Dopamine D1 Receptor

The phosphorylation of the cAMP response element binding (CREB) protein is greatly increased by A68930 (1 μM; 5-60 min; 16HBE14o- or NCI-H292 cells) [1]. NCI-H292 cells exposed to A68930 (1 μM) for 48 hours show increased MUC5AC mRNA expression as well as MUC5AC protein expression [1]. Intracellular cAMP levels are dramatically increased by A68930 (1 μM; 20 min; NCI-H292 cells) [1].

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Dopamine D1 receptor agonist-induced cAMP activity in 16HBE14o- cells and NCI-H292 cells [1]
The dopamine D1 receptor induces production of cAMP through the stimulation of adenylyl cyclase, which is activated by the Gs protein. Therefore, we examined whether activation of the dopamine D1 receptor increases intracellular cAMP levels in human airway epithelial cells (16HBE14o- cells and NCI-H292 cells). Dopamine (1 μM) as well as dopamine D1-like receptor agonists (SKF38393 or A68930; 1 μM) significantly increased intracellular cAMP levels in 16HBE14o- cells (dopamine; P < 0.001, SKF38393; P < 0.001, A68930; P < 0.01, n = 6) and NCI-H292 cells (dopamine; P < 0.01, SKF38393; P < 0.01, A68930; P < 0.01, n = 6) (Fig. 3a). Cyclic AMP production induced by A68930 (1 μM) was significantly reversed by pretreatment with the dopamine D1 receptor antagonists SCH23390 (1 μM) (16HBE14o- cells: P < 0.001, n = 6; NCI-H292 cells: P < 0.05, n = 6) or SCH39166 (1 μM) (16HBE14o- cells: P < 0.001, n = 6; NCI-H292 cells: P < 0.05, n = 6) (Fig. 3b).

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Dopamine D1 receptor agonist-induced CREB phosphorylation in 16HBE14o- cells and NCI-H292 cells [1]
The increase of cAMP after dopamine D1 receptor activation induces the activation of protein kinase A (PKA), which induces phosphorylation of CREB. MEK-ERK signaling also contributes to CREB phosphorylation. In addition, the dopamine D1 receptor activates MEK-ERK signaling through β-arrestin. Therefore, we examined whether the dopamine D1 receptor agonist A68930 phosphorylates CREB through PKA and/or MEK in 16HBE14o- cells and NCI-H292 cells. A68930 (1 μM, 20 min) significantly increased phosphorylation of CREB in 16HBE14o- cells (P < 0.05, n = 3) and NCI-H292 cells (P < 0.05, n = 7) (Fig. 4a and b). The phosphorylation reached maximal levels at 20–30 min and then slowly declined to basal levels within 60 min in 16HBE14o- cells, while the increased phosphorylation was maintained at 60 min in NCI-H292 cells. To confirm that A68930 phosphorylates CREB through PKA or MEK, NCI-H292 cells were pretreated with the PKA inhibitor H89 (10 μM; 30 min) or the MEK inhibitor U0126 (5 μM; 120 min). A68930 (1 μM; 20 min)-stimulated CREB phosphorylation in NCI-H292 cells was significantly inhibited by H89 (P < 0.001, n = 4) or U0126 (P < 0.05, n = 4) (Fig. 4c). These results confirm that the dopamine D1 receptor agonist-induced phosphorylation of CREB proceeds through both PKA and MEK/ERK signaling.

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Dopamine D1 receptor agonist-induced MUC5AC mRNA expression in NCI-H292 cells [1]
CREB was previously shown to mediate the transcriptional regulation of MUC5AC in airway epithelial cells including NCI-H292 cells. We examined whether dopamine or the dopamine D1 receptor agonist A68930 induces MUC5AC mRNA expression in NCI-H292 cells. Dopamine (1 μM), A68930 (1 μM), and cigarette smoke extract (CSE) (10%) significantly increased MUC5AC mRNA expression in NCI-H292 cells. The Gs protein-coupled β2 aderenoceptor agonist isoproterenol (1 μM) also significantly induced MUC5AC mRNA expression (Fig. 5a). Previous studies in airway epithelial cells have employed final CSE concentrations ranging from 1 to 30%. MTT cell viability analyses confirmed that 48 h treatment of NCI-H292 cells with 10% CSE or even higher concentrations (20%) of CSE did not reduce NCI-H292 cell viability, suggesting that treatment with CSE at 10% or 20% has no cytotoxicity (Fig. 5b).

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Dopamine D1 receptor agonist-induced MUC5AC protein expression in NCI-H292 cells [1]
We further investigated the effect of the dopamine D1 receptor agonist A68930 or dopamine on MUC5AC protein expression using an immunofluorescent assay. Consistent with the mRNA data of MUC5AC, MUC5AC protein expression in NCI-H292 cells were increased by dopamine (1 μM), A68930 (1 μM), and CSE (10%) (Fig. 6). These results suggest that activation of dopamine D1 receptor stimulates MUC5AC expression.

Western Blot analysis [1]
Cell Types: 16HBE14o- or NCI-H292 Cell
Tested Concentrations: 1 μM
Incubation Duration: 5~60 minutes
Experimental Results: CREB phosphorylation was Dramatically increased.

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RT-PCR[1]
Cell Types: NCI-H292 Cell
Tested Concentrations: 1 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Induced MUC5AC mRNA expression.

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Immunofluorescence [1]
Cell Types: NCI-H292 cell
Tested Concentrations: 1μM
Incubation Duration: 48 hrs (hours)
Experimental Results: MUC5AC mRNA data and MUC5AC protein expression increased.

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For analysis of CREB phosphorylation, 16HBE14o- cells or NCI-H292 cells were serum-starved for 24 h, and then treated with dopamine D1 receptor agonist A68930 (1 μM) for indicated times (5–60 min). In separate experiments, NCI-H292 cells were initially pretreated with 10 μM H89 (PKA inhibitor; 30 min) or 5 μM U0126 (MEK inhibitor; 120 min) before treatment of the cells with A68930 (1 μM; 20 min). After treatment, the cells were washed twice with ice-cold PBS, and lysed in ice-cold RIPA buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and a 1:200 dilution of protease inhibitor cocktail III. Each lysed cell sample was harvested and centrifuged at 15000 g for 15 min at 4 °C, and an aliquot of the supernatant was subjected to protein analysis. The protein concentration of each sample was determined using Pierce BCA reagents, using BSA as a control, and samples were stored at − 80 °C. [1]

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Cyclic AMP (cAMP) production in 16HBE14o- and NCI-H292 cell lines was measured using a HitHunter™ cAMP Assay for Small Molecules kit according to the manufacturer’s instructions. Briefly, the cells grown in white-walled 96-well plates and were washed twice with warm PBS (37 °C). The cells were incubated with dopamine (1 μM), or the dopamine D1-like receptor agonists (A68930 or SKF38393) (1 μM) for 20 min at 37 °C. The concentration and duration of the dopamine D1-like receptor agonists used in this study was determined based on previous studies. In separate experiments, the cells were pretreated with the dopamine D1-like receptor antagonists [SCH23390 (1 μM) or SCH39166 (1 μM)] or vehicle (PBS) for 30 min followed by incubation with A68930 (1 μM) for 20 min at 37 °C. Then the cAMP antibody reagent followed by the cAMP working solution (mixture of enzyme donor/lysis buffer/Emerald II/Galacton) was added to each well and incubated for 60 min at room temperature. Cells were further incubated with the enzyme acceptor reagent for 3 h at room temperature, and luminescence signals were detected using a multimode microplate reader. [1]

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Total RNA was extracted from cultured NCI-H292 cells after 48 h treatment with dopamine (1 μM), A68930 (1 μM), isoproterenol (1 μM), or CSE (10%) using the RNeasy Mini Kit. Total RNA was transcribed into cDNA using the ReverTra Ace qPCR RT Kit in accordance with the manufacturer’s instructions. Quantitative real-time PCR on the CFX96 Real-Time PCR Detection System was performed using Thunderbird SYBR qPCR kit according to manufacturer’s instruction. Primer sequences for MUC5AC and GAPDH were shown in Table 1. The specificity of amplification was confirmed by melting curve analysis. The Ct value determined by the CFX manager Software for all samples was normalized to the housekeeping gene GAPDH, and the relative fold induction against untreated controls was computed by the comparative Ct (ΔΔCt) method. [1]

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Immunofluorescence staining of MUC5AC protein in NCI-H292 cells was carried out according to the previously described method with some modifications. Briefly, NCI-H292 cells were seeded on an 8-chamber microscope slide and serum-starved for 24 h. After starvation, cells were exposed to A68930 (1 μM), dopamine (1 μM), or CSE (10%) for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed 3 times with PBS. After permeabilization (0.2% Triton X-100 in PBS for 5 min) and blocking (1% bovine serum albumin in 0.1% Triton X-100 in PBS for 15 min), cells were incubated with Alexa Fluor 488-conjugated MUC5AC antibody overnight at 4C. After cells were washed twice with PBS, the slide was coverslipped with ProLong gold antifade-reagent with DAPI, and visualized with an inverted fluorescent microscope. Digitized images were captured with MetaMorph software. When capturing the images, we kept constant the duration of image capture (300 ms), the image intensity gain, the image enhancement, and the image black level among the samples. [1]

[1]. The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium. Respir Res. 2018;19(1):53.

(1R,3S)-1-(aminomethyl)-3-phenyl-3,4-dihydro-1H-2-benzopyran-5,6-diol is a 2-benzopyran.

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Background: Dopamine receptors comprise two subgroups, Gs protein-coupled “D1-like” receptors (D1, D5) and Gicoupled “D2-like” receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression.
Methods: We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively.
Results: The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells.
Conclusions: These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms. [1]

C16H17NO3

271.31108

271.121

C, 70.83; H, 6.32; N, 5.16; O, 17.69

130465-45-1

A68930 hydrochloride;130465-39-3

122324

Typically exists as solid at room temperature

1.273g/cm3

469.5ºC at 760mmHg

237.8ºC

1.94E-09mmHg at 25°C

1.636

3.111

3

4

2

20

319

2

NCC1OC(C2C=CC=CC=2)CC2C(=C(C=CC1=2)O)O

SUHGRZPINGKYNV-GJZGRUSLSA-N

InChI=1S/C16H17NO3/c17-9-15-11-6-7-13(18)16(19)12(11)8-14(20-15)10-4-2-1-3-5-10/h1-7,14-15,18-19H,8-9,17H2/t14-,15-/m0/s1

(1R,3S)-1-(aminomethyl)-3-phenyl-3,4-dihydro-1H-isochromene-5,6-diol

A 68930; 130465-45-1; A68,930; A-70108; A-68,930; 5,6-Dihydroxy-3-phenyl-1-aminomethylisochroman; A 68930; A-70360; A70108; A68930; A-68930

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)