P2X7 receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Abstract

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1beta into mature IL-1beta, which is then secreted. Because both LPS (in vivo) and IL-1beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco's modified Eagle's medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-l-arginine methyl ester or N-([3-(aminomethyl)phenyl]methyl)ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1beta induced iNOS protein expression. Thus, activation of P2X(7) receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1beta-mediated release of nitric oxide by iNOS.

Figure 1. PE-induced vascular contractile responses are attenuated by LPS plus BzATP

Endothelium-intact aortic rings were incubated with DMEM (Control; n=8), LPS (100 μg/ml; n=9), BzATP (150 μM; n=5), and LPS plus BzATP (LPS 100 μg/ml plus BzATP 150 μM; LPS+BzATP; n=7). Data are plotted as the percentage of maximum contractile response to 120 mM KCl. Log EC₅₀ to PE are represented. Data are expressed as mean±SEM of n animals. *P<0.001 vs. Control.

Figure 2. IL-1ra reverses the attenuated vascular contractile response to PE after incubation with LPS plus BzATP

Endothelium-intact aortic rings were incubated with DMEM (Control; n=8), LPS plus BzATP (LPS 100 μg/ml plus BzATP 150 μM; LPS+BzATP; n=7) and LPS plus BzATP plus IL-1ra (LPS 100 μg/ml plus BzATP 150 μM plus IL-1ra 100 ng/ml; LPS+BzATP+IL-1ra; n=7). Data are plotted as the percentage of maximum contractile response to 120 mM KCl. Log EC₅₀ to PE are represented. Data are expressed as mean±SEM of n animals. *P<0.001, LPS+BzATP vs. Control; ^#P<0.01, LPS+BzATP+IL-1ra vs. LPS+BzATP.

Figure 3. NOS inhibitors abolish the effects of LPS plus BzATP on PE-induced vascular reactivity

(A) Endothelium-intact aortic rings were incubated with DMEM (Control; n=8), LPS plus BzATP (LPS 100 μg/ml plus BzATP 150 μM; LPS+BzATP; n=7), l-NAME (l-NAME 100 μM for 40 min in the organ bath after 24 h-incubation with DMEM; n=5), and LPS plus BzATP plus l-NAME (l-NAME 100 μM for 40 min in the organ bath after 24 h-incubation with LPS plus BzATP; LPS+BzATP+l-NAME; n=5). (B) The same curves were represented as (A) in DMEM and LPS plus BzATP. Endothelium-intact aortic rings were incubated with 1400W (1400W 1 μM for 40 min in the organ bath after 24 h-incubation with DMEM; n=5), and LPS plus BzATP plus 1400W (1400W 1 μM for 40 min in the organ bath after 24 h-incubation with LPS plus BzATP; LPS+BzATP+1400W; n=5). Data are plotted as the percentage of maximum contractile response to 120 mM KCl. Data are expressed as mean±SEM of n animals. *P<0.001, LPS+BzATP vs. LPS+BzATP+NOS inhibitor (l-NAME or 1400W).

Figure 4. Oxidized ATP (oATP, P2X₇ receptor antagonist) reverses the attenuated vascular contractile response to PE induced by LPS plus BzATP

Endothelium-intact aortic rings were incubated with DMEM (Control; n=8), LPS plus BzATP (LPS 100 μg/ml plus BzATP 150 μM; LPS+BzATP; n=7), oATP (50 μM for 1 h before incubation with DMEM; n=6) and oATP plus LPS plus BzATP (oATP 50 μM for 1 h before incubation with LPS plus BzATP; oATP+LPS+BzATP; n=6). Data are plotted as the percentage of maximum contractile response to 120 mM KCl. Log EC₅₀ to PE are represented. Data are expressed as mean±SEM of n animals. *P<0.001, LPS+BzATP vs. Control or oATP; ^#P<0.01, oATP+LPS+BzATP vs. LPS+BzATP.

Figure 5. BzATP augments IL-1β production induced by LPS in endothelium-intact vessels

The changes in endothelium-intact and -denuded aortic IL-1β levels were assessed by ELISA, after incubation with DMEM [Control; E(+), n=6; E(-), n=3], LPS [100 μg/ml; E(+), n=3; E(-), n=3], BzATP [150 μM; E(+), n=4], LPS plus BzATP (LPS 100 μg/ml plus BzATP 150 μM; LPS+BzATP; E(+), n=4; E(-), n=3], oATP plus LPS plus BzATP [oATP 50 μM for 1 h before incubation with LPS plus BzATP; oATP+LPS+BzATP; E(+), n=3] and oATP [50 μM for 1 h before incubation with DMEM; E(+), n=3]. Data are expressed as mean±SEM of n animals. *P<0.05 vs. LPS+BzATP in endothelium-intact vessels.

Figure 6. BzATP augments inducible nitric oxide synthase (iNOS) protein expression induced by LPS in endothelium-intact vessels

The figure depicts a typical western blot image of iNOS protein expression (upper figure) and the statistical analysis of changes of iNOS protein expression in mouse endothelium-intact and -denuded aortic rings, which were incubated with DMEM [Control; E(+), n=3; E(-), n=3], LPS [100 μg/ml; E(+), n=3; E(-), n=3], BzATP [150 μM; E(+), n=3], LPS plus BzATP [LPS 100 μg/ml plus BzATP 150 μM; LPS+BzATP; E(+), n=3; E(-), n=3], oATP plus LPS plus BzATP [oATP 50 μM or 250 μM for 1 h before incubation with LPS plus BzATP; oATP (50 μM)+BzATP+LPS; E(+), n=3 or oATP (250 μM) +BzATP+LPS; E(+), n=3] and oATP [50 μM or 250 μM for 1 h before incubation with DMEM; oATP (50 μM); E(+), n=3 or oATP (250 μM); E(+), n=3]. Data are expressed as mean±SEM of n animals. *P<0.05 vs. LPS+BzATP in endothelium-intact vessels.

Figure 7. IL-1β induces iNOS protein expression in endothelium-intact vessels

The figure depicts a typical western blot image of iNOS protein expression (upper figure) and the statistical analysis of changes of iNOS protein expression in mouse endothelium-intact and -denuded aortic rings, which were incubated with DMEM [Control; E(+), n=3; E(-), n=3], IL-1β [20 ng/ml; E(+), n=3; E(-), n=3]. Data are expressed as mean±SEM of n animals. *P<0.01, IL-1β vs. Control in endothelium-intact vessels.