Article Figures & Data
Figures
- Fig. 1.
Agonist-stimulated DMR responses in human SW480 colon carcinoma cells. Raw label-free DMR responses were measured for the nonselective AR agonist norepinephrine (A), the β-AR–selective agonist isoproterenol (B), the α2-AR–selective agonist clonidine (C), the α1-AR–selective agonist phenylephrine (D), and 5-hydroxytryptamine/5-HT (E). (F) Data were used to construct concentration–response curves to calculate agonist potencies and intrinsic activities for stimulating DMR responses at t = 60 minutes (listed in Table 1). Data are mean ± S.E.M. from three to four independent experiments performed with four replicates.
- Fig. 2.
Phenylephrine-stimulated SW480 DMR responses are blocked by α-AR antagonists. Label-free DMR responses were measured for the α1-AR–selective agonist phenylephrine in the absence and presence of (A) the β-AR antagonist propranolol, (B) the α2-AR antagonist rauwolscine, (C) the irreversible α1/α2-AR antagonist phenoxybenzamine, (D) or the competitive α1/α2-AR antagonist phentolamine. For each condition, concentration–response curves were constructed using DMR values obtained at t = 60 minutes, and used to calculate agonist potency. (E) Schild regression analysis of phentolamine affinity from data in (D). Data are mean ± S.E.M. from three independent experiments performed with four replicates.
- Fig. 3.
Phenylephrine-stimulated SW480 DMR responses are blocked with high affinity by α1-AR antagonists. Label-free DMR responses were measured for the α1-AR–selective agonist phenylephrine in the absence and presence of the α1-AR–selective antagonists (A) doxazosin, (C) terazosin, and (E) prazosin. For each condition, concentration–response curves were constructed using DMR values obtained at t = 60 minutes, from which agonist potencies were calculated for subsequent Schild regression analysis of affinity for doxazosin (B), terazosin (D), and prazosin (F). Data are mean ± S.E.M. from three independent experiments performed with four replicates.
- Fig. 4.
Phenylephrine-stimulated SW480 DMR responses are blocked with high affinity by the α1B-AR–selective antagonist cyclazosin. Label-free DMR responses were measured for the α1-AR–selective agonist phenylephrine in the absence and presence of the α1D-AR subtype-selective antagonist BMY7378 [(A) Schild regression analysis in (B)]; the α1A/D-AR subtype-selective antagonist tamsulosin (C); the α1A-AR subtype-selective antagonists 5-methylurapidil (D) and niguldipine (E); and the α1B-AR subtype-selective antagonist cyclazosin [(F) Schild regression analysis in (G)]. For each condition, concentration–response curves were constructed using DMR values obtained at t = 60 minutes. When appropriate, agonist potencies were calculated for subsequent Schild regression analysis of affinity. Data are mean ± S.E.M. from three independent experiments performed with four replicates.
- Fig. 5.
SW480 cells express high levels of α1B-AR mRNA and low densities of functional α1B-ARs. (A) Quantitative reverse-transcriptase polymerase chain reaction was performed on mRNA isolated from SW480 cell lysates using internal primers targeted to the α1A (ADRA1A), α1B (ADRA1B), α1D (ADRA1D), and β2 (ADRB2)-AR subtypes. Data were normalized as cycle threshold (CT) fold change relative to APRT mRNA levels and are as expressed as mean ± S.E.M. (n = 2 with three replicates). (B) Polyacrylamide gel electrophoresis of wild-type SW480 cell lysates (MOCK lane), or SW480 cell lysates following transfection with empty pSNAP vector (SNAP), and 2, 3, or 5 μg N-terminal SNAP-epitope–tagged α1B-AR cDNA. SNAP–α1B-AR protein bands are denoted with black arrow on right (76.6 kDa). (C) Saturation [3H]-prazosin radioligand-binding assays were performed on SW480 cell lysates transfected with empty pSNAP vector (black ▪) or 3 μg SNAP-α1B-AR cDNA (red ▪). Nonspecific binding was determined with 10 μM phentolamine. Data are the mean ± S.E.M. of three experiments with three replicates. (D) Phosphoinositol hydrolysis assays were performed on SW480 cells transfected with empty pSNAP vector or 3 μg SNAP–α1B-AR cDNA. Cells were preincubated with 1 μCi [3H]-myoinositol for 48 hours and treated with HBSS buffer or 100 μM phenylephrine for 1 hour. Data are expressed as the mean ± S.E.M. of three experiments performed in triplicate. **Student’s t test, P < 0.05. (E) Label-free DMR responses were measured for the α1-AR–selective agonist phenylephrine in SW480 cells transfected with empty pSNAP vector (black ▪) or 3 μg SNAP–α1B-AR cDNA (red ▪). Data are the mean ± S.E.M. of two experiments with four replicates.
- Fig. 6.
Stimulation of β-AR decreases cell proliferation and viability, and stimulation of α1B-AR increases cell viability of water soluble tetrazolium salt (SW480) cells. Changes in SW480 cell proliferation and viability in response to drug treatment were measured using bromodeoxyuridine and WST-1 assays, respectively. The β-AR–selective agonist isoproterenol produces concentration-dependent decreases in both cell proliferation and viability (A), whereas the α1-AR–selective agonist phenylephrine increases cell viability without affecting proliferation (B). (C) Phenylephrine-stimulated increases in cell viability (100 μM) were antagonized by 10-minute pretreatment with cyclazosin (CYS, 100 nM) terazosin (TRZ, 1 µM), phenoxybenzamine (PBZ, 300 nM), or phentolamine (PHN, 10 µM). Data are the mean ± S.E.M. of three experiments with four replicates. **P < 0.01 compared with vehicle-treated cells (control) (one-way analysis of variance with Dunnett’s post hoc test).
Tables
- TABLE 1
Pharmacological properties of GPCR agonists in SW480 cells
Agonist-stimulated DMR concentration–response curves were constructed for responses measured at endogenous receptors expressed in SW480 cells. Log molar agonist potency (as stated prior to pEC50) were calculated using the time at which peak DMR response was observed. Intrinsic activities (IA) were calculated by setting norepinephrine maximal DMR responses equal to 1 and then normalizing other observed agonist maximal DMR values to this value. All data were analyzed with GraphPad Prism using nonlinear regression curve analysis and are expressed as mean ± S.E.M. of three to four independent experiments performed with four replicates.
Agonist pEC50 EC50 (μM) Max (pm) IA Norepinephrine −5.96 ± 0.04 1.08 354.74 ± 4.72 1 Isoproterenol −4.27 ± 0.13 54.4 151.35 ± 7.66 0.43 Clonidine −6.52 ± 0.12 0.3 144.47 ± 13.2 0.41 Phenylephrine −5.40 ± 0.03 13.9 238.21 ± 4.52 0.67 5-HT ND ND ND ND IA, intrinsic activity; Max, maximum response observed in pm; ND, not determined.
- TABLE 2
Pharmacological values used for α1-AR subtype–selective antagonist Schild regression analyses
Phenylephrine-stimulated concentration–response curves were calculated for stimulating DMR responses in SW480 cells. Log molar agonist potencies (pEC50) were calculated using the time at which peak DMR response was observed in the absence and presence of various α-AR antagonists (concentration of antagonist used shown in brackets for each phenylephrine pEC50 value). Agonist potencies were subsequently used to calculate antagonist affinity and slope via Schild regression analysis. All data were analyzed with GraphPad Prism and are expressed as mean ± S.E.M. of two to four independent experiments performed with four replicates.
Antagonist Phenylephrine pEC50 ± S.E.M. Schild Analysis Control Dose 1 Dose 2 Dose 3 Dose 4 pA2 Slope Phentolamine −5.12 ± 0.03 −4.52 ± 0.03 (300 nM) −4.19 ± 0.04 (1 μM) −3.54 ± 0.06 (3 μM) −3.11 ± 0.1 (10 μM) −6.93 ± 0.51 1.03 ± 0.08 Doxazosin −5.56 ± 0.05 −5.24 ± 0.05 (10 nM) −5.23 ± 0.03 (30 nM) −4.86 ± 0.04 (100 nM) −4.07 ± 0.08 (300 nM) −7.76 ± 0.27 1.18 ± 0.34 Terazosin −5.28 ± 0.05 −4.98 ± 0.11 (100 nM) −4.52 ± 0.08 (300 nM) −4.06 ± 0.14 (1 μM) — −7.81 ± 0.25 0.93 ± 0.29 Prazosin −5.70 ± 0.06 −5.29 ± 0.05 (100 nM) −4.69 ± 0.05 (300 nM) −4.38 ± 0.07^^ (1 μM) — −7.47 ± 0.32 1.07 ± 0.21 Tamsulosin −5.43 ± 0.06 −4.87 ± 0.07 (300 nM) −4.54 ± 0.07 (1 μM) −4.38 ± 0.1 (3 μM) −4.05 ± 0.1 (10 μM) ND ND 5-Methylurapidil −5.45 ± 0.1 −5.17 ± 0.14 (300 nM) −4.84 ± 0.14 (1 μM) −4.89 ± 0.25 (3 μM) −4.21 ± 0.23 (10 μM) ND ND Niguldipine −5.65 ± 0.08 −5.08 ± 0.11 (3 μM) −3.82 ± 0.15 (10 μM) — — ND ND Cyclazosin −5.42 ± 0.11 −4.86 ± 0.12 (10 nM) −4.62 ± 0.09 (30 nM) −4.01 ± 0.11 (100 nM) — −8.37 ± 0.19 0.97 ± 0.21 BMY7378 −5.14 ± 0.03 −4.57 ± 0.05(300 nM) −4.13 ± 0.03 (1 μM) −3.57 ± 0.07 (3 μM) −3.21 ± 0.1 (10 μM) −6.87 ± 0.47 1.06 ± 0.01 ND, not determined.
^^ Indicates agonist concentration–response curve Hill slope is <1, as determined by nonlinear regression curve four-parameter variable slope analysis.
Data Supplement
Files in this Data Supplement:
- Supplemental Figure - Adrenergic receptor antagonist dynamic mass redistribution responses in human SW480 colon carcinoma cells.
