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Anesth Analg 2003;97:1492-1496
© 2003 International Anesthesia Research Society

CRITICAL CARE AND TRAUMA

Peroxynitrite Decreases Dopamine’s Vasoconstrictive Activity

Ko Takakura, MD PhD*, Wen Xiaohong, MD{dagger}, Kenji Takeuchi, MD{dagger}, and Satoru Fukuda, MD PhD{dagger}

*Department of Anesthesiology, Asahi University, Hozumi, Gifu; and {dagger}Department of Anesthesiology and Reanimatology, Fukui Medical University, Matsuoka, Japan

Address correspondence and reprint requests to Ko Takakura, MD, Department of Anesthesiology, Asahi University, 1851 Hozumi, Hozumi, Gifu 501–0296, Japan. Address e-mail to takakura{at}dent.asahi-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Peroxynitrite (ONOO-1) reacts with dopamine to form an oxidized derivative. To investigate the vasoconstrictive activity of this derivative, we performed functional examinations with dopamine treated with ONOO-1 or 3-morpholinosydonimine-N-ethyl-carbamine (SIN-1; an ONOO-1 producer) on isolated strips of rat thoracic aorta. To exclude the direct effect of ONOO-1, the strips were pretreated with methylene blue, a guanylyl cyclase inhibitor. Dopamine induced concentration-dependent contraction, but dopamine pretreated with ONOO-1 decreased the contraction in an ONOO-1-concentration-dependent manner. Both maximum contractions and 50% effective concentration values for dopamine-induced vasocontraction were significantly decreased by pretreatment with ONOO-1. Dopamine incubated with SIN-1 also decreased the contraction, the decrease being dependent on the incubation time. ONOO-1 formation is a favored reaction and occurs easily when cellular production of both nitric oxide and superoxide increases, as in septic shock. These results may, at least in part, account for dopamine’s limitation as a vasoconstrictor in septic shock.

IMPLICATIONS: Peroxynitrite (ONOO-1) reacts with dopamine to form an oxidized derivative. We investigated the vasoconstrictive activity of this derivative with functional examinations using rat thoracic aorta and found the activity decreased. As ONOO-1 formation increases in septic shock, our results may account for dopamine’s limitation as a vasoconstrictor in septic shock.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The vascular hyporeactivity to catecholamines such as dopamine, norepinephrine, and epinephrine limits successful therapy of the hypotension that is a key feature of the pathophysiology of septic shock. Although the mechanisms of the vascular hyporeactivity have not been elucidated completely, large amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in response to bacterial endotoxin or inflammatory cytokines seem to participate in the hyporeactivity (1,2). iNOS has been identified in many tissues, including vascular endothelium (3), smooth muscle (4), and myocardium (5). Concomitant with the enhanced NO production, bacterial endotoxin, or inflammatory cytokines also increase cellular superoxide anion (O2-1·) production, not only from xanthine oxidase, NAD(P)H oxidase, mitochondria, arachidonic acid metabolism, but also from constitutive NOS and iNOS (6,7).

NO reacts with O2-1· at a near diffusion-limited rate to form the potentially toxic NO metabolite, peroxynitrite (ONOO-1) (8). The second order rate constant for the formation of ONOO-1 is approximately three times greater than the rate of superoxide dismutase-catalyzed dismutation of O2-1·, 2 x 109 M/s (8). Therefore, ONOO-1 formation is a favored reaction and occurs easily when cellular production of both NO and O2-1· increases, as in septic shock (9,10).

Recently, we reported that ONOO-1 oxidized norepinephrine and consequently inactivated its vasoconstrictive actions (11). ONOO-1 also reacts with dopamine to form an oxidized derivative, 6-hydroxyindole-5-one (12). However, the vasoactivities of the oxidized derivative have never been addressed. In this study, we treated dopamine with ONOO-1 and investigated the vasoconstrictive activity of dopamine treated with ONOO-1 on isolated strips of rat thoracic aorta. If the activity of dopamine treated with ONOO-1 decreases, this may account for dopamine’s limitation as a vasoconstrictor in septic shock.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The experimental protocol was approved by the institutional animal care committee of Fukui Medical University. Male Wistar rats weighing 250–300 g were used.

Rats were killed by decapitation under isoflurane anesthesia, and the thoracic aorta was isolated and removed (13). The thoracic aorta was placed in Krebs Henseleit solution (mM; NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, and glucose 10; pH value of 7.4). Helical strips were carefully prepared under a dissecting microscope. To avoid the possible involvement of endothelium-derived relaxing factors in the mechanical response, the absence of a relaxant response to applications of acetylcholine was ascertained after the endothelium was rubbed off with filter paper (13). The strip was carefully suspended in an organ chamber containing 30 mL of Krebs Henseleit solution bubbled with 95% O2/5% CO2 at 37°C. After a resting tension of 0.5 g was applied during an hour equilibration period, changes in the tension were recorded isometrically when the test drugs were added. To investigate dopamine’s vasoconstrictive activity, strips were pretreated with 30 µM of methylene blue, a guanylyl cyclase inhibitor (14), for 5 min. Then cumulative amounts of dopamine or dopamine pretreated with ONOO-1 or 3-morpholinosydonimine-N-ethyl-carbamine (SIN-1; an ONOO-1 producer) were added. To investigate dopamine’s vasorelaxant activity, strips were pretreated with 10 µM of phentolamine, an {alpha} adrenergic antagonist, for 5 min and contracted with 1 µM of prostaglandin F2{alpha}, followed by the additions of dopamine.

Contractions were expressed as milligram contractile tension developed per milligram wet tissue weight. Relaxations were expressed as a percentage of dopamine-induced contraction.

As alkaline conditions are required for ONOO-1 stability, the stock solution (80 mM of ONOO-1 in 0.3 N of NaOH) was diluted to appropriate concentrations with 0.3 N of NaOH. The ONOO-1 was added in a 50 mM of sodium phosphate buffer solution containing dopamine at a pH value of 7.4 and 37°C. To make the ONOO-1 react with dopamine before decomposition of ONOO-1 (half-life < 1 s at pH value of 7.4 (8)), reaction solutions in tubes were rapidly stirred during bolus ONOO-1 additions (15). The final pH value of all reaction solutions was checked to ensure adequate buffering. To examine possible decomposition products of ONOO-1 such as nitrate or nitrite (8), decomposed ONOO-1 was cumulatively applied to the strips precontracted by dopamine. Decomposed ONOO-1 was made by incubation for 10 min in a pH value of 7.4 buffer (15).

Dopamine (1 µM) was incubated with 1 mM of SIN-1 in a 50-mM sodium phosphate buffer solution at a pH value of 7.4 and 23°C for 0, 30, 90, or 180 min. Under these conditions, SIN-1 produced submicromolar ONOO-1 per minute for several hours continuously (16).

ONOO-1 and SIN-1 were purchased from Dojindo Laboratories (Kumamoto, Japan), and the other drugs were purchased from Sigma (St Louis, MO).

The results are expressed as mean ± SD. The maximum response and 50% effective concentration (EC50), the concentration producing a half-maximal response, were determined graphically by Finley’s probit analysis from individual concentration-response curves. Statistical difference was analyzed by one-factor analysis of variance and Fisher’s protected least significant difference as post hoc comparisons for multiple comparison at a significance level of 0.05. Analyses were performed on a personal computer using Stat View II 4.0 software (Abacus Concepts, Berkeley, CA).


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Dopamine induced a concentration-dependent contraction, which was not affected by pretreatment with 30 µM of methylene blue (n = 8; Fig. 1A). Pretreatment of dopamine with ONOO-1 decreased its vas-oconstrictive activity in an ONOO-1-concentration-dependent manner. Representative tracings for contraction by dopamine with or without pretreatment with ONOO-1 are shown in Figure 1B. Both maximum contractions and EC50 values for dopamine-induced vasocontraction were significantly decreased by pretreatment with ONOO-1 (Table).



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  		Figure 1. (A) Contractile forces induced by dopamine pretreated with peroxynitrite (ONOO-1). Dopamine (3 µM–30 mM, 0.1 mL) was added to an organ chamber containing 30 mL of Krebs Henseleit solution, so that the final concentration of dopamine was 10 nM–100 µM. • = dopamine untreated with ONOO-1 on strips untreated with methylene blue. {circ} = dopamine untreated with ONOO-1 on strips pretreated with 30 µM of methylene blue. {blacksquare} = dopamine pretreated with 100 µM of ONOO-1 on strips pretreated with 30 µM of methylene blue. {square} = dopamine pretreated with 300 µM of ONOO-1 on strips pretreated with 30 µM of methylene blue. {blacktriangleup} = dopamine pretreated with 1 mM of ONOO-1 on strips pretreated with 30 µM of methylene blue. Data are expressed as mean ± SD of eight experiments. *P < 0.05 compared with {circ}. ¶P < 0.05 compared with {blacksquare}. {dagger}P < 0.05 compared with {square}. There is no significant difference between • and {circ}. (B) Representative tracings of the contractile forces induced by various concentrations of dopamine (10 nM–100 µM) pretreated with or without 1 mM of ONOO-1 on aortic strips pretreated with methylene blue (30 µM). DOA = dopamine.

 

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  		Table 1. Maximum Contractions and EC50 Values of Dopamine Dose Response Curves in Rat Thoracic Aorta
 
Decomposed ONOO-1 relaxed the strips dose-dependently (Fig. 2A). The relaxations were 4% ± 2%, 20% ± 14%, 57% ± 18%, and 84% ± 17% by 10 µM, 100 µM, 1 mM, and 3 mM decomposed ONOO-1, respectively (n = 8). Relaxation was completely inhibited by pretreatment with 30 µM of methylene blue (0% ± 0%, 0% ± 0%, 2% ± 2%, and 5% ± 3% at 10 µM, 100 µM, 1 mM, and 3 mM decomposed ONOO-1 application, respectively; n = 8; Fig. 2B).



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  		Figure 2. (A) The representative relaxation by peroxynitrite (ONOO-1) decomposed previously on a strip contracted by 1 µM of dopamine. (B) The effects of decomposed ONOO-1 to contractile forces induced by 1 µM of dopamine on a strip pretreated with 30 µM of methylene blue. Decomposition of ONOO-1 (10 µM–3 mM) was performed by incubating for 10 min in a pH-7.4 buffer. As decomposed ONOO-1 (0.1 mL) was added in an organ chamber containing 30 mL of Krebs Henseleit solution, the final concentration of the decomposed ONOO-1 was 33 nM–10 µM. n = 8 experiments each.

 
Incubation of dopamine (1 µM) with SIN-1 (1 mM) decreased its vasoconstrictive activity depending on the incubation time (Fig. 3). Incubation with SIN-1 for 30 min significantly decreased the vasoconstrictive force induced by dopamine, and incubation for 180 min abolished dopamine-induced vasocontraction.



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  		Figure 3. Effects of 3-morpholinosydonimine-N-ethyl-carbamine (SIN-1) on dopamine-induced contraction. Dopamine was incubated with SIN-1 (1 mM) in 50 mM of sodium phosphate buffer at 23°C. Data are expressed as mean ± SD of eight experiments. *P < 0.05 compared with no SIN-1 incubation.

 
Phentolamine (10 µM), an {alpha}-adrenergic antagonist, completely inhibited the contraction induced by 1 µM of dopamine (n = 8; Fig. 4, A and B). Dopamine did not induce relaxation in the strips pretreated with 10 µM of phentolamine and contracted by 1 µM of prostaglandin F2{alpha} (2 ± 2 and 4 ± 8 mg tension/mg wet tissue by 1 µM and 10 µM dopamine, respectively; n = 8; Fig. 4C).



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  		Figure 4. (A) The representative contraction induced by 1 µM of dopamine on an aortic strip. (B) The effect of pretreatment with 10 µM of phentolamine to the contraction induced by 1 µM of dopamine. (C) The effect of dopamine on the strip pretreated with 10 µM of phentolamine for 5 min and contracted by 1 µM of prostaglandin F2 {alpha}. n = 8 experiments each.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Pretreatment of dopamine with ONOO-1 decreased its vasoconstrictive activity in a ONOO-1-concentration-dependent manner in the strips pretreated with methylene blue (Fig. 1, A and B). Both maximum contractions and EC50 values of the dopamine-induced vasoconstrictive response curve were significantly decreased by pretreatment with ONOO-1 (Table). These results may, at least in part, account for the decrease of the vasoactivities of dopamine as a vasoconstrictor in septic shock when ONOO-1 is produced (9,10).

During the pretreatment of dopamine with ONOO-1, ONOO-1 oxidizes dopamine (12), and the rest of ONOO-1 decomposes in a sodium phosphate buffer (8). ONOO-1 or decomposed ONOO-1 stimulates guanylyl cyclase and cyclic guanosine monophosphate synthesis (17) and relaxes vascular smooth muscle (18). Decomposed ONOO-1 relaxed the aortic strips dose-dependently in our study (Fig. 2A). The relaxations by decomposed ONOO-1 via stimulation of guanylyl cyclase prevents dopamine-induced vasocontraction. To avoid the effects of decomposed ONOO-1 in our experiments, we added methylene blue, a guanylyl cyclase inhibitor (14). Methylene blue completely inhibited relaxations by decomposed ONOO-1 (Fig. 2B) but had no effect itself on dopamine-induced vasocontraction (Fig. 1A).

It has been suggested that the net exposure to ONOO-1 should be shown as the area under the curve of time versus concentration (19), because even small concentrations of ONOO-1 may be able to inactivate dopamine if the exposure to ONOO-1 continues for too long. When we incubated dopamine with the ONOO-1 producer SIN-1, dopamine-induced vasoconstriction was inactivated time-dependently (Fig. 3). SIN-1 continuously releases both NO and O2-1· and consequent submicromolar ONOO-1 (16,20). Although ONOO-1 is produced from stimulated vascular smooth muscles (21) and endothelial cells (22), it is unknown how much ONOO-1 is produced in the vascular system during sepsis. However, it may be noteworthy that leukocytes stimulated by endotoxin generate submicromolar ONOO-1 (23), and ONOO-1 continuously generated during sepsis may be enough to deactivate dopamine.

The contraction induced by 1 µM of dopamine (Fig. 4A) was completely inhibited by phentolamine (Fig. 4B), an {alpha}-adrenergic antagonist, which shows that the contraction induced by dopamine was via an {alpha}-adrenergic effect. Dopamine also has ß-adrenergic and dopaminergic effects and relaxes vascular smooth muscle by these mechanisms (24,25). However, there was no relaxation by dopamine on the strips contracted by prostaglandin F2{alpha} in the presence of phentolamine (Fig. 4C). Cohen and Berkowitz (24) reported that aortic strips from adult rats were difficult to relax with dopamine, a finding similar to that in our study.

In conclusion, ONOO-1 decreased dopamine’s vasoconstrictive activity. As ONOO-1 is produced in human septic shock (9,10), these results may account for dopamine’s limitation as a vasoconstrictor in septic shock.


   Acknowledgments
 
Supported, in part, by a grant (KT; no. C2–12671458) from the Ministry of Education, Science, Sports, and Culture, Japan.


   References
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 Introduction
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 Discussion
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Accepted for publication June 3, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2003 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press