D1/dopamine receptor

(R)-SKF 38393. AtT-20 cells, endogenous GIRK currents produced by somatostatin or D3 dopamine receptors are blocked by hydroHClide ((±)-SKF-38393; 0-100 µM) with an IC50 of 268 nM[5]. In vitro activity: SKF-38393 hydrochloride causes a comparable alteration in cytomorphology and raises media cAMP levels[2].
? SKF-38393 hydrochloride (10 μmol/L; 1 hour) increases the threonine-phosphorylation of DA- and cAMP-regulated phosphoprotein of Mr. 32 kD (DARPP-32) in cultured GC cells[2].

---

The catecholamines norepinephrine and dopamine (DA) are present in the human ovary; in particular, in follicular fluid. Norepinephrine activates ovarian alpha- and beta-adrenergic receptors and modulates ovarian steroidogenesis, but the significance of ovarian DA is unclear. We examined whether a DA receptor of the D1-subtype (D1-R) is present in human ovary and in cultured human granulosa luteal cells (GC). Using RT-PCR, we cloned complementary DNAs from adult human ovarian and GC messenger RNAs, which are identical to the human D1-R sequence. In ovarian sections, D1-R protein was identified (by immunohistochemistry) in granulosa cells of large antral follicles, cells of the corpus luteum, as well as in cultured GC. An immunoreactive band of approximately Mr 50,000 was found in cultured luteinized GC using the same antiserum in Western blots. The D1-R in these cells was functional, because DA, alone or in the presence of the beta-receptor antagonist propranolol, caused cellular contraction. The selective D1-R agonist SKF-38393 induced a similar change in cytomorphology and increased the levels of media cAMP. SKF-38393 failed, however, to significantly affect basal and hCG-stimulated progesterone release in vitro, indicating that the activation of the D1-R was not directly linked to synthesis of progesterone, the major steroid of human GC. Estradiol synthesis likewise was not affected. Using RT-PCR and immunohistochemistry, we found that GC express DA- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), a protein typically associated with neurons bearing the D1-R. In cultured GC, DA and SKF-38393 induced increased threonine-phosphorylation of DARPP-32, even in the presence of propranolol but not in the presence of D1-R antagonist SCH-23390. Taken together, the presence of DA and a functional DA receptor and DARPP-32 indicate that a novel, physiological regulatory pathway involving DA exists in the human ovary [2].

SKF 38393(6 mg/kg, i.p.) prevents the scopolamine-induced impairment of performance of a T-maze working memory task. In adult male NMRI mice, SKF38393 (1 μg/mouse) impaires context-dependent fear learning.<
SKF-38393 hydrochloride reduces the dopamine depletion caused by MPTP[3].
SKF-38393 hydrochloride increases superoxide dismutase activity, imitating the effects of selegiline[3].
SKF-38393 hydrochloride increases the tetrodotoxin-resistant excitatory postsynaptic currents' frequency but not their amplitude, supporting a presynaptic locus of D1 action[4].

SKF 38393 hydrochloride is an agonist of D1 with IC50 of 110 nM.
Iodinated SCH 23390, 125I-SCH 23982 (DuPont-NEN), was examined using quantitative autoradiography for its potency, selectivity, and anatomical and neuronal localization of binding to the dopamine D1 receptor in rat brain sections. 125I-SCH 23982 bound to D1 sites in the basal ganglia with very high affinity (Kd values of 55-125 pM), specificity (70-85% of binding was displaced by 5 microM cis-flupenthixol), and in a saturable manner (Bmax values of 65-176 fmol/mg protein). Specific 125I-SCH 23982 binding was displaced by the selective D1 antagonists SCH 23390 (IC50 = 90 pM) and cis-flupenthixol (IC50 = 200 pM) and the D1 agonist SKF 38393 (IC50 = 110 nM) but not by D2-selective ligands (I-sulpiride, LY 171555) or the S2 antagonist cinanserin. Compared with 3H-SCH 23390, the 5- to 10-fold greater affinity for the D1 site and 50-fold greater specific radioactivity of 125I-SCH 23982 makes it an excellent radioligand for labeling the D1 receptor. The concentrations of D1 sites were greatest in the medial substantia nigra and exceeded by over 50% the concentration of D1 sites in the lateral substantia nigra, caudoputamen, nucleus accumbens, olfactory tubercle, and entopeduncular nucleus. Lower concentrations of D1 sites were present in the internal capsule, dorsomedial frontal cortex, claustrum, and layer 6 of the neocortex. D1 sites were absent in the ventral tegmental area. Intrastriatal injections of the axon-sparing neurotoxin, quinolinic acid, depleted by 87% and by 46-58% the concentrations of displaceable D1 sites in the ipsilateral caudoputamen and medial and central pars reticulata of the substantia nigra, respectively. No D1 sites were lost in the lateral substantia nigra. Destruction of up to 94% of the mesostriatal dopaminergic projection with 6-hydroxydopamine did not reduce D1 binding nor, with one exception, increase striatal or nigral D1 receptor concentrations. 125I-SCH 23982 selectively labels D1 binding sites on striatonigral neurons with picomolar affinity, and these neurons contain the majority of D1 sites in rat brain[1].

Cell Line: GC cells
Concentration: 10 μmol/L
Incubation Time: 1 hour
Result: Induced increased threonine-phosphorylation of DA- and cAMP-regulated phosphoprotein of Mr 32 kD (DARPP-32) in cultured GC cells.

---

Western blotting [2]
Western blotting was performed as previously described, with minor modifications. In brief, cells were harvested, frozen, thawed, homogenized in 62.5 mmol/L Tris-HCl buffer (pH 6.8) containing 10% sucrose and 2% SDS, sonicated, and heated (95 C for 5 min) in the presence of 10% mercaptoethanol. Samples (15 μg/lane) were separated electrophoretically on 10% or 12.5% SDS-polyacrylamide gels (SDS-PAGE). Proteins were transferred onto nitrocellulose membranes and probed with the same D1-R antiserum used for immunohistochemistry (1:1,000 dilution, incubation overnight at 4 C).

In addition, a well-characterized monoclonal phospho-DARPP-32 specific antibody was used (1:500) to examine whether treatment of GC (for 1 h, in 2 cases, also 24 h) with DA (1 and 10 μmol/L) or SKF-38393 (1 and 10 μmol/L, RBI, Biotrend, Cologne, Germany, diluted in medium without serum) changed the phosphorylation of DARPP-32. For control purposes, the β-receptor antagonist propranolol (10 μmol/L) or the D1-R antagonist SCH-23390 (10 μmol/L, RBI) were added to the cells treated with DA or SKF-38393 (used at 1 μm). Cell morphology was monitored and documented. Immunoreactivity was detected using peroxidase-labeled antisera (1:3,000) and enhanced chemiluminescence, as described. In some cases, the blots were digitized, and integrated optical densities of the bands were determined using an edited version of the program NIH Image, as described previously.

---

Immunoprecipitation experiments [2]
Immunoprecipitation experiments were carried out to examine whether SKF-38393 (100 μmol/L, diluted in medium without serum) treatment of cultured GC (1 day after isolation) for 1 h increased threonine phosphorylation of DARPP-32. Media were removed, and cells were solubilized in buffer, containing 10 mmol/L NaH2PO4, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X-100, 0.25% SDS, 1% sodium deoxycholate, and 2 mmol/L phenylmethanesulfonylfluoride. For immunoprecipitation, we used magnetic beads labeled with antimouse IgG and magnetic separation. The beads were first incubated with normal mouse serum (5% in PBS containing 10 mmol/L EGTA, 250 mmol/L saccharose, and 0.1% BSA) and were then labeled with 2 μL of the well-characterized monoclonal antibody directed against bovine DARPP-32, which recognizes primate DARPP-32 and which was used for immunocytochemistry, as well. Subsequently, beads were incubated with 150 μL GC cell lysate for 1 h at room temperature and then for 30 min at 4 C. After magnet separation, pellets were washed several times and used for SDS-PAGE, as described. Blots were developed using a monoclonal antibody against phospho-threonine (1:100); and, in some cases, they were evaluated densitometrically.

---

Progesterone and estradiol measurements [2]
The release of progesterone and estradiol into the culture medium by GC incubated for 6 h with SKF-38393 (10μ mol/L) in the absence or presence of hCG (10 IU/mL) was examined using triplicate wells for each treatment (n = 3). Samples were analyzed using commercial enzyme immunoassays, following the instructions of the manufacturer. Intraassay coefficients of variation ranged between 5–8%, and interassay coefficients of variation did not exceed 10%. All incubation and pipetting steps and the calculations of hormone concentrations were carried out in a fully automated immunodiagnos-tic analyzer. Results were corrected for small changes in cellular protein. ANOVA and t test were used to evaluate the results.

---

Determination of cAMP [2]
The levels of cAMP in the media of GC, 1 day after isolation, were examined after incubation for 3 or 6 h with SKF-38393 (1–100μ mol/L) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (1 mmol/L). In a pilot study, SKF-38393 (at a concentration of 1 μmol/L) caused a small, but not statistically significant, increase in cAMP (20% over basal levels). Therefore, for three independent additional experiments, a higher SKF-38393 concentration (100 μmol/L) was used. These samples were measured using an enzyme immunoassay (R&D Systems), according to the instructions of the manufacturer. The sensitivity of the assay was 0.5 pmol/mL, and the intraassay coefficient of variability was smaller than 10%. To correct for small differences in cell density, cAMP results were expressed per microgram of cellular protein. Student’s t test was used to evaluate data.

Balb/c mice were injected intraperitoneally with 5 or 10 mg/kg of SKF-38393 every 16 h with a final dose administered 30 min prior to administration of MPTP. Saline-injected but otherwise identically treated mice served as the control group. Animals were euthanized by decapitation in the morning in order to avoid diurnal variations of the endogenous levels of biogenic amines, enzymes, and antioxidant molecules. SN and NCP were micropunched and homogenized in 0.1 M phosphate buffer, pH 7.8, using a glass-teflon homogenizer. Tissue homogenates were centrifuged at 10 000×g for 60 min at 4°C. The supernatant obtained was assayed for GSH content and the activities of SOD and CAT.[3]

---

Dissolved in saline; 6 mg/kg; i.p. injection

Male Wistar rats

[1]. Picomolar affinity of 125I-SCH 23982 for D1 receptors in brain demonstrated with digital subtraction auto radiography. J Neurosci. 1987 Jan;7(1):213-222.

[2]. Functional Dopamine-1 Receptors and DARPP-32 Are Expressed in Human Ovary and Granulosa Luteal Cells in Vitro. J Clin Endocrinol Metab. 1999 Jan;84(1):257-64.

[3]. SKF-38393, a dopamine receptor agonist, attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity. Brain Res. 2001 Feb 23;892(2):241-7.

[4]. The D1 dopamine receptor agonist SKF-38393 stimulates the release of glutamate in the hippocampus. Neuroscience. 1999;94(4):1063-70.

[5]. Classic D1 dopamine receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) directly inhibits G protein-coupled inwardly rectifying potassium channels. Mol Pharmacol. 2002 Jul;62(1):119-26.

R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzozazepine hydrochloride (SCH23390) is a widely used and highly selective D1 dopamine receptor antagonist. In investigating the interactions between the D1 and D3 dopamine receptor signaling pathways, we found that SCH23390 and related compounds, in addition to acting as D1 receptor antagonists, also inhibit G protein-coupled inward rectifier potassium (GIRK) channels. Our evidence suggests that SCH23390 can block endogenous GIRK currents induced by somatostatin or D3 dopamine receptors in AtT-20 cells (IC50: 268 nM). This inhibition is receptor-independent, as constitutive GIRK currents are also blocked by SCH23390 in GIRK-only hamster ovary cells. Inhibition of GIRK channels exhibits selectivity, as closely associated Kir2.0 family inward rectifying potassium channels and various endogenous cation currents present in AtT-20 cells remain unaffected. Furthermore, in current-clamp recordings, SCH23390 depolarizes the membrane potential and induces action potentials in AtT-20 cells, suggesting that GIRK channel inhibition may have physiological significance. To determine the chemical signature leading to GIRK channel blockade, we tested several structure-related compounds [R(+)-SKF-38393A, R-(+)-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzozazepine hydrochloride (nor-methyl-SCH23390) and R-(+)-2,3,4,5-tetrahydro-8-iodo-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrochloride (iodo-SCH23390)], and the results showed that the halogen atom is crucial for GIRK channel blockade. In summary, our results suggest that SCH23390 and its related compounds may provide a basis for designing novel GIRK channel selective blockers. Perhaps more importantly, some studies that specifically use SCH23390 to explore D1 receptor function or as a D1 receptor in diagnosis may need to be re-evaluated based on these results [1].

---

A selective D1 dopamine receptor agonist primarily used as a research tool.

---

Quantitative autoradiography was used to detect SCH 23390 iodide and 125I-SCH 23982 (DuPont-NEN) to investigate their potency, selectivity, and anatomical and neuronal localization of their binding to dopamine D1 receptors in rat brain slices. 125I-SCH 23982 binds to D1 sites in the basal ganglia with very high affinity (Kd values of 55-125 pM), specificity (70-85% of the binding can be replaced by 5 μM cisfluperidine), and in a saturable manner (Bmax values of 65-176 fmol/mg protein). Selective D1 receptor antagonists SCH 23390 (IC50 = 90 pM) and cis-flupentixol (IC50 = 200 pM), as well as the D1 receptor agonist SKF-38393 (IC50 = 110 nM), can replace the specific binding of 125I-SCH 23982, but D2 receptor selective ligands (125I-sulpiride, LY 171555) or the S2 receptor antagonist cinascorbic acid do not have this effect. Compared with 3H-SCH 23390, 125I-SCH 23982 has a 5-10 times higher affinity for the D1 receptor and a 50-fold higher affinity for radioactivity, making it an excellent radioligand for labeling the D1 receptor. The concentration of D1 receptors was highest in the medial part of the substantia nigra, exceeding that in the lateral part of the substantia nigra, the putamen of the caudate nucleus, the nucleus accumbens, the olfactory tubercle, and the medial globus pallidus by more than 50%. The concentration of D1 receptors was lower in the internal capsule, the dorsomedial frontal cortex, the claustrum, and layer 6 of the neocortex. No D1 receptors were detected in the ventral tegmental area. Intrastriatal injection of the axonoprotective neurotoxin quinolinic acid reduced the concentration of displaceable D1 receptors in the ipsilateral putamen of the caudate nucleus and the medial and central parts of the substantia nigra reticularis by 87% and 46-58%, respectively. No reduction in D1 receptors was observed in the lateral part of the substantia nigra. Disruption of up to 94% of the midbrain striatal dopaminergic projections using 6-hydroxydopamine did not reduce D1 receptor binding, and with one exception, did not increase the concentration of D1 receptors in the striatum or substantia nigra. 125I-SCH 23982 selectively labels D1 receptor binding sites on striatal substantia nigra neurons with picomolar affinity, and these neurons contain most of the D1 receptor sites in the rat brain. [1] Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra striatum. Mitochondrial respiratory depression, the generation of hydroxyl radicals, and the weakening of free radical defense mechanisms leading to oxidative stress are considered to be associated with the degeneration of dopaminergic neurons. Animal models treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are effective experimental models of Parkinson's disease (PD), capable of showing most of the clinical features of the disease as well as the main biochemical and pathological symptoms. This study investigated the potential neuroprotective effect of the dopaminergic (D1) receptor agonist SKF-38393 HCl (SKF) on MPTP-induced dopaminergic neuronal damage. MPTP is converted to its neurotoxic metabolite 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase B (MAO-B), which is then taken up by dopaminergic neurons. SKF-38393 had no effect on total MAO-B or MAO-B in the striatum. SKF-38393 blocked MPTP-induced glutathione depletion and attenuated MPTP-induced dopamine depletion. In addition, it enhanced the activity of superoxide dismutase, thus mimicking the effect of selegiline. These results suggest that SKF-38393 may be an effective drug for the treatment of Parkinson's disease. [3] This study aimed to better evaluate the role of dopamine in exocytosis. Since direct activation of adenylate cyclase (e.g., using foscrixine) enhances neurotransmitter release, we were interested in whether activation of D1 dopamine receptors, which are positively coupled to adenylate cyclase, also regulates the molecular mechanisms of synaptic vesicle fusion and neurotransmitter release. To answer this question, we investigated the effect of the D1 dopamine receptor agonist SKF-38393 on spontaneous glutamate release from cultured rat hippocampal neurons. SKF-38393 enhanced the frequency of tetrodotoxin-resistant excitatory postsynaptic currents but not their amplitude, suggesting that the D1 receptor's site of action is presynaptic. This effect was blocked by the D1 dopamine receptor antagonist SCH-23390, as well as the protein kinase A inhibitors H-7 and Rp-cAMP, while pertussis toxin had no effect on the dopaminergic response. Furthermore, carbachol and ruthenium red also stimulated exocytosis but did not block the SKF-38393-induced regulation. These results indicate that SKF-38393 enhances glutamate release presynaptically through a pertussis toxin-insensitive and protein kinase A-dependent mechanism, which likely involves D1 dopamine receptors. Our results highlight the importance of protein kinase A as a potent regulator of synaptic transmission and suggest that high concentrations of dopamine can significantly enhance glutamate release in the hippocampus. [4]

C16H17NO2.HCL

291.77

291.103

C, 65.86; H, 6.22; Cl, 12.15; N, 4.80; O, 10.97

81702-42-3

SKF 38393 hydrochloride;62717-42-4; SKF 38393 hydrobromide; 20012-10-6; (R)-SKF 38393 hydrochloride; 81702-42-3; 62751-59-1 (R-isomer); 67287-49-4

6852375

Typically exists as solid at room temperature

3.506

4

3

1

20

291

1

OC1C(O)=CC2=C([C@@H](C3C=CC=CC=3)CNCC2)C=1.Cl

YEWHJCLOUYPAOH-PFEQFJNWSA-N

InChI=1S/C16H17NO2.ClH/c18-15-8-12-6-7-17-10-14(13(12)9-16(15)19)11-4-2-1-3-5-11;/h1-5,8-9,14,17-19H,6-7,10H2;1H/t14-;/m1./s1

(5R)-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol;hydrochloride

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)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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