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Pain Medicine  |   August 2000
Propofol Attenuates Acetylcholine-induced Pulmonary Vasorelaxation: Role of Nitric Oxide and Endothelium-derived Hyperpolarizing Factors
Author Affiliations & Notes
  • Mayumi Horibe, M.D.
    *
  • Koji Ogawa, M.D.
    *
  • Ju-Tae Sohn, M.D.
    *
  • Paul A. Murray, Ph.D.
  • *Research Fellow. †Carl E. Wasmuth Endowed Chair and Director.
Article Information
Pain Medicine
Pain Medicine   |   August 2000
Propofol Attenuates Acetylcholine-induced Pulmonary Vasorelaxation: Role of Nitric Oxide and Endothelium-derived Hyperpolarizing Factors
Anesthesiology 8 2000, Vol.93, 447-455. doi:
Anesthesiology 8 2000, Vol.93, 447-455. doi:
NITRIC oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factors (EDHFs) are the three primary mediators of endothelium-dependent vasodilation. 1,2 NO is produced by the l-arginine–NO synthase pathway, and prostacyclin is produced by the arachidonic acid–cyclooxygenase pathway. The chemical nature of the EDHFs has not been fully characterized, but increasing evidence suggests that one form of EDHF is a cytochrome P450-derived metabolite of arachidonic acid. 3–6 The pattern of endothelial dilator mediators depends on the nature of the endothelial stimulus. 7 Several different endothelium-derived mediators, acting alone or in synergy with other mediators, can be the target for inhibitory effects of anesthetic agents on endothelium-dependent vasodilation. 8,9 
We have observed in chronically instrumented dogs that the pulmonary vascular response to the endothelium-dependent vasodilator, acetylcholine, was attenuated during propofol anesthesia compared with the conscious state, whereas the response to another endothelium-dependent vasodilator (bradykinin), as well as the response to an endothelium-independent NO donor (proline/NO) was not altered during propofol anesthesia. The goal of the present study was to investigate the mechanism responsible for this selective effect of propofol on acetylcholine-induced pulmonary vasodilation. Specifically, we tested the hypothesis that propofol exerts its effect by inhibiting one or more of the endothelium-derived relaxing factors that mediate acetylcholine-induced pulmonary vasodilation.
Materials and Methods
All surgical procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee.
Organ Chamber Experiments
Healthy male mongrel dogs weighing 25–30 kg were anesthetized with pentobarbital sodium (30 mg/kg intravenously) and fentanyl citrate (15 μg/kg intravenously) and placed on positive-pressure ventilation. The blood volume was removed by controlled hemorrhage via  a femoral artery catheter, a left lateral thoracotomy was performed, and the dogs were euthanized with electrically induced ventricular fibrillation. The heart and lungs were removed en bloc  . Using aseptic technique, the right and left lower intralobar pulmonary arteries (2–4-mm ID) were dissected free and immersed in cold modified Krebs-Ringer bicarbonate solution of the following composition: 118.3 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 2.5 mm CaCl2, 25.0 mm NaHCO3, 0.016 mm CaEDTA, and 11.1 mm glucose. The arteries were cut into 0.5-cm-wide rings with care taken not to damage the endothelium. In some rings, the endothelium was intentionally removed by inserting forcep tips into the vessel lumen and rolling the rings over damp filter paper. Endothelial denudation was later confirmed by the absence of relaxation to acetylcholine (10−6m). The rings were suspended horizontally between two stainless steel stirrups in organ chambers filled with 25 ml modified Krebs-Ringer bicarbonate solution (37°C) gassed with 95% O2–5% CO2. One of the stirrups was anchored, and the other was connected to a strain gauge (Grass Model FT03, Quincy, MA) for measurement of isometric tension. The rings from the same relative anatomic locations in the right and left lungs were used as paired rings.
Experimental Protocols
Pulmonary arterial rings were stretched at 10-min intervals in increments of 0.5 g to reach optimal resting tone. Optimal resting tone is defined as the minimum level of stretch required to achieve the largest contractile response to KCl (40 mm) and was determined to be 5 g for these studies. After the rings had been stretched to their optimal resting tone, the contractile response to 60 mm KCl was measured. After washout of KCl from the organ chambers and the return of isometric tension to prestimulation values, a concentration–effect curve for the sympathetic α-adrenoreceptor agonist, phenylephrine, was performed in each ring. This was achieved by increasing the concentration of phenylephrine in half-log increments (10−8to 3 × 10−5m) after the response to each preceding concentration had reached a steady state. All rings were pretreated with the β-adrenoreceptor antagonist, propranolol (5 × 10−6m; incubated for 30 min) to inhibit the β-agonist effects of phenylephrine. After washout of phenylephrine from the organ chamber and return to baseline tension, the rings were again pretreated with propranolol and contracted to 50% of their maximal response to phenylephrine (ED50level of tension). When the contractile response was stabilized, concentration–response curves to the endothelial cell activators, acetylcholine, and bradykinin were generated. The rings were exposed to only one endothelial cell activator. Responses to SIN-1 (activates vascular smooth muscle guanylyl cyclase) and papaverine (nonspecific vasorelaxant) were measured in rings denuded of endothelium.
To identify the specific endothelium-derived mediators involved in the relaxation responses to acetylcholine, endothelium-intact rings were incubated with one or more of the following pharmacologic inhibitors: Nω-nitro-l-arginine methyl ester (l-NAME: 3 × 10−5m), an inhibitor of NO synthase; indomethacin (3 × 10−5m), an inhibitor of cyclooxygenase; and either clotrimazole (3 × 10−5m) or SKF 525A (3 × 10−5m), inhibitors of cytochrome P450. Rings were pretreated with these inhibitors, alone or in combination, for 30 min before contraction to the ED50level of tension with phenylephrine. The inhibitors remained in the bath solution for the duration of the experiment. Vasorelaxant responses to acetylcholine in inhibitor-treated rings were compared with responses in untreated rings that were size- and position-matched (right vs.  left lung lobe). None of the inhibitors had an effect on baseline tension.
The effects of propofol (10−6m to 10−4m) on the acetylcholine concentration–effect curve were assessed by comparing vasorelaxant responses to acetylcholine in rings with and without propofol pretreatment. Propofol was added to the organ bath 30 min before phenylephrine precontraction. The intralipid vehicle for propofol had no effect on acetylcholine-induced vasorelaxation. The effects of propofol (10−4m) on the bradykinin and SIN-1 concentration–effect curves were assessed in a similar manner. The effect of propofol (10−4m) on the NO-mediated component of acetylcholine-induced vasorelaxation was assessed in rings pretreated with the combined inhibitors of cyclooxygenase and cytochrome P450. The effect of propofol (10−4m) on the EDHF-mediated component of acetylcholine-induced vasorelaxation was assessed in rings pretreated with the combined inhibitors of cyclooxygenase and NO synthase.
Drugs and Solutions
All drugs were of the highest purity commercially available. The following drugs were obtained from Sigma Chemical (St. Louis, MO): acetylcholine chloride, bradykinin, clotrimazole, indomethacin, l-NAME, papaverine, phenylephrine, propranolol, SKF 525A (proadifen), and SIN-1 (3-morpholinosydnonimine). Propofol and the intralipid vehicle were obtained from the Cleveland Clinic Pharmacy (Cleveland, OH). All drug concentrations are expressed as the final molar concentration in the organ chamber. Stock solutions were prepared on the day of the experiment. Unless stated otherwise, drugs were dissolved in distilled H2O. Indomethacin was dissolved in a NaHCO3solution (final bath concentration of NaHCO3: 0.2 mm). Clotrimazole was dissolved in dimethyl sulfoxide followed by dilution in distilled H2O (final bath concentration of dimethyl sulfoxide: 0.00004% to 0.013% vol/vol). At these concentrations, the vehicles have no effect on isometric tension. 7 
Data Analysis
Values are expressed as mean ± SEM, and n equals the number of dogs from which pulmonary arterial rings were isolated. Vasorelaxant responses of the agonists are expressed as a percentage of phenylephrine precontraction. The effects of the antagonists on the agonist concentration–effect curves were evaluated by comparing the concentration of agonist causing 50% relaxation of the contraction to phenylephrine (inhibitory concentration: IC50). This value was interpolated from the linear portion of the agonist concentration–effect curve by regression analysis and is presented as log IC50. The effects of propofol and the antagonists on the maximum relaxant response (Rmax) to acetylcholine were also measured, with Rmax = 100% indicating complete reversal of phenylephrine contraction. Statistical analysis of the data was performed using the Student t  test for paired comparisons. When more than two mean values were compared, analysis of variance was used. Values were considered to be statistically different at P  < 0.05.
Results
Effect of Propofol on Pulmonary Vasorelaxation
The effects of propofol (10−6to 10−4m) on the acetylcholine concentration–effect curve are summarized in figure 1. The IC50and Rmax values are summarized in table 1. Low-dose propofol (10−6m) had no effect on acetylcholine-induced relaxation (fig. 1A), whereas 10−5.5m and higher doses caused dose-dependent rightward shifts in the acetylcholine concentration–effect curves (figs. 1B–1D). In contrast, propofol (10−4m) had no effect on endothelium-dependent relaxation induced by bradykinin (fig. 2A; IC50: control =−7.90 ± 0.06, propofol =−7.84 ± 0.06) or on endothelium-independent relaxation induced by SIN-1 (fig. 2B; IC50: control =−6.93 ± 0.04, propofol =−6.99 ± 0.13).
Fig. 1. Effect of propofol on the endothelium-dependent relaxation induced by acetylcholine in pulmonary arteries. Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−6m) had no effect (A  ), whereas propofol (10−5.5, 10−5, 10−4m) attenuated (P  < 0.05) vasorelaxation induced by acetylcholine (10−9to 10−5m, n = 8).
Fig. 1. Effect of propofol on the endothelium-dependent relaxation induced by acetylcholine in pulmonary arteries. Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−6m) had no effect (A 
	), whereas propofol (10−5.5, 10−5, 10−4m) attenuated (P 
	< 0.05) vasorelaxation induced by acetylcholine (10−9to 10−5m, n = 8).
Fig. 1. Effect of propofol on the endothelium-dependent relaxation induced by acetylcholine in pulmonary arteries. Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−6m) had no effect (A  ), whereas propofol (10−5.5, 10−5, 10−4m) attenuated (P  < 0.05) vasorelaxation induced by acetylcholine (10−9to 10−5m, n = 8).
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Table 1. Effect of Propofol on Acetylcholine-induced Relaxation
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Table 1. Effect of Propofol on Acetylcholine-induced Relaxation
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Fig. 2. Effect of propofol on the relaxation induced by bradykinin (A  ) and SIN-1 (B  ). Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−4m) had no effect on bradykinin- or SIN-1-induced vasorelaxation (n = 5).
Fig. 2. Effect of propofol on the relaxation induced by bradykinin (A 
	) and SIN-1 (B 
	). Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−4m) had no effect on bradykinin- or SIN-1-induced vasorelaxation (n = 5).
Fig. 2. Effect of propofol on the relaxation induced by bradykinin (A  ) and SIN-1 (B  ). Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−4m) had no effect on bradykinin- or SIN-1-induced vasorelaxation (n = 5).
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Effects of Nitric Oxide Synthase, Cyclooxygenase, and Cytochrome P450 Inhibition on Acetylcholine-induced Pulmonary Vasorelaxation
The effects of the individual inhibitors on the acetylcholine concentration–effect curves are summarized in figures 3 and 4. The IC50and Rmax values are summarized in table 2NO synthase inhibition with l-NAME caused a marked attenuation in the relaxation response to acetylcholine. The acetylcholine concentration–effect curve was rightward shifted (fig. 3A), and the Rmax value was decreased (table 2). Cyclooxygenase inhibition with indomethacin only attenuated the relaxant response to acetylcholine at high concentrations of the agonist (fig. 3B), with no effect on the IC50value and a decrease in Rmax (table 2). The cytochrome P450 inhibitors, SKF 525A and clotrimazole, each attenuated acetylcholine-induced relaxation. Both inhibitors caused rightward shifts in the acetylcholine concentration–effect curves (fig. 4), increased the IC50values, and decreased the Rmax values (table 2). Combined treatment with l-NAME, indomethacin, and clotrimazole abolished acetylcholine-induced relaxation (fig. 5).
Fig. 3. Effect of inhibitions of nitric oxide synthase (l-NAME, 3 × 10−5m;A  ) and cyclooxygenase (indomethacin, 3 × 10−5m;B  ) on vasorelaxation induced by acetylcholine in canine pulmonary arteries (n = 5). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM.
Fig. 3. Effect of inhibitions of nitric oxide synthase (l-NAME, 3 × 10−5m;A 
	) and cyclooxygenase (indomethacin, 3 × 10−5m;B 
	) on vasorelaxation induced by acetylcholine in canine pulmonary arteries (n = 5). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM.
Fig. 3. Effect of inhibitions of nitric oxide synthase (l-NAME, 3 × 10−5m;A  ) and cyclooxygenase (indomethacin, 3 × 10−5m;B  ) on vasorelaxation induced by acetylcholine in canine pulmonary arteries (n = 5). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM.
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Fig. 4. Effect of inhibition of cytochrome P450 monooxygenase (SKF 525A, 3 × 10−5m, A  ; and clotrimazole, 3 × 10−5m, B  ) on vasorelaxation induced by acetylcholine (n = 5).
Fig. 4. Effect of inhibition of cytochrome P450 monooxygenase (SKF 525A, 3 × 10−5m, A 
	; and clotrimazole, 3 × 10−5m, B 
	) on vasorelaxation induced by acetylcholine (n = 5).
Fig. 4. Effect of inhibition of cytochrome P450 monooxygenase (SKF 525A, 3 × 10−5m, A  ; and clotrimazole, 3 × 10−5m, B  ) on vasorelaxation induced by acetylcholine (n = 5).
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Table 2. Effects of Individual Inhibitors on Acetylcholine-induced Relaxation
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Table 2. Effects of Individual Inhibitors on Acetylcholine-induced Relaxation
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Fig. 5. Effect of inhibition of cytochrome P450 monooxygenase (clotrimazole) on l-NAME–indomethacin-resistant component of relaxation induced by acetylcholine (n = 5). Pretreatment with clotrimazole totally abolished acetylcholine-induced relaxation. L = l-NAME pretreated; I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 5. Effect of inhibition of cytochrome P450 monooxygenase (clotrimazole) on l-NAME–indomethacin-resistant component of relaxation induced by acetylcholine (n = 5). Pretreatment with clotrimazole totally abolished acetylcholine-induced relaxation. L = l-NAME pretreated; I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 5. Effect of inhibition of cytochrome P450 monooxygenase (clotrimazole) on l-NAME–indomethacin-resistant component of relaxation induced by acetylcholine (n = 5). Pretreatment with clotrimazole totally abolished acetylcholine-induced relaxation. L = l-NAME pretreated; I = indomethacin pretreated; CLT = clotrimazole pretreated.
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Effect of Propofol on Nitric Oxide–mediated and Endothelium-derived Hyperpolarizing Factor–mediated Acetylcholine-induced Vasorelaxation
The results summarized in figs. 3–5indicate that acetylcholine-induced relaxation is primarily mediated by NO and a P450 metabolite likely to be an EDHF, with only a small contribution from prostacyclin at high concentrations of acetylcholine. To examine the effects of propofol on the NO-mediated component of acetylcholine-induced relaxation, we performed acetylcholine concentration–effect studies after combined cytochrome P450 inhibition and cyclooxygenase inhibition. During these conditions, acetylcholine-induced relaxation is mediated by NO. As summarized in figure 6, combined inhibition caused a rightward shift in the acetylcholine concentration–effect curve. Propofol further attenuated this NO-mediated component of acetylcholine-induced relaxation (fig. 6and table 3). To examine the effects of propofol on the EDHF-mediated component of acetylcholine-induced relaxation, we performed acetylcholine concentration–effect studies after combined NO synthase and cyclooxygenase inhibition. During these conditions, acetylcholine-induced relaxation is mediated by EDHF. As summarized in figure 7, combined inhibition attenuated the relaxation response to acetylcholine. Propofol further reduced this EDHF-mediated component of acetylcholine-induced relaxation (fig. 7and table 3).
Fig. 6. Effect of propofol on the nitric oxide–mediated component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibition of the EDHF-mediated component (by SKF 525A, A  , or by clotrimazole, B  ). I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 6. Effect of propofol on the nitric oxide–mediated component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibition of the EDHF-mediated component (by SKF 525A, A 
	, or by clotrimazole, B 
	). I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 6. Effect of propofol on the nitric oxide–mediated component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibition of the EDHF-mediated component (by SKF 525A, A  , or by clotrimazole, B  ). I = indomethacin pretreated; CLT = clotrimazole pretreated.
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Table 3. Effects of Combined Inhibition on Acetylcholine-induced Relaxation
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Table 3. Effects of Combined Inhibition on Acetylcholine-induced Relaxation
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Fig. 7. Effect of propofol on the nitric oxide–insensitive component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibitions of nitric oxide–mediated component. L = l-NAME pretreated; I = indomethacin pretreated.
Fig. 7. Effect of propofol on the nitric oxide–insensitive component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibitions of nitric oxide–mediated component. L = l-NAME pretreated; I = indomethacin pretreated.
Fig. 7. Effect of propofol on the nitric oxide–insensitive component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibitions of nitric oxide–mediated component. L = l-NAME pretreated; I = indomethacin pretreated.
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Effects of Cytochrome P450 Inhibition on Pulmonary Vasorelaxation Induced by Bradykinin, SIN-1, and Papaverine
The effects of the cytochrome P450 inhibitors, SKF525A and clotrimazole, on the concentration–effect curves for bradykinin, SIN-1, and papaverine are summarized in figure 8and table 4. In endothelium-intact rings, both cytochrome P450 inhibitors attenuated the pulmonary vascular relaxant response to bradykinin, increasing the IC50value and decreasing Rmax. In endothelium-denuded rings, the EDHF inhibitors had no effect on the pulmonary vasorelaxant responses to SIN-1 (guanylyl cyclase activator) or papaverine (nonspecific vasorelaxant).
Fig. 8. Effect of cytochrome P450 inhibitors (SKF525A and clotrimazole, both 3 × 10−5m) on relaxation induced by bradykinin (endothelium intact) and by SIN-1 and papaverine (endothelium denuded). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM (n = 6).
Fig. 8. Effect of cytochrome P450 inhibitors (SKF525A and clotrimazole, both 3 × 10−5m) on relaxation induced by bradykinin (endothelium intact) and by SIN-1 and papaverine (endothelium denuded). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM (n = 6).
Fig. 8. Effect of cytochrome P450 inhibitors (SKF525A and clotrimazole, both 3 × 10−5m) on relaxation induced by bradykinin (endothelium intact) and by SIN-1 and papaverine (endothelium denuded). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM (n = 6).
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Table 4. Effect of Cytochrome P450 Inhibition on Relaxation Induced by Bradykinin, SIN-1, and Papaverine
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Table 4. Effect of Cytochrome P450 Inhibition on Relaxation Induced by Bradykinin, SIN-1, and Papaverine
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Discussion
We observed that propofol selectively attenuates the pulmonary vascular response to the endothelium-dependent vasodilator, acetylcholine, in chronically instrumented dogs. The goal of the present in vitro  study was to investigate the mechanism(s) responsible for this endothelial defect. Our results indicate that acetylcholine-induced relaxation in isolated canine pulmonary arterial rings is mediated primarily by NO and a metabolite of the cytochrome P450 pathway likely to be an EDHF. Propofol attenuates acetylcholine-induced pulmonary vasorelaxation by inhibiting both the NO- and EDHF-mediated components of the response.
Endothelium-dependent relaxation results from the release of multiple substances from the endothelium that decrease vascular smooth muscle tone. Three primary endothelium-derived relaxing factors have been identified: NO, prostacyclin, and EDHFs. 1,2,10 To evaluate the role of each mediator in acetylcholine-induced pulmonary vasorelaxation, we inhibited the production of each of these endothelium-derived relaxing factors. Cyclooxygenase inhibition with indomethacin only attenuated acetylcholine-induced relaxation at high concentrations of the agonist; therefore, prostacyclin does not appear to play a primary role in the response. In contrast, NO synthase inhibition with l-NAME inhibited acetylcholine-induced relaxation by 60–70%, and cytochrome P450 inhibition with clotrimazole or SKF525A inhibited relaxation by 30–40%. These results indicate that NO and EDHFs are the primary mediators of acetylcholine-induced relaxation in canine pulmonary arterial rings. This was confirmed by the observation that combined inhibition abolished the vasorelaxant response to acetylcholine.
Propofol inhibited acetylcholine-induced relaxation in a dose-dependent fashion. In contrast, propofol had no effect on the pulmonary vasorelaxant response to the guanylyl cyclase activator, SIN-1. These results clearly demonstrate that the inhibitory effect of propofol on acetylcholine-induced vasorelaxation is not the result of a defect in pulmonary vascular smooth muscle cyclic guanosine monophosphate production.
To determine whether propofol exerted its inhibitory effect on the NO-mediated component of acetylcholine-induced relaxation, we assessed the effects of propofol on the acetylcholine concentration–effect curve after combined inhibition of the cyclooxygenase and cytochrome P450 pathways. During these conditions, the vasorelaxant response to acetylcholine is mediated by NO and is abolished by NO synthase inhibition. Propofol attenuated, but did not abolish, the NO-mediated component of acetylcholine-induced relaxation, which indicates that propofol exerts a portion of its inhibitory effect on the endothelial signaling pathway for NO production.
To determine whether propofol exerted a generalized inhibitory effect on endothelium-dependent vasodilation, we assessed the effects of propofol on bradykinin-induced vasorelaxation. In canine pulmonary arterial rings, bradykinin-induced vasorelaxation is mediated by a synergistic interaction between NO and prostacyclin. 7 Propofol had no effect on the pulmonary vasorelaxant response to bradykinin. These results suggest that propofol exerts its effect by selectively inhibiting the signaling pathway for acetylcholine-induced NO production, rather than causing a generalized decrease in NO synthesis. The locus of dysfunction would appear to be upstream from NO synthase activity, perhaps involving an effect of propofol on the endothelial muscarinic receptor or the receptor–G-protein interaction. Propofol has been reported to inhibit the rat M1 muscarinic acetylcholine receptor and/or receptor–G-protein interaction in Xenopus  oocytes, 11 although there is a conflicting report. 12 Propofol has also been reported to inhibit neuronal nicotinic acetylcholine receptor-mediated signaling in Xenopus  13,14 oocytes and in a rat pheochromocytoma cell line. 15 The extent to which propofol alters muscarinic receptor function in endothelial cells has not been investigated.
To determine whether propofol exerted its inhibitory effect on the EDHF-mediated component of acetylcholine-induced relaxation, we assessed the effects of propofol on the acetylcholine concentration–effect curve after combined inhibition of the cyclooxygenase and NO synthase pathways. During these conditions, the vasorelaxant response to acetylcholine is mediated by EDHF and is abolished by cytochrome P450 inhibition. The chemical nature of EDHF has not been fully elucidated, and it is likely that there is more than one form of EDHF. 16–18 Recent evidence suggests that an EDHF may be a cytochrome P450 metabolite of arachidonic acid, 4–6 presumably an epoxyeicosatrienoic acid. 5,6,19,20 EDHFs are thought to hyperpolarize vascular smooth muscle cells by activating K+channels. 5,6,19,20 We used both SKF525A and clotrimazole as EDHF inhibitors. SKF525A is an intermediate metabolite of cytochrome P450, whereas clotrimazole directly binds to the cytochrome P450 monooxygenase to specifically inhibit the enzyme. Both of these cytochrome P450 inhibitors attenuated acetylcholine-induced relaxation by 30–40%, which led us to conclude that EDHFs mediate a component of the response. Propofol inhibited this EDHF-mediated component of acetylcholine-induced relaxation. Whether this inhibitory effect is the result of a decrease in the synthesis or activity of EDHFs remains to be elucidated.
The EDHF-mediated vasorelaxant response to acetylcholine has been reported to be attenuated by volatile anesthetics in rabbit carotid artery 21 and by etomidate and thiopental in human renal artery. 22 The importance of EDHFs as modulators of vasomotor tone increases as vessel size decreases. 5,16,23,24 Iranami et al.  23 postulated that the inhibitory effect of halothane on acetylcholine-induced relaxation was related to an effect on NO in rat aorta, whereas it was caused by effects on NO and EDHFs in rat mesenteric artery. Akata et al.  25 suggested that the relative importance of NO and EDHFs in acetylcholine-induced relaxation was dependent on the concentration of the agonist, with EDHFs playing a more prominent role at higher concentrations of acetylcholine. Loeb et al.  26 reported that isoflurane altered the balance between NO and EDHFs in the rat cremaster muscle microcirculation, decreasing the role of EDHFs but increasing the contribution of NO. Thus, the mechanism for anesthesia-induced inhibition of acetylcholine-induced relaxation may depend on vessel type and size.
Additional control experiments were performed to assess the specificity of the cytochrome P450 inhibitors. As previously noted, bradykinin-induced pulmonary vasorelaxation is mediated by a synergistic interaction between NO and prostacyclin that is adenosine triphosphate–sensitive potassium channel dependent (presumably involving an EDHF). As expected, both SKF525A and clotrimazole attenuated the pulmonary vasorelaxant response to bradykinin in endothelium intact rings. In contrast, neither inhibitor had a significant effect on the vasorelaxation responses to SIN-1 or papaverine in endothelium-denuded rings. Thus, the inhibitory effects of SKF525A and clotrimazole on bradykinin (and acetylcholine) relaxation are not caused by a nonspecific effect on pulmonary vascular smooth muscle vasorelaxant activity. The fact that the cytochrome P450 inhibitors attenuated the relaxant responses to acetylcholine and bradykinin, whereas propofol only attenuated the response to acetylcholine, may suggest that these agonists stimulate different forms of EDHF.
The plasma concentration of propofol required to prevent the response to a surgical stimulus is approximately 34 μm in humans and dogs. 27 Because more than 90% of propofol is bound to plasma proteins, the free concentration of propofol is estimated to be 3–10 μm. In our study we observed that 3–10-μm concentrations of propofol attenuated the vasorelaxant response to acetylcholine, although it is acknowledged that higher concentrations (100 μm) were used in some protocols.
In summary, our results indicate that propofol selectively inhibits both the NO- and EDHF-mediated components of acetylcholine-induced relaxation in canine pulmonary arterial rings. These effects are apparent over the full concentration range of acetylcholine.
References
Furchgott RF, Vanhoutte PM: Endothelium-derived relaxing and contracting factors. FASEB J 1989; 3:2007–18Furchgott, RF Vanhoutte, PM
Flavahan NA, Vanhoutte PM: Endothelial cell signaling and endothelial dysfunction. Am J Hypertens 1995; 8:28S–41SFlavahan, NA Vanhoutte, PM
Komori K, Suzuki H: Electrical responses of smooth muscle cells during cholinergic vasodilatation in the rabbit saphenous artery. Circ Res 1987; 61:586–93Komori, K Suzuki, H
Cowan CL, Cohen RA: Two mechanisms mediate the relaxation by bradykinin of pig coronary artery: NO-dependent and -independent responses. Am J Physiol 1991; 261:H830–5Cowan, CL Cohen, RA
Garland CJ, Plane F, Kemp BK, Cocks TM: Endothelium-dependent hyperpolarization: A role in the control of vascular tone. Trends Pharmacol Sci 1995; 16:23–30Garland, CJ Plane, F Kemp, BK Cocks, TM
Bauersachs J, Hecker M, Busse R: Display of the characteristics of endothelium-derived hyperpolarizing factor by a cytochrome P450-derived arachidonic metabolite in the coronary microcirculation. Br J Pharmacol 1994; 113:1548–53Bauersachs, J Hecker, M Busse, R
Gambone LM, Murray PA, Flavahan NA: Synergistic interaction between endothelium-derived NO and prostacyclin in pulmonary artery: Potential role for K+ATPchannels. Br J Pharmacol 1997; 121:271–9Gambone, LM Murray, PA Flavahan, NA
Johns RA: Endothelium, anesthetics, and vascular control. A nesthesiology 1993; 79:1381–91Johns, RA
Gambone LM, Murray PA, Flavahan NA: Isoflurane anesthesia attenuates endothelium-dependent pulmonary vasorelaxation by inhibiting the synergistic interaction between nitric oxide and prostacyclin. A nesthesiology 1997; 86:936–44Gambone, LM Murray, PA Flavahan, NA
Bolton TB, Lang RJ, Takewaki T: Mechanisms of action of noradrenaline and carbachol on smooth muscle of guinea pig anterior mesenteric artery. J Physiol 1984; 351:549–72Bolton, TB Lang, RJ Takewaki, T
Nagase Y, Kaibara M, Uezono Y, Izumi F, Sumikawa K, Taniyama K: Propofol inhibits muscarinic acetylcholine-mediated signal transduction in Xenopus oocytes expressing the rat M1 receptor. Jpn J Pharmacol 1999; 79:319–25Nagase, Y Kaibara, M Uezono, Y Izumi, F Sumikawa, K Taniyama, K
Rossi MA, Chan CK, Christensen JD, DeGuzman EJ, Durieux ME: Interactions between propofol and lipid mediator receptors: Inhibition of lysophosphatidate signaling. Anesth Analg 1996; 83:1090–6Rossi, MA Chan, CK Christensen, JD DeGuzman, EJ Durieux, ME
Violet JM, Downie DL, Nakisa RC, Lieb WR, Franks NP: Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. A nesthesiology 1997; 86:866–74Violet, JM Downie, DL Nakisa, RC Lieb, WR Franks, NP
Flood P, Ramirez-Latorre J, Role L:α4β2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but α7-type nicotinic acetylcholine receptors are unaffected. A nesthesiology 1997; 86:859–65Flood, P Ramirez-Latorre, J Role, L
Furuya R, Oka K, Watanabe I, Kamiya Y, Itoh H, Andoh T: The effects of ketamine and propofol on neuronal nicotinic acetylcholine receptors and P2xpurinoceptors in PC12 cells. Anesth Analg 1999; 88:174–80Furuya, R Oka, K Watanabe, I Kamiya, Y Itoh, H Andoh, T
Feletou M, Vanhoutte PM: Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol 1988; 93:515–24Feletou, M Vanhoutte, PM
Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A: Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery. Br J Pharmacol 1997; 120:439–46Fukao, M Hattori, Y Kanno, M Sakuma, I Kitabatake, A
Bauersachs J, Popp R, Busse R: Nitric oxide and endothelium-derived hyperpolarizing factor: Formation and interactions. Prostaglandins Leukot Essent Fatty Acids 1997; 57:439–46Bauersachs, J Popp, R Busse, R
Quilley J, Fulton D, McGiff JC: Hyperpolarizing factors. Biochem Pharmacol 1997; 54:1059–70Quilley, J Fulton, D McGiff, JC
Corriu C, Feletou M, Canet E, Vanhoutte PM: Endothelium-derived factors and hyperpolarization of the carotid artery of the guinea pig. Br J Pharmacol 1996; 119:959–64Corriu, C Feletou, M Canet, E Vanhoutte, PM
Lischke V, Busse R, Hecker M: Inhalation anesthetics inhibit the release of endothelium-derived hyperpolarizing factor in the rabbit carotid artery. A nesthesiology 1995; 83:574–82Lischke, V Busse, R Hecker, M
Kessler P, Lischke V, Hecker M: Etomidate and thiopental inhibit the release of endothelium-derived hyperpolarizing factor in the human renal artery. A nesthesiology 1996; 84:1485–8Kessler, P Lischke, V Hecker, M
Iranami H, Hatano Y, Tsukiyama Y, Yamamoto M, Maeda H, Mizumoto K: Halothane inhibition of acetylcholine-induced relaxation in rat mesenteric artery and aorta. Can J Anaesth 1997; 44:1196–203Iranami, H Hatano, Y Tsukiyama, Y Yamamoto, M Maeda, H Mizumoto, K
Hwa JJ, Ghibandi L, Williams P, Chatterjee M: Comparison of acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed. Am J Physiol 1994; 266:H952–8Hwa, JJ Ghibandi, L Williams, P Chatterjee, M
Akata T, Nakashima M, Kodama K, Boyle WA, Takahashi S: Effects of volatile anesthetics on acetylcholine-induced relaxation in the rabbit mesenteric resistance artery. A nesthesiology 1995; 82:188–204Akata, T Nakashima, M Kodama, K Boyle, WA Takahashi, S
Loeb AL, Godeny I, Longnecker DE: Anesthetics alter relative contributions of NO and EDHF in rat cremaster muscle microcirculation. Am J Physiol 1997; 273:H618–27Loeb, AL Godeny, I Longnecker, DE
Nolan A, Reid J: Pharmacokinetics of propofol administered by infusion in dogs undergoing surgery. Br J Anaesth 1993; 70:546–51Nolan, A Reid, J
Fig. 1. Effect of propofol on the endothelium-dependent relaxation induced by acetylcholine in pulmonary arteries. Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−6m) had no effect (A  ), whereas propofol (10−5.5, 10−5, 10−4m) attenuated (P  < 0.05) vasorelaxation induced by acetylcholine (10−9to 10−5m, n = 8).
Fig. 1. Effect of propofol on the endothelium-dependent relaxation induced by acetylcholine in pulmonary arteries. Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−6m) had no effect (A 
	), whereas propofol (10−5.5, 10−5, 10−4m) attenuated (P 
	< 0.05) vasorelaxation induced by acetylcholine (10−9to 10−5m, n = 8).
Fig. 1. Effect of propofol on the endothelium-dependent relaxation induced by acetylcholine in pulmonary arteries. Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−6m) had no effect (A  ), whereas propofol (10−5.5, 10−5, 10−4m) attenuated (P  < 0.05) vasorelaxation induced by acetylcholine (10−9to 10−5m, n = 8).
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Fig. 2. Effect of propofol on the relaxation induced by bradykinin (A  ) and SIN-1 (B  ). Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−4m) had no effect on bradykinin- or SIN-1-induced vasorelaxation (n = 5).
Fig. 2. Effect of propofol on the relaxation induced by bradykinin (A 
	) and SIN-1 (B 
	). Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−4m) had no effect on bradykinin- or SIN-1-induced vasorelaxation (n = 5).
Fig. 2. Effect of propofol on the relaxation induced by bradykinin (A  ) and SIN-1 (B  ). Arterial rings were precontracted to the ED50level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM. Propofol (10−4m) had no effect on bradykinin- or SIN-1-induced vasorelaxation (n = 5).
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Fig. 3. Effect of inhibitions of nitric oxide synthase (l-NAME, 3 × 10−5m;A  ) and cyclooxygenase (indomethacin, 3 × 10−5m;B  ) on vasorelaxation induced by acetylcholine in canine pulmonary arteries (n = 5). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM.
Fig. 3. Effect of inhibitions of nitric oxide synthase (l-NAME, 3 × 10−5m;A 
	) and cyclooxygenase (indomethacin, 3 × 10−5m;B 
	) on vasorelaxation induced by acetylcholine in canine pulmonary arteries (n = 5). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM.
Fig. 3. Effect of inhibitions of nitric oxide synthase (l-NAME, 3 × 10−5m;A  ) and cyclooxygenase (indomethacin, 3 × 10−5m;B  ) on vasorelaxation induced by acetylcholine in canine pulmonary arteries (n = 5). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM.
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Fig. 4. Effect of inhibition of cytochrome P450 monooxygenase (SKF 525A, 3 × 10−5m, A  ; and clotrimazole, 3 × 10−5m, B  ) on vasorelaxation induced by acetylcholine (n = 5).
Fig. 4. Effect of inhibition of cytochrome P450 monooxygenase (SKF 525A, 3 × 10−5m, A 
	; and clotrimazole, 3 × 10−5m, B 
	) on vasorelaxation induced by acetylcholine (n = 5).
Fig. 4. Effect of inhibition of cytochrome P450 monooxygenase (SKF 525A, 3 × 10−5m, A  ; and clotrimazole, 3 × 10−5m, B  ) on vasorelaxation induced by acetylcholine (n = 5).
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Fig. 5. Effect of inhibition of cytochrome P450 monooxygenase (clotrimazole) on l-NAME–indomethacin-resistant component of relaxation induced by acetylcholine (n = 5). Pretreatment with clotrimazole totally abolished acetylcholine-induced relaxation. L = l-NAME pretreated; I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 5. Effect of inhibition of cytochrome P450 monooxygenase (clotrimazole) on l-NAME–indomethacin-resistant component of relaxation induced by acetylcholine (n = 5). Pretreatment with clotrimazole totally abolished acetylcholine-induced relaxation. L = l-NAME pretreated; I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 5. Effect of inhibition of cytochrome P450 monooxygenase (clotrimazole) on l-NAME–indomethacin-resistant component of relaxation induced by acetylcholine (n = 5). Pretreatment with clotrimazole totally abolished acetylcholine-induced relaxation. L = l-NAME pretreated; I = indomethacin pretreated; CLT = clotrimazole pretreated.
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Fig. 6. Effect of propofol on the nitric oxide–mediated component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibition of the EDHF-mediated component (by SKF 525A, A  , or by clotrimazole, B  ). I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 6. Effect of propofol on the nitric oxide–mediated component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibition of the EDHF-mediated component (by SKF 525A, A 
	, or by clotrimazole, B 
	). I = indomethacin pretreated; CLT = clotrimazole pretreated.
Fig. 6. Effect of propofol on the nitric oxide–mediated component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibition of the EDHF-mediated component (by SKF 525A, A  , or by clotrimazole, B  ). I = indomethacin pretreated; CLT = clotrimazole pretreated.
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Fig. 7. Effect of propofol on the nitric oxide–insensitive component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibitions of nitric oxide–mediated component. L = l-NAME pretreated; I = indomethacin pretreated.
Fig. 7. Effect of propofol on the nitric oxide–insensitive component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibitions of nitric oxide–mediated component. L = l-NAME pretreated; I = indomethacin pretreated.
Fig. 7. Effect of propofol on the nitric oxide–insensitive component of acetylcholine-induced vasorelaxation in canine pulmonary arteries contracted to the ED50 level of tension with phenylephrine. Relaxation is expressed as the percentage of phenylephrine contraction (n = 5). Propofol further attenuated acetylcholine-induced vasorelaxation after inhibitions of nitric oxide–mediated component. L = l-NAME pretreated; I = indomethacin pretreated.
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Fig. 8. Effect of cytochrome P450 inhibitors (SKF525A and clotrimazole, both 3 × 10−5m) on relaxation induced by bradykinin (endothelium intact) and by SIN-1 and papaverine (endothelium denuded). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM (n = 6).
Fig. 8. Effect of cytochrome P450 inhibitors (SKF525A and clotrimazole, both 3 × 10−5m) on relaxation induced by bradykinin (endothelium intact) and by SIN-1 and papaverine (endothelium denuded). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM (n = 6).
Fig. 8. Effect of cytochrome P450 inhibitors (SKF525A and clotrimazole, both 3 × 10−5m) on relaxation induced by bradykinin (endothelium intact) and by SIN-1 and papaverine (endothelium denuded). Arterial rings were precontracted to the ED50 level of tension with phenylephrine. Relaxations are expressed as percentages of phenylephrine contraction and are presented as mean ± SEM (n = 6).
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Table 1. Effect of Propofol on Acetylcholine-induced Relaxation
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Table 1. Effect of Propofol on Acetylcholine-induced Relaxation
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Table 2. Effects of Individual Inhibitors on Acetylcholine-induced Relaxation
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Table 2. Effects of Individual Inhibitors on Acetylcholine-induced Relaxation
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Table 3. Effects of Combined Inhibition on Acetylcholine-induced Relaxation
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Table 3. Effects of Combined Inhibition on Acetylcholine-induced Relaxation
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Table 4. Effect of Cytochrome P450 Inhibition on Relaxation Induced by Bradykinin, SIN-1, and Papaverine
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Table 4. Effect of Cytochrome P450 Inhibition on Relaxation Induced by Bradykinin, SIN-1, and Papaverine
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