Journal: Investigative Ophthalmology & Visual Science

Article Title: Distinct Transcriptomic Profiles of Cultured Anterior and Posterior Populations of Human Infant Scleral Fibroblasts: Including Dopamine Receptors

doi: 10.1167/iovs.66.5.29

Figure Lengend Snippet: D1-like receptor activity promotes the inhibitory effect of DA on cell contraction, whereas D2-like receptor activity, particularly that of DRD2 in the posterior sclera, impedes it. ( a – p ) The cell contraction rates at 24 hours for anterior ( a – h ) and posterior ( i – p ) scleral fibroblasts from HSF12 and HSF13 were measured following treatment with a range of DA receptor antagonists and agonists, administered 1 hour prior to the addition of DA. One experiment was conducted with three replicate wells for each compound tested, using two biological samples (HSF12 and HSF13; total n = 6). Results from the two cell lines were averaged and presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-way ANOVA).

Article Snippet: The antibodies used were as follows: Dopamine Receptor D1 Rabbit mAb (381747; Zen-Bioscience), DRD2 polyclonal antibody (55084-1-AP; Proteintech, Rosemont, IL, USA), Dopamine Receptor D3 Rabbit mAb (382983; Zen-Bioscience), DRD4 Rabbit polyclonal antibody (28094-1-AP; Proteintech), Dopamine Receptor D5 Rabbit pAb (820522; Zen-Bioscience), GAPDH (7E4) Mouse mAb (200306-7E4; Zen-Bioscience), Beta Tubulin Polyclonal antibody (10094-1-AP; Proteintech), Goat anti-Rabbit IgG(H&L) (HRP conjugate) (511203; Zen-Bioscience), and Goat anti-Mouse IgG (H&L) (HRP conjugate) (511103; Zen-Bioscience).

Techniques: Activity Assay

Journal: Hypertension research : official journal of the Japanese Society of Hypertension

Article Title: Antihypertensive effect of etamicastat in dopamine D2 receptor-deficient mice.

doi: 10.1038/s41440-018-0041-5

Figure Lengend Snippet: Fig. 8 Filtered dopamine, as well as dopamine produced in the renal proximal tubule, stimulates dopamine receptors to increase sodium excretion under conditions of normal or moderate sodium load. Deletion of any dopamine receptor gene increases blood pressure. Etamicastat inhibits dopamine β hydroxylase (DBH), decreasing the conversion of norepinephrine to dopamine, allowing the levels of dopamine to increase. The increase in filtered dopamine and dopamine produced by the kidney (presumably from the renal nerves; DBH is not expressed in renal tubules) stimulate the other dopamine receptor subtypes, D1R, D3R, D4R, and D5R, which overcomes the absence or decreased expression of D2R. The increased production of reactive oxygen species (ROS) is normalized, sodium excretion (UNaV) is increased, and blood pressure is normalized in mice without D2R in the body or mice with decreased renal D2R expression

Article Snippet: We have reported the specificity of our D1R antibody [18, 20], D3R antibody [19], D4R antibody [17], D5R antibody [21, 22], D1R antibody from Origene (Rockville, MD) [22], and D2R from EMD Millipore [13].

Techniques: Produced, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: G protein-coupled receptor 37L1 regulates renal sodium transport and blood pressure

doi: 10.1152/ajprenal.00289.2018

Figure Lengend Snippet: In human renal proximal tubule cells (hRPTCs), overexpression of GPR37L1 increases while silencing of GPR37L1 decreases intracellular sodium. hRPTCs were cultured in 12-well Transwell plates and transfected with the indicated plasmids. Two days later, the cells were serum starved overnight and treated with the indicated drugs at the luminal side for 30 min before loading with the sodium fluorescent dye. A: hRPTCs were transfected with control plasmid (empty vector, pcDNA), plasmid carrying cDNA encoding GPR37L1 (pCMV-GPR37L1), luciferase (LUC), β-galactosidase (GAL), or human DRD1 (pCMV-DRD1). n = 3/group; *P < 0.05 GPR37L1 vs. the rest, **P < 0.05 DRD1 vs. pcDNA. B: hRPTCs transfected with plasmids pcDNA or pCMV-GPR37L1 were treated at the luminal membrane side with vehicle (Veh), nonselective NHE isoform inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 10 μM), or NHE3-selective inhibitor 3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride (S3226, 10 µM). n = 3/group; +P < 0.05 vs. pcDNA-Veh; n = 3/group. C: hRPTCs transfected with nonsilencing shRNA (NS-shRNA) or GPR37L1-shRNA (37L1-shRNA) and treated with Veh or S3226 at the luminal side similar to B. n = 3/group; ++P < 0.05 vs. NS-shRNA-Veh. D: hRPTCs transfected with plasmids pcDNA or pCMV-GPR37L1 were treated at the luminal membrane side with Veh, INM, PMA, FSK, or S3226. n = 3/group; $P < 0.05 vs. pcDNA-Veh, #P < 0.05 vs. GPR37L1-Veh. DRD1, dopamine receptor D1; FSK, forskolin; GPR37L1, G protein-coupled receptor 37L1; INM, ionophore ionomycin; NHE, Na+/H+ exchanger; CMV, cytomegalovirus.

Article Snippet: A plasmid encoding human GPR37L1 (pCMV- GPR37L1 ]) or dopamine receptor D1 (pCMV- DRD1 ), under the control of cytomegalovirus promoter, was obtained from Origene Technology (Rockville, MD; cat. no. GPR37L1 : SC117166; DRD1 : RC210389).

Techniques: Over Expression, Cell Culture, Transfection, Control, Plasmid Preparation, Luciferase, Membrane, shRNA

Journal: Investigative ophthalmology & visual science

Article Title: Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

doi: 10.1167/iovs.65.1.49

Figure Lengend Snippet: FIGURE 1. Cell viability after exposure to IL-1β. Control iMCECs had no additional experimental interventions. (A) The effect of different concentrations of IL-1β (0, 50, 100, 200 ng/mL) at different times (12, 24, 48, 72 hours) of intervention on the cell viability of iMCECs. (B) DRD1 was successfully overexpressed in iMCECs by pcDNA_DRD1. (C) Quantification analysis of iMCEC proliferation during homeostatic conditions via CCK-8 assay. (D) Quantification analysis of iMCEC proliferation in an inflammatory environment via CCK-8 assay. Data are represented as mean ± SEM. Experiments were repeated three or four times, and data in each group are from triplicate or quadruplicate wells. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; ns, not significant.

Article Snippet: Five microliters of 0.1% DRD1 agonist (A68930; MedChemExpress, Monmouth Junction, NJ, USA) or PBS was applied topically to the injured eyes using a micropipette three times a day for up to 5 days post-injury.

Techniques: Control, CCK-8 Assay

Journal: Investigative ophthalmology & visual science

Article Title: Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

doi: 10.1167/iovs.65.1.49

Figure Lengend Snippet: FIGURE 2. Overexpressed DRD1 inhibited NLRP3-related inflammatory factors in iMCECs. (A) Representative confocal microscopic images of the NLRP3+ iMCECs. Scale bars: 100 μm. (B) Quantification analysis of NLRP3 protein expression via immunofluorescence staining. (C) Quantification analysis of IL-1β mRNA expression via real-time qPCR. (D) Quantification analysis of IL-6 mRNA expression via real-time qPCR. Data are represented as mean ± SEM. Experiments were repeated four times, and data in each group are from quadruplicate wells. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; ns, not significant.

Article Snippet: Five microliters of 0.1% DRD1 agonist (A68930; MedChemExpress, Monmouth Junction, NJ, USA) or PBS was applied topically to the injured eyes using a micropipette three times a day for up to 5 days post-injury.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Investigative ophthalmology & visual science

Article Title: Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

doi: 10.1167/iovs.65.1.49

Figure Lengend Snippet: FIGURE 3. DRD1 agonist promoted pMCEC proliferation and inhibited expression of NLRP3. The DRD1 agonist was A68930, and the DRD1 antagonist was SCH23390. (A) The effect of different concentrations of A68930 (0, 10, 20, 30 μM) on the cell viability of pMCECs. (B) Representative immunoblot images of the expression of NLRP3 and GAPDH protein. (C) Quantification analysis of NLRP3 protein expression via western blotting. (D) Representative confocal microscopic images of the Ki-67+ and p63+ pMCECs. Scale bars: 100 μm. (E) Quantification analysis of Ki-67+ pMCECs via immunofluorescent staining. (F) Quantification analysis of p63+ pMCECs via immunoflu- orescent staining. Data are represented as mean ± SEM. Experiments were repeated four or five times, and data in each group are from quadruplicate or quintuplicate wells. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; ns, not significant.

Article Snippet: Five microliters of 0.1% DRD1 agonist (A68930; MedChemExpress, Monmouth Junction, NJ, USA) or PBS was applied topically to the injured eyes using a micropipette three times a day for up to 5 days post-injury.

Techniques: Expressing, Western Blot, Staining

Journal: Investigative ophthalmology & visual science

Article Title: Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

doi: 10.1167/iovs.65.1.49

Figure Lengend Snippet: FIGURE 4. Administration of 0.1% A68930 promoted MCEC proliferation and expression of DRD1 in vivo. (A) Representative confocal micro- scopic images of the Ki-67+ and p63+ cells. Scale bars: 100 μm. (B) Quantification analysis of Ki-67+ cells in corneas via immunofluorescent staining. (C) Quantification analysis of p63+ cells via immunofluorescent staining. (D) Quantification analysis of DRD1 mRNA expression via real-time qPCR. (E) Representative immunoblot images of the expression of DRD1 and GAPDH protein. (F) Quantification analysis of DRD1 protein expression via western blotting. Data are represented as mean ± SEM. Experiments were repeated three to eight times. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; ns, not significant.

Article Snippet: Five microliters of 0.1% DRD1 agonist (A68930; MedChemExpress, Monmouth Junction, NJ, USA) or PBS was applied topically to the injured eyes using a micropipette three times a day for up to 5 days post-injury.

Techniques: Expressing, In Vivo, Staining, Western Blot

Journal: Investigative ophthalmology & visual science

Article Title: Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

doi: 10.1167/iovs.65.1.49

Figure Lengend Snippet: FIGURE 7. Administration of 0.1% A68930 promoted the corneal wound healing process. (A) The PBS treatment group and the 0.1% A68930 treatment group were treated with the same corneal injury. (B) After injury, expression of DRD1 was downregulated and NLRP3, IL-1β, and IL-6 levels were upregulated. (C) The 0.1% A68930 promoted corneal wound healing by upregulation of DRD1 and downregulation of NLRP3-associated inflammatory factors.

Article Snippet: Five microliters of 0.1% DRD1 agonist (A68930; MedChemExpress, Monmouth Junction, NJ, USA) or PBS was applied topically to the injured eyes using a micropipette three times a day for up to 5 days post-injury.

Techniques: Expressing

Journal: Cell reports

Article Title: Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

doi: 10.1016/j.celrep.2022.111431

Figure Lengend Snippet: (A–F) Dispersal of DAT nanodomains is promoted by the D2R antagonist haloperidol and blocked by the agonist quinpirole and by the N-type VGCC blocker ω-conotoxin. Shown are example varicosities from dSTORM of DA neurons subjected to 5-min treatment with haloperidol (10 nM), haloperidol (10 nM) plus quinpirole (50 μM), haloperidol (10 nM) plus ω-conotoxin (1 μM), or haloperidol (10 nM) plus ω-agatoxin (1 μM). (A) DAT clusters with a diameter greater than 75 nm are shown in green and clusters smaller than 75 nm in magenta. (B) Dense DAT clusters are shown in red (>80 localizations; radius, 50 nm). (C) Localizations segmented by Voronoi tessellation. (D and E) Normalized fraction of DAT clusters greater than 75 nm (percent) (D) and normalized fraction of DAT localizations in dense clusters (percent) (E). Means ± S.E.; one-way ANOVA, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. (F) PDFs (curves are color coded as in the bar diagrams) for the Voronoi tessellated areas averaged by varicosity. Data are from 3 cultures and 153 control, 166 haloperidol, 167 quinpirole, 178 ω-conotoxin, and 176 ω-agatoxin varicosities. (G–R) Single-particle tracking dSTORM (SPT-dSTORM) identifies changes in DAT mobility in response to NMDA and D2R antagonism. (G) Example live dSTORM images of DA neurons labeled with the fluorescent cocaine analog DG3–80. (H) Boxed region in (B). (I and J) Detected tracks (I) and distribution of segment lengths (J) in a varicosity from the control SPT-dSTORM experiment (16-ms frame rate). (K) Example traces of a confined and an unconstrained freely moving particle (DG3–80-labeled DAT). (L) Fraction of unconstrained tracks of the total number of tracks. Data are means of 23 tracks ± S.E. (M) PDF for total mean jump distances (MJD) (i.e., the distance an individual molecule/particle has traveled between each image) for control experiments. (N) PDF when separated into static and freely moving particles. Among the freely moving particles, some moved very fast with MJDs of 200–350 nm. (O) NMDA (20 μM, 5 min) increases the population of DAT molecules with MJDs greater than 200 nm, which is blunted by AP5 (100 μM). (P) Fraction of fast-moving particles (>200 nm). Means ± S.E.; data from 13 control, 4 NMDA, and 5 NMDA plus AP5 experiments. (Q) The D2R antagonists haloperidol (10 nM, 5 min) and UH-232 (1 μM) increase the population of DAT molecules with MJDs greater than 200 nm. (R) fraction of fast-moving particles (>200 nm), Means ± S.E.; data are from 10 control, 6 haloperidol, and 5 UH-232 experiments; one-way ANOVA, *p < 0.05; **p < 0.01. See also , and .

Article Snippet: We used the following antibodies and fluorophores for our imaging analysis: DAT, Millipore MAB369 RRID:AB_2190413, Primary culture dSTORM, 1/200, 5 μg/mL Donkey anti-Rat, Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, IHC dSTORM, 1/200, 10 μg/mL Donkey anti-Rat Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, Expansion (ExM), 1/200, 5 μg/mL Donkey anti-Rat Atto 643; TH, Millipore MAB318 RRID:AB_2201528, PALM/dSTORM 1/500, 5 μg/mL Donkey anti-Mouse Alexa647; STX-1, Synaptic Systems 110 011 RRID:AB_887844, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; Clathrin light chain, Synaptic Systems 113 011 RRID:AB_887706, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; PIP2, Abcam ab11039 RRID:AB_442848, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-mouse CF568; D2R, Synaptic Systems 376 205 RRID:AB_2636918, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit, CF568; mKate2, Evrogen AB233 RRID:AB_2571743, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit CF568; Synaptotagmin 1 (luminal domain), Synaptic systems 105 311 RRID:AB_10805653; Active varicosity detection, 1/125, 5 μg/mL Donkey anti-Mouse Alexa 488.

Techniques: Control, Single-particle Tracking, Labeling

Journal: Cell reports

Article Title: Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

doi: 10.1016/j.celrep.2022.111431

Figure Lengend Snippet: (A) Example dual-color dSTORM images of the D2R (red, CF568) and DAT (blue, Alexa 647) in a DA neuronal extension (left) with a close-up image of a varicosity (right) corresponding to the boxed region in the left image. (B) Example dual-color dSTORM images of varicosities from DA neurons showing localizations for D2R (red) and DAT (blue). Left: control. Right: neurons exposed to haloperidol (10 nM, 5 min). (C–F) Effect of NMDA and haloperidol on D2R clustering. Top panels: control, NMDA (20 μM), and NMDA (20 μM) plus AP5 (100 μM). Bottom panels: control, haloperidol (10 nM), and haloperidol (10 nM) plus quinpirole (50 μM). (C and D) Normalized fraction of D2R localizations in large clusters (>75 nm in diameter) (percent) (C) and normalized fraction of D2R localizations in dense clusters (>80 localizations; radius, 50 nm) (percent). Means ± S.E., one-way ANOVA, ***p < 0.001, **p < 0.01, *p < 0.05. (E) PDFs (color-coded as in the bar diagrams) for the Voronoi tessellated areas averaged by varicosity. (F) PDFs (color-coded as in the bar diagrams) of the association of D2R with DAT clusters determined by Voronoi tessellation-based association . Data are from 3 cultures and 112 control, 157 NMDA, and 75 NMDA plus AP5 varicosities and 179 control, 195 haloperidol, and 150 quinpirole varicosities. See also and .

Article Snippet: We used the following antibodies and fluorophores for our imaging analysis: DAT, Millipore MAB369 RRID:AB_2190413, Primary culture dSTORM, 1/200, 5 μg/mL Donkey anti-Rat, Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, IHC dSTORM, 1/200, 10 μg/mL Donkey anti-Rat Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, Expansion (ExM), 1/200, 5 μg/mL Donkey anti-Rat Atto 643; TH, Millipore MAB318 RRID:AB_2201528, PALM/dSTORM 1/500, 5 μg/mL Donkey anti-Mouse Alexa647; STX-1, Synaptic Systems 110 011 RRID:AB_887844, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; Clathrin light chain, Synaptic Systems 113 011 RRID:AB_887706, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; PIP2, Abcam ab11039 RRID:AB_442848, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-mouse CF568; D2R, Synaptic Systems 376 205 RRID:AB_2636918, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit, CF568; mKate2, Evrogen AB233 RRID:AB_2571743, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit CF568; Synaptotagmin 1 (luminal domain), Synaptic systems 105 311 RRID:AB_10805653; Active varicosity detection, 1/125, 5 μg/mL Donkey anti-Mouse Alexa 488.

Techniques: Control

Journal: Cell reports

Article Title: Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

doi: 10.1016/j.celrep.2022.111431

Figure Lengend Snippet:

Article Snippet: We used the following antibodies and fluorophores for our imaging analysis: DAT, Millipore MAB369 RRID:AB_2190413, Primary culture dSTORM, 1/200, 5 μg/mL Donkey anti-Rat, Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, IHC dSTORM, 1/200, 10 μg/mL Donkey anti-Rat Alexa647; DAT, Millipore MAB369 RRID:AB_2190413, Expansion (ExM), 1/200, 5 μg/mL Donkey anti-Rat Atto 643; TH, Millipore MAB318 RRID:AB_2201528, PALM/dSTORM 1/500, 5 μg/mL Donkey anti-Mouse Alexa647; STX-1, Synaptic Systems 110 011 RRID:AB_887844, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; Clathrin light chain, Synaptic Systems 113 011 RRID:AB_887706, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-Mouse CF568; PIP2, Abcam ab11039 RRID:AB_442848, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-mouse CF568; D2R, Synaptic Systems 376 205 RRID:AB_2636918, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit, CF568; mKate2, Evrogen AB233 RRID:AB_2571743, Primary culture dSTORM, 1/500, 5 μg/mL Donkey anti-rabbit CF568; Synaptotagmin 1 (luminal domain), Synaptic systems 105 311 RRID:AB_10805653; Active varicosity detection, 1/125, 5 μg/mL Donkey anti-Mouse Alexa 488.

Techniques: Virus, Recombinant, Synthesized, Generated, Software