Characterization of Antibodies to Identify Cellular Expression of Dopamine Receptor 4.

The dopamine receptor D4 (DRD4) plays an important role in vision. In order to study the DRD4 expression in vivo, it is important to have antibodies that are specific for DRD4 for both immunoblot and immunohistochemical (IHC) applications. In this study, six antibodies raised against DRD4 peptides were tested in vitro, using transfected mammalian cells, and in vivo, using mouse retinas. Three Santa Cruz (SC) antibodies, D-16, N-20, and R-20, were successful in IHC of transfected DRD4; however, N-20 was the only one effective on immunoblot analysis in DRD4 transfected cells and IHC of mouse retinal sections, while R-20, 2B9, and Antibody Verify AAS63631C were non-specific or below detection.

Roles of dopamine receptor subtypes in mediating modulation of T lymphocyte function.

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.

Should we be cautious on the use of commercially available antibodies to dopamine receptors?

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.

Cutting Edge: Stimulation of dopamine D4 receptors induce T cell quiescence by up-regulating Kruppel-like factor-2 expression through inhibition of ERK1/ERK2 phosphorylation.

The neurotransmitter dopamine (DA) is an important regulator of human T cell functions. Although it has been observed that DA, by acting through the D1/D5, D2, and D3 receptors, can activate resting T cells by stimulating the release of cytokines and the expression of surface integrins and also inhibit the proliferation of activated T cells by down-regulating nonreceptor tyrosine kinases, there is not yet a report indicating the functional significance of the D4 DA receptors present in these cells. The present work, for the first time, demonstrates that the stimulation of D4 DA receptors in human T cells induces T cell quiescence by up-regulating lung Krüppel-like factor-2 expression through the inhibition of ERK1/ERK2 phosphorylation. These results reveal a new link between the nervous system and T cell quiescence and indicate that D4 DA receptor agonists may have a therapeutic value in diseases with uncontrolled T cell proliferation.

Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound.

The blood-brain barrier (BBB) is a persistent obstacle for the local delivery of macromolecular therapeutic agents to the central nervous system (CNS). Many drugs that show potential for treating CNS diseases cannot cross the BBB and there is a need for a non-invasive targeted drug delivery method that allows local therapy of the CNS using larger molecules. We developed a non-invasive technique that allows the image-guided delivery of antibody across the BBB into the murine CNS. Here, we demonstrate that subsequent to MRI-targeted focused ultrasound induced disruption of BBB, intravenously administered dopamine D(4) receptor-targeting antibody crossed the BBB and recognized its antigens. Using MRI, we were able to monitor the extent of BBB disruption. This novel technology should be useful in delivering macromolecular therapeutic or diagnostic agents to the CNS for the treatment of various CNS disorders.

D3 dopamine receptor mRNA is elevated in T cells of schizophrenic patients whereas D4 dopamine receptor mRNA is reduced in CD4+ -T cells.

The expression of dopamine receptors was examined in purified human neutrophils, monocytes, B cells, natural killer cells and CD4+ - and CD8+ -T lymphocytes by RT-PCR. In healthy subjects, D1 and D2 receptors were not expressed in leukocytes. Real Time PCR for dopamine receptors D3 and D4 disclosed that D3 receptors are expressed in T cells and natural killer cells and D4 receptors in CD4+ -T cells. The comparison of schizophrenic patients with sex- and age-matched controls revealed a significantly higher expression of D3 receptor mRNA in T cells of schizophrenic patients, whereas D4 receptor mRNA in CD4+ -T cells was downregulated.