ChemiSCREEN™ Human Recombinant α1D Adrenergic Receptor Calcium-Optimized Stable Cell Line with N-terminal truncation

ChemiSCREEN™ Human Recombinant α1D Adrenergic Receptor Calcium-Optimized Stable Cell Line with N-terminal truncation

Chemicon (Millipore)

2 vials

Truncated human ADRA1D cDNA encoding α1D lacking residues 2-79

The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the α- and β-adrenoceptors (Bylund et al., 1994). The three members of the α₁ subclass of adrenoceptors, α1A, α1B and α1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue. The α1D adrenergic receptor mediates smooth muscle contraction in several tissues. In the vasculature, activation of α1D increases blood pressure (Tanoue et al., 2002; Hosoda et al., 2005). In the urinary tract, α1D promotes bladder contraction. Antagonists of α1 receptors are used to treat bladder outlet obstruction, and this effect is thought to be mediated by α1D (Chen et al., 2005). The α1D adrenergic receptors has a relatively long N-terminal extracellular domain, and truncation of this domain has been shown to increase expression of the receptor at the cell surface (Pupo et al., 2003). Millipore’s cloned human α1D -expressing cell line contains a version of α1D lacking residues 2-79. The cell line is made in the Chem-1 host, which supports high levels of recombinant α1D expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between α1D and its ligands.

Calcium flux assay, ligand binding assays

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Human

Table I. Comparison of EC50 values of α1D(Δ2-79)–expressing Chem-1 cells with values described in the literature.

ligand	assay	potency (nM)	Efficacy (% of maximum)	Reference
epinephrine	Calcium	EC50 = 4.8	100	Figure 1
norepinephrine	Calcium	EC50 = 2.3	94.9	Figure 1
cirazoline	Calcium	EC50 = 190	32	Figure 1
norepinephrine	Calcium	EC50 = 3.2	100	Horie et al., 1995
cirazoline	Calcium	EC50 = 240	47	Horie et al., 1995
BMY 7378	Calcium	IC50 = 3.5	N/A	Figure 2
prazocin	Calcium	IC50 = 1.4	N/A	Figure 2
BMY 7378	Radioligand binding	Ki = 2.5	N/A	Pupo et al., 2003
prazocin	Radioligand binding	Ki = 0.22	N/A	Pupo et al., 2003

Chemicon

Chem-1

Cells are frozen at 2 x 10⁶ cells/mL in 90% fetal bovine serum/10% DMSO. Cell line tests negative for mycoplasma.

alpha1D

non-peptide

1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen.

2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37ºC with 5% CO₂.

3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator.

4. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca⁺⁺ and Mg⁺⁺ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37ºC with 5% CO₂ until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Chem-1 Growth Media per 1 mL trypsin.

5.Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator.

6.Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Step 4). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10⁶ cells/mL in Chem-1 Freezing Media (cell densities of 2-10 x 10⁶ are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen.

7. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Chem-1 Plating Media for plating for calcium assay.

Adrenergic

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A

2 x 10⁶ cells/vial

ChemiSCREEN™ Human Recombinant α1D Adrenergic Receptor Calcium-Optimized Stable Cell Line with N-terminal truncation

NM_000678

Chem-1 Growth Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine (Millipore SLM-020-A)
10% heat-inactivated FBS
1x Nonessential amino acids (from 100x stock, Millipore TMS-001-C)
10mM HEPES (from 1 M HEPES, Millipore TMS-003-C)
1x Pen-Strep (from 100x stock, Millipore TMS-AB2-C)
250μg/mL Genetecin/G-418



Chem-1 Plating Media:
DMEM with 4.5 g/L glucose and 4 mM glutamine
10% heat-inactivated FBS
1x NEAA
10mM HEPES
1x Pen-Strep

Chem-1 Freezing Media:
90% heat-inactivated FBS
10% DMSO (cell culture grade)