Vascular actions of the Pseudomonas aeruginosa quorum sensing molecule, N-3-(oxododecanoyl)-L-homoserine lactone

FA Alassaf, M Camara, P Williams, WR Dunn, VG Wilson. University of Nottingham, Nottingham, UK

Many bacteria communicate using intercellular messengers called quorum sensing molecules. These molecules can influence eukaryotic cells with the capacity to supress host immunity (1). We have shown that N-3-(oxododecanoyl)-L-homoserine lactone (3OC12-HSL), a quorum-sensing molecule produced by Pseudomonas aeruginosa, caused vasorelaxation in the porcine coronary artery (2). This study further examined the effect of 3OC12-HSL on the vasoconstrictor tone and its potential targets in porcine coronary arteries. Pig hearts were obtained from the abattoir. Contraction-based studies using isometric tension recording were used. Each organ bath contained Krebs-Henseleit solution, maintained at 37 °C and gassed with 95% O2/ 5% CO2. Each preparation was exposed to KCl (60 mM) to assess viability then pre-contracted with U46619 (5-30 nM) to produce 40-70% of the response to KCl. Inhibitor drugs used in the experiments were incubated for 30 min before U46619 was applied. The drugs used were: the nitric oxide synthase inhibitor, L-NAME (100 µM); the cyclooxygenase (COX) inhibitor, indomethacin (1 µM); the non-selective potassium channel inhibitor, TEA (1 mM); the PPAR-gamma antagonist, GW9662 (1 µM) and the mitochondrial respiration inhibitors, rotenone (1 µM) and antimycin A (1 µM). When the appropriate level of U46619-induced tone had been obtained, 3OC12-HSL (1-30 µM) was added cumulatively, at 20 min intervals, or after the response to any given concentration had achieved steady state. The negative logarithm of the 3OC12-HSL concentration that resulted in a 50% decrease in vasoconstrictor tone (pIC50) was determined. All values were expressed as the mean ± standard error of the mean (S.E.M) in tissues from different animals (n). A student’s unpaired t-test was used to assess the statistical significance of any differences between means. A P value of less than 0.05 was considered to be statistically significant (P<0.05). In control experiments, the relaxation response to the maximum used 3OC12-HSL concentration (30 µM) was -83.5 ± 3.8 %, while the solvent (acetonitrile) produced a change of -7.7 ± 3.3%, (n=6), (P <0.0001). Removing the endothelium caused a leftward displacement of responses to 3OC12-HSL with a statistically significant change in pIC50 to -5.0 ± 0.06, (P<0.01) compared to -4.7 ± 0.04, (n=6) (endothelium-denuded versus endothelium intact vessels respectively).

Table 1 The effects of inhibitory drugs on 3OC12-HSL relaxation responses in porcine coronary artery.

We have shown that the vasorelaxant response to 3OC12-HSL does not involve COX, K+ channels, PPAR-gamma receptor activation since the inhibitors had no effect, nor does it involve inhibition of mitochondrial function (Table 1). The endothelium is not a prerequisite for 3OC12-HSL-induced vasorelaxation since removal of endothelium and L-NAME did not attenuate responses. Rather, the endothelium may modulate 3OC12-HSL vasorelaxation since responses were larger in the absence of the endothelium and nitric oxide.

(1) Ritchie et al. (2003) Infect Immun, 71, 4421-31.

(2) Lawrence et al (1999) Br J Pharmacol, 128, 845-8.