Journal: Scientific reports

Article Title: Dopamine D1-like receptor blockade and stimulation decreases operant responding for nicotine and food in male and female rats.

doi: 10.1038/s41598-022-18081-3

Figure Lengend Snippet: Figure 2. Effects of the D1-like receptor agonist A77636 on nicotine and food intake and locomotor activity in male and female rats. The effects of A77636 on nicotine (A 15 min; B 24 h; C 48 h) and food intake (D 15 min; E 24 h; F 48 h) and locomotor activity (G 15 min; H 24 h; I 48 h) were investigated. Treatment with A77636 decreased nicotine intake 15 min, 24 h, and 48 h later. A77636 decreased food intake at the 15 min and 24 h time point and locomotor activity at the 48 h time point. Nicotine self-administration (males n = 9, females, n = 9), operant responding for food and small open field test (males n = 8, females, n = 8). Asterisks indicate a significant difference from rats of the same sex that received vehicle. *P < 0.05, **P < 0.01. Data are expressed as means ± SEM.

Article Snippet: Drugs and treatment. (−)-Nicotine hydrogen tartrate (Sigma-Aldrich), SCH 23390 hydrochloride (Tocris bioscience), and A77636 hydrochloride (Tocris bioscience) were dissolved in sterile saline (0.9% sodium chloride).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

doi: 10.1101/2024.04.15.589637

Figure Lengend Snippet: D1R-mediated cAMP accumulation (black) was measured using the Glosensor assay in HEK293 cells stably expressing the D1R and β-arrestin recruitment (blue) was measured with the Tango assay as described in methods. Agonist structures are shown in upper left corner of each panel. A) SKF-81297 is a full agonist for both G protein/cAMP (black) and β-arrestin recruitment (blue). All agonists were normalized to SKF-81297. B) SKF-38393 is a high partial agonist for G protein/cAMP but does not recruit β-arrestin. C) SKF-77434 is a low partial agonist for G protein/cAMP activity compared to SKF-81297 but has no activity in the β-arrestin recruitment assay. D) A-77636 is a full agonist for G protein/cAMP signaling but, interestingly, was a super agonist for β-arrestin recruitment. Representative plots shown from at least three independent experiments performed in technical triplicate. Light counts per second (bioluminescence) from both assays was normalized to 100% SKF-81297 response. EC 50 and E max values were obtained by non-linear regression analysis for all experiments and average values are summarized in .

Article Snippet: The commercially available compounds were purchased from the following suppliers: dopamine (Sigma, St. Louis, MO), SKF-81297 (Sigma, St. Louis, MO), SKF-38393 (Tocris, Bristol, UK), SKF-77434 (Tocris, Bristol, UK), A-77636 (Tocris, Bristol, UK), ascorbic acid (Sigma, St. Louis, MO).

Techniques: Stable Transfection, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

doi: 10.1101/2024.04.15.589637

Figure Lengend Snippet: The Glosensor assay was used to measure D5R-mediated cAMP accumulation (black) in transiently transfected HEK293 cells while the Tango assay measured β-arrestin recruitment (blue). All responses in both assays were normalized to the full agonist, SKF-81297. Agonist structures are shown in the upper left corner of the corresponding panel. A) SKF-81297 is a full agonist in both cAMP and β-arrestin recruitment assays. B) A-77636 is a full agonist in both cAMP and β-arrestin recruitment assays. In the β-arrestin recruitment assay, A-77636 is more potent compared to SKF-81297. C) PF-1119 is a partial agonist for D5R-mediated cAMP accumulation and has very low partial agonist activity in the β-arrestin recruitment assay for the D5R. D) Cmpd 19 is a full agonist for D5R-mediated cAMP accumulation and is a partial agonist for β-arrestin recruitment. Representative plots shown from at least three independent experiments performed in technical triplicate. Light counts per second (bioluminescence) from both assays was normalized to 100% SKF-81297 response. EC 50 and E max values were obtained by non-linear regression analysis for all experiments and average values are summarized in .

Article Snippet: The commercially available compounds were purchased from the following suppliers: dopamine (Sigma, St. Louis, MO), SKF-81297 (Sigma, St. Louis, MO), SKF-38393 (Tocris, Bristol, UK), SKF-77434 (Tocris, Bristol, UK), A-77636 (Tocris, Bristol, UK), ascorbic acid (Sigma, St. Louis, MO).

Techniques: Transfection, Activity Assay

Journal: bioRxiv

Article Title: Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

doi: 10.1101/2024.04.15.589637

Figure Lengend Snippet: HEK293 cells were transfected with HA-D1R and β-arrestin2-GFP. The cells were then treated with 3 µM of the indicated agonist and fixed after 10 minutes. The D1R was then detected using antibodies against the HA tag and visualized with alexa594 secondary antibodies. TIRF microscopy was used to image a thin slice of the cells at the plasma membrane. Punctate GFP signal indicates β-arrestin recruitment to the plasma membrane. A) Vehicle treatment did not recruit β-arrestin to the plasma membrane. B) SKF-81297 strongly recruited β-arrestin to the plasma membrane. β-arrestin colocalized in puncta with HA-D1R. C) A-77636 treatment induced β-arrestin recruitment to the plasma membrane that colocalized with the HA-D1R. D) PF-1119 weakly recruited β-arrestin to the plasma membrane. E) Cmpd 41 moderately recruited β-arrestin to the plasma membrane. F) Cmpd 19 strongly recruited β-arrestin to the plasma membrane and colocalized with HA-D1R. Similar results observed in >21 cells across three independent experiments. White boxes indicate the area that is enlarged in the corner of merged images. Scale bar = 10 μm.

Article Snippet: The commercially available compounds were purchased from the following suppliers: dopamine (Sigma, St. Louis, MO), SKF-81297 (Sigma, St. Louis, MO), SKF-38393 (Tocris, Bristol, UK), SKF-77434 (Tocris, Bristol, UK), A-77636 (Tocris, Bristol, UK), ascorbic acid (Sigma, St. Louis, MO).

Techniques: Transfection, Microscopy, Membrane

Journal: bioRxiv

Article Title: Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

doi: 10.1101/2024.04.15.589637

Figure Lengend Snippet: A) A schematic representation of the Antibody Feeding assay. HEK293 cells were transfected with N-terminally HA tagged D1R. In living cells, the surface D1Rs were labeled with an anti-HA antibody conjugated to Alexa488 on ice to prevent endocytosis. The indicated agonists were added at saturating concentrations (10 μM) and the cells warmed to 37°C for 60 minutes, then fixed and imaged as described for Antibody feeding in the methods. A punctate appearance inside the plasma membrane indicates receptor/antibody endocytosis. B) Cells treated with vehicle for 60 minutes undergo trivial amounts of D1R endocytosis. Cells treated with the balanced catechol agonists, C) dopamine and D) SKF-81297, had punctate and perinuclear HA-D1R localization indicating dopamine and SKF-81297 strongly induced D1R endocytosis. E) HEK293 cells treated with A-77636 had numerous puncta and perinuclear antibody localization indicating robust D1R endocytosis. F) In contrast, the non-catechol agonist PF-1119 did not induce D1R endocytosis. G) PF-2334 induced less D1R endocytosis than SKF-81297 as seen by puncta without perinuclear signal. H) PF-6142 did not induce D1R endocytosis. I) Cmpd 19 induced robust D1R endocytosis. Representative images shown from three independent experiments. Similar results observed in >20 cells across three independent experiments. White boxes indicate the area that is enlarged in the lower right corner. White arrows indicate endocytosis. Scale bar = 10 μm.

Article Snippet: The commercially available compounds were purchased from the following suppliers: dopamine (Sigma, St. Louis, MO), SKF-81297 (Sigma, St. Louis, MO), SKF-38393 (Tocris, Bristol, UK), SKF-77434 (Tocris, Bristol, UK), A-77636 (Tocris, Bristol, UK), ascorbic acid (Sigma, St. Louis, MO).

Techniques: Feeding Assay, Transfection, Labeling, Membrane

Journal: bioRxiv

Article Title: Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

doi: 10.1101/2024.04.15.589637

Figure Lengend Snippet: A) HEK293 cells transfected with HA-D1R were treated with saturating concentrations (10 μM) of the indicated agonist for 0-60 minutes and fixed. Surface HA-D1Rs were detected using an ELISA assay conducted under non-permeabilizing conditions with an anti-HA antibody as described in the methods. B) Balanced/β-arrestin super agonists, SKF-81297, dopamine, and A-77636 induced D1R endocytosis after agonist treatment. A-77636 induced a maximum of 47% of the surface HA-D1R to be endocytosed compared to SKF-81297 and dopamine which were 33% and 29%, respectively. C) The non-catechol agonists induced varying levels of D1R endocytosis. PF-2334 induced a maximum of 16% HA-D1R endocytosis while PF-6142 induced a maximum of 13% HA-D1R endocytosis. PF-1119 induced the lowest HA-D1R endocytosis with a maximum of 5% HA-D1R endocytosis. Cmpd 19, the balanced non-catechol agonist, induced D1R endocytosis at similar levels to the balanced catechol agonists with a maximum D1R endocytosis of 28%. D) Total HA-D1R endocytosis (Area under the curve, A.U.) analysis was conducted to determine the total amount of HA-D1R endocytosis across the 120 minute treatment. A-77636 induced significantly more D1R endocytosis than SKF-81297. On the other hand, PF-1119, PF-2334, and PF-6142 induced significantly less total HA-D1R endocytosis than SKF-81297. Both dopamine and Cmpd 19 were not significantly different from SKF-81297. Data presented as Mean ± SD, n=3, *, p<0.05 vs. SKF-81297; Two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: The commercially available compounds were purchased from the following suppliers: dopamine (Sigma, St. Louis, MO), SKF-81297 (Sigma, St. Louis, MO), SKF-38393 (Tocris, Bristol, UK), SKF-77434 (Tocris, Bristol, UK), A-77636 (Tocris, Bristol, UK), ascorbic acid (Sigma, St. Louis, MO).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay