrophoresis. A single UV-absorbing peak was obtained by HPLC using three different columns and solvent systems. DBI has a molecular mass of approximately equal to 11,000 daltons. Carboxyl-terminus analysis shows that tyrosine is the only residue while the amino-terminus was blocked. Cyanogen bromide treatment of DBI yields three polypeptide fragments, and the sequences of two of them have been determined for a total of 45 amino acids. DBI is a competitive inhibitor for the binding of [3H]diazepam, [3H]flunitrazepam, beta-[3H]carboline propyl esters, and 3H-labeled Ro 15-1788. The Ki for [3H]-diazepam and beta-[3H]carboline binding were 4 and 1 microM, respectively. Doses of DBI that inhibited [3H]diazepam binding by greater than 50% fail to change [3H]etorphine, gamma-amino[3H]butyric acid, [3H]-quinuclidinyl benzilate, [3H]dihydroalprenolol, [3H]adenosine, and [3H]imipramine binding tested at their respective Kd values. DBI injected intraventricularly at doses of 5-10 nmol completely reversed the anticonflict action of diazepam on unpunished drinking and, similar to the anxiety-inducing beta-carboline derivative FG 7142 (beta-carboline-3-carboxylic acid methyl ester), facilitated the shock-induced suppression of drinking by lowering the threshold for this response.

Amino Acids↗
PubMed1983

Phosphorylation induces a decrease in the biological activity of the protein inhibitor (GABA-modulin) of gamma-aminobutyric acid binding sites.

gamma-Aminobutyric acid (GABA)-modulin is a brain protein of Mr 16,500 that down-regulates the high-affinity binding site for GABA which is located in crude synaptic membranes. This protein can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase and by a partially purified preparation of calmodulin-sensitive Ca2+-dependent protein kinase. The GABA-modulin sites that are phosphorylated by the two enzymes are different, as revealed by HPLC analysis of tryptic digests. The capacity of GABA-modulin to decrease the number of sites that bind [3H]muscimol was completely abolished by phosphorylation of this protein with the cAMP-dependent protein kinase but not with the Ca2+-dependent enzyme. GABA-modulin present in crude synaptic membranes prepared from rat cortex also was shown to be phosphorylated by endogenous protein kinases activated by cAMP, Ca2+ and calmodulin, and Ca2+ and phosphatidylserine. These results suggest a potentially important role for protein kinase and GABA-modulin in the regulation of the number of GABA recognition sites.

Animals↗
PubMed1983

beta-Carbolines enhance shock-induced suppression of drinking in rats.

By using Vogel's method to test the anxiolytic action of benzodiazepines and reducing the intensity of the current delivered to the drinking tube, it is possible to distinguish the pharmacological activity of three types of ligands for the benzodiazepine recognition site. An anticonflict action typical of anxiolytic benzodiazepines, a proconflict action typical of many beta-carbolines, including FG 7142 (beta-carboline-3-carboxylic acid ethyl ester methyl amide), and an antagonistic action of the proconflict and anticonflict actions typical of RO 15-1788 (ethyl-8-fluoro-5, 6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-alpha]-[1, 4]-benzodiazepine-3-carboxylate) and CGS 8216 (2-phenylpyrazolo[4,3-c]quinolin-3-(5H)-one). Pentylenetetrazole, which causes convulsions by interacting with a subunit of the gamma-aminobutyric acid receptor that is different from the benzodiazepine recognition site, also induces a proconflict action that is antagonized by anxiolytic benzodiazepines but not by RO 15-1788.

Animals↗
PubMed1983

Modulation of GABA receptor binding by Ca2+.

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.

Animals↗
PubMed1983

Cotransmitters: pharmacological implications.

The discovery that two or more neuroactive substances coexist in the same nerve terminal suggests that two or more neuroactive compounds can be released by nerve impulses simultaneously and probably act cooperatively at postsynaptic sites. This interaction changes the models of synaptic transmission we have used in the past and imposes a reevaluation of current understanding of synaptic pharmacology. Neuroactive substances co-existing in the same axon terminal can function as "primary transmitter" if they activate the receptor-transducer system or as "cotransmitter" if they modulate the gain of the system. Two examples of synaptic mechanisms in which two neuroactive substances coexisting in the same axon terminal appear to function as primary transmitter and cotransmitter are discussed. These examples are: 1. the modulation of the function of nicotinic receptors of chromaffin cells by endogenous opiate peptides stored in the splanchnic nerve and 2. the modulation of GABA receptor function by benzodiazepines. The understanding of the mechanisms by which primary transmitter and cotransmitter interact at the postsynaptic site may be of obvious importance in elucidating the integrative and discriminative function of the nervous system, in interpreting the action of drugs and in developing new therapeutic agents devoid of untoward side effects.

Animals↗