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The neurotransmitter dopamine is a key factor in central nervous system (CNS) function, regulating many processes including reward, movement, and cognition. Dopamine also regulates critical functions in peripheral organs, such as blood pressure, renal activity, and intestinal motility. Beyond these functions, a growing body of evidence indicates that dopamine is an important immunoregulatory factor. Most types of immune cells express dopamine receptors and other dopaminergic proteins, and many immune cells take up, produce, store, and/or release dopamine, suggesting that dopaminergic immunomodulation is important for immune function. Targeting these pathways could be a promising avenue for the treatment of inflammation and disease, but despite increasing research in this area, data on the specific effects of dopamine on many immune cells and disease processes remain inconsistent and poorly understood. Therefore, this review integrates the current knowledge of the role of dopamine in immune cell function and inflammatory signaling across systems. We also discuss the current understanding of dopaminergic regulation of immune signaling in the CNS and peripheral tissues, highlighting the role of dopaminergic immunomodulation in diseases such as Parkinson's disease, several neuropsychiatric conditions, neurologic human immunodeficiency virus, inflammatory bowel disease, rheumatoid arthritis, and others. Careful consideration is given to the influence of experimental design on results, and we note a number of areas in need of further research. Overall, this review integrates our knowledge of dopaminergic immunology at the cellular, tissue, and disease level and prompts the development of therapeutics and strategies targeted toward ameliorating disease through dopaminergic regulation of immunity. SIGNIFICANCE STATEMENT: Canonically, dopamine is recognized as a neurotransmitter involved in the regulation of movement, cognition, and reward. However, dopamine also acts as an immune modulator in the central nervous system and periphery. This review comprehensively assesses the current knowledge of dopaminergic immunomodulation and the role of dopamine in disease pathogenesis at the cellular and tissue level. This will provide broad access to this information across fields, identify areas in need of further investigation, and drive the development of dopaminergic therapeutic strategies., (Copyright © 2023 by The Author(s).)

Objectives: To extend the symptomatic spectrum of acute neurologic syndrome associated with dopamine-2 receptor (D2R) antibodies., Methods: A 13-year-old adolescent boy was admitted to the Neurology Department with abnormal jaw movements. The initial evaluation included laboratory examinations of blood, chest radiography, brain MRI, EEG, and neuropsychologic tests. Serum and CSF samples were collected for immunologic studies. The clinical outcome of the patient was followed up for 18 months after the first hospitalization., Results: Paroxysmal jaw clonus, blepharospasm, and sialorrhea were observed in the patient with a history of Tourette syndrome and obsessive-compulsive disease and with an acute neurologic syndrome associated with D2R antibodies. The symptoms responded to IV methylprednisolone (IVMP), relapsed twice during prednisone reduction, and, finally, improved after the combined treatment of IVMP and IV immunoglobulin., Discussion: Recognizing paroxysmal jaw clonus (possibly with blepharospasm and sialorrhea) and considering the relationship between these episodes and D2R antibodies will be helpful in the early diagnosis and treatment of immune neurologic syndromes., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Dopamine is a neurotransmitter that mediates neuropsychological functions of the central nervous system (CNS). Recent studies have shown the modulatory effect of dopamine on the cells of innate and adaptive immune systems, including Th17 cells, which play a critical role in inflammatory diseases of the CNS. This article reviews the literature data on the role of dopamine in the regulation of neuroinflammation in multiple sclerosis (MS). The influence of dopaminergic receptor targeting on experimental autoimmune encephalomyelitis (EAE) and MS pathogenesis, as well as the therapeutic potential of dopaminergic drugs as add-on pathogenetic therapy of MS, is discussed.

The role of inflammation and immune cells has been demonstrated in neurological diseases, including epilepsy. Leukocytes, as well as inflammatory mediators, contribute to abnormal processes that lead to a reduction in seizure threshold and synaptic reorganization. In this sense, identifying different phenotypes of circulating immune cells is essential to understanding the role of these cells in epilepsy. Immune cells can express a variety of surface markers, including neurotransmitter receptors, such as serotonin and dopamine. Alteration in these receptors expression patterns may affect the level of inflammatory mediators and the pathophysiology of epilepsy. Therefore, in the current study, we evaluated the expression of dopamine and serotonin receptors on white blood cells from patients with temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Blood samples from 17 patients with TLE-HS and 21 controls were collected. PBMC were isolated and stained ex vivo for flow cytometry. We evaluated the expression of serotonin (5-HT 1A , 5-HT 1B , 5-HT 2 , 5-HT 2B , 5-HT 2C , 5-HT 3 , 5-HT 4 ), and dopamine receptors (D 1 , D 2 , D 3 , D 4 , and D 5 ) on the cell surface of lymphocytes and innate immune cells (monocytes and granulocytes). Our results demonstrated that innate cells and lymphocytes from patients with TLE-HS showed high mean fluorescent intensity (MFI) for 5-HT 1A , 5-HT 1B , 5-HT 2A, and 5-HT 4 compared to controls. No difference was observed for 5-HT 2B . For dopamine receptors, the expression of D 1 , D 2 , D 4, and D 5 receptors was higher on innate cells from patients with TLE-HS when compared to controls for the MFI. Regarding lymphocytes population, D 2 expression was increased in patients with TLE-HS. In conclusion, there are alterations in the expression of serotonin and dopamine receptors on immune blood cells of patients with TLE-HS. Although the biological significance of these findings still needs to be further investigated, these changes may contribute to the understanding of TLE-HS pathophysiology., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The overwhelming prevalence of obesity is a priority for public health compromising human lifespan and representing important economic burden worldwide. Obesity is characterized by a state of chronic low-grade inflammation associated to metabolic dysfunction. Although the efforts for unravelling the complex immunometabolic signaling pathways to explain the association of obesity with type 2 diabetes, cardiovascular diseases, cancer, neurodegenerative diseases and psychiatric disorders, we still do not have all the picture to design effective therapeutic to fight these immunometabolic disease clusters. Dopaminergic pathways apart from having a major role in the regulation of appetite and feeding behaviors are important immunoregulators in inflammation; thus, dopaminergic regulation is suggested to impact obesity- associated inflammation. Dopamine (DA) has been reported to modulate immune function and immune cells themselves produce endogenous DA. DA-induced immunomodulation is currently the focus of intense experimental research and dopaminergic pathways are increasingly considered a target for drug development in immune diseases. While the role of dopaminergic pathways in immune-mediated diseases has been intensively investigated in neurodegenerative diseases, dopaminergic immunomodulation in obesity-associated inflammation is largely unknown. This review will integrate the actual knowledge about dopaminergic pathways involved in obesity-associated inflammation with special focus on immune innate key cell players. We present an explanatory hypothesis with a model that integrate central and peripheral dopaminergic circuits in the relationship between neuroimmune and metabolic systems in obesity-associated inflammation. A perspective on the potential role of dopaminergic drugs in the context of obesity will be given. Graphical Abstract Graphical representation of central and peripheral dopaminergic pathways in obesity-associated inflammation.

Dopamine has emerged as a fundamental regulator of inflammation. In this regard, it has been shown that dopaminergic signalling pathways are key players promoting homeostasis between the central nervous system and the immune system. Dysregulation in the dopaminergic system affects both innate and adaptive immunity, contributing to the development of numerous autoimmune and inflammatory pathologies. This makes dopamine receptors interesting therapeutic targets for either the development of new treatments or repurposing of already available pharmacological drugs. Dopamine receptors are broadly expressed on different immune cells with multifunctional effects depending on the dopamine concentration available and the pattern of expression of five dopamine receptors displaying different affinities for dopamine. Thus, impaired dopaminergic signalling through different dopamine receptors may result in altered behaviour of immunity, contributing to the development and progression of autoimmune pathologies. In this review we discuss the current evidence involving the dopaminergic system in inflammatory bowel disease, multiple sclerosis and Parkinson's disease. In addition, we summarise and analyse the therapeutic approaches designed to attenuate disease development and progression by targeting the dopaminergic system. Graphical Abstract Targetting the dopaminergic system in autoimmunity. Effector T-cells (Teff) orchestrate inflamamtion involved in autoimmunity, whilst regulatory T-cells (Tregs) suppress Teff activity promoting tolerance to self-constituents. Dopamine has emerged as a key regulator of Teff and Tregs function, thereby dopamine receptors have becoming important therapeutic targets in autoimmune disorders, especially in those affecting the brain and the gut, where dopamine levels strongly change with inflammation.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) with an autoimmune mechanism of development. Currently, one of the most promising directions in the study of MS pathogenesis are the neuroimmune interactions. Dopamine is one of the key neurotransmitters in CNS. Furthermore, dopamine is a direct mediator of interactions between the immune and nervous systems and can influence MS pathogenesis by modulating immune cells activity and cytokine production. Recent studies have shown that dopamine can enhance or inhibit the functions of innate and adaptive immune system, depending on the activation of different dopaminergic receptors, and can therefore influence the course of experimental autoimmune encephalomyelitis (EAE) and MS. In this review, we discuss putative dopaminergic therapeutics in EAE and MS with focus on Th17-cells, which are thought to play crucial role in MS pathogenesis. We suggest that targeting dopaminergic receptors could be explored as a new kind of disease-modifying treatment of MS. Graphical Abstract.

The dopamine (DA) D2 receptor is a member of the G protein-coupled receptors of organisms and plays an important role in immune system regulation. The presence of DA receptors has been widely reported in vertebrates, but few studies have been conducted in shellfish. Here, we identified a novel DA-D2 receptor gene, ScDopR2-1, in the razor clam Sinonovacula constricta. ScDopR2-1 belongs to the family of G protein-coupled receptors, containing seven hydrophobic transmembrane domains, along with 16 predicted N-glycosylation sites and 69 phosphorylation sites. A longer third intracellular loop and a shorter C-terminus in ScDopR2-1 are characteristic features of D2 receptors. ScDopR2-1 is widely expressed in tissues from adult clams, showing high expression in siphon and foot tissues. Furthermore, in response to Vibrio anguillarum challenge, ScDopR2-1 expression levels are significantly increased in liver tissue. Moreover, changes in the activities of catalase (CAT) and superoxide dismutase (SOD) also indicate that the organism causes an immune response. In summary, the results indicate that ScDopR2-1 plays a pivotal role in antioxidant responses in S. constricta., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hormones are known to influence various body systems that include skeletal, cardiac, digestive, excretory, and immune systems. Emerging investigations suggest the key role played by secretions of endocrine glands in immune cell differentiation, proliferation, activation, and memory attributes of the immune system. The link between steroid hormones such as glucocorticoids and inflammation is widely known. However, the role of peptide hormones and amino acid derivatives such as growth and thyroid hormones, prolactin, dopamine, and thymopoietin in regulating the functioning of the immune system remains unclear. Here, we reviewed the findings pertinent to the functional role of hormone-immune interactions in health and disease and proposed perspective directions for translational research in the field., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Neuroinflammation is a general pathological feature of central nervous system (CNS) diseases, primarily caused by activation of astrocytes and microglia, as well as the infiltration of peripheral immune cells. Inhibition of neuroinflammation is an important strategy in the treatment of brain disorders. Dopamine (DA) receptor, a significant G protein-coupled receptor (GPCR), is classified into two families: D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptor families, according to their downstream signaling pathways. Traditionally, DA receptor forms a wide variety of psychological activities and motor functions, such as voluntary movement, working memory and learning. Recently, the role of DA receptor in neuroinflammation has been investigated widely, mainly focusing on nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, renin-angiotensin system, αB-crystallin, as well as invading peripheral immune cells, including T cells, dendritic cells, macrophages and monocytes. This review briefly outlined the functions and signaling pathways of DA receptor subtypes as well as its role in inflammation-related glial cells, and subsequently summarized the mechanisms of DA receptors affecting neuroinflammation. Meaningfully, this article provided a theoretical basis for drug development targeting DA receptors in inflammation-related brain diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Activated T cells are pathological in various autoimmune and inflammatory diseases including Psoriasis, and also in graft rejection and graft-versus-host-disease. In these pathological conditions, selective silencing of activated T cells through physiological receptors they express remains a clinical challenge. In our previous studies we found that activation of dopamine receptors (DRs) in resting human T cells activates these cells, and induces by itself many beneficial T cell functions. In this study, we found that normal human T cells express all types of DRs, and that expression of D1R, D4R and D5R increases profoundly after T cell receptor (TCR) activation. Interestingly, DR agonists shift the membrane potential (V m ) of both resting and activated human T cells, and induces instantaneous T cell depolarization within 15 seconds only. Thus, activation of DRs in T cells depolarize these immune cells, alike activation of DRs in neural cells. The skin of Psoriasis patients contains 20-fold more D1R + T cells than healthy human skin. In line with that, 25-fold more D1R + T cells are present in Psoriasis humanized mouse model. Highly selective D1-like receptor agonists, primarily Fenoldopam (Corlopam) - a D1-like receptor agonist and a drug used in hypertension, induced the following suppressive effects on activated T cells of Psoriasis patients: reduced chemotactic migration towards the chemokine SDF-1/CXCL12; reduced dramatically the secretion of eight cytokines: tumor necrosis factor-α, interferon-γ, interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8 and IL-10; and reduced three T cell activation proteins/markers: CD69, CD28 and IL-2. Next, we invented a novel topical/dermal Fenoldopam formulation, allowing it to be spread on, and providing prolonged and regulated release in, diseased skin. Our novel topical/dermal Fenoldopam: reduced secretion of the eight cytokines by activated human T cells; reduced IL-1β and IL-6 secretion by human lipopolysaccharide-inflamed skin; eliminated preferentially >90% of live and large/proliferating human T cells. Together, our findings show for the first time that both resting and activated T cells are depolarized instantaneously via DRs, and that targeting D1-like receptors in activated T cells and inflamed human skin by Fenoldopam, in Psoriasis, and potentially in other T cell-mediated diseases, could be therapeutic. Validation in vivo is required., (© 2019 John Wiley & Sons Ltd.)

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated., Competing Interests: The authors have declared that no competing interests exist.

Parkinson's disease (PD) is a progressive neurodegenerative disorder implicitly marked by the substantia nigra dopaminergic neuron degeneration and explicitly characterized by the motor and non-motor symptom complexes. Apart from the nigrostriatal dopamine depletion, the immune and endocrine study findings are also frequently reported, which, in fact, have helped to broaden the symptom spectrum and better explain the pathogenesis and progression of PD. Nevertheless, based on the neural, immune, and endocrine findings presented above, it is still difficult to fully recapitulate the pathophysiologic process of PD. Therefore, here, in this review, we have proposed the neuroimmunoendocrine (NIE) modulatory network in PD, aiming to achieve a more comprehensive interpretation of the pathogenesis and progression of this disease. As a matter of fact, in addition to the classical motor symptoms, NIE modulatory network can also underlie the non-motor symptoms such as gastrointestinal, neuropsychiatric, circadian rhythm, and sleep disorders in PD. Moreover, the dopamine (DA)-melatonin imbalance in the retino-diencephalic/mesencephalic-pineal axis also provides an alternative explanation for the motor complications in the process of DA replacement therapy. In conclusion, the NIE network can be expected to deepen our understanding and facilitate the multi-dimensional management and therapy of PD in future clinical practice.

The neuropathogenesis of HIV-associated neurocognitive disorders (HAND) remains puzzling. We interrogated several levels of data (host genetic, histopathology, brain viral load, and neurocognitive) to identify histopathological changes most relevant to HAND. The design of the study is a clinicopathological study employing genetic association analyses. Data and brain tissue from 80 HIV-infected adults were used. Markers in monocyte chemoattractant protein-1 (MCP-1), interleukin 1-alpha (IL1-α), macrophage inflammatory protein 1-alpha (MIP1-α), DRD3, DRD2, and apolipoprotein E (ApoE) were genotyped. Microtubule associated protein 2 (MAP2), synaptophysin (SYP), human leukocyte antigen-DR (HLA-DR), glial fibrillary acidic protein (GFAP), amyloid beta (A-Beta), and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactivity were quantified in the frontal cortex, putamen, and hippocampus. A composite score for each marker (mean of the three brain regions) was used. Neurocognitive functioning and other clinical variables were determined within 1 year of death. Brain HIV RNA viral load was available for a subset of cases. MAP2 and SYP proved most relevant to neurocognitive functioning. Immunoreactivity of these markers, as well as A-Beta and Iba-1, was correlated with brain HIV RNA viral load. Several genetic markers in combination with other factors predicted histopathology: HIV blood viral load, MIP1-α genotype, and DRD3 genotype predicted Iba-1 immunoreactivity; the duration of infection and IL1-α genotype predicted GFAP immunoreactivity; ApoE genotype and age at death predicted A-Beta immunoreactivity. These data indicate that HIV replication in the brain is the primary driving force leading to neuroinflammation and dysfunctional protein clearance, as reflected by A-Beta and Iba-1. Downstream to these changes are synaptodendritic degeneration, which is the immediate histopathological substrate of the neurocognitive impairment characteristic of HAND. These intermediate histopathological phenotypes are influenced by host genetic polymorphisms in genes encoding cytokines/chemokines, neuronal protein clearance pathways, and dopaminergic factors.

Antineuronal autoantibodies are associated with the involuntary movement disorder Sydenham chorea (SC) and paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) which are characterized by the acute onset of tics and/or obsessive compulsive disorder (OCD). In SC and PANDAS, autoantibodies signal human neuronal cells and activate calcium calmodulin-dependent protein kinase II (CaMKII). Animal models immunized with group A streptococcal antigens demonstrate autoantibodies against dopamine receptors and concomitantly altered behaviours. Human monoclonal antibodies (mAbs) derived from SC target and signal the dopamine D2L (long) receptor (D2R). Antibodies against D2R were elevated over normal levels in SC and acute-onset PANDAS with small choreiform movements, but were not elevated over normal levels in PANDAS-like chronic tics and OCD. The expression of human SC-derived anti-D2R autoantibody V gene in B cells and serum of transgenic mice demonstrated that the human autoantibody targets dopaminergic neurones in the basal ganglia and other types of neurones in the cortex. Here, we review current evidence supporting the hypothesis that antineuronal antibodies, specifically against dopamine receptors, follow streptococcal exposures and may target dopamine receptors and alter central dopamine pathways leading to movement and neuropsychiatric disorders., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Dopamine is a key transmitter in the neuroimmune network, acting through five dopaminergic receptors (DR): the D1-like D1 and D5 and the D2-like D2, D3 and D4. Several DR gene variants exist and may affect DR expression and activity. We assessed total lymphocytes, CD3+, CD4+ and CD8+ T lymphocytes in peripheral blood of healthy subjects and their association with selected DR gene variants (DRD1 rs4532 and rs686, DRD5 rs6283, DRD2 rs1800497 and rs6277, DRD3 rs6280 and rs1800828, DRD4 rs747302 and 7 48-base pair VNTR). DRD1 rs4532 and rs686 and DRD5 rs6283 were associated with total lymphocytes, and with CD3+ and CD4+ (but not CD8+) T lymphocytes, while none of the D2-like DR gene variants showed any association with lymphocyte counts. An arbitrary score based on the activity of D1-like vs D2-like DR correlated with total lymphocytes, CD3+ and CD4+ T cells (but not with CD8+ T cells). The association between D1-like DR gene variants and lymphocyte count, and in particular with CD4+ (but not CD8+) T lymphocytes, may imply a functional prevalence of D1-like over D2-like DR in CD4+ T cells. This is the first study showing an influence of DR gene polymorphisms on lymphocyte count, and in particular on CD4+ T cells. Future studies should address the possible association between DR gene variants and the immune function in health and disease. The relevance of these findings for the immune effects of dopaminergic agents should be also carefully examined., (Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The group A streptococcus, Streptococcus pyogenes, and its link to autoimmune sequelae, has acquired a new level of understanding. Studies support the hypothesis that molecular mimicry between the group A streptococcus and heart or brain are important in directing immune responses in rheumatic fever. Rheumatic carditis, Sydenham chorea and a new group of behavioral disorders called pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections are reviewed with consideration of autoantibody and T cell responses and the role of molecular mimicry between the heart, brain and group A streptococcus as well as how immune responses contribute to pathogenic mechanisms in disease. In rheumatic carditis, studies have investigated human monoclonal autoantibodies and T cell clones for their crossreactivity and their mechanisms leading to valve damage in rheumatic heart disease. Although studies of human and animal sera from group A streptococcal diseases or immunization models have been crucial in providing clues to molecular mimicry and its role in the pathogenesis of rheumatic fever, study of human monoclonal autoantibodies have provided important insights into how antibodies against the valve may activate the valve endothelium and lead to T cell infiltration. Passive transfer of anti-streptococcal T cell lines in a rat model of rheumatic carditis illustrates effects of CD4+ T cells on the valve. Although Sydenham chorea has been known as the neurological manifestation of rheumatic fever for decades, the combination of autoimmunity and behavior is a relatively new concept linking brain, behavior and neuropsychiatric disorders with streptococcal infections. In Sydenham chorea, human mAbs and their expression in transgenic mice have linked autoimmunity to central dopamine pathways as well as dopamine receptors and dopaminergic neurons in basal ganglia. Taken together, the studies reviewed provide a basis for understanding streptococcal sequelae and how immune responses against group A streptococci influence autoimmunity and inflammatory responses in the heart and brain.

Dopamine (DA) is a crucial transmitter in the neuroimmune network, where it contributes to the nervous system-immune system interplay as well as in the communication among immune cells. DA acts through five different dopaminergic receptors (DR) grouped into two families: the D1-like (D1 and D5) and the D2-like (D2, D3 and D4). By use of 5-color flow cytometric analysis, we examined the expression of DR on human CD4+ naive T lymphocytes (CD3+CD4+CD45RA+CCR7+), central memory (TCM, CD3+CD4+CD45RA-CCR7+) and effector memory T cells (TEM, CD3+CD4+CD45RA-CCR7-). In addition, in cultured CD4+ T cells we investigated the changes in DR expression induced by stimulation with antiCD3/antiCD28 antibodies. Results showed that CD4+ T cells always expressed all the five DR: D1-like DR were identified on average on 11.6-13.1 % and D2-like DR on 3.1-8.1 % of the cells. DR on CD4+ naive T cells, TCM, and TEM had distinct expression patterns: naive T cells expressed more D1-like than D2-like DR, which on the contrary were increased in TCM and TEM cells. In cultured CD4+ T cells stimulation with anti-CD3/anti-CD28 antibodies increased the expression of D1-like DR by 71-84 % and of D2-like DR by 55-97 %. The frequency of DR was higher in apoptotic cells in comparison to viable cells, however stimulation increased all DR on viable cells, without affecting their expression on apoptotic cells. The present results contribute to unravel the complexity of dopaminergic pathways in human CD4+ T lymphocytes, suggesting their involvement in memory functions as well as in apoptotic processes. In view of the role of CD4+ memory T cells in neuroinflammation and neurodegeneration during Parkinson's disease, the relevance of these findings must be assessed in the clinical setting.

Protein domains involved in receptor heteromer formation are disordered and rich in the amino acids necessary for the formation of noncovalent complexes (NCX). We present mass spectral NCX data from proteins and protein receptors' epitopes obtained by combining ion mobility (IM) and MALDI. We focus on NCX involved in heteromer formation occurring between epitopes of the Dopamine D2 (D2R) and Adenosine A2A receptors (A2AR) as well as D2R and the α2 nicotinic (NR) receptor's subunit. The IM data yield information on the gas phase conformation of the singly charged NCX which are observed either directly from MALDI or as codesorbed neutrals that are subsequently postionized by a time-delayed excimer laser pulse directed onto a portion of the neutral plume created by the MALDI desorption laser. Imaging mass spectrometry of the matrix/epitope dried droplet surface shows that the acidic and basic epitopes and their NCX are found to be spatially collocated within regions as small as 25 × 50 μm(2). Subtle differences in the relative abundance of protonated and cationized NCX and epitopes are measured in spatial regions near the sodium-rich outer border of the droplet.

Recent reports of autoantibodies that bind to neuronal surface receptors or synaptic proteins have defined treatable forms of autoimmune encephalitis. Despite these developments, many cases of encephalitis remain unexplained. We have previously described a basal ganglia encephalitis with dominant movement and psychiatric disease, and proposed an autoimmune aetiology. Given the role of dopamine and dopamine receptors in the control of movement and behaviour, we hypothesized that patients with basal ganglia encephalitis and other putative autoimmune basal ganglia disorders harboured serum autoantibodies against important dopamine surface proteins. Basal ganglia encephalitis sera immunolabelled live surface cultured neurons that have high expression of dopamine surface proteins. To detect autoantibodies, we performed flow cytometry cell-based assays using human embryonic kidney cells to express surface antigens. Twelve of 17 children (aged 0.4-15 years, nine males) with basal ganglia encephalitis had elevated immunoglobulin G to extracellular dopamine-2 receptor, compared with 0/67 controls. Immunofluorescence on wild-type mouse brain showed that basal ganglia encephalitis sera immunolabelled microtubule-associated protein 2-positive neurons in striatum and also in cultured striatal neurons, whereas the immunolabelling was significantly decreased in dopamine-2 receptor knock-out brains. Immunocytochemistry confirmed that immunoreactivity localized to the surface of dopamine-2 receptor-transfected cells. Immunoabsorption of basal ganglia encephalitis sera on dopamine-2 receptor-transfected human embryonic kidney cells decreased immunolabelling of dopamine-2 receptor-transfected human embryonic kidney cells, neurons and wild-type mouse brain. Using a similar flow cytometry cell-based assay, we found no elevated immunoglobulin G binding to dopamine 1, 3 or 5 receptor, dopamine transporter or N-methyl-d-aspartate receptor. The 12 dopamine-2 receptor antibody-positive patients with encephalitis had movement disorders characterized by parkinsonism, dystonia and chorea. In addition, the patients had psychiatric disturbance with emotional lability, attention deficit and psychosis. Brain magnetic resonance imaging showed lesions localized to the basal ganglia in 50% of the patients. Elevated dopamine-2 receptor immunoglobulin G was also found in 10/30 patients with Sydenham's chorea, 0/22 patients with paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection and 4/44 patients with Tourette's syndrome. No dopamine-1 receptor immunoglobulin G was detected in any disease or control groups. We conclude that assessment of dopamine-2 receptor antibodies can help define autoimmune movement and psychiatric disorders.

Neuromediators such as dopamine (DA), serotonin (5-HT) and neuroopioids are known to be involved in the immune response control. This review has summarized the evidence supporting roles for brain DA (D(1), D(2)), 5-HT (5-HT(1A), 5-HT(2A)) and opioid (mu, delta, kappa) receptor subtypes in regulating immune function. Activation of postsynaptic D(1)- and D(2)-receptors produced immunostimulation while their blockade or activation of presynaptic D(2)-receptors mediated an immunoinhibitory effect. Activation of mu- and delta(1)-opioid receptors also increased the immune reaction intensity. Activation of postsynaptic 5-HT(1A)-, 5-HT(2A)-receptors, delta(2)- and kappa-opioid receptors resulted in immunosuppression while the blockade of postsynaptic 5-HT(1A)-, 5-HT(2A)-receptors or activation of somatodendritic 5-HT(1A)-autoreceptors resulted in the immune response stimulation. Immunomodulating effects of drugs acting at various mediator and opioid receptor subtypes depend on the initial psychoemotional state of animals (aggression, submission, depression). The presented data may have implications for the treatment of emotional stress and mental disorders associated with changes in activity of brain DA, 5-HT, opioid systems and their receptor function as well as immune reactivity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To observe the effect of repeated electroacupuncture (EA) on the expression of cannabinoid receptor-1 (CB 1) mrRNA and dopamine 1 receptor (D 1) mRNA in Nucleus Accumbens (NAC)-Caudate Nucleus (CN) region in inflammatory-pain rats, so as to study its underlying mechanism in analgesia., Methods: A total of 30 SD rats were randomized into normal control, model, EA, EA+ AM 251 and WIN 55212-2 groups, with 6 cases in each group. EA (2 Hz/100 Hz, 1 -3 mA) was applied to "Zusanli"(ST 36) and "Kunlun"(BL 60) for 30 min, once every other day, and 4 sessions all together. Arthritis model was established by injection of Freund's complete adjuvant 0.05 mL in the rat's left ankle. Thermal pain threshold (paw withdrawal latency, PWL) was detected before and after modeling and after repeated EA and/or intraperitoneal injection of AM 251(an inverse antagonist at the CB 1 cannabinoid receptor, 0. 1 mg/100 g) and WIN 55212-2 (a potent cannabinoid receptor agonist, 0. 2 mg/100 g). The expression of CB 1 receptor mRNA and D 1 receptor mRNA in the NAC-CN region was measured by real time fluorescence quantitative-polymerase chain reaction., Results: Compared with the control group, the pain threshold values of the model group was decreased significantly (P<0.01). In comparison with the model group, the pain threshold values of the EA group and WIN 55212-2 group were increased considerably on day 10 (P<0. 01). No significant differences were found between the EA+ AM 251 and model groups and between the EA and WIN 55212-2 groups in PWL after the treatment (P>0.05). Compared with the control group, both CB 1 R mRNA and D 1 R mRNA expression levels in the model group were increased slightly, while in comparison with the model group and EA+ AM 251 group, CB 1 R mRNA and D 1 R mRNA expression levels in the EA group and WIN 55212-2 group were upregulated obviously. No significant differences were found between the EA + AM 251 and model groups and between the EA and WIN 55212-2 groups in CB 1 R mRNA and D 1 R mRNA expression levels.

The neurotransmitter dopamine (DA) is an important molecule bridging the nervous and immune systems. DA through autocrine/paracrine manner modulates the functions of immune effector cells by acting through its receptors present in these cells. DA also has unique and opposite effects on T cell functions. Although DA activates naïve or resting T cells, but it inhibits activated T cells. In addition, changes in the expression of DA receptors and their signaling pathways especially in T cells are associated with altered immune functions in disorders like schizophrenia and Parkinson's disease. These results suggest an immunoregulatory role of DA. Therefore, targeting DA receptors and their signaling pathways in these cells by using DA receptor agonists and antagonists may be useful for the treatment of diseases where DA induced altered immunity play a pathogenic role., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objective: Dopamine exists in the immune system and has obvious immunomodulating action. However, receptor mechanism underlying the dopamine immunomodulation remains to be clarified. In the present study, we provide the evidence for existence of dopamine receptor subtypes in T lymphocytes and show the roles of the receptors and the receptor-coupled signaling in mediating the dopamine immunomodulation., Methods: The purified T lymphocytes from the mesenteric lymph nodes of mice were detected for expressions of all five subtypes of dopamine receptor mRNAs by reverse transcription-polymerase chain reaction. Lymphocyte proliferation and production of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in response to concanavalin A (Con A) were measured by colorimetric methyl-thiazole-tetrazolium assay and cytometric bead array, respectively, after the cells were exposed to dopamine D1-like or D2-like receptor agonists and antagonists. Meanwhile, content of cAMP and phosphorylation of cAMP-response element-binding (CREB) in the lymphocytes were examined by 125I-cAMP radioimmunoassay and Western blot assay, respectively., Results: T lymphocytes expressed all the five subtypes of dopamine receptor mRNAs, i.e., D1, D2, D3, D4 and D5 receptors. SKF38393, an agonist of dopamine D1-like receptors (D1 and D5 receptors) only reduced the IFN-γ production, but did not significantly affect the proliferative response, IL-4 production, cAMP content or CREB activation of the lymphocytes. The SKF38393-induced decrease in IFN-γ level was blocked by the D1-like receptor antagonist SCH23390. Quinpirole, an agonist of dopamine D2-like receptors (D2, D3 and D4 receptors) attenuated the lymphocyte proliferation to Con A, and decreased the IFN-γ but increased the IL-4 production. Meanwhile, the quinpirole diminished the cAMP content and the phosphorylated CREB level in the lymphocytes. All the quinpirole-induced changes were reversed by dopamine D2-like receptor antagonist haloperidol., Conclusions: Five dopamine receptor subtypes of the two families, D1-like and D2-like receptors, exist on T lymphocytes of mice. Of the two families, D2-like receptors are more important in mediating modulation of T cell function than D1-like receptors. D2-like receptors are involved in suppression of T helper 1 (Th1) cell function and enhancement of Th2 cell function through negative link to cAMP-CREB pathway.

Evidence indicate that it is difficult to obtain specific antibodies to G protein-coupled receptors and different technical difficulties may allow the generation of antibodies that lack specificity. We conducted experiments to validate the specificity of commercially available antibodies raised against dopamine (DA) receptors hD(1), hD(4), and hD(5) using a transfection approach: we studied whether, in HEK 293 cells selectively transfected with the various cloned subtypes, each antibody generates bands only in cells expressing its cognate receptor but not in those expressing the other DA receptors. Our results demonstrated that hD(1) and hD(4) receptor antibodies recognize not only their respective epitope, but also other DA receptor subtypes, while for the hD(5) receptor detection, we observed a signal only in the lane loaded with hD(5)-transfected HEK 293 cells, although with a lack of purity. Therefore, we recommend caution on the use of commercially available DA receptor antibodies.

A cluster of manuscripts in this issue of the Journal highlights a lack of selectivity of 49 antibodies against 19 subtypes of alpha(1)- and beta-adrenoceptors, muscarinic, dopamine and galanin receptors as well as vanilloid (TRPV1) receptors. Taken together these data demonstrate that lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors. Thus, the previously often applied validation of such antibodies by the disappearance of staining in the presence of blocking peptide, i.e. the antigen against which the antibody was raised, alone is insufficient to demonstrate specificity. We propose that receptor antibodies should be validated by at least one of the following techniques: a) disappearance of staining in knock-out animals of the target receptor, b) reduction of staining upon knock-down approaches such as siRNA treatment, c) selectivity of staining in immunoblots or immunocytochemistry for the target receptor vs. related subtypes when expressed in the same cell line and/or d) antibodies raised against multiple distinct epitopes of a receptor yielding very similar staining patterns. Other issues of consideration to obtain reliable results based on receptor antibodies in applications such as immunohistochemistry or immunoblotting are also being discussed.

Here we review the evidence for immune cells expressing multiple components of the serotonergic and dopaminergic systems that are more commonly associated with the central nervous system (CNS). We discuss where and how peripheral encounters with these biogenic monoamines occur and posit reasons as to why the immune system would wish to deploy these pathways. A full taxonomy of serotonergic and dopaminergic constituents and their workings in component cells of the immune system should facilitate the formulation of novel therapeutic approaches in diseases characterized by immune dysfunction and potentially provide a range of surrogate peripheral markers for registering and monitoring disturbances within the CNS.

The human polyomavirus, JCV, causes the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised patients. We found that the serotonergic receptor 5HT2AR could act as the cellular receptor for JCV on human glial cells. The 5HT2A receptor antagonists inhibited JCV infection, and monoclonal antibodies directed at 5HT2A receptors blocked infection of glial cells by JCV, but not by SV40. Transfection of 5HT2A receptor-negative HeLa cells with a 5HT2A receptor rescued virus infection, and this infection was blocked by antibody to the 5HT2A receptor. A tagged 5HT2A receptor colocalized with labeled JCV in an endosomal compartment following internalization. Serotonin receptor antagonists may thus be useful in the treatment of progressive multifocal leukoencephalopathy.

Studies reported here demonstrated that activation of the dopaminergic system induces increases in the immune response regardless of the type of behavior in mice (line CBA), i.e., in aggressive mice, submissive mice, and mice lacking experience of victory or defeat (controls). Changes in the activity of the dopaminergic system were induced with SKF-38393, a selective agonist of dopamine D1 receptors, and with p-chlorophenylalanine (PCPA), which we have previously shown to activate D2 receptors. In the aggressive form of behavior, which was characterized by strong (compared with controls) immune responses, SKF-38393 and PCPA led to further increases in the immune response. In submissive mice, activation of the dopaminergic system altered the nature of the immune response, with immunostimulation, as in aggression. It is suggested that activation of the dopaminergic system in conditions of defined psychoemotional status fixed by acquisition of opposite types of behavior, induces the formation of a new neurochemical pattern--the dopaminergic set--which led to changes in the nature and intensity of the immune response.

It was shown that activation of dopaminergic (Daergic) system induced an increase of the immune responsiveness independent of the CBA mice behaviour typeanimals without experience of victories and defeats (control), with aggression and submission. Administration of SKF-38393, a selective agonist of DA D1-receptors, resulted in enhanced immune response as tested by plaque-forming cells and rosette-forming cells number. Similar immunostimulation was observed after injection of p-chlorophenylalanine realizing its influence on the immune response through DA D2-receptors as shown by us elsewhere. It was suggested that activation of Da-ergic system produces a new neurochemical pattern (Daergic neurochemical set) which are responsible for character and intensity changes of the immune response in mice with alternative form of social behaviour.

The expression of dopamine receptors by human platelets was investigated by Western blot analysis and immunocytochemical techniques using antibodies raised against dopamine D1-D5 receptor protein. The influence of dopamine D1-like and D2-like receptor agonists on adrenaline-induced platelet aggregation was also investigated. Western blot analysis revealed that platelet membranes bind anti-dopamine D3 or D5 receptor protein antibodies, but not anti-D1, D2 or D4 receptor protein antibodies. Cytospin centrifuged human platelets exposed to anti-dopamine D3 or D5 receptor protein antibodies developed a specific immune staining, whereas no positive staining was noticeable in platelets exposed to other antibodies tested. Both the D1-like receptor agonist 1-phenyl2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF 38393) and the D2-like receptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) dose-dependently inhibited adrenaline-induced platelet aggregation. These effects were decreased respectively by the D-like and D2-like receptor antagonists R(+)-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride (SCH 23390) and (-)sulpiride. The above findings indicate that human platelets express dopamine D3 and D5 receptors probably involved in the regulation of platelet function.

The possible correction of the immune response affecting the mechanisms of neuroimmunomodulation in the animal model of immobilization stress is considered. Immobilization (3 h on the back) of CBA mice caused a suppression of the immune response. The number of plaque-forming cell (PFC) on the 4th day as well as rosette-forming cell (RFC) number on the 5th day of the immune reaction to sheep red blood cells (SRBC) were found to be reduced as compared to the control. Immunoinhibition in the stressed mice was reversed by the depletion of the cerebral serotonin (5-HT) with p-chlorophenylalanine (PCPA) 2 days before immobilization at a dose of 500 mg/kg. Activation of postsynaptic D-1 and D-2 dopamine (DA) receptors with apomorphine administered 30 min before stress attenuated stress-induced immunosuppression as well. Thus, the immunosuppression elicited by a stressor can be modified by drugs influencing the 5-HT and DAergic systems. It is suggested that the stress-provoked alterations of the immune response can be a consequence of changing neurochemical pattern of the brain and the disturbances of the mechanisms of psychoneuroimunomodulation.

The stimulatory and inhibitory effects of dopamine (DA) upon avian prolactin (PRL) secretion suggest that, in birds, these actions are mediated by multiple DA receptors. To test this hypothesis, combined intracranial infusions of DA and selective D1 or D2 DA receptor blockers, plus electrical stimulation (ES) of the brain and vasoactive intestinal peptide (VIP) immunoneutralization, were used to characterize the actions of DA on PRL secretion in the turkey. Blockade of D1 DA receptors prevented the increase in circulating PRL observed in response to infusion of stimulatory concentrations of DA or to ES. Stimulatory infusions of DA also failed to increase circulating PRL in birds immunized against VIP. Results from infusion of the D2 DA receptor antagonist were unclear. Low concentrations had no effect, while the highest concentration (100 nmol/min) produced an increase in plasma PRL. At the high concentration the drug may be affecting PRL secretion by (1) acting nonspecifically, (2) acting as a partial DA agonist on D1 DA receptors, or (3) diffusion to the pituitary and blockade of D2 receptors there. These data suggest that avian PRL secretion is mediated by D1 DA receptors within the brain and that the stimulatory effect of DA upon PRL secretion requires an intact VIPergic system.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

We have developed a range of specific anti-peptide antibodies directed against the D2 and D3 dopamine receptors, and their alternatively spliced isoforms. To demonstrate the subtype- and isoform-specificities of these antibodies we have expressed the receptors in an in vitro translation system and have immunoprecipitated the expressed proteins. We have shown that the D2 and D3 receptors unexpectedly exhibit different migration properties on SDS-PAGE, in addition to displaying a significant difference between their predicted and apparent molecular weights. We have used D2/D3 chimaeric receptors to show that the differential migration between the subtypes can be attributed to the N-terminal half of the receptor and propose that the highly hydrophobic nature of the receptor proteins underlies their anomalous migration properties.

Anti-peptide antibodies were generated against amino acid sequences of intracellular and extracellular portions of the native proteins for the cloned rat D1A and D3 dopamine receptor subtypes in order to determine the cellular distribution of these specific forms in the brain. These polyclonal antisera exhibited high specific titers, assessed by ELISA and immunofluorescent detection of functional recombinant receptor proteins expressed in stably transfected Chinese hamster ovary (CHO) cells. Central nervous system (CNS) areas of the male rat were examined using standard immunofluorescent methods in fresh frozen tissues. This paradigm detected D1A-like and D3-like dopamine receptor staining primarily in larger-sized neurons throughout layers 3 and 5 of the cortex, in medium-diameter somata of the striatum, and in the densely packed cells of the olfactory tubercle and hippocampal formation. More attenuated immunoreactivity for both dopamine receptor subtypes was noted in the substantia nigra, not associated with perikarya. Differences in cellular staining patterns and intensity were evident between the D1A-like and D3-like dopamine receptor subtypes. Equivalent morphological elements exhibited dopamine receptor expression following incubation using antisera generated against either extracellular or intracellular epitopes of either the D1A or D3 native proteins. Dopamine receptor immunoreactivity could not be detected in the cerebellum at equivalent antisera dilutions used to discriminate cellular staining patterns within the forebrain. Fluorescent-labeled latex microspheres were infused into the substantia nigra terminal fields to retrogradely identify the cell bodies of the striatonigral projection system. This paradigm showed that 80% of striatonigral neurons expressed D1A-like receptors, while 65% demonstrated D3-like dopamine receptor staining. This distribution for the D1A-like and D3-like receptor subtypes suggests that overlap may occur in the expression of the receptors in the striatonigral neuron population. Our previous results localizing cellular D2-like receptor expression patterns in this projection system of the rat neostriatum implies that all three of these dopamine receptor subtypes may be co-expressed in this efferent system.

Organotypic cultures established from the third thoracic ganglion of locust embryo have been used to investigate dopamine receptors. In this in vitro system, neurites emerge directly from the explants and form a dense network around the explants, presenting cell surface freely exposed for experimental labelling. Polyclonal anti-idiotypic antibodies raised in rabbits to antibodies against dopamine conjugate, and previously found to bind to dopamine receptors, have been used to investigate putative dopamine receptors in these neurites. Immunocytochemical detection by light microscopy employing immunofluorescence labelling, was correlated with electron microscopy, using peroxidase staining. In addition to a location for dopamine receptors on the neurite surface, intracellular binding sites were also found in neurites. This internal labelling might represent an intracellular pool of dopamine receptor precursors. The labelling was specific in that it was not present when the anti-idiotypic dopamine antibodies were replaced with non-immune serum or when preincubation with conjugated dopamine preceded incubation with anti-idiotypic dopamine antibodies.

A baculovirus expression system provided an enriched source of biologically and immunologically active D3 dopamine receptors. Receptors expressed in Spodoptera frugiperda insect (Sf9) cells at a density of 5 to 15 pmol/mg of protein displayed high affinity for the antagonists, eticlopride, fluphenazine and spiroperidol, and the agonist, N-propylnorapomorphine. The binding of agonists was not sensitive to GTP. Antisera raised against synthetic peptides in the third intracellular loop of the D3 dopamine receptor immunoprecipitated binding sites for (S)-3-[125I]-iodo-2-hydroxy-5,6-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)- methyl]-benzamide from solubilized extracts of infected Sf9 cells and detergent extracts of rat caudate. These antisera specifically recognized a single band on immunoblots of Sf9 cells infected with recombinant D3 baculovirus. Both the immunoprecipitation and immunoblot reactions were blocked by preincubation of the antisera with the immunization peptide. These results suggest that the D3 receptor protein is expressed in rat brain.

Antisera have been raised against two peptides from the sequence of D2 dopamine receptors: peptide 1 from the predicted second extracellular loop and peptide 2 from the predicted third intracellular loop. The antisera recognize specifically a 95 kDa band in Western blots of several bovine brain regions, which corresponds to the denatured D2 dopamine receptor, whereas in recombinant CHO cells expressing D2 dopamine receptors a 80 kDa band is seen. The antisera immunoprecipitate 10-20% of the D2 dopamine receptors from soluble preparations of bovine brain. The antisera recognize D2 dopamine receptors in immunofluorescence analyses of recombinant CHO cells bearing the receptor gene. The antisera directed against the third intracellular loop, but not those against the second extracellular loop, will interfere with the coupling of D2 dopamine receptors and G-proteins in bovine brain preparations.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.

The precise distribution of the dopamine type D2 receptor has been mapped for the first time in rat brain using an antibody to D2 receptor protein. Polyclonal antisera were collected from rabbits inoculated with an undecapeptide identical to residues 24-34 of the D2 protein sequence. Rat brain slices, 40 microns in thickness, were incubated with either primary antiserum, the antiserum plus free peptide antigen, or pre-immune serum. Antibody binding was visualized by peroxidase-antiperoxidase (PAP) reaction followed by light microscopy. PAP complex bound moderately-to-densely throughout the medial forebrain bundle, and was seen in more discrete regions in the midbrain, consistent with the binding of D2 radioligands. There were some unexpected results, namely in the cerebral cortex and nucleus accumbens, there were unexpectedly steep gradients in binding density, decreasing caudally; no binding was detected in the hippocampus or the substantia nigra pars reticulata. In all positive-staining regions examined, the antibody was highly localized to neuronal cell bodies, except in the frontal cortex where antibody was also evident on basilar dendrites. These data confirm that the polyclonal antibody recognized dopamine D2 receptor protein throughout the rat brain, and suggest that the D2 receptor is distributed more abundantly on somata than on cellular processes.

Hemorrhagic pancreatitis was induced in cats by perfusing pancreatic enzymes through a pancreatic duct after the administration of intragastric ethanol. Dimethyl prostaglandin E2 was administered concurrently. In the first study, dopamine's antiinflammatory effect on the pancreas was determined in the presence of haloperidol, propranolol, or both. Next, dopamine's effects on blood flow in the normal and inflamed pancreas were compared using a hydrogen gas-clearance technique. In the final study, the effect of dopamine on fluorescein isothiocyanate-labeled dextran leakage from the pancreatic duct to portal venous blood was investigated. It was found that blockade of either dopamine or beta-adrenergic receptors reduced, and blockade of both receptors completely eliminated, the antiinflammatory effect. Dopamine had no effect on pancreatic blood flow in normal cats. In pancreatitis, although dopamine transiently reduced blood flow, after an hour flow had returned to normal. Dopamine reversed the leakage of fluorescein isothiocyanate-labeled dextran from the pancreatic duct caused by ethanol and by ethanol and prostaglandin E2. It was concluded that dopamine ameliorated pancreatitis by reducing pancreatic ductal and/or microvascular permeability rather than by altering pancreatic blood flow. The antiinflammatory effect was mediated by both dopamine and beta-adrenergic receptors.

Antibodies against synthetic peptides of the D2 dopamine receptor were used, in combination with photoaffinity labeling, to localize the region of ligand binding in the receptor. Specific antibodies to peptide sequences 221-234 and 259-272 and to the carboxyl-terminal peptide 402-415, all corresponding to cytoplasmic regions in the D2 dopamine receptor, were elicited. After photoaffinity labeling with N-(4-azido-3-[125I]iodophenethyl)spiperone ([125I]NAPS), all three antibodies specifically immunoprecipitated the 90-kDa D2 dopamine receptor. Differential reactivity of the antipeptide antibodies with various proteolytic fragments indicates that [125I]NAPS binds covalently to a 13-kDa fragment of the D2 dopamine receptor. This fragment is immunoprecipitated with anti-peptide 402-415 and not with the other two antipeptide antibodies, indicating that the photoaffinity ligand binds to a fragment that begins beyond amino acid 272 and extends through the carboxyl-terminal end of the receptor.

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed specific antibodies directed against two synthetic peptides corresponding to defined sequences in the D2 dopamine receptor. One peptide is from a region that is present only in the 'long' isoform of the receptor, whereas the other is from a region that is common to both. These antibodies are able to recognize the native receptor as judged by immunocytochemical staining of cells transfected with dopamine receptor DNA. One antibody was shown to be specific for the 'long' form of the receptor and reacts only with cells transfected with the 'long' DNA subtype and not with those transfected with the 'short' DNA subtype. This recognition is specific and can be inhibited by the corresponding free peptide and not by a non-relevant peptide.