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E Bronzetti

Publications and source records attributed to E Bronzetti.

At least 19 recordsLinked to original sources
PubMed1999

alpha1-adrenergic receptor subtypes in human peripheral blood lymphocytes.

We investigated the expression of alpha1-adrenergic receptor subtypes in intact human peripheral blood lymphocytes using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding assay techniques combined with antibodies against the three subtypes of alpha1-adrenergic receptors (alpha1A, alpha1B, and alpha1D). RT-PCR amplified in peripheral blood lymphocytes a 348-bp alpha1A-adrenergic receptor fragment, a 689-bp alpha1B-adrenergic receptor fragment, and a 540-bp alpha1D-adrenergic receptor fragment. Radioligand binding assay with [3H]prazosin as radioligand revealed a high-affinity binding with a dissociation constant value of 0. 65+/-0.05 nmol/L and a maximum density of binding sites of 175. 3+/-20.5 fmol/10(6) cells. The pharmacological profile of [3H]prazosin binding to human peripheral blood lymphocytes was consistent with the labeling of alpha1-adrenergic receptors. Antibodies against alpha1A-, alpha1B-, and alpha1D-receptor subtypes decreased [3H]prazosin binding to a different extent. This indicates that human peripheral blood lymphocytes express the three alpha1-adrenergic receptor subtypes. Of the three different alpha1-adrenergic receptor subtypes, the alpha1B is the most represented and the alpha1D, the least. Future studies should clarify the functional relevance of alpha1-adrenergic receptors expressed by peripheral blood lymphocytes. The identification of these sites may represent a step for evaluating whether they represent a marker of alpha1-adrenergic receptors in cardiovascular disorders or for assessing responses to drug treatment on these receptors.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1998

Labeling of dopamine D3 and D4 receptor subtypes in human peripheral blood lymphocytes with [3H]7-OH-DPAT: a combined radioligand binding assay and immunochemical study.

Molecular biology studies have demonstrated that human peripheral blood lymphocytes express dopamine D2-like receptors belonging to the D3 and D4 receptor subtypes, whereas the characterization of these receptors using radioligand binding assay techniques provided conflicting results. The preferential dopamine D3 receptor agonist [3H]7-hydroxy-N, N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) was used recently for labeling lymphocyte dopamine D3 receptor. However, the selectivity of this compound for the D3 receptor was questioned. In this study we have investigated human peripheral blood lymphocyte dopamine receptor subtypes labeled by [3H]7-OH-DPAT using a conventional radioligand binding assay technique and antibodies against dopamine D2-like receptor subtypes. [3H]7-OH-DPAT was specifically bound to intact human peripheral blood lymphocytes with a dissociation constant (Kd) value of 0.32 + 0.03 nM and a maximum density of binding sites (Bmax) of 18.2 + 0.8 fmol/2 x 10(6) cells. [3H]7-OH-DPAT binding was unaffected by antibodies against dopamine D2 and D2S receptors. Anti-dopamine D3 and D4 receptor antibodies reduced [3H]7-OH-DPAT binding by about 53% and 32% respectively. Combination of anti D3 and D4 receptor antibodies reduced remarkably [3H]7-OH-DPAT binding. The above results suggest that the dopamine receptor agonist [3H]7-OH-DPAT labels dopamine D3 and D4 receptor subtypes in human peripheral blood lymphocytes. The use of antibodies raised against dopamine receptor subtypes in combination with radioligand binding assay may contribute to define receptor subtypes expressed by human peripheral blood lymphocytes in health and disease.

Adult↗
PubMed1998

Astrocyte changes in aging cerebral cortex and hippocampus: a quantitative immunohistochemical study.

Glial cells are sensitive to aging, but the real extent of age-related quantitative and qualitative changes of these brain cellular elements has not yet been clarified. Brain volume undergoes age-related changes, but several studies on the number of glial cells have not taken this important variable into account. In this study we quantitatively evaluated the number and morphology of glial fibrillary acidic protein (GFAP)-immunoreactive astroglia in the frontal cortex and in the CA1 subfield of the hippocampus of male Sprague-Dawley rats of aged 12 and 24 months, considered adult and aged, respectively. The volume of frontal cortex was unchanged in the two age groups investigated, whereas the volume of hippocampus was higher in aged rats. An increase in the number and size of GFAP-immunoreactive astrocytes was observed in the frontal cortex and in the CA1 subfield of the hippocampus of aged rats. The numeric increase in astrocytes was more pronounced in the hippocampus than in the frontal cortex, whereas age-related hypertrophy of astroglia was more accentuated in the frontal cortex. The possible significance of hyperplasia and hypertrophy of GFAP-immunoreactive astrocytes in the frontal cortex and in the CA1 subfield of the hippocampus of aged rats is discussed.

Age Factors↗
PubMed1998

Pharmacological characterization and autoradiographic localization of a putative dopamine D4 receptor in the heart.

1. The pharmacological profile and the anatomical localization of a putative dopamine D4 receptor were assessed in sections of rat and human atria and ventricles using combined radioligand binding and autoradiographic techniques with [3H]-spiperone as a ligand. 2. [3H]-Spiperone was bound specifically to sections of human and rat atria and ventricles. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high-affinity sites. In atria, the dissociation constant value (Kd) was 0.45 nM in rats and 0.32 nM in humans, and the maximum density of binding sites (Bmax) was 31.6+/-2.9 fmol mg(-1) tissue in rats and 18.8+/-0.7 fmol mg(-1) tissue in humans. In ventricles, Kd was 0.38 nM in rats and 0.39 nM in humans, and the Bmax was 43.5+/-3.0 fmol mg(-1) tissue in rats and 56.4+/-3.2 fmol mg(-1) tissue in humans. 3. The pharmacological profile of [3H]-spiperone binding to sections of both rat and human atria and ventricles was consistent with the labelling of dopamine D2-like receptors. [3H]-Spiperone binding was more sensitive to displacement by the neuroleptic clozapine in sections of atria than of ventricles, suggesting the expression of a dopamine D4 receptor in atrial tissue. Moreover, preincubation of some sections with a dopamine D4 receptor antibody and subsequent exposure to [3H]-spiperone caused a remarkable decrease of radioligand binding to sections of atria, but only a slight reduction of binding to sections of ventricles. 4. Light microscope autoradiography revealed the accumulation of silver grains over atrial tissue within atrial myocardiocytes. A higher density of silver grains was developed in rat than in human atria. In ventricles, silver grains were accumulated primarily in cluster areas both in rats and in humans. 5. The above findings suggest the expression of a dopamine D4 receptor in rat atria, but not in ventricles. A similar site was identified in human atria. The possible relevance of a dopamine D4 receptor in the heart is discussed.

Adolescent↗
PubMed1998

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1. Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [3H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2. [3H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (Kd) values of [3H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (Bmax) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3. The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4. This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.

Adult↗
PubMed1998

Density and localization of calcium channels of the L-type in human pulmonary artery.

The pharmacological profile and the anatomical localization of Ca2+ channels of the L-type were investigated in the human pulmonary artery to identify possible mechanisms involved in the regulation of the pulmonary vascular tone. Analysis was performed on slide-mounted frozen sections of human pulmonary artery using radioligand binding assay techniques associated with light microscope autoradiography. [3H]-Nicardipine was used as ligand. Human renal and right coronary arteries also were used as systemic reference arteries. Binding of [3H]-nicardipine to sections of human pulmonary artery was time-, temperature- and concentration-dependent, saturable and reversible. In the human pulmonary artery, the apparent equilibrium dissociation constant (Kd) was 0.12+/-0.02 nM and the maximum density of binding sites (Bmax) was 38.15+/-2.25 fmol/mg tissue. Kd values were 0.3+/-0.01 nM and 0.5+/-0.02 in the human renal artery and right coronary artery respectively. Bmax values were 248+/-16 fmol/mg tissue and 173+/-9.5 fmol/mg tissue in the human renal artery and right coronary artery respectively. The pharmacological profile of [3H]-nicardipine binding to sections of human pulmonary artery was consistent with the labeling of Ca2+ channels of the L-type. It was similar in the pulmonary artery and in the human renal and right coronary arteries. Light microscope autoradiography revealed a high density of [3H]-nicardipine binding sites within smooth muscle of the tunica media of human pulmonary artery as well as of human renal and right coronary arteries. A lower accumulation of the radioligand occurred in the tunica adventitia. No specific binding was noticeable in the tunica intima. Our data suggest that human pulmonary artery expresses Ca2+ channels of the L-type sensitive to dihydropyridines. These sites have similar affinity and lower density than those expressed by systemic arteries. The presence of Ca2+ channels of the L-type in human pulmonary artery suggests that their pharmacological manipulation may be considered in the treatment of pulmonary hypertension.

Aged↗
PubMed1997

Dopamine D4 receptor in human peripheral blood lymphocytes: a radioligand binding assay study.

The expression of dopamine D4 receptor was investigated in human peripheral blood lymphocytes with a radioligand binding assay technique, using [3H]clozapine as radioligand. [3H]Clozapine was specifically bound to human peripheral blood lymphocytes. The binding was time-, temperature-, and concentration-dependent and of high affinity, with a dissociation constant (K(d)) value of 0.34 +/- 0.02 nM and a maximum density of binding sites (B(max)) value of 27 +/- 1.4 fmol/10(6) cells. The pharmacological profile of [3H]clozapine binding to human peripheral blood lymphocytes was similar to that found in Chinese hamster ovary (CHO) cells transfected with the D4 clone (D4.2 variant). The above results are consistent with molecular biology studies demonstrating the expression of a dopamine D4 receptor in immune cells and in human peripheral blood lymphocytes. The availability of a rapid and sensitive radioligand binding assay technique for the dopamine D4 receptor in human peripheral blood lymphocytes may contribute to better define the role of this dopamine receptor subtype in neurological and psychiatric disorders.

Adult↗
PubMed1997

Use of [3H]-clozapine as a ligand of the dopamine D4 receptor subtype in peripheral tissues.

1. Molecular biology studies have documented the presence of peripheral dopamine D4 receptors. This site has not been characterized yet with classical radioligand binding assay techniques because of the lack of selective radioligands. 2. The atypical neuroleptic clozapine labelled with tritium ([3H]-clozapine) has been proposed and sold as a radioligand for brain dopamine D4 receptors. However, the selectivity of [3H]-clozapine for D4 receptor subtypes, and its specificity for brain dopamine receptors, have been questioned. 3. In this study dopamine D4 receptors were assayed in peripheral organs known to express them, such as rat atria and kidney, by using a radioligand binding assay technique with [3H]-clozapine as the radioligand. Parallel experiments were performed using Chinese hamster ovary (CHO) cells transfected with the D4 receptor clone (variant D4.2). 4. [3H]-Clozapine was bound to sections of rat atria and kidney. After appropriate blockade of sites other than dopamine receptors to which it can bind (i.e. muscarinic cholinergic, serotonergic and alpha-adrenergic receptors), the radioligand was bound to a site displaying a pharmacological profile similar to that expressed by CHO cells transfected with the D4 receptor. 5. The above findings indicate that with appropriate protocols, [3H]-clozapine may represent a radioligand for peripheral dopamine D4 receptors.

Animals↗
PubMed1997

Dopamine D3 receptor in peripheral mononuclear cells of essential hypertensives.

Dopamine D3 receptor was studied in peripheral mononuclear cells of high-normal, stage 1, stage 2, and stage 3 essential hypertensives using a radioligand binding assay technique with [3H]-7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT) as a radioligand. A group of de novo Parkinsonian patients was also examined as a reference group of impaired dopaminergic function. [3H]-7-OH-DPAT was bound specifically to human peripheral mononuclear cells in a manner consistent with the labeling of a dopamine D3 receptor. No changes in free dopamine, norepinephrine, epinephrine and aldosterone levels, renin activity, dissociation constant of [3H]-7-OH-DPAT binding, or the pharmacological profile of [3H]-7-OH-DPAT binding were found between normotensive control subjects and essential hypertensives or Parkinsonians. The density of peripheral mononuclear cell [3H]-7-OH-DPAT binding sites increased in essential hypertensives parallel to blood pressure value augmentation. A higher density of [3H]-7-OH-DPAT binding sites was found in Parkinsonians. In these patients, the density of [3H]-7-OH-DPAT binding sites was similar to that observed in high-normal subjects and in stage 1 essential hypertensives. The increased density of peripheral mononuclear cell dopamine D3 receptor in hypertension as well as in Parkinson's disease may represent an upregulation mechanism consequent to impaired dopaminergic function. In view of the difficulty in identifying markers of peripheral dopamine function, analysis of dopamine D3 receptor in peripheral mononuclear cells may help evaluate whether the dopaminergic system is involved in hypertension.

Adult↗
PubMed1996

Muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes.

The subtypes of muscarinic cholinergic receptors were studied in human peripheral blood lymphocytes with radioligand binding techniques and the non-selective muscarinic cholinergic receptor antagonist [3H]quinuclidinyl benzylate (QNB) as a ligand. [3H]QNB was bound to human peripheral lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.60 +/- 0.08 nM and the maximum density of binding sites (Bmax) was 2.33 +/- 0.03 fmol/2.2 x 10(6) cells. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high affinity sites. Analysis of the pharmacological profile of [3H]QNB binding in the presence of compounds specific for the different muscarinic receptor subtypes suggests that human peripheral blood lymphocytes express mainly muscarinic cholinergic M2 and M3 receptor subtypes and to a lesser extent muscarinic M4 receptors. The characterization of the subtypes of muscarinic cholinergic recognition sites expressed by human peripheral blood lymphocytes may represent a tool for investigating the possible relationships between immune and cholinergic systems in normal and pathologic conditions.

Adult↗
PubMed1996

Pharmacological characterisation of Ca2+ channels of the L-type in human peripheral blood lymphocytes.

Ca2+ channels of the L-type were characterised in intact human peripheral blood lymphocytes using a radioligand binding technique and the dihydropyridine-type Ca2+ channel antagonist [3H](+)-PN 200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbon yl-2, 6-dimethyl-3-pyridine carboxylate) as a ligand. [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was time-, temperature-, concentration-dependent and of high affinity. The dissociation constant (Kd) value was 0.4 +/- 0.02 nM and the maximum binding capacity (Bmax) was 33.5 +/- 1.6 fmol/10(6) cells. Pharmacological analysis of [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was consistent with the labelling of a Ca2+ channel of the L-type. In fact, dihydropyridine derivatives were the most potent competitors of [3H](+)-PN 200-110 binding, whereas phenylalkylamine and benzothiazepine compounds or non-selective Ca2+ channel modulators were weak or ineffective displacers. These findings are the first observation that human peripheral blood lymphocytes express Ca2+ channels of the L-type. The possibility that Ca2+ channel antagonists may interfere with immune system function is discussed.

Adult↗
PubMed1996

Increased density of dopamine D5 receptor in peripheral blood lymphocytes of migraineurs: a marker for migraine?

The expression of dopamine D5 receptor was investigated in peripheral blood lymphocytes of 11 migraine patients and of ten healthy control subjects using a radioligand binding technique with [3H]SCH 23390 as a ligand. [3H]SCH 23390 is a benzazepine derivative with potent antagonist properties at the dopamine D1-like receptors. [3H]SCH 23390 was specifically bound to peripheral blood lymphocytes of migraineurs and control subjects in a manner consistent with the labelling of a dopamine D5 receptor. In migraineurs a statistically significant higher density of lymphocyte dopamine D5 receptor compared with controls was noticeable, whereas the affinity of the radioligand was unchanged. The increased density of dopamine D5 receptor in peripheral blood lymphocytes may reflect the dopaminergic hypersensitivity displayed by migraineurs and may represent a relatively simple and reliable peripheral marker of altered dopaminergic function.

Adult↗
PubMed1996

Distribution of p75 and trk-neurotrophin receptor proteins in adult human sympathetic ganglia.

We investigated the expression of immunoreactivity (IR) for low- (p75) and high-affinity (trk proteins) neurotrophin-receptor proteins in adult human paravertebral-sympathetic ganglion neurons. Mouse monoclonal antibodies against the pan-neurotrophin-receptor p75, and rabbit polyclonal antibodies against specific epitopes of the intracytoplasmic domain on trk neurotrophin-receptor proteins were used in fresh unfixed and formaldehyde-fixed paraffin-embedded sympathetic ganglia. All adult human paravertebral-sympathetic neurons displayed trkA neurotrophin-receptor-like protein IR, 10% express trkC neurotrophin-receptor-like protein IR, 37-44% show p75 IR, and no IR was obtained for trkB neurotrophin-receptor-like protein. The intensity of immunostaining was independent of the neuron size. Labelling of non-neuronal tissues, especially blood-vessel walls, was observed for p75, trkA and trkC neurotrophin-receptor proteins. These results indicate that overlapping exists in the expression of p75 and trk neurotrophin-receptor proteins in adult human paravertebral-sympathetic neurons, and suggest that neurotrophins might act on these neurons.

Adult↗
PubMed1996

Calretinin immunoreactivity in human sympathetic ganglia.

Calretinin is an "EF-hand" calcium-binding protein involved in the maintenance of intracellular calcium ion homeostasis. This study was undertaken to investigate the presence of calretinin in human lumbar paravertebral sympathetic ganglia from subjects of different ages (26-85 years) using immunohistochemical and immunoblotting methods. Calretinin-like immunoreactivity was found in a subpopulation of postganglionic sympathetic neurons, whose percentage decreased progressively with aging by about 50% (63% of immunoreactive neurons at < or = 40 years; 29% at > or = 81 years) whereas the neuronal density remained basically unchanged. Calretinin-like immunoreactivity showed a granular pattern of cytoplasmic distribution suggesting preferential localization of this protein associated with intracellular membranes. Occasionally diffuse cytosolic labelling was also observed. The immunoblotting demonstrated a protein band with an estimated molecular weight of 30 kDa, approximately. Present results provide, for the first time, evidence for the presence of calretinin in human paravertebral sympathetic ganglia. Since the number of calretinin-like immunoreactive neurons decreased significantly with aging our findings suggest an involvement of this protein in the age-dependent impairment of sympathetic function.

Adult↗
PubMed1996

Muscarinic cholinergic receptor subtypes in the pigeon bursa of Fabricius: a radioligand binding and autoradiographic study.

The pharmacological profile and the anatomical localisation of muscarinic cholinergic receptor subtypes were studied in the pigeon bursa of Fabricius, using radioligand binding and autoradiographic techniques with [3H]quinuclidinyl benzilate (QNB) as a ligand. [3H]QNB was specifically bound to sections of bursa of Fabricius. The binding was time-, temperature- and concentration-dependent. The dissociation constant was 0.31 +/- 0.02 nM, and the maximum density of binding sites averaged 38 +/- 2.5 fmol/mg protein. The pharmacological profile of [3H]QNB binding to sections of pigeon bursa of Fabricius was consistent with the labelling of M2, M3 and M4 muscarinic receptor subtypes. Light microscope autoradiography showed the localisation of [3H]QNB binding sites in the medulla, in follicular septa, in the cortico-medullary border and in lesser amounts in the cortical layer. The functional significance of these receptors should be clarified in future studies.

Animals↗
PubMed1996

Age-dependent changes in the expression of dopamine receptor subtypes in human peripheral blood lymphocytes.

The pharmacological profile and the density of dopamine D3 and D5 receptor subtypes expressed by human peripheral blood lymphocytes of subjects of different ages (ranging from 20 to 75 years) were assessed using radioligand binding techniques. Dopamine D3 receptor was assayed with [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]7-OH-DPAT) as a ligand. Dopamine D5 receptor was assayed using [3HIR]-(+)-(-chloro-2,3,4,5, tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate) ([3H]SCH 23390) as a ligand. The affinity and the pharmacological profile of [3H]7-OH-DPAT and [3H]SCH 23390 at dopamine D3 and D5 receptor, respectively, were similar in subjects of different ages. The density of dopamine D3 receptor binding sites was slightly decreased in subjects of 30-39 years in comparison with younger individuals. A remarkable loss of dopamine D3 receptor was then found between 40 and 49 years of age in comparison with younger subjects. A further slight decrease was noticeable between 50 and 59 years of age. The number of [3H]7-OH-DPAT binding sites was then stabilized after 60 years of age. The density of dopamine D5 receptor binding sites did not show age-dependent changes. The above findings indicate the occurrence of a decline in the density of lymphocyte dopamine D3 but not D5 receptor between adult and mature subjects. The possibility that dopamine D3 receptor assay in peripheral blood lymphocytes may represent a tool for investigating dopamine receptor function in aging and age-related neurological disorders is discussed.

Adult↗
PubMed1995

Effect of long-term treatment with L-deprenyl on the age-dependent microanatomical changes in the rat hippocampus.

Chronic treatment with L-deprenyl increases both mean and maximum life span and improves cognitive functions in the aged rat. The present study was designed to evaluate whether long-term treatment with L-deprenyl at a dosage not inhibiting the monoamine oxidase-B (MAO-B) (1.25 mg/kg/day) or inhibiting the enzyme activity (5 mg/kg/day) had any effect on the age-dependent microanatomical changes in the rat hippocampus. The hippocampus was chosen in view of its key role in learning and memory functions. Treatment with L-deprenyl started at 19 months and lasted until the 24th month of age. Age-matched untreated rats were used as a control, whereas 11-month-old rats were used as an adult reference group. The number of nerve cell and glial fibrillary acidic protein-immunoreactive astrocyte profiles in the CA1 and CA3 fields of the hippocampus and in the dentate gyrus was decreased and increased, respectively in aged compared with adult rats. Treatment with 5 mg/kg/day, but not with 1.25 mg/kg/day L-deprenyl increased the number of neuronal profiles and decreased the number of astrocytes in the hippocampus of aged rats. The density of zinc stores in the associative intrahippocampal pathway of mossy fibres, which was decreased in aged animals, was increased after treatment with the two doses of L-deprenyl. Lipofuscin accumulation within the cytoplasm of pyramidal neurons of the hippocampus was reduced dose dependently by L-deprenyl treatment. These results suggest that long-term treatment with L-deprenyl is able to counter the expression of age-dependent microanatomical changes in the rat hippocampus. These effects seem only partially correlated with the MAO-B inhibitory activity of L-deprenyl.

Aging↗