fetal-calfserum, penicillin, streptomycin (Gibco, Paisley,. Renfrewshire, Scotland, U.K.) and glutamine (480 mg/ litre). When confluent, (8-12) x 106 cells (0.5 ml) ...
921
Biochem. J. (1990) 266, 921-923 (Printed in Great Britain)
Nitric oxide is inactivated by the bacterial pigment pyocyanin John B. WARREN, Rashpal LOI, Nigel B. RENDELL, and Graham W. TAYLOR Department of Clinical Pharmacology, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, U.K.
Pyocyanin is a phenazine pigment produced by the bacterium Pseudomonas aeruginosa and found in human lung secretions. Micromolar concentrations of pyocyanin inhibited the bioactivity of endotheliumderived relaxing factor (EDRF) generated from bovine pulmonary-artery endothelium in response to bradykinin. This inhibition was reversed by perfusing the EDRF-bioassay system with pyocyanin-free buffer for 15 min, but persisted in the presence of superoxide dismutase (20 units/ml). When nitric oxide, the major component of EDRF, was passed into an aqueous solution of pyocyanin in the absence of 02, a rapid colour change occurred from blue to pink; m.s. analysis of the products showed that the pyocyanin had been converted into a nitrosylated species. INTRODUCTION
Endothelium-derived relaxing factor (EDRF), first described by Furchcott & Zawadzki [1], is now known to be mainly nitric oxide (NO) [2]. Endogenous NO is a major influence in the control of blood flow, blood pressure and immune function [3]. Many compounds inhibit the activity of EDRF, including haemoglobin, superoxide anion and Methylene Blue; a number of compounds interact with NO through a redox mechanism [4]. The bacterium Pseudomonas aeruginosa synthesizes a phenazine pigment, pyocyanin (Fig. 1), which has been detected in concentrations above 10-4 M in the sputum of infected patients [5]. Phenazine pigments such as pyocyanin are electron acceptors and are known to inhibit mitochondrial respiration by this mechanism [6]. We therefore investigated whether pyocyanin would inactivate EDRF in vitro and determined the products of the pyocyanin-NO interaction. METHODS Pyocyanin and 1-hydroxyphenazine were synthesized by photolysis of phenazine methosulphate and phenazine (Aldrich Chemical Co., Gillingham, Dorset, U.K.) respectively, as previously described [5,7,8]. Bovine pulmonary-artery endothelial cells were prepared by nonenzymic methods [9]. A pure cell line was used between passages 10 and 15, which had been shown to have normal cobblestone morphology, Factor VIII immunoreactivity and high levels of angiotensin-converting-
CH3
Fig. 1. Structure of pyocyanin Abbreviation used: EDRF, endothelium-derived relaxing factor. Vol. 266
enzyme activity. The cells were grown on Cytodex 3 microcarrier beads (Pharmacia, Uppsala, Sweden) in Dulbecco's modified Eagle's medium with 10% (v/v) fetal-calf serum, penicillin, streptomycin (Gibco, Paisley, Renfrewshire, Scotland, U.K.) and glutamine (480 mg/ litre). When confluent, (8-12) x 106 cells (0.5 ml) was packed into a column. This was maintained at 37 °C by a water jacket and perfused at 2 ml/min with KrebsHenseleit buffer (NaCl, 118 mM; KCI, 4.7 mM; MgSO4 7H20, 1.2 mm; NaH2PO4, 1.2 mM;NaHCO3, 25 mM;CaCl2, 1.5 mM), bubbled with C02/air (1:19). The effluent of the column was dripped on to a bioassay tissue of 3 mm segments of lower thoracic aorta from male Sprague-Dawley rats, pre-contracted with phenylephrine (10-6 M). The rings had been previously deendothelialized by gently rotating around a closed pair of fine forceps and then mounted on hooks attached to a Grass model 79D force transducer and recorder (Grass Instrument Co., Quincy, MA, U.S.A.). They were perfused with Krebs-Henseleit buffer at 2 ml/min by a second pump, which by-passed the column of cells, and set to a resting tension of 1.1-1.5 g. The removal of the endothelium from the bioassay was confirmed by the lack of relaxation of the pre-contracted ring on stimulating with bradykinin, which was passed through the pump which by-passed the column. The cell column was stimulated by 1 min pulses of bradykinin to give a dose response over the range 10-1o-10-6 M, doses being given in random order. Pyocyanin was then added via the pump, which by-passed the column, to give a tissue concentration of 10-6 M. After 15 min the dose response to bradykinin was repeated. When this had been completed, the pyocyanin was switched off and 15 min later the column was again stimulated with 10-6 M-bradykinin. In a further four preparations, superoxide dismutase (from bovine erythrocytes; 20 units/ml; Sigma, Poole, Dorset, U.K.) was included in the buffer perfusing the bioassay tissue, by-passing the endothelial column. NO (BDH, Poole, Dorset, U.K.) and helium (British Oxygen Company) were bubbled through a solution of pyocyanin, 1-hydroxyphenazine or 1-naphthol in the absence of 02 The compounds were initially dissolved in a minimum amount of ethanol and diluted to 1 mg/ml in helium-sparged water. Samples were removed at
J. B. Warren and others
922 r-
suitable intervals for analysis by u.v. spectroscopy in a Perkin-Elmer 555 instrument and also by m.s., in a Finnigan 4500 instrument, in the desorption-electronimpact and fast-atom-bombardment modes.
0
1 125-
._-
>. 100-
-o
RESULTS Bradykinin released EDRF from the endothelial-cell column, causing relaxation of the phenylephrine-contracted rat aorta (Figs. 2 and 3). At 10-6 M-bradykinin this relaxation was 59 + 9 % (mean+ S.D., n = 9), which was used to standardize all bradykinin responses to overcome bioassay variation. Pyocyanin (10-8-10-6 M) did not constrict the bioassay tissue under resting tension. When the bioassay tissue was continuously contracted with phenylephrine, the addition of pyocyanin (10-6 M) did not alter the contraction, although the response to EDRF released by bradykinin was abolished [P < 0.001 (analysis of variance), Fig. 2]. A 10-fold lower concentration of pyocyanin did not significantly inhibit the action of EDRF. When the bioassay tissue was washed for 15 min with buffer containing phenylephrine alone, the response to EDRF returned, with 97+500O relaxation caused by 1O-6 Mbradykinin. In the presence of superoxide dismutase (Fig. 3) the inhibitory effect of pyocyanin persisted, although the effect was slightly diminished. NO bubbled through an 02-free aqueous solution of pyocyanin caused an immediate colour change from blue to pink. The pH of the solution was unaffected. The u.v. spectrum of the product in water (pH 4.5) showed a Amax. of 304 nm (cf. pyocyanin, which has a Amax. of 278 nm [10]). The spectrum shifted bathochromically in 0.1 M-
Control
.0
75-.
(D
0 m
50-
-
25-
/
1 0-6 M - Pyocyanin T -
C
0
xm
Av
I
I
t
10
9
8
7
6
-log{[Bradykinin] (M)} Fig. 2. Effect of pyocyanin of ERDF-induced relaxation of a
precontracted
rat
aortic-ring preparation
Bradykinin causes a dose-dependent release of EDRF as determined on a rat aortic-ring bioassay (@). The effect of EDRF was abolished by the presence of 10-6 M-pyocyanin on the bioassay tissue (0). Data are shown as means +S.E.M. (n = 9; five preparations). When pyocyanin was removed from the bioassay tissue, the EDRF response returned to normal (97 + 5 %).
NaOH to a Amax of 342 nm. Fast-atom-bombardment m.s. showed that pyocyanin (ions at m/z 211/212) had disappeared completely. Under desorption-electron-im-
SOD
)-(20 units/ml)--
w
PE 10-6 M-PYO
+SOD (20 units/ml)
I
4
4
4
4
10-8 M
10-8 M
[BK]... 10-7 M 1 0-8 M
A
4 10-8 M
H 10min-I PE
T
, I
4 [BK]
-/40 min I
1
4 10-8 M
ig
0-6 M-Pyo
10-8 M
4
lo-8
M
1
g
resting tension
Fig. 3. Effect of superoxide dismutase (SOD) on the interaction between pyocyanin (PYO) and EDRF The experiment was performed with four preparations, and a representative trace from one preparation is shown. The bioassay tissue was contracted with 10-6 M-phenylephri-ne (PE),~and the EDRF responses to 10- and 10-8 M-bradykinin were determined; superoxide dismutase in the buffer perfusing the bioassay tissue did not diminish this response. In the presence of both pyocyanin and superoxide dismutase the response to EDRF was inhibited almost to the same extent as with pyocyanin alone.
1990
Bacterial pigment pyocyanin inactivates NO
pact ionization, small amounts of 1 -hydroxyphenazine and 1-methoxyphenazines were observed, together with a major species generating intense ions at m/z 221 (M+*) and 193 (M+`-CO). This could be explained by nitrosylation of pyocyanin, followed by dehydration (perhaps in the mass spectrometer) to form a substituted oxazoline. 1 -Hydroxyphenazine, the demethyl analogue of pyocyanin, also reacted with NO to form a nitroso derivative (m/z 225 M+). l-Naphthol (ions at m/z 144 M+, 115) was treated with NO under the same condition and was also nitrosylated (m/z 173 M+, 156, 115), whereas 1-methoxyphenazine was unaffected by NO, suggesting that the presence of a phenolic hydroxy group, rather than the phenazine nucleus, was required for reaction.
DISCUSSION We have shown that pyocyanin is capable of inhibiting the activity of EDRF released from pulmonary endothelial cells. This occurred at a pyocyanin concentration which is over 100-fold less than that which has been reported in the sputum of patients with cystic fibrosis who are infected with Pseudomonas aeruginosa [5]. It is unlikely that pyocyanin is acting intracellularly in this system, because the antagonism of EDRF was easily reversed by washing the bioassay tissue with pyocyanin-free buffer. There are three basic mechanisms by which EDRF could be inactivated by pyocyanin: either by direct nitrosation of the activated aromatic nucleus, through electron transfer to pyocyanin (an electron acceptor [11]), generating the electrophile NO+, or through increased superoxide formation. Moncada and colleagues [4] were the first to suggest that the inactivation of EDRF by a number of compounds in vitro depended on their redox potential and the formation of superoxide. Pyocyanin could generate superoxide by interfering with the respiratory chain, or through intracellular reduction to leucopyocyanin, which then reoxidizes spontaneously in air to form superoxide [12]. In our system, superoxide dismutase did not abolish the inhibitory effect of pyocyanin on the bioassay. A small effect was noted, either because pyocyanin acts partially through superoxide formation or, more likely, because superoxide dismutase increases the half-life of EDRF [4]. Further, as the reaction between NO and pyocyanin can take place in the absence of 02 and tissue, we conclude that superoxide generation is not necessary to explain the inhibitory effect of pyocyanin on EDRF in vitro. The nitrosylation of hydroxylated (activated) aromatic nuclei, such as 1 -naphthol, provides further evidence that the inactivation of NO may occur independently of either superoxide formation or via a redox mechanism and NO' formation. The presence of 02 in vivo may convert NO into NO2 with the consequent hydration to HNO3 and HNO2; the latter can also nitrosylate activated aromatic nuclei. As NO production occurs in both the pulmonary endothelium and in inflammatory cells, our findings raise the possibility that the reaction between pyocyanin and NO may occur in vivo in patients infected with Pseudomonas aeruginosa (e.g. those with severe bronchiectasis or cystic fibrosis). Reduced endothelium-dependent Received 27 October 1989/2 January 1990; accepted 16 January 1990
Vol. 266
923
relaxation has been observed in pulmonary arteries from patients with cystic fibrosis, although the mechanism is unexplained [13]. Clearly, for pyocyanin to be a contributory factor in pulmonary hypertension, then it would need to diffuse from the airway lumen (where it can be present in mucus at concentrations of 10-i M). It is not possible to estimate the concentration to which the pulmonary endothelial cell would be exposed during the infective episode, but the concentration of pyocyanin would have to reach 1O-6'M at the vessel wall for a biologial effect to be observed. Neutrophils also synthesize NO, although its importance to function has not been elucidated [14]. It is known that pyocyanin stimulates the production of superoxide from neutrophils [15], and this may be a further factor in neutralizing neutrophil-derived NO. The cytotoxic activity of macrophages has been shown to be dependent upon NO formation from L-arginine [16,17]. The effect of pyocyanin on NO production by. these inflammatory cells is not known. Pyocyanin is a low-molecular-mass species present in high concentration of purulent secretions from patients colonized with Pseudomonas aeruginosa. The importance of its reaction with NO to both the pulmonary circulation and inflammatory cells needs to be assessed in vivo. We thank the Medical Research Council for financial support.
REFERENCES 1. Furchgott, R. F. & Zawadzki, J. V. (1980) Nature (London) 288, 373-376 2. Palmer, R. M. J., Ferrige, A. G. & Moncada, S. (1987) Nature (London) 327, 524-526 3. Moncada, S., Palmer, R. M. & Higgs, E. A. (1989) Biochem. Pharmacol. 38, 1709-1715 4. Moncada, S., Palmer, R. M. & Gryglewski, R. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9164-9168 5. Wilson, R., Sykes, D. A., Watson, D., Rutman, A., Taylor, G. W. & Cole, P. J. (1988) Infect. Immun. 56, 2515-2517 6. Armstrong, A. V., Stewart-Tull, E. S. & Roberts, J. S. (1970) J. Med. Microbiol. 4, 249-262 7. Knight, M., Hartman, P. E., Hartman, Z. & Young, A. (1979) Anal. Biochem. 95, 19-23 8. Flood, M. E., Herbert, R. B. & Holliman, R. (1972) J. Chem. Soc. Perkin Trans. I, 4, 622-666 9. Ryan, U. S., Mortara, M. & Whitaker, C. (1980) Tissue Cell 12, 619-635 10. Watson, D., MacDermot, J., Wilson, R., Cole, P. J. & Taylor, G. W. (1986) Eur. J. Biochem. 159, 309-313 11. King, T. E. (1963) J. Biol. Chem. 230, 4032-4036 12. Hassan, H. M. & Fridovich, I. (1980) J. Bacteriol. 141,
156-163 13. Dinh Xuan, A. T., Higgenbottam, T. W., Pepke-Zaba, J., Clelland, C. & Wallwork, J. (1989) Eur. J. Pharmacol. 163, 401-403 14. Wright, C. D., Mulsch, A., Busse, R., Osswald, H. (1989) Biochem. Biophys. Res. Commun. 160, 813-819 15. Miller, K. M., Dearborn, D. G. & Sorensen, R. U. (1987) Infect. Immun. 55, 559-563 16. Stuer, D., Gross, S., Sakuma, I., Levi, R. & Nathan, C. (1989) J. Exp. Med. 169, 1011-1020 17. Marletta, M. A., Yoon, P. S., Iyengar, R., Leaf, C. D. & Wishnok, J. 5. (1988) Biochemistry 27, 87068711
