Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H(2) synthases

Abstract

Acetaminophen has antipyretic and analgesic properties yet differs from the nonsteroidal antiinflammatory drugs and inhibitors of prostaglandin H synthase (PGHS)-2 by exhibiting little effect on platelets or inflammation. We find parallel selectivity at a cellular level; acetaminophen inhibits PGHS activity with an IC(50) of 4.3 microM in interleukin (IL)-1 alpha-stimulated human umbilical vein endothelial cells, in contrast with an IC(50) of 1,870 microM for the platelet, with 2 microM arachidonic acid as substrate. This difference is not caused by isoform selectivity, because acetaminophen inhibits purified ovine PGHS-1 and murine recombinant PGHS-2 equally. We explored the hypothesis that this difference in cellular responsiveness results from antagonism of the reductant action of acetaminophen on the PGHSs by cellular peroxides. Increasing the peroxide product of the PGHS-cyclooxygenase, prostaglandin G(2) (PGG(2)), by elevating the concentration of either enzyme or substrate reverses the inhibitory action of acetaminophen, as does the addition of PGG(2) itself. 12-Hydroperoxyeicosatetraenoic acid (0.3 microM), a major product of the platelet, completely reverses the action of acetaminophen on PGHS-1. Inhibition of PGHS activity by acetaminophen in human umbilical vein endothelial cells is abrogated by t-butyl hydroperoxide. Together these findings support the hypothesis that the clinical action of acetaminophen is mediated by inhibition of PGHS activity, and that hydroperoxide concentration contributes to its cellular selectivity.

Figure 1

ApAP does not inhibit prostacyclin synthase activity in HUVECs. HUVECs were grown in the presence of 15% FBS until confluence, medium was exchanged with 0.75% HBSS/BSA, and ApAP (final concentration 330 μM) or ethanol vehicle was added. After 2 h at 37°C, the medium was replaced with the same medium, [¹⁴C]PGH₂, added for 15 min, and the medium was analyzed by reversed-phase HPLC coupled to a radioactive detector. As a control, [¹⁴C]PGH₂ was added to the medium without HUVECs. 6-Keto-PGF_1α is indicated by a star. The results are expressed as the ratio of 6-keto-PGF_1α to PGE₂ (the rearrangement product of PGH₂).

Figure 2

Comparison of inhibition by ApAP of PGHS activity in HUVECs and platelets. Synthesis of TxB₂ in washed human platelets and of 6-keto-PGF_1α in HUVECs was assayed in the presence of increasing concentrations of ApAP (Material and Methods). AA (20 μM) was added as substrate. The prostanoid concentration present in the medium was determined by GC/NICI/MS and normalized to the value of the control (no ApAP was added) for each cell type. The data points represent the average of at least three independent experiments, each performed in triplicate, ± SD.

Figure 3

Effect of the concentration of AA on the inhibition by ApAP of TxB₂ synthesis in human platelets. Washed human platelets were incubated at room temperature with ApAP at the indicated concentrations. After 20 min, AA was added (final concentrations, 0.5, 2.0, and 20 μM). After 15 min, TxB₂ was extracted with 400 μl of diethyl ether/methanol/4 M citric acid (30:4:1) and derivatized for analysis by GC/NICI/MS. The concentration of TxB₂ present in the medium is represented as a percentage of the control to which no ApAP was added. Experiments were performed in triplicate.

Figure 4

Effect of the concentration of AA on the inhibition by ApAP of 6-keto-PGF_1α synthesis by IL-1α-activated HUVECs. HUVECs were starved for 24 h in the presence of 5% FBS before activation for 24 h with 1 ng/ml IL-1α. The medium was exchanged with 0.75% HBSS/BSA and ApAP at the concentrations indicated. After 20 min at 37°C, 2 (♦) or 20 (★) μM AA was added for 15 min, and the medium was collected for analysis by GC/NICI/MS. The concentrations of 6-keto-PGF_1α present in the medium are represented as a percentage of the control to which no ApAP was added. 100% represents 3.5 ng of 6-keto-PGF_1α per ml of medium.

Figure 5

Effect of the concentration of enzyme and substrate on the inhibition of PGHS-2 by ApAP. PGHS-2 was reconstituted (Material and Methods). The reaction was started by adding [¹⁴C]AA and stopped after 8 sec. The data points represent the average of triplicates. NS, not significant. (A) PGHS-2 at the concentrations indicated was preincubated at 37°C with 1 mM ApAP. [¹⁴C]AA was added (final concentration, 10 μM). (B) PGHS-2 (10 nM) was preincubated at 37°C with 1 mM ApAP. [¹⁴C]AA was added at the concentrations indicated. PGHS-2 activity is expressed as a percentage of the control to which no ApAP was added.

Figure 6

Effect of t-butyl hydroperoxide (t-ButOOH) on the inhibition by ApAP of 6-keto-PGF_1α synthesis by IL-1α-activated HUVECs. HUVECs were starved for 24 h in the presence of 5% FBS before activation for 24 h with 1 ng/ml IL-1α. The medium was exchanged with 0.75% HBSS/BSA, and 666 μM ApAP or vehicle was added. After 20 min at 37°C, t-butyl hydroperoxide was added at the indicated concentration, immediately followed by AA. After 15 min, the medium was collected for analysis of 6-keto-PGF_1α.

Figure 7

Effect of PGG₂ concentration on inhibition of PGHS-1 by ApAP. PGHS-1 was reconstituted (Material and Methods) in the presence of 0.5 mM ApAP. PGG₂ was added at the concentrations indicated, immediately followed by [¹⁴C]AA (final concentration, 0.5 μM). The reaction was stopped after 8 sec. PGHS activity is expressed as a percentage of the control to which no ApAP was added. The significance of differences between each concentration of substrate and the control was determined (n = 4: *, P < 0.01; **, P < 0.001).

Figure 8

Effect of 12-HPETE on the inhibition of PGHS by ApAP. PGHS-1 and PGHS-2 were reconstituted (Material and Methods). 12-HPETE was added at the indicated final concentrations, immediately followed by [¹⁴C]AA (final concentration, 0.5 μM). The reaction was stopped after 8 sec. PGHS activity is expressed as a percentage of the control with the indicated concentration of 12-HPETE, to which no ApAP was added. (A) PGHS-1 (5.4 nM) was preincubated at 37°C with 1.0 mM ApAP. The data points represent the average of six values. (B) PGHS-2 (10 nM) was preincubated at 37°C with 1 mM ApAP. The data points represent the average of at least three values. The significance of differences between each concentration of 12-HPETE was determined (*, P < 0.001 when compared with the two other concentrations).