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Potency of authentic nitric oxide in inducing aortic relaxation
Pharmacological Research 55 (2007) 329–334
Potency of authentic nitric oxide in inducing aortic relaxation Qingtao Yan, Qihui Liu, Jay L. Zweier, Xiaoping Liu ∗ Davis Heart and Lung Research Institute, The Division of Cardiovascular Medicine, Department of Internal Medicine, and Biomedical Engineering Department, The Ohio State University, 473 W. 12th Avenue, Columbus, OH 43210, United States Accepted 8 January 2007
Abstract The reported EC50 of vasorelaxation for authentic nitric oxide (NO) is from the nM to M range. The cause of this large difference is undetermined. In this study, NO electrodes were used to monitor the actual NO concentration in the organ chamber during the recording of the relaxation of rat aortic rings. It was demonstrated that both the O2 concentration in the solution and the rate of stirring the solution markedly affected the actual NO concentration, while the vasorelaxation response to NO was also changed with the composition of the buffer solution. It was observed that the apparent EC50 of aortic relaxation for authentic NO is 340 ± 40 nM and 81 ± 4 nM in the conventional organ chamber containing Krebs–Ringer buffer bubbled with a high content (95% O2 + 5% CO2 ) and a low content (20% O2 + 5% CO2 + 75% N2 ) of O2 gas mixture, respectively. The apparent EC50 was further reduced to 45 ± 2 nM after the Krebs–Ringer buffer, a bicarbonate buffer, was replaced by a phosphate buffer solution bubbled with a low content of O2 gas mixture (20% O2 + 80% N2 ) or with air. By using an organ chamber, which makes low concentrations of NO more stable in the solution, it was determined that the apparent EC50 was 9.7 ± 0.4 nM and the threshold of NO concentration in dilating the aortas was ∼0.3 nM. This modified organ chamber will be useful for quantitatively measuring EC50 of vasorelaxation for authentic NO under physiological and pathological conditions. © 2007 Elsevier Ltd. All rights reserved. Keywords: EC50 ; Organ chamber; Rat aorta; NO electrode
1. Introduction The endothelium derived relaxing factor was identified as nitric oxide (NO) 20 years ago [1–3]. This endotheliumgenerated NO needs to diffuse into blood vessel walls to activate the soluble guanylate cyclase (sGC) in smooth muscle cells [4–6]. The activated sGC accelerates the formation of cyclic GMP and initiates a series of events that dilate the blood vessel. NO plays an important role in regulating vascular tone. Lack of NO bioavailability is associated with certain cardiovascular diseases such as hypertension, diabetes and atherosclerosis [7,8]. NO bioavailability can be assessed by organ chamber experimental method in vitro. A reduced percentage of aortic relaxation response to exogenous NO indicates a lack of NO bioavailability due to the destruction of NO in the vascular wall. Although the relaxation response of blood vessels to NO donors or to endothelial NO agonists such as acetylcholine (ACh) and bradykinin (BK) has been well documented, the vasorelaxation
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Corresponding author. Tel.: +1 614 292 1305; fax: +1 614 292 8778. E-mail address: [email protected] (X. Liu).
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response to authentic NO is still inconsistent in published papers. For example, it was observed that the EC50 of aortic relaxation for NO was in the low nM range if NO is generated at its sites of action from caged NO [9]. However, when experiments were performed in the organ chamber or the small myograph bath with injections of authentic NO, the reported EC50 value is much higher, which was in the sub M to M range [10–12]. In this study, we examined the effect of solution stirring, oxygen partial pressure and the composition of buffer solution on the apparent potency of authentic NO in mediating aortic relaxation by simultaneously measuring the aortic tension and the exogenous NO in the organ chambers. A modified organ chamber method was used to characterize the relaxation response of rat aortas to authentic NO. 2. Materials and methods 2.1. Preparation of NO solution NO gas was scrubbed of higher nitrogen oxides by passage first through a U-tube containing NaOH pellets and then through a 1 M deaerated (bubbled with 100% argon) KOH solution in
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a custom-designed apparatus using only glass or stainless steel tubing and fittings [13,14]. To prepare the saturated NO solution, the purified NO gas was bubbled into the deaerated buffer (0.2 M potassium phosphate, pH 7.4), which was contained in a glass sampling flask with a septum purchased from Kimble/Kontes (Vineland, NJ), for 15 min. The saturated NO concentration was nearly 2 mM. This high concentration of NO stock solution was used for bolus injections of NO concentrations greater than 300 nM into the organ chambers. To prepare NO stock solution with a lower concentration (50–200 M), the purified NO gas was bubbled to the deaerated phosphate buffer in a sampling flask for less than 1 min. The final stock NO concentration was calibrated by the oxyhaemoglobin (oxyHb) method. This NO stock solution was used for the bolus injection of NO concentrations between 10 nM and 300 nM. NO stock solution at the M range was prepared in a commercial 4-port electrochemical chamber purchased from World Precision Instruments (WPI, Florida) right before it was used. The protocol was similar to that described in our previous paper [13]. Briefly, 2 ml phosphate buffer (pH 7.4) was loaded into the chamber. The buffer solution in the chamber was stirred by a magnetic bar, and the chamber was closed with a cap. The cap could be freely moved down or up in the chamber to regulate the distance between the solution surface and the cap bottom. The distance between the surface of the buffer solution and the bottom of the cap was initially held at ∼1 cm. A gas tube was inserted into the solution through one of three holes in the cap. Pure argon gas was bubbled into the solution through the tube for at least 15 min. After the gas tube was removed from the chamber, the cap was pushed down into the solution until the solution completely filled the three holes in the cap. This was followed by injecting 2 l of high concentration (2 mM) NO solution into the electrochemical chamber. NO concentration in the electrochemical chamber was monitored by an ISO-NOP Clark-type NO electrode (WPI, Florida) that was installed in a port on the side wall of the chamber. This low concentration (∼2 M) of NO stock solution was used for bolus injections of NO concentrations below 10 nM into the organ chamber. 2.2. Harvest of rat aortas WKY rats at ages 6–8 weeks were anaesthetized with pentobarbital (100 mg kg−1 , i.p.). The thoracic aorta was rapidly dissected and placed into an ice-cold tissue phosphate buffer solution with glucose (PBSG, pH 7.4) of the following composition: 137 mM NaCl, 2.5 mM KCl, 0.9 mM CaCl2 , 0.5 mM MgSO4 , 1.5 mM KH2 PO4 , 0.8 mM Na2 HPO4 and 5.6 mM glucose. The blood in the aorta was immediately washed out and the loosely adhering fat and connective tissue were then removed. The use of animals and the animal protocol were approved by the Institutional Lab Animal Care and Use Committee of The Ohio State University. 2.3. Conventional and modified organ chambers The experiments for recording aortic relaxation and NO concentration were performed in conventional organ chambers
and modified organ chambers at 37 ◦ C separately. There is a gas inlet port on the bottom of conventional organ chamber, which was used for bubbling a gas mixture (usually 95% O2 + 5% N2 ) into the chamber containing 15 ml Krebs–Ringer bicarbonate solution (37 ◦ C, pH 7.4) of the following composition: 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.17 mM MgSO4 , 1.15 mM KH2 PO4 , 25.1 mM NaHCO3 and 11 mM glucose. Unlike the conventional organ chambers, the modified organ chambers do not have the gas bubbling system. Instead, the solution (PBSG) in the chambers was rapidly stirred so that air can be efficiently dissolved into solution to maintain the equilibrium of oxygen between the solution and the environment. In this study, each organ chamber (conventional or modified) was installed with a Clark-type NO electrode or an ISO-NOPF carbon fibre NO electrode (WPI, Florida) for monitoring NO concentration in the solution. 2.4. Simultaneous measurements of aortic relaxation and exogenous NO concentration The isolated and cleaned rat aorta was cut into several ∼5 mm long rings. The rings were mounted on stainless steel hooks connected to force transducers, which were linked to a data acquisition unit MP100A (Biopac Systems, California). The rings were allowed to equilibrate in the buffer solution for 60 min under a resting tension of 2 g at 37 ◦ C. During this period, the buffer solution was exchanged three times, and then the aortic rings were precontracted with 10 M phenylephrine (Phe) prior to cumulative injections of different concentrations (1 nM to 10 M) of ACh. After measurements of [ACh]-vasorelaxation response curves were completed, the tissue buffer in each chamber was changed three times in 1 h. Then the aortic relaxation response to different concentrations of NO (0.1 nM to 10 M) was recorded, and the NO concentration in the chamber was monitored simultaneously by a Clark-type NO electrode or a carbon fibre NO electrode connected to a four-channel Apollo 4000 Free Radical Analyzer (WPI, Florida). In conventional organ chamber experiments, an O2 -containing gas mixture flows from the bottom to the top of the chamber. In some measurements, we needed to change the flow rate of the bubbling gas in the conventional organ chamber. We use the word “high flow rate” to represent a normal flow rate (∼10 ml min−1 ) and “low flow rate” to represent a flow rate in the range of 0.3 to 0.5 ml min−1 . 2.5. Data analysis The values of the aortic relaxation response to different authentic NO concentrations were given by mean ± standard error (S.E.). To determine the aortic relaxation response at a certain NO concentration, we plotted the values (mean) of aortic relaxation versus log[NO] and fitted these data points with a sigmoid curve using SigmaPlot 9.0 software. The mean and S.E. of the vasorelaxation were obtained from the best fitting results.
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Fig. 1. [ACh]-vasorelaxation response curves. Measurements were performed in the conventional organ chambers. During the measurements, the gas (95% O2 + 5% CO2 ) was bubbled through the gas inlet on the bottom of the chamber (A) and flowed over the solution surface (B). ACh was added into the solution in cumulative concentrations between 1 nM and 10 M at the times shown by the arrows (from left to right): 1, 3, 10, 30, 100, 300, 1000, 3000, and 10,000 nM.
3. Results 3.1. Stirring of the buffer in the conventional organ chamber by the bubbling gas Aortic rings in the organ chambers were precontracted using 10 M Phe while each chamber was bubbled with a gas mixture (95% O2 + 5% CO2 ). Different concentrations of ACh were cumulatively added into the chambers. The relaxation response curve of an aortic ring is demonstrated in Fig. 1A. To observe the stirring effect of the bubbling gas, the gas flow through the bottom of the chamber was closed with a gas tube placed above the solution and a parafilm membrane covering the chamber. The gas mixture (95% O2 + 5% CO2 ) flowed over the solution and exited the chamber through open ports in the membrane during the measurements. The same experimental protocol was repeated with different ACh concentrations. It was observed that the aortic ring had no response to [ACh] < 100 nM. For [ACh] > 100 nM, the aortic ring responded to the ACh added, but the response was sometimes small, sometimes large or sometimes delayed (Fig. 1B). 3.2. The effect of the flow rate and O2 content of the bubbling gas on the NO concentration in the conventional organ chamber Compared to stable chemicals such as ACh, NO is unstable. In the conventional organ chamber, NO can be consumed by reacting with O2 and can also be brought out of the solution by the bubbling of the gas. Thus, it is necessary to test how the bubbling gas and its O2 content affect NO concentration in the solution. Experiments were performed to test the response of the aortic ring to different concentrations of NO at a low flow rate and a high flow rate of bubbling gas. It was observed that the current of the electrode and the aortic tension response to each NO addition was irregular at low flow rate and did not change immediately after each NO injection (Fig. 2A). In contrast, when a high flow rate of bubbling gas was used, the detected NO concentration was approximately proportional to the amount of injected NO
and the aortic tension changed immediately following each NO injection (Fig. 2B). However, NO decay was much faster at a high flow rate of bubbling gas compared to that predicted from the rate constant of NO autoxidation. To examine the effect of O2 concentration on the effective NO concentration, we used a high content of O2 -containining gas mixtures (95% O2 + 5% CO2 ) and a low content of O2 containing gas mixtures (either 20% O2 + 75% N2 + 5% CO2 or 20% O2 + 80% N2 ). Experiments were performed with a high flow rate of gas mixture. It was observed that when a low content of O2 -containing gas mixtures was used, NO concentrations were 2 or 3 times higher and the aortic relaxation was more sensitive to the same amount of injected NO (Fig. 2C) than when a high content of O2 -containing gas mixture was used (Fig. 2B). The NO concentration dependent aortic relaxation under two different O2 partial pressures and in two different solutions were shown in Fig. 2D. The apparent EC50 of aortic relaxation for authentic NO in Krebs–Ringer solution bubbled with 95% O2 + 5% CO2 gas mixture (a) and with 20% O2 + 5% CO2 + 75% N2 gas mixture (b) was 340 ± 40 nM (n = 8) and 81 ± 4 nM (n = 8), respectively. The apparent EC50 of aortic relaxation for authentic NO in PBSG solution bubbled either with 20% O2 + 80% N2 gas mixture or with air (c) was 45 ± 2 nM (n = 5). 3.3. The rate of NO decay and the vasorelaxation response to authentic NO in the modified organ chambers In the modified organ chamber, O2 -containing gas mixture was not directly bubbled into the solution. Instead, the solution in the chamber was rapidly stirred with a magnetic bar so that environmental O2 could be effectively dissolved into the solution and the equilibrium between the O2 concentration in solution and the O2 partial pressure in the environment was maintained. It was observed that the rate of NO decay in the modified chamber was much slower than that in the conventional organ chamber (Fig. 3), especially when NO concentration was 300 nM or less. In Fig. 4, we demonstrate the NO concentration and the vasorelaxation response of the aortic rings to different concentrations
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Fig. 2. The effect of the flow rate, O2 content of bubbling gas and the composition of buffer solution on NO concentration and aortic relaxation. In (A)–(C), experiments were performed in Krebs–Ringer solution. The downward arrows indicate the times that different concentrations of NO were added. The amounts of added NO from left to right are 1, 3, 10, 30, 100, 300, 1000, 3000 and 10,000 nM. (A) Aortic relaxation (a) and NO concentration (b) with a slow flow rate of bubbling gas (95% O2 + 5% CO2 ). (B) Aortic relaxation (a) and NO concentration (b) with a high flow rate of bubbling gas (95% O2 + 5% CO2 ). (C) Aortic relaxation (a) and NO concentration (b) with a high flow rate of bubbling gas (20% O2 + 75% N2 + 5% CO2 ). (D) The aortic relaxation responses were measured in the Krebs–Ringer buffer bubbled either with 95% O2 + 5% CO2 (䊉) or with 20% O2 + 75% N2 + 5% CO2 (), and in the PBSG solution bubbled either with 20% O2 + 80% N2 or with air ().
of authentic NO in the modified organ chambers. The apparent EC50 of aortic relaxation for NO (Fig. 4B) was found to be 9.7 ± 0.4 nM (n = 6), and the threshold of NO concentration for dilating the aorta was ∼0.3 nM in the modified organ chamber.
Fig. 3. The rate of NO decay in the conventional organ chamber and the modified organ chamber as O2 (20% O2 + 80% N2 ) gas mixture or air (21% O2 + 79% N2 ) was used in the experiments. Different amounts of NO were injected at the times designated by the arrows. Measurements were performed in the modified organ chamber (a) and the conventional organ chamber (b). The curve a and b in the inset are magnified (50×) from curve a and b, respectively. The arrows indicate the times that different concentrations of NO were added. The amounts of added NO from left to right are 1, 3, 10, 30, 100, 300, 1000 and 3000 nM for curves a and b, and 1, 3, 10, 30 nM for curves a and b .
4. Discussion To understand what may affect the vasorelaxation response to NO in the conventional organ chamber, we examined the effect of the flow rate of the bubbling gas mixture, O2 partial pressure and buffer composition on aortic relaxation. Our results show that bubbling a gas mixture (95% O2 + 5% CO2 ) into the tissue buffer in the conventional organ chamber not only provided O2 to the solution but also stirred the solution. Without the bubbling gas, even though O2 was provided to the chamber, the [ACh]vasorelaxation response curves could not be obtained correctly (Fig. 1). Although the mode of stirring with the bubbling gas is suitable for non-volatilizable substances such as ACh, it may bring a marked experimental error when determining the actual NO concentration in the solution because NO cannot only be oxidized by O2 but also be purged from the solution by the bubbling gas. Under a constant O2 partial pressure, the rate of NO reaction with O2 is second order in [NO] [13,15–18]. This means that NO decay rate will increase 100 times if its concentration increases 10 times. In this case, rapidly stirring the solution is important for the bolus injection of authentic NO. If the injected NO is not quickly diluted into the whole solution, there will be a relatively higher NO concentration in some localized areas of the solution. A high NO concentration in some areas of the solution will decay much faster, causing a marked consumption of NO by O2 . Furthermore, these areas with localized high
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Fig. 4. Aortic relaxation response to different concentrations of authentic NO in the modified organ chamber. (A) Simultaneous measurements of the [NO]vasorelaxation response curve (a) and NO concentration (b) in the solution. The aortic ring was contracted by 10 M Phe (designated by the downward arrow), then different NO concentrations were added at the times shown by the upward arrows (from left to right): 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10,000 nM. Curve c in the inset was magnified (4×) from the low NO concentration portion of curve b. The added NO concentrations designated by the upward arrows (from left to right) in the inset are 0.1, 0.3, 1, 3, 10, 30 and 100 nM. (B) NO concentration dependent relaxation response of aortas from 6–8 week-old WKY rats. The experimental points (closed circles with error bars) were fitted by a sigmoidal curve (solid line).
NO concentration may move around in the solution. This would explain the detected irregular currents by the NO electrodes and the currents that usually did not immediately increase following an injection of NO (Fig. 2A). Correspondingly, the vasorelaxation response to the injected NO was also irregular. To solve this problem, it was necessary to increase the stirring speed in the conventional chamber by increasing the flow rate of the bubbling gas. As shown in Fig. 2B, at high flow rate of bubbling gas, the response currents of electrodes were more regular and tended to be approximately proportional to the injected NO amounts. However, the rate of NO decay markedly increased as the flow rate of bubbling gas increased. It can be seen that the half life of 100 nM NO in the conventional chamber was only 30–40 s, whereas the half-live of NO autoxidation predicted from published rate constants at this concentration should have been more than 15 min [13,15–18]. These results indicate that the conventional organ chamber, in which the solution is stirred with the bubbling gas, is not the best experimental setup for measuring vasorelaxation to authentic NO. If a low flow rate of bubbling gas were used, a large portion of NO could be oxidized by O2 before NO is uniformly diluted into the whole solution; on the other hand, if a high flow rate of bubbling gas were used, NO decay in the solution would be markedly faster because it could be rapidly purged out by the bubbling gas. Since the flow rate of bubbling gas is usually set by experience, the flow rate used in the conventional organ chamber experiments varies from laboratory to laboratory. Thus, it would not be surprising to find that an EC50 value of vasorelaxation for authentic NO reported from one laboratory is largely different from another in the literature. In Fig. 2C, we showed that NO concentration in the solution was markedly affected by O2 partial pressure. When 20% O2 gas was used, the resulting current detected by the NO electrode to each NO injection was 2 to 3 times higher than the corresponding currents to the same amount of injected NO when 95% O2 gas was used, indicating that the actual NO concentration in the conventional chamber bubbling with 95% O2 gas would be at least 2 times lower than the NO concentration calculated from
the dilution factor in a bolus injection. Correspondingly, the apparent EC50 of aortic relaxation was reduced more than 4fold from 340 nM to 81 nM. This decrease in EC50 is 1.5 to 2 times more than that in the effective NO concentration detected by the electrodes. This difference can be caused by two different factors. The first factor is that it takes time for NO diffusion from the aortic surface to the smooth muscle cell layer in the aortic wall, while the NO concentration in the high O2 concentration solution decreases more rapidly than that in the low O2 concentration solution. Thus, even if NO concentration in the high O2 concentration solution is initially equal to the NO concentration in the low O2 concentration solution, when NO diffuses into the smooth muscle layer, the actual amount of NO diffusion into the aortic wall in the high O2 concentration solution will be less than that in the low O2 concentration solution. The second factor is that the rate of superoxide generation in the high O2 concentration solution is higher than that in the low concentration of O2 solution, resulting in a faster NO decay rate in the aortic wall. When the organ chamber experiments were performed in the PBSG solution bubbled with 20% O2 + 80% N2 or air, the apparent EC50 was found to be 45 nM, which is less than a 2fold decrease. It is unlikely that this decrease was caused by the difference in concentration of a single chemical such as glucose, Ca2+ , or Mg2+ from our preliminary observation (data not shown). To solve the bubbling gas-caused problem in the conventional organ chambers, we used modified organ chambers in our experiments. Because the solution in the modified organ chamber was stirred by a stir bar and not the bubbling gas, the rate of NO decay after each NO injection was markedly slower than that in the conventional organ chamber. Since NO can stay in the solution much longer, a relatively higher effective NO concentration can be built up in the aortic wall. Using this modified organ chamber containing PBSG solution equilibrated with 20% O2 + 80% N2 gas or air, we observed that the threshold of NO concentration for inducing a minimal aortic relaxation was as low as 0.3 nM, and the EC50 of aortic relaxation for NO was ∼10 nM. As shown in Fig. 3, the low concentration of NO (<300 nM)
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is much more stable in the modified organ chamber than that in conventional chamber. Correspondingly, the vasorelaxation curves recorded in the two kinds of organ chambers have some notable differences. The typical aortic relaxation response curve to each NO injection in the modified organ chamber exhibited a “stair” shape (see curve a in Fig. 4A), which declined and then reached a plateau before the next NO injection. In contrast, the vasorelaxation curve to each NO injection recorded in the conventional chamber formed a downward peak (Fig. 2B and C). Although we (in this study) and others [9] have shown that the EC50 of vasorelaxation for NO was around 10 nM, the actual NO concentration required to induce 50% vasorelaxation in the vascular wall has not been directly measured. Strong evidence showed that NO can be consumed in cells and blood vessels by O2 species or O2 -dependent reactions [14,19,20]. Thus, even if experiments are performed in the solution equilibrated with 20% O2 gas, the reactive O2 species may still have a marked influence on the effective NO concentration in the aortic wall. From this viewpoint, the actual effective NO concentration in the vascular wall may be appreciably lower than 10 nM whether NO is generated outside or inside of the vascular wall, because the rate of NO consumption in the vascular wall is significantly higher than that in the solution. Therefore, the value of EC50 of rat aorta, 10 nM, for authentic NO that is reported in this study should be considered as an apparent EC50 , meaning that the aortic relaxation response to authentic NO reaches 50% of maximal relaxation while the NO concentration at the aortic surface rather than inside of the vascular wall is equal to 10 nM. In summary, the conventional organ chamber is not the best setup for measuring the vasorelaxation response to authentic NO. By using an organ chamber modified to provide rapid stirring under ambient air or 20% O2 , we observed that the EC50 for aortic relaxation was 10 nM. The NO concentration that caused a minimal relaxation was ∼0.3 nM. This modified organ chamber method enables a better quantitative characterization of the vasorelaxation response to authentic NO in organ chambers under different physiological and pathological conditions. Acknowledgement This work was supported by HL63744, HL65608, and HL38324 (J.L.Z.) and the American Heart Association Scientific Development Grant 0130367N (X.L.). References [1] Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288(5789):373–6.
[2] Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endotheliumderived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 1987;84(24):9265–9. [3] Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327(6122):524–6. [4] Arnold WP, Mittal CK, Katsuki S, Murad F. Nitric oxide activates guanylate cyclase and increases guanosine 3 ,5 -cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci USA 1977;74(8):3203–7. [5] Gruetter CA, Barry BK, McNamara DB, Gruetter DY, Kadowitz PJ, Ignarro L. Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine. J Cyclic Nucleotide Res 1979;5(3):211–24. [6] Lancaster Jr JR. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc Natl Acad Sci USA 1994;91(17):8137–41. [7] Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension 2003;42(6):1075–81. [8] Heistad DD. Oxidative stress and vascular disease: 2005 Duff lecture. Arterioscler Thromb Vasc Biol 2006;26(4):689–95. [9] Carter TD, Bettache N, Ogden D. Potency and kinetics of nitric oxidemediated vascular smooth muscle relaxation determined with flash photolysis of ruthenium nitrosyl chlorides. Br J Pharmacol 1997;122(6): 971–3. [10] Wanstall JC, Jeffery TK, Gambino A, Lovren F, Triggle CR. Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxylion. Br J Pharmacol 2001; 134(3):463–72. [11] Lynch SM, Frei B, Morrow JD, Roberts 2nd LJ, Xu A, Jackson T, et al. Vascular superoxide dismutase deficiency impairs endothelial vasodilator function through direct inactivation of nitric oxide and increased lipid peroxidation. Arterioscler Thromb Vasc Biol 1997;17(11):2975–81. [12] Soloviev AI, Stefanov AV, Tishkin SM, Khromov AS, Parshikov AV, Ivanova IV, et al. Saline containing phosphatidylcholine liposomes possess the ability to restore endothelial function damaged resulting from gamma-irradiation. J Physiol Pharmacol 2002;53(4 Part 1):701–12. [13] Liu X, Liu Q, Gupta E, Zorko N, Brownlee E, Zweier JL. Quantitative measurements of NO reaction kinetics with a Clark-type electrode. Nitric Oxide 2005;13(1):68–77. [14] Thomas DD, Liu X, Kantrow SP, Lancaster Jr JR. The biological lifetime of nitric oxide: implications for the perivascular dynamics of NO and O2 . Proc Natl Acad Sci USA 2001;98(1):355–60. [15] Wink DA, Darbyshire JF, Nims RW, Saavedra JE, Ford PC. Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction. Chem Res Toxicol 1993;6(1):23–7. [16] Lewis RS, Deen WM. Kinetics of the reaction of nitric oxide with oxygen in aqueous solutions. Chem Res Toxicol 1994;7(4):568–74. [17] Goldstein S, Czapski G. Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates. J Am Chem Soc 1995;117(49):12078–84. [18] Kharitonov VG, Sundquist AR, Sharma VS. Kinetics of nitric oxide autoxidation in aqueous solution. J Biol Chem 1994;269(8):5881–3. [19] Gardner PR, Martin LA, Hall D, Gardner AM. Dioxygen-dependent metabolism of nitric oxide in mammalian cells. Free Radic Biol Med 2001;31(2):191–204. [20] Liu X, Cheng C, Zorko N, Cronin S, Chen YR, Zweier JL. Biphasic modulation of vascular nitric oxide catabolism by oxygen. Am J Physiol Heart Circ Physiol 2004;287(6):H2421–6.
Potency of authentic nitric oxide in inducing aortic relaxation Qingtao Yan, Qihui Liu, Jay L. Zweier, Xiaoping Liu ∗ Davis Heart and Lung Research Institute, The Division of Cardiovascular Medicine, Department of Internal Medicine, and Biomedical Engineering Department, The Ohio State University, 473 W. 12th Avenue, Columbus, OH 43210, United States Accepted 8 January 2007
Abstract The reported EC50 of vasorelaxation for authentic nitric oxide (NO) is from the nM to M range. The cause of this large difference is undetermined. In this study, NO electrodes were used to monitor the actual NO concentration in the organ chamber during the recording of the relaxation of rat aortic rings. It was demonstrated that both the O2 concentration in the solution and the rate of stirring the solution markedly affected the actual NO concentration, while the vasorelaxation response to NO was also changed with the composition of the buffer solution. It was observed that the apparent EC50 of aortic relaxation for authentic NO is 340 ± 40 nM and 81 ± 4 nM in the conventional organ chamber containing Krebs–Ringer buffer bubbled with a high content (95% O2 + 5% CO2 ) and a low content (20% O2 + 5% CO2 + 75% N2 ) of O2 gas mixture, respectively. The apparent EC50 was further reduced to 45 ± 2 nM after the Krebs–Ringer buffer, a bicarbonate buffer, was replaced by a phosphate buffer solution bubbled with a low content of O2 gas mixture (20% O2 + 80% N2 ) or with air. By using an organ chamber, which makes low concentrations of NO more stable in the solution, it was determined that the apparent EC50 was 9.7 ± 0.4 nM and the threshold of NO concentration in dilating the aortas was ∼0.3 nM. This modified organ chamber will be useful for quantitatively measuring EC50 of vasorelaxation for authentic NO under physiological and pathological conditions. © 2007 Elsevier Ltd. All rights reserved. Keywords: EC50 ; Organ chamber; Rat aorta; NO electrode
1. Introduction The endothelium derived relaxing factor was identified as nitric oxide (NO) 20 years ago [1–3]. This endotheliumgenerated NO needs to diffuse into blood vessel walls to activate the soluble guanylate cyclase (sGC) in smooth muscle cells [4–6]. The activated sGC accelerates the formation of cyclic GMP and initiates a series of events that dilate the blood vessel. NO plays an important role in regulating vascular tone. Lack of NO bioavailability is associated with certain cardiovascular diseases such as hypertension, diabetes and atherosclerosis [7,8]. NO bioavailability can be assessed by organ chamber experimental method in vitro. A reduced percentage of aortic relaxation response to exogenous NO indicates a lack of NO bioavailability due to the destruction of NO in the vascular wall. Although the relaxation response of blood vessels to NO donors or to endothelial NO agonists such as acetylcholine (ACh) and bradykinin (BK) has been well documented, the vasorelaxation
∗
Corresponding author. Tel.: +1 614 292 1305; fax: +1 614 292 8778. E-mail address: [email protected] (X. Liu).
1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2007.01.001
response to authentic NO is still inconsistent in published papers. For example, it was observed that the EC50 of aortic relaxation for NO was in the low nM range if NO is generated at its sites of action from caged NO [9]. However, when experiments were performed in the organ chamber or the small myograph bath with injections of authentic NO, the reported EC50 value is much higher, which was in the sub M to M range [10–12]. In this study, we examined the effect of solution stirring, oxygen partial pressure and the composition of buffer solution on the apparent potency of authentic NO in mediating aortic relaxation by simultaneously measuring the aortic tension and the exogenous NO in the organ chambers. A modified organ chamber method was used to characterize the relaxation response of rat aortas to authentic NO. 2. Materials and methods 2.1. Preparation of NO solution NO gas was scrubbed of higher nitrogen oxides by passage first through a U-tube containing NaOH pellets and then through a 1 M deaerated (bubbled with 100% argon) KOH solution in
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a custom-designed apparatus using only glass or stainless steel tubing and fittings [13,14]. To prepare the saturated NO solution, the purified NO gas was bubbled into the deaerated buffer (0.2 M potassium phosphate, pH 7.4), which was contained in a glass sampling flask with a septum purchased from Kimble/Kontes (Vineland, NJ), for 15 min. The saturated NO concentration was nearly 2 mM. This high concentration of NO stock solution was used for bolus injections of NO concentrations greater than 300 nM into the organ chambers. To prepare NO stock solution with a lower concentration (50–200 M), the purified NO gas was bubbled to the deaerated phosphate buffer in a sampling flask for less than 1 min. The final stock NO concentration was calibrated by the oxyhaemoglobin (oxyHb) method. This NO stock solution was used for the bolus injection of NO concentrations between 10 nM and 300 nM. NO stock solution at the M range was prepared in a commercial 4-port electrochemical chamber purchased from World Precision Instruments (WPI, Florida) right before it was used. The protocol was similar to that described in our previous paper [13]. Briefly, 2 ml phosphate buffer (pH 7.4) was loaded into the chamber. The buffer solution in the chamber was stirred by a magnetic bar, and the chamber was closed with a cap. The cap could be freely moved down or up in the chamber to regulate the distance between the solution surface and the cap bottom. The distance between the surface of the buffer solution and the bottom of the cap was initially held at ∼1 cm. A gas tube was inserted into the solution through one of three holes in the cap. Pure argon gas was bubbled into the solution through the tube for at least 15 min. After the gas tube was removed from the chamber, the cap was pushed down into the solution until the solution completely filled the three holes in the cap. This was followed by injecting 2 l of high concentration (2 mM) NO solution into the electrochemical chamber. NO concentration in the electrochemical chamber was monitored by an ISO-NOP Clark-type NO electrode (WPI, Florida) that was installed in a port on the side wall of the chamber. This low concentration (∼2 M) of NO stock solution was used for bolus injections of NO concentrations below 10 nM into the organ chamber. 2.2. Harvest of rat aortas WKY rats at ages 6–8 weeks were anaesthetized with pentobarbital (100 mg kg−1 , i.p.). The thoracic aorta was rapidly dissected and placed into an ice-cold tissue phosphate buffer solution with glucose (PBSG, pH 7.4) of the following composition: 137 mM NaCl, 2.5 mM KCl, 0.9 mM CaCl2 , 0.5 mM MgSO4 , 1.5 mM KH2 PO4 , 0.8 mM Na2 HPO4 and 5.6 mM glucose. The blood in the aorta was immediately washed out and the loosely adhering fat and connective tissue were then removed. The use of animals and the animal protocol were approved by the Institutional Lab Animal Care and Use Committee of The Ohio State University. 2.3. Conventional and modified organ chambers The experiments for recording aortic relaxation and NO concentration were performed in conventional organ chambers
and modified organ chambers at 37 ◦ C separately. There is a gas inlet port on the bottom of conventional organ chamber, which was used for bubbling a gas mixture (usually 95% O2 + 5% N2 ) into the chamber containing 15 ml Krebs–Ringer bicarbonate solution (37 ◦ C, pH 7.4) of the following composition: 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.17 mM MgSO4 , 1.15 mM KH2 PO4 , 25.1 mM NaHCO3 and 11 mM glucose. Unlike the conventional organ chambers, the modified organ chambers do not have the gas bubbling system. Instead, the solution (PBSG) in the chambers was rapidly stirred so that air can be efficiently dissolved into solution to maintain the equilibrium of oxygen between the solution and the environment. In this study, each organ chamber (conventional or modified) was installed with a Clark-type NO electrode or an ISO-NOPF carbon fibre NO electrode (WPI, Florida) for monitoring NO concentration in the solution. 2.4. Simultaneous measurements of aortic relaxation and exogenous NO concentration The isolated and cleaned rat aorta was cut into several ∼5 mm long rings. The rings were mounted on stainless steel hooks connected to force transducers, which were linked to a data acquisition unit MP100A (Biopac Systems, California). The rings were allowed to equilibrate in the buffer solution for 60 min under a resting tension of 2 g at 37 ◦ C. During this period, the buffer solution was exchanged three times, and then the aortic rings were precontracted with 10 M phenylephrine (Phe) prior to cumulative injections of different concentrations (1 nM to 10 M) of ACh. After measurements of [ACh]-vasorelaxation response curves were completed, the tissue buffer in each chamber was changed three times in 1 h. Then the aortic relaxation response to different concentrations of NO (0.1 nM to 10 M) was recorded, and the NO concentration in the chamber was monitored simultaneously by a Clark-type NO electrode or a carbon fibre NO electrode connected to a four-channel Apollo 4000 Free Radical Analyzer (WPI, Florida). In conventional organ chamber experiments, an O2 -containing gas mixture flows from the bottom to the top of the chamber. In some measurements, we needed to change the flow rate of the bubbling gas in the conventional organ chamber. We use the word “high flow rate” to represent a normal flow rate (∼10 ml min−1 ) and “low flow rate” to represent a flow rate in the range of 0.3 to 0.5 ml min−1 . 2.5. Data analysis The values of the aortic relaxation response to different authentic NO concentrations were given by mean ± standard error (S.E.). To determine the aortic relaxation response at a certain NO concentration, we plotted the values (mean) of aortic relaxation versus log[NO] and fitted these data points with a sigmoid curve using SigmaPlot 9.0 software. The mean and S.E. of the vasorelaxation were obtained from the best fitting results.
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Fig. 1. [ACh]-vasorelaxation response curves. Measurements were performed in the conventional organ chambers. During the measurements, the gas (95% O2 + 5% CO2 ) was bubbled through the gas inlet on the bottom of the chamber (A) and flowed over the solution surface (B). ACh was added into the solution in cumulative concentrations between 1 nM and 10 M at the times shown by the arrows (from left to right): 1, 3, 10, 30, 100, 300, 1000, 3000, and 10,000 nM.
3. Results 3.1. Stirring of the buffer in the conventional organ chamber by the bubbling gas Aortic rings in the organ chambers were precontracted using 10 M Phe while each chamber was bubbled with a gas mixture (95% O2 + 5% CO2 ). Different concentrations of ACh were cumulatively added into the chambers. The relaxation response curve of an aortic ring is demonstrated in Fig. 1A. To observe the stirring effect of the bubbling gas, the gas flow through the bottom of the chamber was closed with a gas tube placed above the solution and a parafilm membrane covering the chamber. The gas mixture (95% O2 + 5% CO2 ) flowed over the solution and exited the chamber through open ports in the membrane during the measurements. The same experimental protocol was repeated with different ACh concentrations. It was observed that the aortic ring had no response to [ACh] < 100 nM. For [ACh] > 100 nM, the aortic ring responded to the ACh added, but the response was sometimes small, sometimes large or sometimes delayed (Fig. 1B). 3.2. The effect of the flow rate and O2 content of the bubbling gas on the NO concentration in the conventional organ chamber Compared to stable chemicals such as ACh, NO is unstable. In the conventional organ chamber, NO can be consumed by reacting with O2 and can also be brought out of the solution by the bubbling of the gas. Thus, it is necessary to test how the bubbling gas and its O2 content affect NO concentration in the solution. Experiments were performed to test the response of the aortic ring to different concentrations of NO at a low flow rate and a high flow rate of bubbling gas. It was observed that the current of the electrode and the aortic tension response to each NO addition was irregular at low flow rate and did not change immediately after each NO injection (Fig. 2A). In contrast, when a high flow rate of bubbling gas was used, the detected NO concentration was approximately proportional to the amount of injected NO
and the aortic tension changed immediately following each NO injection (Fig. 2B). However, NO decay was much faster at a high flow rate of bubbling gas compared to that predicted from the rate constant of NO autoxidation. To examine the effect of O2 concentration on the effective NO concentration, we used a high content of O2 -containining gas mixtures (95% O2 + 5% CO2 ) and a low content of O2 containing gas mixtures (either 20% O2 + 75% N2 + 5% CO2 or 20% O2 + 80% N2 ). Experiments were performed with a high flow rate of gas mixture. It was observed that when a low content of O2 -containing gas mixtures was used, NO concentrations were 2 or 3 times higher and the aortic relaxation was more sensitive to the same amount of injected NO (Fig. 2C) than when a high content of O2 -containing gas mixture was used (Fig. 2B). The NO concentration dependent aortic relaxation under two different O2 partial pressures and in two different solutions were shown in Fig. 2D. The apparent EC50 of aortic relaxation for authentic NO in Krebs–Ringer solution bubbled with 95% O2 + 5% CO2 gas mixture (a) and with 20% O2 + 5% CO2 + 75% N2 gas mixture (b) was 340 ± 40 nM (n = 8) and 81 ± 4 nM (n = 8), respectively. The apparent EC50 of aortic relaxation for authentic NO in PBSG solution bubbled either with 20% O2 + 80% N2 gas mixture or with air (c) was 45 ± 2 nM (n = 5). 3.3. The rate of NO decay and the vasorelaxation response to authentic NO in the modified organ chambers In the modified organ chamber, O2 -containing gas mixture was not directly bubbled into the solution. Instead, the solution in the chamber was rapidly stirred with a magnetic bar so that environmental O2 could be effectively dissolved into the solution and the equilibrium between the O2 concentration in solution and the O2 partial pressure in the environment was maintained. It was observed that the rate of NO decay in the modified chamber was much slower than that in the conventional organ chamber (Fig. 3), especially when NO concentration was 300 nM or less. In Fig. 4, we demonstrate the NO concentration and the vasorelaxation response of the aortic rings to different concentrations
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Fig. 2. The effect of the flow rate, O2 content of bubbling gas and the composition of buffer solution on NO concentration and aortic relaxation. In (A)–(C), experiments were performed in Krebs–Ringer solution. The downward arrows indicate the times that different concentrations of NO were added. The amounts of added NO from left to right are 1, 3, 10, 30, 100, 300, 1000, 3000 and 10,000 nM. (A) Aortic relaxation (a) and NO concentration (b) with a slow flow rate of bubbling gas (95% O2 + 5% CO2 ). (B) Aortic relaxation (a) and NO concentration (b) with a high flow rate of bubbling gas (95% O2 + 5% CO2 ). (C) Aortic relaxation (a) and NO concentration (b) with a high flow rate of bubbling gas (20% O2 + 75% N2 + 5% CO2 ). (D) The aortic relaxation responses were measured in the Krebs–Ringer buffer bubbled either with 95% O2 + 5% CO2 (䊉) or with 20% O2 + 75% N2 + 5% CO2 (), and in the PBSG solution bubbled either with 20% O2 + 80% N2 or with air ().
of authentic NO in the modified organ chambers. The apparent EC50 of aortic relaxation for NO (Fig. 4B) was found to be 9.7 ± 0.4 nM (n = 6), and the threshold of NO concentration for dilating the aorta was ∼0.3 nM in the modified organ chamber.
Fig. 3. The rate of NO decay in the conventional organ chamber and the modified organ chamber as O2 (20% O2 + 80% N2 ) gas mixture or air (21% O2 + 79% N2 ) was used in the experiments. Different amounts of NO were injected at the times designated by the arrows. Measurements were performed in the modified organ chamber (a) and the conventional organ chamber (b). The curve a and b in the inset are magnified (50×) from curve a and b, respectively. The arrows indicate the times that different concentrations of NO were added. The amounts of added NO from left to right are 1, 3, 10, 30, 100, 300, 1000 and 3000 nM for curves a and b, and 1, 3, 10, 30 nM for curves a and b .
4. Discussion To understand what may affect the vasorelaxation response to NO in the conventional organ chamber, we examined the effect of the flow rate of the bubbling gas mixture, O2 partial pressure and buffer composition on aortic relaxation. Our results show that bubbling a gas mixture (95% O2 + 5% CO2 ) into the tissue buffer in the conventional organ chamber not only provided O2 to the solution but also stirred the solution. Without the bubbling gas, even though O2 was provided to the chamber, the [ACh]vasorelaxation response curves could not be obtained correctly (Fig. 1). Although the mode of stirring with the bubbling gas is suitable for non-volatilizable substances such as ACh, it may bring a marked experimental error when determining the actual NO concentration in the solution because NO cannot only be oxidized by O2 but also be purged from the solution by the bubbling gas. Under a constant O2 partial pressure, the rate of NO reaction with O2 is second order in [NO] [13,15–18]. This means that NO decay rate will increase 100 times if its concentration increases 10 times. In this case, rapidly stirring the solution is important for the bolus injection of authentic NO. If the injected NO is not quickly diluted into the whole solution, there will be a relatively higher NO concentration in some localized areas of the solution. A high NO concentration in some areas of the solution will decay much faster, causing a marked consumption of NO by O2 . Furthermore, these areas with localized high
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Fig. 4. Aortic relaxation response to different concentrations of authentic NO in the modified organ chamber. (A) Simultaneous measurements of the [NO]vasorelaxation response curve (a) and NO concentration (b) in the solution. The aortic ring was contracted by 10 M Phe (designated by the downward arrow), then different NO concentrations were added at the times shown by the upward arrows (from left to right): 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10,000 nM. Curve c in the inset was magnified (4×) from the low NO concentration portion of curve b. The added NO concentrations designated by the upward arrows (from left to right) in the inset are 0.1, 0.3, 1, 3, 10, 30 and 100 nM. (B) NO concentration dependent relaxation response of aortas from 6–8 week-old WKY rats. The experimental points (closed circles with error bars) were fitted by a sigmoidal curve (solid line).
NO concentration may move around in the solution. This would explain the detected irregular currents by the NO electrodes and the currents that usually did not immediately increase following an injection of NO (Fig. 2A). Correspondingly, the vasorelaxation response to the injected NO was also irregular. To solve this problem, it was necessary to increase the stirring speed in the conventional chamber by increasing the flow rate of the bubbling gas. As shown in Fig. 2B, at high flow rate of bubbling gas, the response currents of electrodes were more regular and tended to be approximately proportional to the injected NO amounts. However, the rate of NO decay markedly increased as the flow rate of bubbling gas increased. It can be seen that the half life of 100 nM NO in the conventional chamber was only 30–40 s, whereas the half-live of NO autoxidation predicted from published rate constants at this concentration should have been more than 15 min [13,15–18]. These results indicate that the conventional organ chamber, in which the solution is stirred with the bubbling gas, is not the best experimental setup for measuring vasorelaxation to authentic NO. If a low flow rate of bubbling gas were used, a large portion of NO could be oxidized by O2 before NO is uniformly diluted into the whole solution; on the other hand, if a high flow rate of bubbling gas were used, NO decay in the solution would be markedly faster because it could be rapidly purged out by the bubbling gas. Since the flow rate of bubbling gas is usually set by experience, the flow rate used in the conventional organ chamber experiments varies from laboratory to laboratory. Thus, it would not be surprising to find that an EC50 value of vasorelaxation for authentic NO reported from one laboratory is largely different from another in the literature. In Fig. 2C, we showed that NO concentration in the solution was markedly affected by O2 partial pressure. When 20% O2 gas was used, the resulting current detected by the NO electrode to each NO injection was 2 to 3 times higher than the corresponding currents to the same amount of injected NO when 95% O2 gas was used, indicating that the actual NO concentration in the conventional chamber bubbling with 95% O2 gas would be at least 2 times lower than the NO concentration calculated from
the dilution factor in a bolus injection. Correspondingly, the apparent EC50 of aortic relaxation was reduced more than 4fold from 340 nM to 81 nM. This decrease in EC50 is 1.5 to 2 times more than that in the effective NO concentration detected by the electrodes. This difference can be caused by two different factors. The first factor is that it takes time for NO diffusion from the aortic surface to the smooth muscle cell layer in the aortic wall, while the NO concentration in the high O2 concentration solution decreases more rapidly than that in the low O2 concentration solution. Thus, even if NO concentration in the high O2 concentration solution is initially equal to the NO concentration in the low O2 concentration solution, when NO diffuses into the smooth muscle layer, the actual amount of NO diffusion into the aortic wall in the high O2 concentration solution will be less than that in the low O2 concentration solution. The second factor is that the rate of superoxide generation in the high O2 concentration solution is higher than that in the low concentration of O2 solution, resulting in a faster NO decay rate in the aortic wall. When the organ chamber experiments were performed in the PBSG solution bubbled with 20% O2 + 80% N2 or air, the apparent EC50 was found to be 45 nM, which is less than a 2fold decrease. It is unlikely that this decrease was caused by the difference in concentration of a single chemical such as glucose, Ca2+ , or Mg2+ from our preliminary observation (data not shown). To solve the bubbling gas-caused problem in the conventional organ chambers, we used modified organ chambers in our experiments. Because the solution in the modified organ chamber was stirred by a stir bar and not the bubbling gas, the rate of NO decay after each NO injection was markedly slower than that in the conventional organ chamber. Since NO can stay in the solution much longer, a relatively higher effective NO concentration can be built up in the aortic wall. Using this modified organ chamber containing PBSG solution equilibrated with 20% O2 + 80% N2 gas or air, we observed that the threshold of NO concentration for inducing a minimal aortic relaxation was as low as 0.3 nM, and the EC50 of aortic relaxation for NO was ∼10 nM. As shown in Fig. 3, the low concentration of NO (<300 nM)
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is much more stable in the modified organ chamber than that in conventional chamber. Correspondingly, the vasorelaxation curves recorded in the two kinds of organ chambers have some notable differences. The typical aortic relaxation response curve to each NO injection in the modified organ chamber exhibited a “stair” shape (see curve a in Fig. 4A), which declined and then reached a plateau before the next NO injection. In contrast, the vasorelaxation curve to each NO injection recorded in the conventional chamber formed a downward peak (Fig. 2B and C). Although we (in this study) and others [9] have shown that the EC50 of vasorelaxation for NO was around 10 nM, the actual NO concentration required to induce 50% vasorelaxation in the vascular wall has not been directly measured. Strong evidence showed that NO can be consumed in cells and blood vessels by O2 species or O2 -dependent reactions [14,19,20]. Thus, even if experiments are performed in the solution equilibrated with 20% O2 gas, the reactive O2 species may still have a marked influence on the effective NO concentration in the aortic wall. From this viewpoint, the actual effective NO concentration in the vascular wall may be appreciably lower than 10 nM whether NO is generated outside or inside of the vascular wall, because the rate of NO consumption in the vascular wall is significantly higher than that in the solution. Therefore, the value of EC50 of rat aorta, 10 nM, for authentic NO that is reported in this study should be considered as an apparent EC50 , meaning that the aortic relaxation response to authentic NO reaches 50% of maximal relaxation while the NO concentration at the aortic surface rather than inside of the vascular wall is equal to 10 nM. In summary, the conventional organ chamber is not the best setup for measuring the vasorelaxation response to authentic NO. By using an organ chamber modified to provide rapid stirring under ambient air or 20% O2 , we observed that the EC50 for aortic relaxation was 10 nM. The NO concentration that caused a minimal relaxation was ∼0.3 nM. This modified organ chamber method enables a better quantitative characterization of the vasorelaxation response to authentic NO in organ chambers under different physiological and pathological conditions. Acknowledgement This work was supported by HL63744, HL65608, and HL38324 (J.L.Z.) and the American Heart Association Scientific Development Grant 0130367N (X.L.). References [1] Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288(5789):373–6.
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