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The Protein Ocular Albinism 1 Is the Orphan GPCR GPR143 and Mediates Depressor and Bradycardic Responses to DOPA in the Nucleus Tractus Solitarii

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The Protein Ocular Albinism 1 Is the Orphan GPCR GPR143 and Mediates Depressor and Bradycardic Responses to DOPA in the Nucleus Tractus Solitarii

Y Hiroshima et al. Br J Pharmacol.

Abstract

Background and purpose: L-DOPA is generally considered to alleviate the symptoms of Parkinson's disease by its conversion to dopamine. We have proposed that DOPA is itself a neurotransmitter in the CNS. However, specific receptors for DOPA have not been identified. Recently, the gene product of ocular albinism 1 (OA1) was found to exhibit DOPA-binding activity. Here, we have investigated whether OA1 is a functional receptor of DOPA in the nucleus tractus solitarii (NTS).

Experimental approach: We examined immunohistochemical expression of OA1 in the NTS, and the effects of DOPA microinjected into the depressor sites of NTS on blood pressure and heart rate in anaesthetized rats, with or without prior knock-down of OA1 in the NTS, using shRNA against OA1.

Key results: Using a specific OA1 antibody, OA1-positive cells and nerve fibres were found in the depressor sites of the NTS. OA1 expression in the NTS was markedly suppressed by microinjection into the NTS of adenovirus vectors carrying the relevant shRNA sequences against OA1. In animals treated with OA1 shRNA, depressor and bradycardic responses to DOPA, but not those to glutamate, microinjected into the NTS were blocked. Bilateral injections into the NTS of DOPA cyclohexyl ester, a competitive antagonist against OA1, suppressed phenylephrine-induced bradycardic responses without affecting blood pressure responses.

Conclusion and implications: OA1 acted as a functional receptor for DOPA in the NTS, mediating depressor and bradycardic responses. Our results add to the evidence for a central neurotransmitter role for DOPA, without conversion to dopamine.

Keywords: 4-dihydroxyphenylalanine; G-protein coupled receptor; L-3; baroreceptor reflex; neurotransmitter; nucleus tractus solitarii.

Figures

Figure 1
Figure 1
(A) Immunoblot analysis of COS-7 cells expressing Myc-tagged mouse OA1 expression vector (OA1) or empty vector (V) with anti-OA1 rabbit antibody or anti-Myc antibody. The anti-OA1 antibody recognized the 42, 80 kDa and higher molecular weight bands in the OA1-expressing cell lysates without heat denaturation. In samples boiled for 5 min at 100°C, several bands higher than 200 kDa were detected, suggesting that heat denaturation caused aggregation of OA1. These immunoreactive bands were blocked by synthetic OA1 C-terminal peptide (314-405AA) including the immunogen sequence. Similar immunoreactive bands appeared with anti-Myc antibody. The size of 42 kDa correlates well with the deduced molecular weight of mouse OA1 protein from the cDNA sequence. The higher molecular weight bands may reflect glycosylation and/or homo- and hetero-oligomeric complexes of OA1. The similar amount of protein loading was confirmed by tubulin immunoblotting (lower panels). (B) OA1-mCherry expression in COS7 cells was detected by anti-OA1 antibody and mCherry fluorescence. Immunofluorescence of OA1 detected in mCherry-positive cells was blocked by the synthetic OA1 peptide. (C) Immunohistochemical localization of OA1 in rat retinae infected with oa1 and scramble vectors. cGFP was monitored to track the transfection efficiency (left). The expression of OA1 in retinal pigment epithelium (RPE) indicated by the asterisk (*) was significantly suppressed by oa1-Ad injection. IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, PL: photoreceptor layer, Ch: choroid, Sc: sclera. Immunohistochemical examination was performed 48–72 h after infection of rat retinae with the adenoviral constructs. Scale bar, 50 μm.
Figure 2
Figure 2
(A) Diagram of rat medulla oblongata in a coronal section. Image of boxed area in (A) is shown in (B). CC: central canal, AP: area postrema, SolM: nucleus of solitary tract medial, SolC: commissural, sol: solitary tract. (B) The coronal section of rat medulla oblongata immunostained with anti-OA1 antibody. Magnified image of boxed area in (B) is shown in (C). (D) The OA1 signals were inhibited by the peptide immunogen against the OA1 antibody. (E) The coronal section of rat medulla oblongata was immunostained with anti-TH antibody. Arrowheads in a magnified image (right lower panel) of boxed area indicate the cells positively stained with anti-TH antibody. (F) Diagram of rat hypothalamus in a coronal view. Image of boxed area in (F) is shown in (G). VMHDM: nucleus of ventromedial hypothalamus dorsomedial, VMHC: central, VMHVL: ventrolateral, Arc: arcuate nucleus, ME: median eminence, 3V: third ventricle. (G) The coronal section of rat hypothalamus was immunostained with anti-OA1 antibody. Magnified image of boxed area in (G) is shown in (H). Arrowheads indicate the cells positively stained with anti-OA1 antibody. These signals were inhibited by the peptide immunogen (data not shown).
Figure 3
Figure 3
(A) Quantitative RT-PCR analysis of the expression of OA1 mRNA in the NTS infected with scramble- and oa1-adenovirus vectors. The expression of OA1 was suppressed in the side injected with oa1-adenovirus (oa1-Ad). *P < 0.05, compared with scramble-adenovirus (scramble-Ad); (n = 5). (B) Immunohistochemical localization of OA1 in the NTS injected with scramble- and oa1-adenovirus vectors. The immunofluorescence levels of OA1 in the NTS injected with oa1-adenovirus vector were suppressed, compared to that with scramble-adenovirus vector. (C) Quantitative analysis of the effect of oa1-adenovirus vector injection. The ratio between OA1 and cGFP immunofluorescence level was calculated in each cell expressing OA1 and cGFP. **P < 0.01, compared with the levels of the scramble side (n = 11–13).
Figure 4
Figure 4
(A,B) Typical traces of the effects of DOPA (60 ng) and glutamate (Glu) (100 ng) microinjected into the right or left NTS infected with scramble- (scramble-Ad) or oa1-adenovirus (oa1-Ad) vectors on BP and HR in anaesthetized rats. In the scramble side, the depressor and bradycardic responses to DOPA were detected, whereas these responses were suppressed in the side injected with oa1-adenovirus. Responses to glutamate were detected in the oa1-adenovirus-injected side as well as in the scramble-adenovirus-injected side. Scale bar, 1 min. (C,D) Summarized effects of scramble- and oa1-adenovirus vectors on the depressor (ΔMBP) and bradycardic (ΔHR) responses to DOPA and glutamate. *P < 0.05, compared with the levels of the scramble side (n = 5).
Figure 5
Figure 5
(A,B) [3H]-DOPA binding on CHO cells expressing OA1-EGFP. Specific binding was obtained by the subtraction of the binding value with 10 mM DOPA CHE from the total binding. One typical example is shown. (A) Saturation curve. (B) Scatchard plot. The estimated KD is 81.3 μM from this experiment. (C) Competitive inhibition of 10 μM [3H]-DOPA binding to OA1-EGFP with various concentration of DOPA CHE. Results indicate that DOPA CHE and DOPA compete for the same OA1 binding site. The estimated Ki for DOPA CHE from this experiment is 155.1 μM. (D–G) Representative traces of [Ca2+]i during the time course of the standard experimental protocol in ARPE-19 cells transfected with mouse OA1-mCherry. After establishing a stable baseline, the cells were stimulated with 10 μM DOPA. DOPA CHE (10 μM), when used, was applied 10 min before DOPA application. DOPA CHE alone produced no effect. (D,E: upper panels) were inhibited by DOPA CHE pre-incubation (E,F: lower panels). Calcium ionophore (4-Br A23187) was added to confirm the Ca2+ imaging (D,F). (G) Summary data for [Ca2+]i in response to DOPA and DOPA CHE inhibition. Each bar represents the mean change from baseline [Ca2+]i after DOPA stimulation. Data shown are mean ± SEM (n = 5). *P < 0.01, compared with no pre-treatment.
Figure 6
Figure 6
(A) A typical trace of the effect of DOPA (60 ng) before and after microinjection of DOPA CHE (1 μg) into the NTS of anaesthetized rats on BP and HR. (B) Antagonism by bilateral microinjection (Rt, right; Lt, left) of DOPA CHE (1 μg) against bradycardic responses to i.v. infusion of phenylephrine (Phe) (15 μg 10 mL−1·min−1) for 15 s. Scale bar, 1 min. (C,D) Summarized data on Phe-induced changes in BP (C, ΔBP) and HR (D, ΔHR) before and after DOPA CHE. **P < 0.01, compared with the levels before DOPA CHE microinjection (n = 6).

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