Thromboxane A2 from Kupffer cells contributes to the hyperresponsiveness of hepatic portal circulation to endothelin-1 in endotoxemic rats
Hongzhi Xu,
Katarzyna Korneszczuk,
Amel Karaa,
Tian Lin,
Mark G. Clemens, and
Jian X. Zhang
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina
Submitted 14 June 2004
; accepted in final form 30 September 2004
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ABSTRACT
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We examined the role of thromboxane A2 (TXA2) in LPS-induced hyperresponsiveness of hepatic portal circulation to endothelins (ETs) and whether Kupffer cells are the primary source of TXA2 release in response to ET-1 in endotoxemia. After 6 h of LPS (1 mg/kg body wt ip) or saline (control), liver was isolated and perfused with recirculating Krebs-Henseleit bicarbonate buffer at a constant flow rate (100 ml·min1·kg body wt1). ET-1 (10 pmol/min) was infused for 10 min. Portal pressure (PP) was continuously monitored during perfusion. Perfusate was sampled for enzyme immunoassay of thromboxane B2 (TXB2; the stable metabolite of TXA2) and lactate dehydrogenase (LDH) assay. ET-1 infusion resulted in a significantly greater increase of PP in the LPS group than in controls. Both TXA2 synthase inhibitor furegrelate (Fureg) and TXA2 receptor antagonist SQ-29548 (SQ) substantially blocked enhanced increase of PP in the LPS group (4.9 ± 0.4 vs. 3.6 ± 0.5 vs. 2.6 ± 0.6 mmHg for LPS alone, LPS + Fureg, and LPS + SQ, respectively; P < 0.05) while having no significant effect on controls. GdCl3 for inhibition of Kupffer cells had similar effects (4.9 ± 0.4 mmHg vs. 2.9 ± 0.4 mmHg for LPS alone and GdCl3 + LPS, respectively; P < 0.05). In addition, the attenuated PP after ET-1 was found concomitantly with significantly decreased releases of TXB2 and LDH in LPS rats treated with Fureg, SQ, and GdCl3 (886.6 ± 73.4 vs. 110.8 ± 0.8 vs. 114.8 ± 54.7 vs. 135.2 ± 45.2 pg/ml, respectively; P < 0.05). After 6 h of LPS, Kupffer cells in isolated cell preparations released a significant amount of TXA2 in response to ET-1. These results clearly indicate that hyperresponsiveness of hepatic portal circulation to ET-1 in endotoxemia is mediated at least in part by TXA2-induced receptor activation, and Kupffer cells are likely the primary source of increased TXA2 release.
isolated liver perfusion; furegrelate; SQ-29548
ONE OF THE PATHOLOGICAL CHANGES in the early stage of endotoxemia is hepatic portal circulatory dysfunction, which leads to a reduction in sinusoidal perfusion and may ultimately result in hepatic failure (18). Growing evidence has shown that endothelin-1 (ET-1), a potent vasoconstrictive mediator, plays an important role under these conditions (17, 21, 29). Exogenous ET-1 causes reductions in sinusoid diameter and sinusoidal flow as well as increases in total portal resistance in the normal rat liver (3). In LPS-induced endotoxemia, ET-1 has been reported to be elevated in the portal and the systemic circulations (20). Studies from our laboratory and others' have also demonstrated an upregulation of mRNA levels of ET-1 in the rat liver tissue in endotoxemia (17, 25). In addition, the study by Pannen et al. (22) showed that the contractile response of the portal circulation to ET-1 was significantly potentiated by LPS-induced acute endotoxemia in the isolated perfused liver model, suggesting that enhanced ET-1 response, which occurs at sinusoidal and presinusoidal levels, may also contribute to endotoxin-induced hepatic microcirculatory failure. Indeed, our recent studies (4) have provided evidence showing that this portal hyperresponsiveness to ET-1 was responsible for severely reduced sinusoidal blood flow as well as impaired oxygen delivery in LPS-pretreated liver in rats. However, the underlining mechanism of this hyperresponsiveness is not fully understood. In the present study, we hypothesized that ET-1 triggers release of other vasoconstrictive mediators such as thromboxane A2 (TXA2) in the endotoxemic liver, which mediate the hyperresponsiveness of the portal circulation to endothelins.
TXA2 has been shown to be a potent vasoconstrictor in the portal circulation (8, 27). Various studies have reported the increased release of TXA2 in the liver (14, 16, 19) and other tissues (7) in endotoxemia. Inhibition of TXA2 synthase and TXA2 receptors provided a protective effect on the LPS-primed liver, suggesting an important role of TXA2 in the pathogenesis of liver injuries during endotoxemia (13, 14). In addition, several studies have provided the evidence linking endothelins and the release of TXA2. Stanimirovic et al. (26) showed that ET-1 induced a time-dependent release of TXA2 in human brain capillary endothelial cells. Rodrigue et al. (24) suggested a possible involvement of TXA2 in the ET-1-induced vascular effect in recombinant human erythropoietin-induced hypertension in uremic rats. Furthermore, previous studies have showed that Kupffer cells are the major sources of TXA2 production in diseased livers (30), and cultured Kupffer cells isolated from the rat liver were able to release TXA2 in response to LPS administration (5, 15, 28).
Therefore, the goals of the present study were 1) to investigate whether TXA2 plays any role in mediating the hyperresponsiveness of the portal circulation to ET-1 in endotoxemia and 2) to determine whether Kupffer cells are the major source of TXA2 production in the endotoxemic liver in response to ET-1.
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MATERIALS AND METHODS
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Animals.
Male Sprague-Dawley rats weighing 200250 g were housed in a temperature-controlled animal facility with alternating 12:12-h light-dark cycles and were fed standard lab chow ad libitum with free access to water. All procedures were performed in accordance with the National Institutional Animal Care and Use Committee of the University of North Carolina at Charlotte.
Experimental groups.
Escherichia coli LPS (1 mg/kg body wt; Sigma, St. Louis, MO) was injected intraperitoneally in vivo 6 h before experiments. In control groups, the same amounts of saline were injected. Saline- and LPS-treated animals were then assigned to 1) nontreatment groups: control (n = 9) and LPS (n = 10); 2) furegrelate-pretreatment groups: saline + Fureg (n = 5) and LPS + Fureg (n = 9); 3) SQ-29548-pretreatment groups: saline + SQ-29548 (n = 6) and LPS + SQ-29548 (n = 5); and 4) GdCl3-pretreatment groups: saline + GdCl3 (n = 5) and LPS + GdCl3 (n = 5).
Isolated liver perfusion.
Six hours after pretreatment with saline or LPS, rats were subjected to isolated liver perfusion. The liver was exposed through a side transverse incision, and the portal vein was isolated. After the portal vein was cannulated with a PE-240 catheter, the liver was perfused with Krebs-Henseleit bicarbonate buffer (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.1 EDTA, and 2.5 CaCl2, pH 7.4, 37°C, saturated with 95% O2 and 5% CO2) for 10 min to wash out the blood. In furegrelate-pretreatment groups, the specific TXA2 synthase inhibitor furegrelate [5-(3-pyridinyl-methyl) bensofurancarboxylic acid, 1 mg/100 g body wt; Cayman Chemical, Ann Arbor, MI] was injected intraperitoneally 14 h before the experiment, and a boosting dose (5 µM) was added in the perfusion buffer during perfusion to inhibit TXA2 production. In SQ-29548 pretreatment groups, SQ-29548 (5-heptenoic acid, 7-[3-[[2-[(phenylamino) carbonyl]hydrazine]methyl]-7-oxabicyclo; Cayman Chemical) was added into the perfusion buffer (1 µM) 10 min before ET-1 infusion to block the TXA2 receptor activity. In GdCl3 pretreatment groups, 7.5 mg/kg body wt GdCl3 in 0.5 ml saline was injected through the penile vein 24 h before the perfusion to inhibit the function of Kupffer cells. The isolated liver perfusion was performed by using a constant flow rate (100 ml·min1·kg body wt 1), as described previously (6) with minor modification. Briefly, warmed perfusate was pumped from a reservoir into an overflow chamber and was oxygenated via a silicone tubing oxygenator (95% O2-5% CO2). The temperature of perfusate was maintained at 3637°C by warming the reservoir in a water bath. A pressure transducer was placed in line immediately before the portal inlet cannula to monitor portal pressure. The liver was perfused for 10 min in a nonrecirculating fashion to wash out the blood and stabilize the pressure. Then the perfusion was switched to recirculation in a total perfusate volume of 100 ml, and ET-1 was infused simultaneously at 10 pmol/min for 10 min. Perfusate was sampled before and after the infusion of ET-1 for the measurement of thromboxane B2 (TXB2) levels and lactate dehydrogenase (LDH) activity. LDH activity measurements were conducted spectrophotometrically by using diagnostic kits from Sigma.
Kupffer cell isolation and culture.
Male Sprague-Dawley rats weighing 350400g were used for all experiments. The rat liver was perfused in situ through the portal vein with Gey's balanced salt solution (GBSS; in mM: 137 NaCl, 2.7 NaHCO3, 5 KCl, 1.5 CaCl2·2H2O, 1 MgCl2·6H2O, 0.7 Na2HPO4, 0.2 KH2PO4, 0.3 MgSO4·7H2O, 5.5 glucose, and 25 HEPES) equilibrated with 95% O2-5% CO2 at 37°C at a flow rate of 30 ml/min for 4 min. The liver was then perfused with Ca2+- and Mg2+-free Krebs-Ringer-HEPES solution (in mM: 250 HEPES, 1,150 NaCl, 50 KCl, 1 KH2PO4) containing 0.5 mM EGTA at a flow rate of 18 ml/min for 6 min. Subsequently, the liver was digested with GBSS containing 0.025% type IV collagenase (Sigma) and 20 µg/ml DNase I (Sigma) at a flow rate of 15 ml/min for 12 min. In the digestion procedure, we omitted pronase because pronase has been reported to destroy the LPS receptor CD14 on Kupffer cells during cell isolations (12). After digestion and removal of the capsule, the liver was excised and cut into small pieces in the GBSS-containing collagenase. Cells were suspended in GBSS and were centrifuged at 50 g at 4°C for 3 min to remove the hepatocyte fraction, followed by centrifugation at 700 g for 7 min to separate the nonparenchymal cell fraction. Kupffer cells were separated from the nonparenchymal cell fraction via centrifugal elutriation using a JB6 rotor (Beckman Instruments, Fullerton, CA). Kupffer cells were eluted at a constant rotor speed of 2,500 rpm and flow rate of 45 ml/min. Kupffer cell viability as assessed by trypan blue exclusion was >95%.
Cells were plated in 24-well plastic culture dishes (Corning, Corning, NY) at a concentration of 1 x 106 cells/well and were cultured in 500 µl RPMI 1640 medium (Sigma) supplemented with 25 mM HEPES, 20% fetal bovine serum and antibiotics (0.05% gentamycin sulfate). Nonadherent cells were removed by changing culture medium 2 h after plating. The purity of Kupffer cells is >95% of adherent cells, as indicated by a latex bead phagocytosis test. Cells were cultured at 37°C in 5% CO2 for 18 h. Then culture medium was replaced with fresh medium containing either PBS or LPS (final concentration, 50 ng/ml). After 6 h of incubation, cell medium was removed and cells were carefully washed with PBS three times, then 500 µl PBS containing either vehicle (PBS) or ET-1 at a final concentration of 10 nM was added to each well. After 10 min of incubation at 37°C, the medium was sampled for TXB2 assay. In this experiment, cells were divided into four groups (n = 4 for each group). In the LPS/ET group, the cells were treated with PBS for 6 h, followed by vehicle incubation for 10 min. In the LPS/ET+ group, the cells were treated with PBS for 6 h, followed by ET-1 incubation for 10 min. In the LPS+/ET group, the cells were treated with LPS for 6 h, followed by vehicle incubation for 10 min. Finally, in the LPS+/ET+ group, the cells were treated with LPS for 6 h, followed by ET-1 incubation for 10 min.
Enzyme immunoassay for TXB2.
Enzyme immunoassay kits (Assay Designs, Ann Arbor, MI) were used to determine the concentration of TXB2 (a stable metabolite of TXA2 as an indicator of TXA2 release) in the perfusate. The samples were diluted 1:2 with Krebs buffer so that the highest concentration of TXB2 in the perfusate fell in the linear range of the standard curve. The level of TXB2 was expressed as picograms per milliliter.
Statistical analysis.
Statistical significance in each group was tested by using one-way ANOVA, with individual means detected by Student-Newman-Keuls test. When criteria for parametric testing were violated, the appropriate nonparametric test (Mann-Whitney U-test) was used. The portal pressures within each group and among the groups were analyzed by using two-way ANOVA. A P value <0.05 was considered significant. All results are presented as means ± SE.
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RESULTS
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Portal hemodynamic change in response to ET-1 after 6 h of LPS pretreatment.
After 6 h of LPS, administration of ET-1 for 10 min induced a peak increase in portal pressure in both LPS-pretreated and saline-pretreated groups during the isolated liver perfusion (Fig. 1). However, the response to ET-1 was much more pronounced in livers from animals pretreated with LPS compared with livers from saline-pretreated animals (9.1 ± 0.3 mmHg for LPS vs. 7.6 ± 0.4 mmHg for control at 10 min; P < 0.05).

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Fig. 1. Portal pressure response to endothelin (ET) -1 infusion in isolated rat liver perfusion. Liver from a control (n = 9) or LPS-treated (LPS; n = 10) animal was isolated and perfused with Krebs-Henseleit bicarbonate buffer by a flow-controlled system. In the LPS group, LPS (1 mg/kg body wt) was injected intraperitoneally in vivo 6 h before experiments. ET-1 was infused for 10 min, and the portal pressure was continuously monitored during the entire experiment period. *P < 0.05 vs. control (saline) group at the same time point.
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TXA2 released into the perfusate in response to ET-1 following LPS pretreatment.
TXA2 release was estimated by measuring the levels of TXB2 in the perfusate. Before ET-1 infusion, TXB2 levels in the perfusate of the LPS group were slightly higher than in the control group, but the difference did not reach statistical significance (P > 0.05; Fig. 2). After 10 min of ET-1 infusion, a dramatic increase of perfusate TXB2 (>8-fold) was observed in the LPS group compared with the initial level at 0 min (886.5 ± 73.3 vs. 144.9 ± 41.1 pg/ml at 0 and 10 min, respectively; P < 0.001; Fig. 2), whereas no change in TXB2 in the control group was observed.

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Fig. 2. Perfusate thromboxane B2 (TXB2) levels following ET-1 infusion. LPS (1 mg/kg body wt) or saline was injected intraperitoneally in vivo 6 h before isolated liver perfusion. Perfusate samples were taken right before and 10 min after administration of ET-1 at a 10 pmol/min infusion rate; TXB2 levels were determined for control (n = 9) and LPS-treated (n = 10) groups. *P < 0.05 vs. control (saline) group at 10 min and LPS group at 0 min.
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Effect of furegrelate on the responsiveness of the LPS-primed liver to ET-1.
Furegrelate, a specific inhibitor for TXA2 synthase, was applied to block the production of TXA2 in response to ET-1. Pretreatment of furegrelate significantly attenuated the ET-1-induced increase in portal pressure in the LPS group (absolute increase at 10 min: 4.9 ± 0.4 mmHg for LPS alone vs. 3.6 ± 0.5 mmHg for LPS + Furegrelate; P < 0.05). The portal pressure response with furegrelate showed no significant difference from that of the normal control group (saline only). Clearly, the TXA2 inhibitor completely prevented the LPS-induced portal pressure hyperresponsiveness to ET-1 (Fig. 3A). In contrast, the portal pressure in the saline + Fureg group was not significantly different from that of the saline-only group, indicating that furegrelate did not affect the portal resistance in the normal control group in response to ET-1.

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Fig. 3. A: effect of furegrelate on ET-1-induced increase in portal pressure. In both saline- and LPS-pretreated rat livers, a specific TXA2 synthase inhibitor furegrelate (Fureg, 1 mg/100 g body wt) was injected intraperitoneally 14 h before the experiment, and a boosting dose (5 µM) was added in the perfusate during perfusion to inhibit TXA2 production. Absolute changes in portal pressure in response to ET-1 are shown. The potentiated portal pressure increase induced by ET-1 was significantly blocked by Fureg in the LPS-primed rat liver. *P < 0.05 vs. saline + Fureg group at 10 min and saline (control) group at 10 min. B: effect of Fureg on perfusate TXB2 levels. Perfusate samples were taken right before and 10 min after administration of ET-1, and TXB2 levels were determined by ELISA. The significant increase of perfusate TXB2 levels after 10 min of ET-1 infusion was markedly attenuated by Fureg in the LPS-primed rat liver. *P < 0.05 vs. saline + Fureg group at 10 min and saline (control) group at 10 min; n = 9 for saline; n = 10 for LPS; n = 5 for saline + Fureg; n = 9 for LPS + Fureg.
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At the end of the 10-min infusion of ET-1, the markedly increased perfusate TXB2 in LPS-primed livers was completely abolished by the pretreatment of furegrelate (886.6 ± 73.4 pg/ml for LPS alone vs. 110.8 ± 0.8 pg/ml LPS + Furegrelate; P < 0.05). The perfusate TXB2 levels after ET-1 in LPS livers treated with furegrelate were not significantly different from those of the saline + Fureg group or the levels before ET-1 in either group (Fig. 3B). The perfusate TXB2 levels of the control group (saline only) didn't show any significant change after the administration of furegrelate.
Effects of SQ-29548 on the responsiveness of the LPS-primed liver to ET-1.
To confirm the specific action of TXA2 in mediating the enhanced portal response by ET-1 in LPS primed livers, SQ-29548, a selective TXA2 receptor antagonist, was employed. The baseline portal pressure of both saline- and LPS-pretreated livers was not significantly altered during the 10-min preperfusion with SQ-29548 (data not shown). After 10 min of ET-1 infusion, the increase in portal pressure did not change significantly in control compared with saline + SQ-29548. However, the enhanced increase of portal pressure in response to ET-1 in LPS-primed livers was completely abolished by the administration of SQ-29548 (absolute increase at 10 min: 4.9 ± 0.4 mmHg for LPS vs. 2.6 ± 0.6 mmHg for LPS + SQ-29548; P < 0.05; Fig. 4A). Surprisingly, the measurement of perfusate TXB2 showed that SQ-29548 significantly inhibited the ET-1-induced TXB2 increase in LPS-primed livers (at 10 min: 886.6 ± 73.4 pg/ml for LPS alone vs. 114.8 ± 54.6 pg/ml for LPS + SQ-29548; Fig. 4B)

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Fig. 4. A: effect of SQ-29548 on ET-1-induced increase in portal pressure. In both saline- and LPS-pretreated rat livers, SQ-29548 was added into the perfusion buffer (1 µM) 10 min before ET-1 to block the thromboxane A2 (TXA2) receptor activity. The enhanced portal pressure increase in response to ET-1 infusion was significantly blocked by SQ-29548 in the LPS-primed rat liver, whereas no significant effect on portal pressure of the control group was observed. Absolute changes in portal pressure in response to ET-1 are shown. *P < 0.05 vs. saline group and saline + SQ-29548 group at 10 min. B: effect of SQ-29548 on perfusate TXB2 levels in response to ET-1. Perfusate samples were taken right before and 10 min after administration of ET-1, and TXB2 levels were determined by ELISA. *P < 0.05 vs. saline group and saline + SQ-29548 group at 10 min; n = 9 for saline; n = 10 for LPS; n = 6 for saline + SQ-29548; n = 5 for LPS + SQ-29548.
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Effects of GdCl3 on the responsiveness of the LPS-primed liver to ET-1.
To determine whether Kupffer cells are the major source of TXA2 release, we pretreated animals with GdCl3 to inhibit the Kupffer cell function. As shown in Fig. 5A, the hyperresponse of portal pressure to ET-1 in LPS-primed livers was significantly attenuated following the pretreatment with GdCl3 (absolute increases 10 min: 4.9 ± 0.4 mmHg for LPS alone vs. 2.9 ± 0.4 mmHg for LPS + GdCl3; P < 0.05), and the change of portal pressure in response to ET-1 in the LPS + GdCl3 group was not significantly different from that of the control (saline-only) group. No significant difference in the change of the response in portal pressure to ET-1 was observed between control and saline + GdCl3 groups.

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Fig. 5. A: effects of GdCl3 on ET-1-induced increase in portal pressure. GdCl3 (7.5 mg/kg body wt in 0.5 ml saline) was injected through the penile vein 24 h before the perfusion to inhibit the function of Kupffer cells. ET-1 was infused for 10 min during isolated liver perfusion following in vivo intraperitoneal injection of LPS 6 h earlier. The enhanced portal pressure increase in response to ET-1 was significantly attenuated by GdCl3 in the LPS-primed rat liver. Absolute changes in portal pressure in response to ET-1 are shown. *P < 0.05 vs. saline group and saline + GdCl3 group at 10 min. B: effect of GdCl3 on perfusate TXB2 levels in response to ET-1 during isolated liver perfusion. Perfusate samples were taken right before and 10 min after administration of ET-1, and TXB2 levels were determined by ELISA. The significant increase of perfusate TXB2 after 10 min of ET-1 infusion was markedly decreased by GdCl3 in the LPS-primed rat liver. *P < 0.05 vs. saline group and saline + GdCl3 group at 10 min; n = 9 for saline; n = 10 for LPS; n = 5 for saline + GdCl3; n = 5 for LPS + GdCl3.
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GdCl3 pretreatment completely prevented the increase of TXB2 in the perfusate following ET-1 infusion in LPS-primed livers (10 min: 886.6 ± 73.4 pg/ml for LPS alone vs. 135.2 ± 45.2 pg/ml for LPS + GdCl3; P < 0.05; Fig. 5B).
LDH releases.
In response to ET-1, LDH levels in the perfusate were significantly elevated in LPS-primed livers compared with control livers (10 min: 192.0 ± 86.8 vs. 32.8 ± 4.0 U/l; P < 0.05); the administration of furegrelate completely prevented the ET-1-induced elevation in LDH levels in LPS-primed livers. Inhibition of ET-1-induced LDH increase was also observed with SQ-29548 and GdCl3, respectively (Fig. 6).

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Fig. 6. Lactate dehydrogenase (LDH) releases in response to ET-1 infusion during the isolated liver perfusion. Perfusate samples were taken right before and 10 min after administration of ET-1. In LPS-primed livers, ET-1 resulted in a significant increase in perfusate LDH levels, which were completely prevented by furegrelate, SQ-29548, or GdCl3. *P < 0.05 vs. saline, saline + Fureg, saline + SQ-29548, and saline + GdCl3 groups at 10 min.
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Release of TXA2 from isolated Kupffer cells in response to LPS and ET-1.
To verify whether LPS-primed Kupffer cells release TXA2 in response to ET-1, we performed an in vitro study using isolated Kupffer cells. When Kupffer cells were pretreated with PBS for 6 h, no significant increase of TXA2 release was observed in response to 10 min of ET-1 treatment compared with the baseline level. However, for the Kupffer cells primed with LPS, incubation with ET-1 for 10 min induced a significantly greater production of TXA2, as indicated by higher TXB2 levels in the media compared with that from Kupffer cells pretreated without LPS (LPS/ET and LPS/ET+ groups) or with LPS but without ET-1 (LPS+/ET group) (Fig. 7).

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Fig. 7. TXB2 levels of Kupffer cell media after 10 min of ET-1 treatment. Isolated Kupffer cells were cultured for 18 h, followed by pretreatment with either PBS or LPS (50 ng/ml) for 6 h. Cells were then washed 3 times with PBS and treated with either vehicle or ET-1 (10 nM) for 10 min. Medium samples were taken for TXB2 measurement. After Kupffer cells were incubated with ET-1 for 10 min, TXB2 levels in the LPS+/ET+ group were significantly higher than in the other 3 groups. *P < 0.05 vs. LPS/ET, LPS/ET+, and LPS+/ET groups; n = 4 in each group.
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DISCUSSION
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Hepatic microcirculatory dysfunction is a severe and early complication in endotoxemia (18). It has been suggested that ET-1 plays a significant role in mediating hepatic microcirculatory dysfunction in endotoxemia (17, 21, 29). The study by Pannen et al. (22) showed that portal venous contractile response to ET-1 was significantly enhanced in the rat liver following the pretreatment with endotoxin. The sinusoids constricted with a greater magnitude in response to ET-1, which contributed to a dramatic decrease in the sinusoidal flow in LPS-primed animals compared with control animals. Our recent studies have also demonstrated that the enhanced response of the portal vascular system to ET-1 results in functional impairment in oxygen delivery, which causes focal areas of hypoxia in the liver, leading to local mismatches between the oxygen delivery and metabolic demands (4). Although the deleterious effect induced by the enhanced response to ET-1 on the hepatic microcirculation during endotoxemia has been recognized, the mechanism underlining LPS-induced hyperresponsiveness to ET-1 is still not clear. In the present study, we hypothesized that ET-1 triggers releases of other vasoconstrictive mediators such as TXA2 in the endotoxemic liver, which mediate the hyperresponsiveness of the portal circulation to endothelins.
To explore the mechanism of the hyperresponsiveness of the portal circulation to ET-1 in acute endotoxemia, we employed the isolated liver perfusion system. After 6 h of pretreatment with LPS, the liver showed a slightly higher baseline portal pressure and significantly enhanced portal pressure in response to the exogenous ET-1 infusion compared with the control liver. The results confirmed Pannen et al.'s observation (22). This enhanced portal pressure increase in response to ET-1 in the LPS-pretreated liver was accompanied by more severe liver injury, as indicated by higher levels of LDH in the perfusate at the end of 10 min of ET-1 infusion, suggesting the detrimental effect of this potentiated portal response to ET-1 on hepatocellular function.
Several possible mechanisms may be responsible for the hyperresponsiveness of the portal circulation to ET-1 as a result of LPS priming. For example, releases of other vasoconstrictors such as TXA2 in response to ET-1 could explain the enhanced portal pressor response seen in the LPS liver. In the present study, we sought to determine the possible role of TXA2 in this LPS-induced hyperresponsiveness. A number of studies (911) have suggested the important role of TXA2 in the portal system. Our recent study (30) has also shown that hepatic release of TXA2 contributes to the early development of portal hypertension in the bile duct-ligated (BDL) liver (30). Interestingly, in the present study the levels of perfusate TXB2, a stable metabolite of TXA2, were found dramatically increased after 10 min of ET-1 infusion in LPS-pretreated livers but not in control livers, indicating that the hyperresponse to ET-1 was associated with the TXA2 release into the portal circulation. These observations suggested that TXA2 might play a role in the hyperresponsiveness of the portal circulation to ET-1.
TXA2, derived from arachidonic acid, is synthesized by a specific enzyme: TXA2 synthase. To determine the role of TXA2 in the hyperresponsiveness of portal circulation to ET-1 during endotoxemia, we used furegrelate, a potent selective TXA2 synthase inhibitor, to block the production of TXA2 in the liver. Our recent data showed that furegrelate effectively suppressed the TXA2-mediated increase in portal pressure in BDL-induced early portal hypertension (30). In the present study, administration of furegrelate significantly attenuated the portal pressure elevation in response to 10 min of ET-1 infusion to a level similar to control, i.e., furegrelate eliminated the potentiated portal pressure response to ET-1 in the LPS-pretreated livers. The attenuated portal pressure response was accompanied by a substantial decrease of TXB2 release. LDH release measured at the end of 10 min of ET-1 infusion was also significantly decreased in the furegrelate-pretreated liver. These observations indicated that increased release of TXA2 into the portal system may account for the enhanced ET-1-induced portal pressure increase as well as liver injury in LPS-primed livers.
TXA2 and other vasoactive prostaglandins share a common biosynthesis pathway. It is possible that the inhibition of TXA2 synthesis with a TXA2 synthase inhibitor may affect the production of other vasoactive prostaglandins, thus confounding the results of our furegrelate experiment. To confirm the role of TXA2, we specifically blocked TXA2 receptors by using the TXA2 receptor antagonist SQ-29548. As a result, the inhibition of the TXA2 receptor eliminated the hyperresponsiveness of portal pressure to ET-1 in the LPS-pretreated liver. Similarly, the diminished portal pressure response was accompanied by amelioration of hepatic injury, as indicated by reduced perfusate levels of LDH. Clearly, the data demonstrate that TXA2 released from the liver in response to ET-1 plays a role in mediating the hyperresponsiveness of portal pressure during acute endotoxemia.
It is, however, noteworthy that SQ-29548 not only suppressed the enhanced portal response to ET-1 in endotoxemic rat livers, it also significantly reduced perfusate TXB2 levels (Fig. 4B). SQ-29548 has been shown to block TXA2 receptor activation by competing the receptor binding with TXA2 (1). This was an unexpected finding. We initially suspected that the compound SQ-29548 in the perfusate samples might cross-react with the antibodies of the enzyme immunoassay kits, thus interfering with the measurement of TXB2. We performed additional experiments using SQ-29548 only in the samples and found no cross-relativities of the enzyme immunoassay kits with the compound. Therefore, we currently do not have an explanation of why SQ-29548 inhibited TXB2 levels in the perfusate other than to speculate that TXA2 might provide positive feedback to regulate its production. Clearly, whether our speculation is true or not requires further investigation.
The role of TXA2 in the potentiated portal system's response to ET-1 during acute endotoxemia raises the question of the source of the increased release of TXA2. A number of studies have demonstrated that activated Kupffer cells were able to produce TXA2 in response to LPS stimulation in vitro (5, 15, 28). We recently have shown (30) that an increased amount of TXA2 is likely released from Kupffer cells in the early BDL-induced portal hypertension model. With Kupffer cell inhibitor GdCl3 in the present study, we were able to completely eliminate the increased production of TXA2 in the portal circulation, indicating that Kupffer cells are the major source of the TXA2 release. Indeed, our in vitro study provided direct evidence showing that Kupffer cells pretreated with LPS release a significantly elevated amount of TXA2 in response to ET-1 stimulation. Furthermore, inhibition of TXA2 production from Kupffer cells also abolished the enhanced portal response to ET-1 and decreased hepatocellular injury, as indicated by reduced perfusate levels of LDH, suggesting a pathogenic role of Kupffer cells in the ET-1-induced microcirculatory dysfunction during endotoxemia.
The precise mechanism of the significantly increased production of TXA2 by Kupffer cells during 10 min of ET-1 infusion is not clear. Although several studies have shown that LPS pretreatment itself may induce TXA2 synthesis (10, 14), and our data with Western blot analysis also revealed upregulations of PLA2 and COX-2 expression after 6 h of LPS (data not shown), the measurement of TXB2 levels in perfusate before ET-1 administration, which indicates the baseline release of TXA2 induced by LPS, was not significantly higher than in the control group, suggesting that at the early stage of LPS-induced endotoxemia, LPS may only "prime" the TXA2 synthesis pathway. The question is how the LPS-primed liver responds to ET-1 and rapidly increases the production of TXA2 within minutes. Apparently, activation of the key enzymes (such as PLA2, COX, or TXA2 synthase) by ET-1 likely precedes the rapid increased release of TXA2. However, the precise mechanism of ET-1-induced activation of the TXA2 synthesis pathway remains to be elucidated.
Nevertheless, our results demonstrate that release of TXA2 during acute endotoxemia likely plays an important role in the hyperresponsiveness of the portal circulation to ET-1. However, other mechanisms most likely also contribute to the LPS-induced hyperresponsiveness to ET-1. Recent work from our lab (2) has shown that although ETB receptors that can mediate dilation through activation of endothelial nitric oxide synthase (eNOS) in endothelial cells are upregulated, their coupling to eNOS activation is impaired. Moreover, inhibition of eNOS sensitizes the liver microcirculation to ET-1 and impairs oxygen delivery in a manner similar to that seen with LPS treatment (23). These results indicate that suppression of eNOS activation also contributes to the hypersensitivity to endothelin following LPS. Further studies will be needed to elucidate the interaction between increased thromboxane production and decreased nitric oxide release in the hypersensitivity to ET-1.
In summary, the present study demonstrates that the hyperresponse of portal pressure to ET-1 in acute endotoxemia is at least partly mediated by TXA2, which suggests an important role of thromboxane in the pathogenesis of portal circulatory dysfunction during acute endotoxemia. In addition, Kupffer cells are likely the major source of thromboxane production in LPS-induced endotoxemia.
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GRANTS
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This study was supported by grants DK-60606 and DK-38201 from National Institute of Diabetes and Digestive and Kidney Diseases.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. X. Zhang, Dept. of Biology, Univ. of North Carolina at Charlotte, 9201 Univ. City Blvd., Charlotte, NC 28223 (E-mail: jxzhang{at}uncc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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