Elena Bronzetti

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.

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. Microsc. Res. Tech. 43:29–33, 1998.

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.

DOPAMINE D1-LIKE RECEPTOR SUBTYPES IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES

Molecular biology studies have shown that human peripheral blood lymphocytes express a dopamine D5 receptor, whereas no information is available on dopamine D receptor, the other dopamine D1-like receptor subtype. Radioligand binding assay investigations with the nonsubtype selective dopamine D1-like receptor antagonist [3H]SCH 23390 as radioligand have suggested the presence of a dopamine D5 receptor in human peripheral blood lymphocytes. However, so far no evidence was provided as whether or not human peripheral blood lymphocytes express a dopamine D1 receptor. In this study, we have investigated dopamine D1 and D5 receptor mRNA and the influence of antibodies against dopamine D1 and D5 receptors on [3H]SCH 23390 binding to intact human peripheral blood lymphocytes. The two receptors were also analyzed by immunocytochemistry. Dopamine D5 receptor, but not D1 mRNA, was detected in human peripheral blood lymphocytes. Anti-dopamine D5 receptor antibodies, but not anti-dopamine D1 receptor antibodies, significantly decreased [3H]SCH 23390 binding to human peripheral blood lymphocytes. A dark-brown immunoreactivity was visualized in cytospin centrifuged human peripheral blood lymphocytes exposed to anti-dopamine D5, but not to anti-dopamine D1 receptor antibodies. These data collectively indicate that dopamine D5 receptor is the only dopamine D1-like receptor subtype expressed by human peripheral blood lymphocytes.

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.

Reduced density of dopamine D2-like receptors on peripheral blood lymphocytes in Alzheimer's disease

Clinical and pathological evidence points to an involvement of dopamine in Alzheimers disease (AD). The present study was designed to assay dopamine D1-like and D2-like receptors on peripheral blood lymphocytes (PBL) in 20 patients with AD and in 25 healthy controls by radioligand binding assay techniques with [3H][R]-(+)-(-)chloro-2,3,4,5 tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate (SCH 23390) and [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7OH-DPAT) as radioligands. The density of dopamine D1-like receptors and the affinity of [3H]SCH 23390 and [3H]7OH-DPAT binding to PBL were similar in both groups investigated. AD patients revealed a lower density of dopamine D2-like receptors on PBL than controls (P=0. 0016). The pharmacological profile of [3H]SCH 23390 and [3H]7OH-DPAT binding to PBL was consistent with the labeling of dopamine D5 and D3 receptor subtypes, respectively. The reduced density of dopamine D2-like receptors on PBL is consistent with the observation of changes in the expression of D2-like receptors in dopaminergic brain areas in AD. Our findings support the hypothesis of an involvement of dopamine in AD, even in those patients with no evidence of Parkinsonism, behavioral abnormalities or psychosis.

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.

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.

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.