Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells.

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

Improved preservation of warm ischemic livers by hypothermic machine perfusion with supplemented University of Wisconsin solution.

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Endothelin 1 impairs oxygen delivery in livers from LPS-primed animals.

Endothelin 1 (ET-1) is a potent vasoactive peptide that acts at sinusoidal and extrasinusoidal sites in the liver. Sensitivity to ET-1 increases in LPS-primed animals and is associated with impaired liver microcirculation in these animals. We hypothesized that LPS priming leads to an exacerbation in the impaired oxygen delivery in response to intraportal infusion of ET-1. Rats were studied 24 h after LPS injection (1 mg/kg, i.p.). Surface PO2 was determined using a recently developed technology of O2 mapping. The baseline portal pressure was higher in LPS-primed animals (P < 0.05), and increased to'similar magnitude as sham animals after a 10-min infusion of ET-1. The resultant portal pressure remained elevated in LPS compared to sham animals. There was no significant difference in baseline mean arterial pressure, and no significant systemic response to ET-1 in either group. In contrast to the macrohemodynamic, the decrease in tissue surface PO2 in response to ET-1 infusion was potentiated by LPS treatment (increased from baseline levels 33.8+/-9 to 46.8+/-8.3 in sham; 42.3+/-9.1 to 69+/-6.5 gray scale units in LPS; P < 0.01, sham vs. LPS) at end of infusion of ET-1 for 10 min. This indicates tissue hypoxia in response to ET-1, which is exacerbated in livers from LPS-primed animals compared to sham. Frequency distribution analysis showed a shift in mode from lower intensity (higher PO2) to areas with higher fluorescent intensity ranges (lower PO2), indicating areas with shut down in perfusion in LPS-treated animals. In the whole liver, ET-1 suppressed oxygen consumption, and this response was potentiated by LPS pretreatment. We propose that ET-1 impairs oxygen delivery in the liver during endotoxemia, resulting in areas of focal hypoxia. This response is possibly due to potentiated action of ET-1 at both sinusoidal and extrasinusoidal sites in the liver during endotoxemia.

Remote trauma sensitizes hepatic microcirculation to endothelin via caveolin inhibition of eNOS activity.

This study addresses the microvascular mechanisms by which a remote, mild stress such as blunt trauma sensitizes the liver to injury. Rats received closed femur fracture (FFx), and 24 h later livers were isolated and perfused at a similar starting flow rate for assessment of vascular response to endothelin-1 (ET-1). Sinusoidal volumetric flow (QS), red blood cell velocity (VRBC), and sinusoidal diameter (Ds) were determined by intravital microscopy. Baseline portal resistance in livers from FFx rats was not changed. The FFx group showed a lower baseline VRBC (322.9 +/- 26.4 and 207.3 +/- 17.2 microm/s in sham and FFx,) and QS (28.4 +/- 4.2 and 17.6 +/- 2.1 pL/s in sham and FFx, P < 0.05). ET-1 caused a decrease in the VRBC in sham but no change after FFx. In contrast, Ds was unchanged by ET-1 in sham but decreased in FFx (10.3 +/- 0.4 to 10.7 +/- 0.5 vs. 10.6 +/- 0.4 to 9.0 +/- 0.4 microm at 10 min in sham and FFx groups, P < 0.05). The overall result of these changes was a greater decrease in sinusoidal flow in FFx compared with sham. There was no significant change in mRNA for ET-1, endothelin A (ETA) receptor, or iNOS (inducible nitric oxide synthase) in FFx compared with sham. However, endothelin B (ETB) receptor mRNA and eNOS (endothelial nitric oxide synthase) mRNA were increased in the FFx group (ETB, 54.81 +/- 8.08 in sham vs. 83.28 +/- 8.19 in FFx; eNOS, 56.11 +/- 2.53 in sham vs. 83.31 +/- 5.51 in FFx; P < 0.05) while the levels of these proteins remained unchanged. Caveolin-1 (cav-1) protein levels were elevated in FFx, and coimmunoprecipitation with both ETB and eNOS showed increased associations with these proteins, suggesting a possible inactivation of eNOS. The eNOS activity was also blunted in FFx animals in the presence of increased cav-1 expression. Taken together, these results demonstrate that remote trauma sensitizes the liver to the sinusoidal constrictor effect of ET-1. We propose that this hyperresponsiveness occurs as a result of uncoupling of the ETB receptor from eNOS activity mediated by interaction of eNOS and possibly the ETB receptor with increased caveolin-1. This vascular sensitization that occurs after FFx may contribute to the exacerbation of injury during subsequent stresses.

Differential mechanisms of hepatic vascular dysregulation with mild vs. moderate ischemia-reperfusion.

Endotoxemia produces hepatic vascular dysregulation resulting from inhibition of endothelin (ET)-stimulated NO production. Mechanisms include overexpression of caveolin-1 (Cav-1) and altered phosphorylation of endothelial nitric oxide (NO) synthase (NOS; eNOS) in sinusoidal endothelial cells. Since ischemia-reperfusion (I/R) also causes vascular dysregulation, we tested whether the mechanisms are the same. Rats were exposed to either mild (30 min) or moderate (60 min) hepatic ischemia in vivo followed by reperfusion (6 h). Livers were harvested and prepared into precision-cut liver slices for in vitro analysis of NOS activity and regulation. Both I/R injuries significantly abrogated both the ET-1 (1 microM) and the ET(B) receptor agonist (IRL-1620, 0.5 microM)-mediated stimulation of NOS activity. 30 min I/R resulted in overexpression of Cav-1 and loss of ET-stimulated phosphorylation of Ser1177 on eNOS, consistent with an inflammatory response. Sixty-minute I/R also resulted in loss of ET-stimulated Ser1177 phosphorylation, but Cav-1 expression was not altered. Moreover, expression of ET(B) receptors was significantly decreased. This suggests that the failure of ET to activate eNOS following 60-min I/R is associated with decreased protein expression consistent with ischemic injury. Thus hepatic vascular dysregulation following I/R is mediated by inflammatory mechanisms with mild I/R whereas ischemic mechanisms dominate following more severe I/R stress.

LPS inhibits endothelin-1-induced endothelial NOS activation in hepatic sinusoidal cells through a negative feedback involving caveolin-1.

During endotoxemia, liver microcirculation disruption is characterized by a hypersensitivity to the constrictor effects of endothelin 1 (ET-1). The shift of ET-1-mediated effects toward vasoconstriction may result from depressed ET-1-mediated vasodilation through decreased ET-1-induced nitric oxide (NO) production. We have previously shown that lipopolysaccharide (LPS) pretreatment abrogates ET-1-induced endothelial nitric oxide synthase (eNOS) translocation, but its effects on eNOS activation are yet to be determined. Our aim was to assess the effects of LPS on ET-1-mediated eNOS activation in hepatic sinusoidal endothelial cells (SECs) and to investigate the molecular mechanisms involved. SECs were treated with LPS (100 ng/mL) for 6 hours followed by 30 minutes ET-1 (10 nmol/L) stimulation. LPS significantly inhibited ET-1-mediated eNOS activation. This inhibition was associated with upregulation of Caveolin-1 (CAV-1) and a shift in ET-1-mediated eNOS phosphorylation from an activation (Ser1177) to an inhibition (Thr495). LPS treatment has been shown to induce ET-1 expression and secretion from endothelial cells. We therefore investigated the role of endogenous ET-1 in the inhibition of ET-1 activation of eNOS after LPS. Antagonizing ET-1 effects and blocking its activation in LPS pretreated SECs decreased the LPS-induced overexpression of CAV-1 as well as the inhibition of ET-1-induced NOS activity. Furthermore, 6 hours of ET-1 treatment exerted the same effects on eNOS activity, phosphorylation, and CAV-1 expression as LPS treatment. In conclusion, LPS-induced suppression of ET-1-mediated eNOS activation is ET-1 dependent and suggest a pivotal role of CAV-1 in eNOS induction inhibition under stress.

LPS inhibits endothelin-1-mediated eNOS translocation to the cell membrane in sinusoidal endothelial cells.

OBJECTIVE: The objectives of this study were to develop a model for studying endothelin-1-mediated eNOS regulation in cultured sinusoidal endothelial cells and determine the effect of endothelin-1 and endotoxin (LPS) on eNOS localization. METHODS: Changes in caveolin-1, calmodulin, and eNOS expression were determined by western blot and densitometric analysis. Endothelin receptor expression and localization and the intracellular localization of eNOS and caveolin-1 were assessed by confocal microscopy. RESULTS: Sinusoidal endothelial cells expressed caveolin-1 and calmodulin, and expression was altered in cultured and passaged cells. eNOS expression decreased significantly in 24-h cultured cells, with expression dropping below the level of detection in passaged cells. Both endothelin A and endothelin B receptors were expressed on the cell surface after 24 h in culture. In 24-h cultured cells, caveolin-1 was localized in the perinuclear region and cell membrane, while eNOS was predominantly localized in the perinuclear region, where it co-localized with caveolin-1. Endothelin-1 stimulated eNOS translocation to the cell membrane. Pretreatment with LPS markedly inhibited the endothelin-1-mediated eNOS translocation. CONCLUSIONS: These studies demonstrate an LPS-mediated uncoupling of endothelin receptor activation and eNOS translocation. This functional uncoupling may, in part, account for the hyperconstrictive effects of endothelin-1 during inflammatory conditions.

Thromboxane A2 from Kupffer cells contributes to the hyperresponsiveness of hepatic portal circulation to endothelin-1 in endotoxemic rats.

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.min(-1).kg body wt(-1)). 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.

Functional link between ETB receptors and eNOS maintain tissue oxygenation in the normal liver.

OBJECTIVE: Endothelins and their receptors play a crucial role in regulating liver microcirculation in pathophysiological conditions. The authors investigated the functional significance of the coupling of ET(B) receptors and eNOS in maintaining regional perfusion and tissue oxygenation in the normal liver. METHODS: The effect of endothelin-1 or the ET(B) agonist IRL1620 on oxygen consumption was determined in isolated perfused liver and isolated hepatocytes. Oxygen delivery to the liver tissue was determined in vivo. Following eNOS or iNOS blockade, either ET-1 or IRL1620 was infused via the portal vein. Hepatic tissue oxygenation, redox state, and microcirculation were investigated by intravital microscopy. Injury was estimated by serum LDH. RESULTS: Although IRL1620 and endothelin-1 increased oxygen consumption in isolated hepatocytes, in intact liver, endothelin decreased oxygen consumption while IRL1620 produced no change. In vivo, ET(B) stimulation modestly altered hepatic tissue P(O(2)), redox potential, and microcirculation. eNOS inhibition and ET(B) activation dramatically reduced microcirculatory blood flow, oxygen supply, and increased LDH release. Inhibition of iNOS resulted in small but not significant changes in these parameters. Concomitant ET(A)/ET(B) receptor activation increased microcirculatory failure and decreased tissue oxygen even without NOS inhibition. In contrast, hepatocellular injury was significantly increased following eNOS inhibition. CONCLUSIONS: Coupling between ET(B) receptor stimulation and eNOS activation decreases sinusoidal constriction and plays a functionally important role in maintaining microcirculation and tissue oxygenation in the normal liver.