Laura Felici

Identification of dopamine plasma membrane and vesicular transporters in human peripheral blood lymphocytes.

Plasma membrane dopamine transporter (DAT), vesicular monoamine transporters (VMAT) type-1 and -2 and the expression of the dopaminergic markers dopamine and tyrosine hydroxylase were assessed in membranes and/or in cytospin centrifuged human peripheral blood lymphocytes. The radiolabeled DAT ligand [3H]GBR12935 was bound to peripheral lymphocytes in a manner consistent with the specific binding to a dopamine uptake system, with a dissociation constant similar to that found in striatum, but with a lower density of binding sites. On the other hand, no specific binding occurred in cerebellum used as a test tissue not expressing DAT. Western blot analysis using antibodies raised against amino or carboxy terminus of DAT or against VMAT-1 or VMAT-2 revealed labeling of single bands of approximately 76, 55 or 68 KDa, respectively, displaying similar migration characteristics in lymphocytes and test tissues used for comparison. Immunofluorescence revealed that anti-dopamine, anti-tyrosine hydroxylase, anti-DAT, anti-VMAT-1 and anti-VMAT-2 antibodies labeled the total population of cytospin-centrifuged lymphocytes mounted on microscope slides. Confocal laser microscopy demonstrated that dopamine and VMAT-2 immunoreactivity was developed mainly in cytoplasmic punctiform areas likely corresponding to vesicles and to a lower extent was associated to plasma membrane. Tyrosine hydroxylase immunoreactivity was diffused to cytoplasm and to plasma membrane of lymphocytes, whereas DAT and VMAT-1 immunoreactivity were located almost exclusively in lymphocyte plasma membrane and cytoplasm, respectively. Lymphocyte DAT characterized in this study has probably functional relevance as [3H]dopamine was taken up by intact lymphocytes and uptake was inhibited specifically by compounds known to affect dopamine transport. These findings indicate that human peripheral blood lymphocytes possess DAT plasma membrane and VMAT-1 and VMAT-2 transporters. Increasing evidence indicates that dopamine transporter changes may be related to neuronal injury. In view of this assessment of lymphocyte DAT and VMAT transporters can be considered for identifying pathologies characterized by impaired dopaminergic neurotransmission.

Increased expression of dopamine receptors on lymphocytes in Parkinson's disease

Dopamine D1‐like and D2‐like receptors on peripheral blood lymphocytes (PBL) were assayed in 50 de novo patients with idiopathic Parkinsons disease (PD), in 36 neurologic control subjects (multiple‐system atrophy, n = 16; essential tremor, n = 10; other neurodegenerative diseases, n = 10), and in 26 healthy control subjects by radioligand binding assay techniques using [3H]SCH 23390 and [3H]7OH‐DPAT as ligands. Patients with PD revealed a higher density (Bmax) of dopamine D1‐like (p <0.001) and D2‐like (p <0.00001) receptors on PBL than either neurologic or healthy control subjects, whereas no differences in Bmax were observed among patients affected by other neurologic diseases and healthy control subjects. The affinity (Kd) of both radioligands was similar in the groups investigated. The pharmacologic profile of [3H]SCH 23390 and [3H]7OH‐DPAT binding was consistent with the labeling of dopamine D5 and D3 receptor subtypes, respectively. Twenty‐five of the 50 patients with PD were retested after 3 months of therapy with levodopa or bromocriptine. Both treatments reduced the density of D1‐like (p <0.001) and D2‐like (p <0.001) receptors on PBL to values comparable to those of control subjects. The increased density of D1‐like and D2‐like receptors on PBL in de novo PD patients may represent an upregulation mechanism resulting from the diffuse impairment of the dopaminergic system in PD.

Neurotrophin and neurotrophin receptor expression in alveolar macrophages: an immunocytochemical study.

Abstract Alveolar macrophages play a crucial role in regulating lung immune responses and in maintaining the integrity of the respiratory tract. Neurotrophins (NTs), besides to their neurotrophic activities, exhibit physiological effects in the immune system. In this study, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3 and low- (p75) and high affinity (Trks) NT receptors were investigated by immunocytochemistry in cytospin centrifuged preparations of human alveolar macrophages. Approximately 2.5% alveolar macrophages were immunoreactive for NGF, whereas no macrophages displaying immunoreactivity for BDNF or NT-3 were observed. A 3.5% macrophages displayed immunoreactivity for TrkA-receptor protein, 10% for TrkB-receptor protein (full length isoform), and 2% for TrkC-receptor protein. No low-affinity p75 NT and TrkB[-] truncated isoform receptor immunoreactive macrophages were found. These findings support the hypothesis that NTs and the corresponding receptors may play a role in regulating immunological and functional activity of alveolar macrophages via paracrine/autocrine mechanisms.

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.

LOCALIZATION OF DOPAMINE RECEPTOR SUBTYPES IN SYSTEMIC ARTERIES

Dopamine D1-D5 receptor protein immunoreactivity was investigated in different sized pial, renal and mesenteric artery branches using immunohistochemical techniques and anti-dopamine D1-D5 receptor protein antibodies. Faint dopamine D1 receptor protein immunoreactivity was observed in smooth muscle of tunica media of pial, renal and mesenteric artery branches. Dopamine D2 receptor protein immunoreactivity was located in the adventitia and adventitia-media border of pial and renal artery branches and to a lesser extent of mesenteric artery branches. No dopamine D3 receptor protein immunoreactivity was observed in pial and mesenteric arteries. In renal arteries a moderate dopamine D3 receptor immunoreactivity was detectable in the adventitia and adventitia-media border. A strong dopamine D4 receptor protein immunoreactivity displaying the same localization of dopamine D2 receptor protein was observed in pial and mesenteric arteries, but not in renal artery branches. Moderate dopamine D5 receptor protein immunoreactivity was observed in smooth muscle of the tunica media of pial, renal and mesenteric artery branches. Bilateral removal of superior cervical ganglia, from which sympathetic supply to cerebral circulation originate abolished dopamine D2 and D4 receptor protein immunoreactivity in pial arteries but was without effect on dopamine D1 and D5 receptor protein immunoreactivity. These findings indicate that systemic arteries express dopamine D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptor subtypes displaying respectively a muscular (postjunctional) and prejunctional localization. The specific distribution of dopamine D2-like receptor subtypes in systemic arteries suggests that they may have a different role in regulating blood flow through the vascular beds investigated.

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.

Neurotrophins and Neurotrophin Receptors in Human Pulmonary Arteries

The localization of neurotrophins (NTs) and NT receptors was analyzed in sections of human extra- and intrapulmonary arteries by Western blot analysis and immunohistochemistry. In extrapulmonary branches of human pulmonary artery, NT and NT receptor immunoreactivity was located in the tunica intima, within endothelium, in the tunica media, within smooth muscle and in the tunica adventitia. In different sized intrapulmonary arteries, NT and NT receptor immunoreactivity was observed primarily in the tunica adventitia. A faint NT and NT receptor immunoreactivity was observed in the tunica media of large-sized branches of intrapulmonary arteries, but not within medium- or small-sized intrapulmonary vessels or in tunica intima of different sized intrapulmonary arteries. These findings suggest that NTs may have a role in the control of vascular responses in the pulmonary system acting as local paracrine or autocrine mediators. The possible relevance of the NT system in human pulmonary vasculature identified in this study is discussed.

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.

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.

Quantitative image analysis of the density and pattern of adrenergic innervation of blood vessels of rat spinal cord

The density and pattern of the noradrenergic innervation of the main extramedullary blood vessels of the rat spinal cord was studied using combined glyoxylic acid histofluorescence and quantitative image analysis techniques. The anterior spinal artery has a perivascular plexus of fine noradrenergic nerves rich in varicosities; the central artery has nerve fibers of various sizes; and the posterior spinal vein has a limited plexus of noradrenergic nerve fibers. The possible significance of the noradrenergic innervation of extramedullary spinal cord blood vessels is discussed.