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Meeting Abstracts  |   August 2005
Fentanyl Attenuates α1B-Adrenoceptor–Mediated Pulmonary Artery Contraction
Author Affiliations & Notes
  • Ju-Tae Sohn, M.D.
    *
  • Xueqin Ding, M.D. Ph.D.
  • Daniel F. McCune, Ph.D.
  • Dianne M. Perez, Ph.D.
    §
  • Paul A. Murray, Ph.D.
  • * Associate Professor, Department of Anesthesia and Pain Medicine, Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju, Republic of Korea. † Research Fellow in Anesthesiology Research, ‡ Research Fellow in Molecular Cardiology, § Full Staff in Molecular Cardiology, ∥ Carl E. Wasmuth Endowed Chair and Director, Center for Anesthesiology Research, The Cleveland Clinic Foundation, Cleveland, Ohio.
Article Information
Meeting Abstracts   |   August 2005
Fentanyl Attenuates α1B-Adrenoceptor–Mediated Pulmonary Artery Contraction
Anesthesiology 8 2005, Vol.103, 327-334. doi:
Anesthesiology 8 2005, Vol.103, 327-334. doi:
INTRAVENOUS fentanyl has been used widely as a general anesthetic for patients undergoing cardiac surgery. Lower concentrations of fentanyl slightly decrease systemic blood pressure,1 whereas higher concentrations significantly decrease systemic blood pressure and peripheral vascular resistance.2 In laboratory studies of the systemic vasculature, fentanyl has been shown to relax isolated rabbit and rat aorta by an α-adrenoceptor–blocking action.3,4 However, the extent and mechanism of action by which fentanyl alters the pulmonary vascular response to α-adrenoceptor activation is entirely unknown. In the current study, we tested the hypothesis that fentanyl would attenuate the pulmonary vasoconstrictor response to the α1-adrenoceptor agonist phenylephrine by binding to α1adrenoceptors. Moreover, we investigated the specific α1-adrenoceptor subtypes that mediate the effects of fentanyl on phenylephrine contraction.
α1-Adrenoceptors are a heterogenous group of receptors that have been classified into three subtypes—α1A, α1B, and α1D—based on radioligand binding, molecular biology, and isolated tissue experiments.5 All three subtypes are expressed in vascular smooth muscle,6 including pulmonary vascular smooth muscle.7 These subtypes can also be identified with selective and nonselective antagonists. For example, 5-methylurapidil has a 50- to 100-fold higher affinity for the α1Asubtype, BMY 7378 has a 100-fold higher affinity for the α1Dsubtype, and chloroethylclonidine inactivates the α1Bsubtype preferentially, although at a lower rate it can inactivate the other two α1subtypes.8 In the current study, we used these antagonists to confirm the presence of these α1-adrenoceptor subtypes in pulmonary vascular smooth muscle. Moreover, we performed “receptor protection” studies to identify the α1-adrenoceptor subtype that mediates the effect of fentanyl on phenylephrine-induced contraction. Finally, we performed competitive binding studies in rat-1 fibroblasts that were stably transfected with human α1-adrenoceptor complementary DNAs (cDNAs) corresponding to the α1A, α1B, and α1Dsubtypes to assess the direct effect of fentanyl on these α1-adrenoceptor subtypes.
Materials and Methods
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of The Cleveland Clinic Foundation (Cleveland, Ohio).
Organ Chamber Experiments
Healthy mongrel dogs weighing 20–30 kg were anesthetized with intravenous pentobarbital sodium (30 mg/kg) and fentanyl citrate (15 μg/kg). After tracheal intubation, the lungs were mechanically ventilated. A catheter was placed in the femoral artery, and the dogs were exsanguinated by controlled hemorrhage. A left lateral thoracotomy was performed through the fifth intercostal space, and the heart was arrested with induced ventricular fibrillation. The heart and lungs were removed from the thorax en bloc  . Lower right and left intralobar pulmonary arteries were dissected free. Intralobar pulmonary arteries (2–4 mm ID) were carefully dissected and immersed in cold modified Krebs-Ringer's bicarbonate (KRB) solution composed of 118.3 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 2.5 mm CaCl2, 25 mm NaHCO3, 0.016 mm Ca-EDTA, and 11.1 mm glucose. The arteries were cleaned of connective tissue and cut into ring segments 4–5 mm in length. In all rings, the endothelium was removed by gently rubbing the intimal surface with a cotton swab. Removal of the endothelium was later verified by the absence of a vasorelaxant response to acetylcholine (10−6m).
Isometric Tension Experiments
Pulmonary arterial rings were vertically mounted between stainless steel hooks in organ chambers filled with 25 ml KRB solution (37°C) gassed with 95% oxygen and 5% carbon dioxide. One of the hooks was anchored, and the other was connected to a strain gauge (model FT03 force displacement transducer; Grass Instruments, West Warwick, RI) to measure isometric tension. The rings were stretched at 10-min intervals in increments of 1 g to achieve optimal resting tension. Optimal resting tension was defined as the minimum amount of stretch required to achieve the largest contractile response to 60 mm KCl and was determined to be 5 g for the size of arteries used in these experiments. After the rings had been stretched to their optimal resting tension, the contractile response to 60 mm KCl was measured. All rings were pretreated with the β-adrenoceptor antagonist propranolol (5 × 10−6m, incubated for 30 min) before phenylephrine administration to avoid any β-agonist effect of phenylephrine.
Effects of Fentanyl on Phenylephrine-induced Contraction
The effects of fentanyl (0.297 × 10−6, 7.86 × 10−6, 1.57 × 10−5m) on the phenylephrine concentration–response relation were assessed in size and positioned-matched pulmonary arterial rings. Fentanyl was added to the organ chambers 30 min before phenylephrine administration. The contractile responses to phenylephrine in fentanyl-pretreated rings were compared with responses in matched untreated rings.
Effects of α-Adrenoceptor Antagonists on Phenylephrine Contraction
To determine whether α2-adrenoceptor activation is involved in phenylephrine-induced contraction, pulmonary arterial rings were pretreated for 30 min with the α2-adrenoceptor antagonist rauwolscine (3 × 10−7m). The contractile responses to phenylephrine in rauwolscine-pretreated rings were compared with responses in matched untreated rings.
To assess the role of α1-adrenoceptor activation in phenylephrine contraction and to identify the α1-adrenoceptor subtypes involved in the response, pulmonary arterial rings were pretreated for 30 min with the following α1-adrenoceptor antagonists: the non–subtype-selective α1-adrenoceptor antagonist prazosin (3 × 10−10to 3 × 10−9m), the α1A-adrenoceptor–selective antagonist 5-methylurapidil (10−7, 3 × 10−7, 10−6m), the α1B-adrenoceptor–selective alkylating agent chloroethylclonidine (10−6, 2 × 10−5, 10−4m), and the α1D-adrenoceptor–selective antagonist BMY 7378 (10−7, 3 × 10−7, 10−6m). Contractile responses to phenylephrine in antagonist-pretreated rings were compared with responses in matched untreated rings. In experiments with the irreversible α1B-adrenoceptor–selective alkylating agent chloroethylclonidine, the antagonist was incubated for 30 min at 37°C, followed by six successive washouts with fresh KRB to remove chloroethylclonidine hydrolysis products. The phenylephrine concentration–response relation was then measured.
Interaction between Fentanyl and Chloroethylclonidine
To determine whether fentanyl-induced changes in phenylephrine contraction involve α1B-adrenoceptors, the rings were pretreated with fentanyl (7.86 × 10−6m), followed by a 30-min treatment with chloroethylclonidine (2 × 10−5m). Non–fentanyl-treated rings were suspended for 30 min in KRB and then were treated for 30 min with chloroethylclonidine (2 × 10−5m). After 30 min of exposure to chloroethylclonidine, all rings were repeatedly washed with fresh KRB to remove chloroethylclonidine hydrolysis products as described above, and concentration–response curves for phenylephrine were then measured.
Competition Binding Studies for Fentanyl
Rat-1 fibroblasts were stably transfected with human α1-adrenoceptor cDNA corresponding to the α1A-, α1B-, or α1D-adrenoceptor subtypes (gift from GlaxoSmithKline, Uxbridge, United Kingdom). These cells were derived from clonal isolates. Cells were propagated in 75-cm2flasks in a humidified atmosphere (37°C) in Dulbecco's Modified Eagle Medium containing 5% fetal bovine serum, 10 U/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml of the selection antibiotic G418. Culture plates were scraped, and the stably transfected rat-1 fibroblast cells were collected in a sterile conical tube containing Hank's balanced salt solution. The cells were pelleted and then resuspended in a 5-ml volume of 0.25 m sucrose. After further centrifugation, the cells were resuspended in 10 ml of water containing protease inhibitors (leupeptin, phenylmethylmethylsulfonylfluoride, bacitracin, and benzamidine) and frozen at −70°C for 30 min. Cell membranes, disrupted by freezing and the hypotonic action of water, were processed further by Dounce homogenization. Nuclear debris was removed by pelleting under low-speed centrifugation (30,000g  for 15 min). Membranes were washed further by repeating the above step. Membranes were finally resuspended in HEM buffer (20 mm HEPES, pH 7.4, 1.4 mm EGTA, 12.5 mm MgCl2) containing 10% glycerol, aliquoted and stored at −70°C until use. Competition binding studies were performed to determine the affinity with which fentanyl inhibits the binding of the nonselective α1-adrenoceptor antagonist 125I-HEAT to the three α1-adrenoceptor subtypes. Nonspecific binding of 125I-HEAT was accounted for by the addition of 100 μm phentolamine and subtracted from total binding to achieve specific binding. Competition binding isotherms were generated by incubating a Ki (a measure of affinity of a ligand for a receptor) concentration of 125I-HEAT with a fixed amount of stably transfected rat-1 fibroblast cell membranes together with various concentrations of fentanyl. All incubations were performed at 22°C in HEM buffer, in a total volume of 0.5 ml. Upon reaching steady state (approximately 60 min), bound radioactivity was separated from unbound by vacuum filtration through Whatman GF/C filter paper (Whatman Inc., Florham Park, NJ) using a Brandel Cell Harvestor (Brandel, Gaithersburg, MD). Filters were quickly washed with 20 ml ice-cold HEM buffer to remove further unbound radioligand. Bound radioactivity was counted on a gamma counter operating at 79.8% efficiency.
Drugs and Solutions
The following drugs and chemicals were used: phenylephrine, acetylcholine, propranolol, prazosin, phentolamine (Sigma Chemical, St. Louis, MO); 5-methylurapidil, chloroethylclonidine, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride), rauwolscine (Research BioChemical Incorporated, Natick, MA); fentanyl citrate (Abbott Laboratories, Abbott Park, IL); and 125I-HEAT (Amersham Bioscience, Chicago, IL). All concentrations are expressed as the final molar concentration in the organ chamber. All drugs were dissolved in distilled water.
Data Analyses
Values are expressed as mean ± SEM. Contractile responses to phenylephrine were calculated as a percentage of the maximum contractile response to 60 mm KCl.
The effects of fentanyl and the α-adrenoceptor antagonists on the phenylephrine concentration–response relation were assessed by calculating the phenylephrine concentrations that produced 50% of the maximal contractile response to 60 mm KCl (phenylephrine ED50) using Graph Pad Prism version 3.0 (Graph Pad Software Inc., San Diego, CA). The phenylephrine ED50values in the presence or absence of the antagonists were used to calculate the concentration ratio (CR). The pA2values represent the concentration of the antagonists necessary to displace the concentration–response curve of the agonist by twofold. The pA2values (−log M) were calculated by linear regression using Graph Pad Prism and were obtained from the x-intercept of the plot of log (CR-1) against log molar antagonist concentration, where the slope was not different from unity.9 
Competition binding data were analyzed by nonlinear regression using a noniterative curve-fitting program (GraphPad Prism). The best fit of the data was determined by comparison of the least squares value resulting from one- and two-site competition models in the F test from three to four individual experiments. The data fit best to the one-site competition model using the algorithm y = Bottom + (Top − Bottom)/(1 + 10(X − LogEC50)).
Statistical analysis was performed using the Student t  test for paired comparisons. Two-way analysis of variance, followed by the Tukey post hoc  test, was used when more than two means were compared. A P  value less than 0.05 was considered a statistically significant change. N refers to the number of dogs from which pulmonary arterial rings were studied in the isometric tension experiments.
Results
Effect of Fentanyl on Phenylephrine Concentration–Response Relation
We tested the hypothesis that fentanyl attenuates phenylephrine-induced contraction. Fentanyl alone had no effect on resting tension. Compared with the control condition, fentanyl caused a dose-dependent rightward shift (P  < 0.05) in the phenylephrine concentration–response relation (fig. 1), i.e.  , fentanyl attenuated phenylephrine contraction in canine pulmonary artery.
Fig. 1. Effect of fentanyl on phenylephrine concentration–response curve in endothelium–denuded pulmonary arterial rings. Fentanyl caused a rightward shift in the phenylephrine concentration–response curve compared with control. Values represent mean ± SEM in all figures. n = 6. * Statistically significant increase (  P  < 0.05) in the phenylephrine ED50values after fentanyl treatment compared with control  .
Fig. 1. Effect of fentanyl on phenylephrine concentration–response curve in endothelium–denuded pulmonary arterial rings. Fentanyl caused a rightward shift in the phenylephrine concentration–response curve compared with control. Values represent mean ± SEM in all figures. n = 6. * Statistically significant increase (  P  < 0.05) in the phenylephrine ED50values after fentanyl treatment compared with control 
	.
Fig. 1. Effect of fentanyl on phenylephrine concentration–response curve in endothelium–denuded pulmonary arterial rings. Fentanyl caused a rightward shift in the phenylephrine concentration–response curve compared with control. Values represent mean ± SEM in all figures. n = 6. * Statistically significant increase (  P  < 0.05) in the phenylephrine ED50values after fentanyl treatment compared with control  .
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Effect of Rauwolscine on Phenylephrine-induced Contraction
The selective α2-adrenoceptor antagonist rauwolscine had no effect on resting tension. Pretreatment of rings with rauwolscine had no effect on phenylephrine-induced contraction (fig. 2), which indicates that α2-adrenoceptor activation is not involved in phenylephrine contraction in canine pulmonary artery.
Fig. 2. Effect of the α2-adrenoceptor antagonist rauwolscine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Rauwolscine had no effect on phenylephrine-induced contraction. n = 6  .
Fig. 2. Effect of the α2-adrenoceptor antagonist rauwolscine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Rauwolscine had no effect on phenylephrine-induced contraction. n = 6 
	.
Fig. 2. Effect of the α2-adrenoceptor antagonist rauwolscine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Rauwolscine had no effect on phenylephrine-induced contraction. n = 6  .
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Effect of Prazosin on Phenylephrine Concentration–Response Relation
The selective α1-adrenoceptor antagonist prazosin had no effect on resting tension. However, prazosin caused a rightward shift (P  < 0.05) in the phenylephrine concentration–response curve (fig. 3A). The slope (0.905 ± 0.181) of the Arunlakshana and Schild plot for prazosin (fig. 3B) did not differ significantly from unity, and the pA2value for prazosin was 9.286 ± 0.095. This pA2value confirms that phenylephrine-induced contraction is mediated by α1-adrenoceptors in canine pulmonary artery.10 
Fig. 3. (  A  ) Effect of the α1-adrenoceptor antagonist prazosin on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Prazosin caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 5. (  B  ) Arunlakshana and Schild plot for prazosin was constructed using the phenylephrine concentration ratio (CR; phenylephrine ED50in presence and absence of prazosin) for individual experiments  . r  2= 0.625  .
Fig. 3. (  A  ) Effect of the α1-adrenoceptor antagonist prazosin on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Prazosin caused a parallel rightward shift ( 
	*P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 5. (  B  ) Arunlakshana and Schild plot for prazosin was constructed using the phenylephrine concentration ratio (CR; phenylephrine ED50in presence and absence of prazosin) for individual experiments 
	. r  2= 0.625 
	.
Fig. 3. (  A  ) Effect of the α1-adrenoceptor antagonist prazosin on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Prazosin caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 5. (  B  ) Arunlakshana and Schild plot for prazosin was constructed using the phenylephrine concentration ratio (CR; phenylephrine ED50in presence and absence of prazosin) for individual experiments  . r  2= 0.625  .
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Effect of 5-Methylurapidil on Phenylephrine Concentration–Response Relation
We investigated the role of the α1A-adrenoceptor subtype in phenylephrine-induced contraction. 5-Methylurapidil, which has a higher affinity for the α1A-adrenoceptor subtype, had no effect on resting tension. 5-Methylurapidil caused a parallel rightward shift (P  < 0.05) in the phenylephrine concentration–response curve (fig. 4A). The slope (0.972 ± 0.113) of the Arunlakshana and Schild plot for 5-methylurapidil (fig. 4B) did not differ significantly from unity. The pA2for 5-methylurapidil was 7.185 ± 0.099, approximately 100-fold lower than the reported negative logarithm of the inhibitory constant6 at α1Aadrenoceptors, which suggests that the α1A-adrenoceptor subtype does not play a primary role in phenylephrine-induced contraction in canine pulmonary artery.
Fig. 4. (  A  ) Effect of the α1A-adrenoceptor–selective antagonist 5-methylurapidil on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. 5-Methylurapidil caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for 5-methylurapidil  .r  2= 0.803. CR = concentration ratio  .
Fig. 4. (  A  ) Effect of the α1A-adrenoceptor–selective antagonist 5-methylurapidil on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. 5-Methylurapidil caused a parallel rightward shift ( 
	*P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for 5-methylurapidil 
	.r  2= 0.803. CR = concentration ratio 
	.
Fig. 4. (  A  ) Effect of the α1A-adrenoceptor–selective antagonist 5-methylurapidil on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. 5-Methylurapidil caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for 5-methylurapidil  .r  2= 0.803. CR = concentration ratio  .
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Effect of BMY 7378 on Phenylephrine Concentration–Response Relation
We investigated the role of the α1D-adrenoceptor subtype in phenylephrine-induced contraction. BMY 74378, which has a higher affinity for the α1D-adrenoceptor subtype, had no effect on resting tension. BMY 74378 caused a rightward shift (P  < 0.05) in the phenylephrine concentration–response curve (fig. 5A). The slope (0.919 ± 0.162) of the Arunlakshana and Schild plot (fig. 5B) did not differ significantly from unity, and the pA2value for BMY 7378 was 6.879 ± 0.098, approximately 100-fold lower than the reported pKi6 at α1Dadrenoceptors, which suggests that the α1D-adrenoceptor subtype does not play a primary role in phenylephrine-induced contraction in canine pulmonary artery.
Fig. 5. (  A  ) Effect of the α1D-adrenoceptor–selective antagonist BMY 7378 on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. BMY 7378 caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for BMY 7378.  r  2= 0.667. CR = concentration ratio  .
Fig. 5. (  A  ) Effect of the α1D-adrenoceptor–selective antagonist BMY 7378 on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. BMY 7378 caused a parallel rightward shift ( 
	*P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for BMY 7378.  r  2= 0.667. CR = concentration ratio 
	.
Fig. 5. (  A  ) Effect of the α1D-adrenoceptor–selective antagonist BMY 7378 on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. BMY 7378 caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for BMY 7378.  r  2= 0.667. CR = concentration ratio  .
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Effect of Chloroethylclonidine on Phenylephrine Concentration–Response Relation
We investigated the role of the α1B-adrenoceptor subtype in phenylephrine-induced contraction. Chloroethylclonidine, which selectively inactivates the α1B-adrenoceptor subtype, had no effect on resting tension. Chloroethylclonidine at 10−6m had no significant effect on the phenylephrine concentration–response curve. However, chloroethylclonidine at 2 × 10−5m significantly decreased (P  < 0.05) the maximal contractile response to phenylephrine (fig. 6), and chloroethylclonidine at 10−4m abolished phenylephrine-induced contraction. Together with the other subtype-selective antagonists, these results suggest that the α1B-adrenoceptor subtype plays a primary role in phenylephrine-induced contraction in canine pulmonary artery.
Fig. 6. Effect of the α1B-adrenoceptor alkylating agent chloroethylclonidine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. The maximal response to phenylephrine in rings treated with 2 × 10−5m chloroethylclonidine was significantly decreased (#  P  < 0.05  vs.  control), and the phenylephrine ED50was increased (  *P  < 0.05  vs.  control). Complete inhibition (#  P  < 0.05  vs.  control) of phenylephrine contraction was observed when rings were treated with 10−4m chloroethylclonidine. n = 6  .
Fig. 6. Effect of the α1B-adrenoceptor alkylating agent chloroethylclonidine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. The maximal response to phenylephrine in rings treated with 2 × 10−5m chloroethylclonidine was significantly decreased (#  P  < 0.05  vs.  control), and the phenylephrine ED50was increased ( 
	*P  < 0.05  vs.  control). Complete inhibition (#  P  < 0.05  vs.  control) of phenylephrine contraction was observed when rings were treated with 10−4m chloroethylclonidine. n = 6 
	.
Fig. 6. Effect of the α1B-adrenoceptor alkylating agent chloroethylclonidine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. The maximal response to phenylephrine in rings treated with 2 × 10−5m chloroethylclonidine was significantly decreased (#  P  < 0.05  vs.  control), and the phenylephrine ED50was increased (  *P  < 0.05  vs.  control). Complete inhibition (#  P  < 0.05  vs.  control) of phenylephrine contraction was observed when rings were treated with 10−4m chloroethylclonidine. n = 6  .
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Interaction between Fentanyl and Chloroethylclonidine
If fentanyl attenuates phenylephrine-induced contraction via  an effect on the α1B-adrenoceptor subtype, pretreatment with fentanyl before the administration of chloroethylclonidine should “protect”α1Badrenoceptors from chloroethylclonidine and thereby potentiate phenylephrine contraction. As summarized in fig. 7, phenylephrine-induced contraction was potentiated (P  < 0.05) in fentanyl-pretreated rings exposed to 2 × 10−5m chloroethylclonidine compared with the response observed with chloroethylclonidine alone.
Fig. 7. Effect of fentanyl (7.86 × 10−6m) pretreatment on chloroethylclonidine (2 × 10−5m)–induced attenuation of phenylephrine contraction in endothelium-denuded pulmonary arterial rings. Rings pretreated with fentanyl before exposure to chloroethylclonidine exhibited a potentiated contractile response to phenylephrine (  *P  < 0.05, decrease in phenylephrine ED50, and #  P  < 0.05, increase in maximal contractile response) compared with rings exposed to chloroethylclonidine alone. n = 6  .
Fig. 7. Effect of fentanyl (7.86 × 10−6m) pretreatment on chloroethylclonidine (2 × 10−5m)–induced attenuation of phenylephrine contraction in endothelium-denuded pulmonary arterial rings. Rings pretreated with fentanyl before exposure to chloroethylclonidine exhibited a potentiated contractile response to phenylephrine ( 
	*P  < 0.05, decrease in phenylephrine ED50, and #  P  < 0.05, increase in maximal contractile response) compared with rings exposed to chloroethylclonidine alone. n = 6 
	.
Fig. 7. Effect of fentanyl (7.86 × 10−6m) pretreatment on chloroethylclonidine (2 × 10−5m)–induced attenuation of phenylephrine contraction in endothelium-denuded pulmonary arterial rings. Rings pretreated with fentanyl before exposure to chloroethylclonidine exhibited a potentiated contractile response to phenylephrine (  *P  < 0.05, decrease in phenylephrine ED50, and #  P  < 0.05, increase in maximal contractile response) compared with rings exposed to chloroethylclonidine alone. n = 6  .
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Effect of Fentanyl on α1A-, α1B-, and α1D-Adrenoceptor Binding
Because fentanyl can have effects at the α1Badrenoceptor through indirect mechanisms or cross-talk, we explored the possibility that fentanyl directly binds and occupies the activational pocket of α1-adrenoceptor subtypes. Structural analysis of fentanyl reveals that it has pharmacophores similar to both α1-adrenoceptor agonists and antagonists (fig. 8). Binding studies on cell membranes containing transfected individual α1-adrenoceptor subtypes would determine whether fentanyl was subtype selective without the confounding effects of multiple subtypes as found in native tissue. As summarized in figure 9, fentanyl can compete with 125I-HEAT binding at a single site of high affinity at each of the α1-adrenoceptor subtypes. pKi values were 5.92 ± 0.032, 5.28 ± 0.031, and 5.53 ± 0.036, respectively, for the α1B-, α1D-, and α1A-adrenoceptor subtypes, similar in potency to α1-adrenoceptor agonists. The Ki of fentanyl for the α1Badrenoceptor was approximately fivefold higher than that of the α1Dadrenoceptor (P  < 0.05) but was not significantly different from that of the α1Aadrenoceptor.
Fig. 8. Chemical structures for the α1A-adrenoceptor–selective antagonist 5-methylurapidil, the nonselective α1-adrenoceptor full agonist phenylephrine, and fentanyl. Note that fentanyl contains pharmacophores similar to both α1-adrenoceptor agonists and antagonists  .
Fig. 8. Chemical structures for the α1A-adrenoceptor–selective antagonist 5-methylurapidil, the nonselective α1-adrenoceptor full agonist phenylephrine, and fentanyl. Note that fentanyl contains pharmacophores similar to both α1-adrenoceptor agonists and antagonists 
	.
Fig. 8. Chemical structures for the α1A-adrenoceptor–selective antagonist 5-methylurapidil, the nonselective α1-adrenoceptor full agonist phenylephrine, and fentanyl. Note that fentanyl contains pharmacophores similar to both α1-adrenoceptor agonists and antagonists  .
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Fig. 9. Effect of fentanyl on displaced α1-adrenoceptor nonselective radiolabeled antagonist 125I-HEAT binding sites in competition binding assays. The negative logarithm of the inhibitory constant for the α1B-adrenoceptor subtype was fivefold higher (  *P  < 0.05) than the α1D-adrenoceptor subtype but not significantly different at the α1A-adrenoceptor subtype. B/Bo = % specific binding  .
Fig. 9. Effect of fentanyl on displaced α1-adrenoceptor nonselective radiolabeled antagonist 125I-HEAT binding sites in competition binding assays. The negative logarithm of the inhibitory constant for the α1B-adrenoceptor subtype was fivefold higher ( 
	*P  < 0.05) than the α1D-adrenoceptor subtype but not significantly different at the α1A-adrenoceptor subtype. B/Bo = % specific binding 
	.
Fig. 9. Effect of fentanyl on displaced α1-adrenoceptor nonselective radiolabeled antagonist 125I-HEAT binding sites in competition binding assays. The negative logarithm of the inhibitory constant for the α1B-adrenoceptor subtype was fivefold higher (  *P  < 0.05) than the α1D-adrenoceptor subtype but not significantly different at the α1A-adrenoceptor subtype. B/Bo = % specific binding  .
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Discussion
Our laboratory is systematically examining the effects of general anesthetics on vascular mechanisms that regulate the pulmonary circulation. If an anesthetic agent either directly alters the pulmonary vasculature or alters agonist-induced changes in pulmonary vasomotor tone, this could have either a beneficial or deleterious effect on right ventricular function. The major finding in the current study is that fentanyl attenuates the pulmonary vasoconstrictor response to α1-adrenoceptor activation via  a direct effect on the α1B-adrenoceptor subtype. All other things being equal, this effect would decrease right ventricular afterload in the setting of sympathetic nervous system activation, which could improve right ventricular function.
Molecular pharmacology studies in rats and humans have demonstrated the expression of three subtypes of α1-adrenoceptors (α1A, α1B, and α1D).5 However, only one study has assessed the relative contributions of these α1-adrenoceptor subtypes to mediate phenylephrine-induced contraction in canine pulmonary artery7 and reported that only one subtype (likely to be α1B) had a high affinity for prazosin and was activated by phenylephrine. In the current study, pretreatment with the α1-adrenoceptor antagonist prazosin caused a rightward shift in the phenylephrine concentration–response curve, which confirms that phenylephrine-induced contraction in canine pulmonary artery is mediated by α1-adrenoceptor activation. Then, we investigated the contribution of each α1-adrenoceptor subtype to phenylephrine-induced contraction. 5-Methylurapidil is a selective α1A-adrenoceptor antagonist. The estimated affinity of 5-methylurpidil for the α1Aadrenoceptor is approximately 50 times greater than for the α1Dadrenoceptor and 100 times greater than for the α1Badrenoceptor.11,12 In the current study, the estimated affinity of 5-methylurapidil (pA2= 7.185 ± 0.099) was close to the value expected for an interaction at the cloned human α1Bor α1Dadrenoceptors rather than α1Aadrenoceptors.11,12 This suggests that phenylephrine contraction is not mediated by α1A-adrenoceptor activation in canine pulmonary artery.
To determine whether the phenylephrine-mediated contraction in canine pulmonary artery was the α1Bor the α1Dadrenoceptor, BMY 7378 was used. BMY 7378 is a selective α1D-adrenoceptor antagonist whose selectivity for α1Dadrenoceptors is 100-fold greater than α1Aor α1Badrenoceptors.13 In the current study, the estimated affinity of BMY 7378 was 6.879 ± 0.098, which is lower than the affinity reported for human recombinant α1Dadrenoceptors expressed in rat-1 fibroblast (9.39) or in rat aorta (8.88), a tissue that exhibits a contractile function mediated predominantly by the α1D-adrenoceptor subtype.14 The pKivalue reported for BMY 7378 for cloned human α1Badrenoceptors stably expressed in rat-1 fibroblast was 6.7 ± 0.11, which is close to the pA2value for BMY 7378 in the current study.15 Therefore, the inappropriately low affinity for BMY 7378 indicates that phenylephrine-induced contraction in canine pulmonary artery is not mediated by α1D-adrenoceptor activation.
Chloroethylclonidine is an irreversible antagonist that preferentially alkylates and completely inactivates α1Badrenoceptors, but it can also inactivate α1Aor α1Dadrenoceptors, depending on the treatment time and concentration used.8,11,12 Chloroethylclonidine can be used to confirm the functional presence of α1Badrenoceptors when used in conjunction with other more selective antagonists. In the current study, chloroethylclonidine at 10−6m had no effect on the phenylephrine concentration–response curve. However, chloroethylclonidine at 2 × 10−5m caused significant inhibition of phenylephrine contraction, and chloroethylclonidine at 10−4m completely abolished phenylephrine contraction. Taken together with the low affinity of BMY 7378 and 5-methylurapidil for α1Badrenoceptors noted above, our results indicate that phenylephrine-induced contraction is primarily mediated by α1Badrenoceptors in canine pulmonary artery. Chloroethylclonidine has been reported to cause concentration-dependent contraction in dog saphenous vein by stimulating α2adrenoceptors.16 However, the α2-adrenoceptor antagonist rauwolscine had no effect on phenylephrine contraction in the current study, and treatment with chloroethylclonidine alone did not cause contraction. These results confirm the absence of functional α2adrenoceptors in canine pulmonary artery.
Fentanyl has been reported to attenuate phenylephrine contraction in isolated rabbit and rat aorta via  antagonism of α1adrenoceptors.3,4 However, the α1-adrenoceptor subtypes involved in the fentanyl-induced attenuation of phenylephrine contraction have not been identified. We also observed that fentanyl attenuated phenylephrine contraction. Moreover, preincubation with fentanyl before exposure to chloroethylclonidine produced greater maximal phenylephrine-induced contraction compared with the response observed in rings pretreated with chloroethylclonidine alone, i.e.  , fentanyl effectively protected α1Badrenoceptors from the effects of chloroethylclonidine. These results suggest that fentanyl inhibits α1Badrenoceptors in canine pulmonary artery by directly competing with chloroethylclonidine in the α1-adrenoceptor activation pocket.
Fentanyl is a potent analgesic of the 4-anilidopiperidine class of opioids. It is a cationic selective full agonist at μ-opioid receptors.17 As illustrated in figure 8, fentanyl is composed of both an α1-adrenoceptor agonist functional group (pharmacophore) and an antagonist pharmacophore, suggesting that fentanyl should also bind and block the α1-adrenoceptor activation pocket. In fact, common functional pharmacophores do exist between α1adrenoceptors and opioids, because metabolites of some α1-adrenoceptor antagonists form agonists at μ-opioid receptors.18 Many ligands of the rhodopsin class of biogenic amine receptor family (i.e.  , adrenergic, dopamine, serotonin, histamine) have similar binding pockets and can cross-activate or antagonize. This binding pocket similarity is also true of the μ-opioid receptor and the rhodopsin class of biogenic amine receptors.19,20 We found that fentanyl can bind and compete for the activational binding pocket for all three α1-adrenoceptor subtypes (fig. 9). The pKi values are similar to those of other α1-adrenoceptor ligands. There was significant fivefold selectivity of fentanyl for the α1Badrenoceptor over the α1Dadrenoceptor.
Despite widespread distribution of the three α1-adrenoceptor subtypes in many tissues, the α1Badrenoceptor has not been linked to the activation of contraction in any of the systemic arteries in which it is expressed.21 This has led to a theory that an α1adrenoceptor can be expressed but cannot participate in contractile regulation, but may regulate other important functions in arterial smooth muscle (i.e.  , growth). Our study is the first report that the α1Badrenoceptor can predominantly regulate the contraction of pulmonary arterial smooth muscle. In contrast, the α1Badrenoceptor is known to regulate venule contraction,22 perhaps because of the higher receptor reserve in veins than in arteries.23 Two studies have indicated that both the α1Aand α1Badrenoceptors regulate the human saphenous and umbilical veins.24,25 The pulmonary artery shares functional similarity with veins in that both are low-pressure systems with relatively low blood oxygenation, which may influence the regulation of genes in those vessels. In fact, hypoxia is known to increase the expression of the α1Badrenoceptor, which contains a hypoxia regulatory element on the α1B-adrenoceptor promoter.26 
Concentrations of fentanyl used in this study are higher than typically encountered in clinical settings. However, rapid redistribution of fentanyl (octanol:water partition coefficient = 813)27 to lipid-rich tissue, which also contain lipophilic G protein–coupled receptors, may create a discrepancy between serum concentration and actual tissue concentration. Because cerebrospinal fluid contains very little protein compared with plasma,28 the active fentanyl concentration in cerebrospinal fluid averages approximately 46% of total plasma fentanyl concentration, which is more than twice the free fraction of plasma fentanyl.29 Moreover, small changes in the amount or binding capacity of proteins in certain pathologic conditions (e.g.  , liver disease, hemodilution, hypoproteinemia) could result in an increase in the free fraction of fentanyl. Because fentanyl is highly lipophilic, we believe the fentanyl concentrations (7.86 × 10−6, 1.57 × 10−5m) used in this in vitro  experiment are justified by the fact that fentanyl (6.9 ng/ml) in human blood corresponds to the fentanyl concentration (2.96 × 10−3m) calculated in the brain lipid.30 
In summary, our results indicate that phenylephrine-induced contraction in canine pulmonary artery is mediated primarily by α1Badrenoceptors. Fentanyl attenuates phenylephrine contraction by binding to and directly inhibiting α1Badrenoceptors.
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Fig. 1. Effect of fentanyl on phenylephrine concentration–response curve in endothelium–denuded pulmonary arterial rings. Fentanyl caused a rightward shift in the phenylephrine concentration–response curve compared with control. Values represent mean ± SEM in all figures. n = 6. * Statistically significant increase (  P  < 0.05) in the phenylephrine ED50values after fentanyl treatment compared with control  .
Fig. 1. Effect of fentanyl on phenylephrine concentration–response curve in endothelium–denuded pulmonary arterial rings. Fentanyl caused a rightward shift in the phenylephrine concentration–response curve compared with control. Values represent mean ± SEM in all figures. n = 6. * Statistically significant increase (  P  < 0.05) in the phenylephrine ED50values after fentanyl treatment compared with control 
	.
Fig. 1. Effect of fentanyl on phenylephrine concentration–response curve in endothelium–denuded pulmonary arterial rings. Fentanyl caused a rightward shift in the phenylephrine concentration–response curve compared with control. Values represent mean ± SEM in all figures. n = 6. * Statistically significant increase (  P  < 0.05) in the phenylephrine ED50values after fentanyl treatment compared with control  .
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Fig. 2. Effect of the α2-adrenoceptor antagonist rauwolscine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Rauwolscine had no effect on phenylephrine-induced contraction. n = 6  .
Fig. 2. Effect of the α2-adrenoceptor antagonist rauwolscine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Rauwolscine had no effect on phenylephrine-induced contraction. n = 6 
	.
Fig. 2. Effect of the α2-adrenoceptor antagonist rauwolscine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Rauwolscine had no effect on phenylephrine-induced contraction. n = 6  .
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Fig. 3. (  A  ) Effect of the α1-adrenoceptor antagonist prazosin on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Prazosin caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 5. (  B  ) Arunlakshana and Schild plot for prazosin was constructed using the phenylephrine concentration ratio (CR; phenylephrine ED50in presence and absence of prazosin) for individual experiments  . r  2= 0.625  .
Fig. 3. (  A  ) Effect of the α1-adrenoceptor antagonist prazosin on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Prazosin caused a parallel rightward shift ( 
	*P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 5. (  B  ) Arunlakshana and Schild plot for prazosin was constructed using the phenylephrine concentration ratio (CR; phenylephrine ED50in presence and absence of prazosin) for individual experiments 
	. r  2= 0.625 
	.
Fig. 3. (  A  ) Effect of the α1-adrenoceptor antagonist prazosin on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. Prazosin caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 5. (  B  ) Arunlakshana and Schild plot for prazosin was constructed using the phenylephrine concentration ratio (CR; phenylephrine ED50in presence and absence of prazosin) for individual experiments  . r  2= 0.625  .
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Fig. 4. (  A  ) Effect of the α1A-adrenoceptor–selective antagonist 5-methylurapidil on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. 5-Methylurapidil caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for 5-methylurapidil  .r  2= 0.803. CR = concentration ratio  .
Fig. 4. (  A  ) Effect of the α1A-adrenoceptor–selective antagonist 5-methylurapidil on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. 5-Methylurapidil caused a parallel rightward shift ( 
	*P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for 5-methylurapidil 
	.r  2= 0.803. CR = concentration ratio 
	.
Fig. 4. (  A  ) Effect of the α1A-adrenoceptor–selective antagonist 5-methylurapidil on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. 5-Methylurapidil caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for 5-methylurapidil  .r  2= 0.803. CR = concentration ratio  .
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Fig. 5. (  A  ) Effect of the α1D-adrenoceptor–selective antagonist BMY 7378 on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. BMY 7378 caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for BMY 7378.  r  2= 0.667. CR = concentration ratio  .
Fig. 5. (  A  ) Effect of the α1D-adrenoceptor–selective antagonist BMY 7378 on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. BMY 7378 caused a parallel rightward shift ( 
	*P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for BMY 7378.  r  2= 0.667. CR = concentration ratio 
	.
Fig. 5. (  A  ) Effect of the α1D-adrenoceptor–selective antagonist BMY 7378 on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. BMY 7378 caused a parallel rightward shift (  *P  < 0.05, phenylephrine ED50  vs.  control) in the phenylephrine concentration–response curve. n = 6. (  B  ) Arunlakshana and Schild plot for BMY 7378.  r  2= 0.667. CR = concentration ratio  .
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Fig. 6. Effect of the α1B-adrenoceptor alkylating agent chloroethylclonidine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. The maximal response to phenylephrine in rings treated with 2 × 10−5m chloroethylclonidine was significantly decreased (#  P  < 0.05  vs.  control), and the phenylephrine ED50was increased (  *P  < 0.05  vs.  control). Complete inhibition (#  P  < 0.05  vs.  control) of phenylephrine contraction was observed when rings were treated with 10−4m chloroethylclonidine. n = 6  .
Fig. 6. Effect of the α1B-adrenoceptor alkylating agent chloroethylclonidine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. The maximal response to phenylephrine in rings treated with 2 × 10−5m chloroethylclonidine was significantly decreased (#  P  < 0.05  vs.  control), and the phenylephrine ED50was increased ( 
	*P  < 0.05  vs.  control). Complete inhibition (#  P  < 0.05  vs.  control) of phenylephrine contraction was observed when rings were treated with 10−4m chloroethylclonidine. n = 6 
	.
Fig. 6. Effect of the α1B-adrenoceptor alkylating agent chloroethylclonidine on phenylephrine concentration–response curve in endothelium-denuded pulmonary arterial rings. The maximal response to phenylephrine in rings treated with 2 × 10−5m chloroethylclonidine was significantly decreased (#  P  < 0.05  vs.  control), and the phenylephrine ED50was increased (  *P  < 0.05  vs.  control). Complete inhibition (#  P  < 0.05  vs.  control) of phenylephrine contraction was observed when rings were treated with 10−4m chloroethylclonidine. n = 6  .
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Fig. 7. Effect of fentanyl (7.86 × 10−6m) pretreatment on chloroethylclonidine (2 × 10−5m)–induced attenuation of phenylephrine contraction in endothelium-denuded pulmonary arterial rings. Rings pretreated with fentanyl before exposure to chloroethylclonidine exhibited a potentiated contractile response to phenylephrine (  *P  < 0.05, decrease in phenylephrine ED50, and #  P  < 0.05, increase in maximal contractile response) compared with rings exposed to chloroethylclonidine alone. n = 6  .
Fig. 7. Effect of fentanyl (7.86 × 10−6m) pretreatment on chloroethylclonidine (2 × 10−5m)–induced attenuation of phenylephrine contraction in endothelium-denuded pulmonary arterial rings. Rings pretreated with fentanyl before exposure to chloroethylclonidine exhibited a potentiated contractile response to phenylephrine ( 
	*P  < 0.05, decrease in phenylephrine ED50, and #  P  < 0.05, increase in maximal contractile response) compared with rings exposed to chloroethylclonidine alone. n = 6 
	.
Fig. 7. Effect of fentanyl (7.86 × 10−6m) pretreatment on chloroethylclonidine (2 × 10−5m)–induced attenuation of phenylephrine contraction in endothelium-denuded pulmonary arterial rings. Rings pretreated with fentanyl before exposure to chloroethylclonidine exhibited a potentiated contractile response to phenylephrine (  *P  < 0.05, decrease in phenylephrine ED50, and #  P  < 0.05, increase in maximal contractile response) compared with rings exposed to chloroethylclonidine alone. n = 6  .
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Fig. 8. Chemical structures for the α1A-adrenoceptor–selective antagonist 5-methylurapidil, the nonselective α1-adrenoceptor full agonist phenylephrine, and fentanyl. Note that fentanyl contains pharmacophores similar to both α1-adrenoceptor agonists and antagonists  .
Fig. 8. Chemical structures for the α1A-adrenoceptor–selective antagonist 5-methylurapidil, the nonselective α1-adrenoceptor full agonist phenylephrine, and fentanyl. Note that fentanyl contains pharmacophores similar to both α1-adrenoceptor agonists and antagonists 
	.
Fig. 8. Chemical structures for the α1A-adrenoceptor–selective antagonist 5-methylurapidil, the nonselective α1-adrenoceptor full agonist phenylephrine, and fentanyl. Note that fentanyl contains pharmacophores similar to both α1-adrenoceptor agonists and antagonists  .
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Fig. 9. Effect of fentanyl on displaced α1-adrenoceptor nonselective radiolabeled antagonist 125I-HEAT binding sites in competition binding assays. The negative logarithm of the inhibitory constant for the α1B-adrenoceptor subtype was fivefold higher (  *P  < 0.05) than the α1D-adrenoceptor subtype but not significantly different at the α1A-adrenoceptor subtype. B/Bo = % specific binding  .
Fig. 9. Effect of fentanyl on displaced α1-adrenoceptor nonselective radiolabeled antagonist 125I-HEAT binding sites in competition binding assays. The negative logarithm of the inhibitory constant for the α1B-adrenoceptor subtype was fivefold higher ( 
	*P  < 0.05) than the α1D-adrenoceptor subtype but not significantly different at the α1A-adrenoceptor subtype. B/Bo = % specific binding 
	.
Fig. 9. Effect of fentanyl on displaced α1-adrenoceptor nonselective radiolabeled antagonist 125I-HEAT binding sites in competition binding assays. The negative logarithm of the inhibitory constant for the α1B-adrenoceptor subtype was fivefold higher (  *P  < 0.05) than the α1D-adrenoceptor subtype but not significantly different at the α1A-adrenoceptor subtype. B/Bo = % specific binding  .
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