Background
Sinusoidal perfusion is highly variable and regulated by different humoral substances including nitric oxide and endothelin [
1‐
3]. Endothelin-1 (ET-1), one of the most potent endogenous vasoconstrictors [
4], has extra- and intra-sinusoidal actions, the latter being more important at low endothelin concentrations [
1,
5] This effect has been associated to hepatic stellate cell contraction [
6]. The resulting increase in shear stress activates endothelial nitric oxide (NO) production
via ET
B receptors [
7].
Intrahepatic vascular resistance is also regulated by vasoactive substances that may act locally or systemically. An excess of vasoconstrictors increases the vascular tone and may lead to an exaggerated response of the hepatic vascular bed. These factors include noradrenaline, angiotensin II and leukotrienes [
8], but ET-1 seems to be the most potent one. In the rat liver, the ET
A receptor subtype causes vasoconstriction, while the ET
B receptor subtype is associated with a dual vascular response. ET
B on hepatic stellate cells mediates their constriction, but this is normally countered by the vasodilatory effect of NO, released under the regulation of ET
B on endothelial sinusoidal cells [
1].
The vascular balance is maintained by the availability of vasodilators. NO is the best known, but other molecules such as carbon monoxide [
9] and adrenomedullin contribute to intrahepatic vasodilation [
10].
The release of NO has been demonstrated to be a crucial regulatory mechanism counteracting the action of ET-1 in the kidney of ET-1 transgenic mice, highlighting the
in vivo interaction between NO and ET-1 [
11]. Additionally, an increased NO bioavailability has been shown to improve endothelium-dependent relaxation of aortic rings from mice overexpressing ET-1, suggesting that, in the presence of an activated ET system, NO production may essentially contribute to maintain a normal vascular pressure [
12].
Mice in which the key enzyme catalyzing the release of NO, endothelial constitutive NO synthase, has been knocked out have arterial hypertension but are otherwise phenotypically normal [
13]. We argued that the hepatic vasculature of such mice should be more sensitive to exogenous endothelin-1 since the compensatory vasodilatation could not occur. Therefore, vascular resistance in response to ET-1 was studied in the perfused mouse liver of ecNOS knockout mice and their wild type counterparts.
Surprisingly, sensitivity to ET-1 was decreased in ecNOS knockout mice, suggesting the presence of compensatory mechanisms counteracting the absence of NO. The decreased hepatic expression of ETA receptors in endothelial constitutive nitric oxide synthase (ecNOS) knockout mice may contribute to the observed decreased vascular sensitivity.
Discussion
Contrary to our expectations, the present investigation demonstrates a decreased sensitivity of the liver from ecNOS knockout mice to exogenous ET-1. This appears to be achieved independent of changes in other major vasodilatory systems including inducible nitric oxide synthase (iNOS), heme oxygenase and adrenomedullin.
Different factors that could compensate for the lack of ecNOS were evaluated. The prime candidate being obviously the inducible isoform [
15], it was not detectable by Western blotting excluding its participation in the reduced response to endothelin in ecNOS knock-out mice. Direct assessment of NO formation by the citrulline assay [
16] is not reliable in liver tissue owing to competing enzymatic reactions. Hence, other potential candidates were examined.
Carbon monoxide (CO) has recently been described as another factor modulating sinusoidal tone [
9]. NO and CO may complement each other as signaling molecules in some physiological situations [
17‐
19]. Heme oxygenase catalyses the production of CO from heme [
17]. However, heme oxygenase isoforms 1 and 2 were similarly expressed in the two strains on the protein and mRNA level, making unlikely a contribution from this system. The same held true for adrenomedullin, a potent vasodilator peptide [
20] which acts in part through cAMP and in part through ecNOS [
21‐
26]. However, decreased NO may theoretically reduce adrenomedullin receptor availability even if it increases adrenomedullin mRNA, since in a rat mesangial cell culture system, NO donors increased binding of adrenomedullin to its receptor but reduced adrenomedullin mRNA levels [
27].
Finally, we looked at the endothelin system itself. Although endothelin was originally described as a potent vasoconstrictor [
4], depending on the particular receptor involved it can also have vasodilatory properties. It would have been conceivable that endothelin would be altered in the absence of ecNOS-derived NO; this was clearly not the case since at the protein and the mRNA level there was no difference between the two strains.
Vasoconstriction is mediated mainly by ET
A receptors [
28], but also by ET
B receptors present on smooth muscle cells [
29]. In contrast, vasodilatation is evoked via ET
Breceptors through release of endothelium derived vasodilators such as NO and prostacyclin [
30,
31] ET
Breceptor mRNA was unchanged as shown by quantitative PCR. This suggests that ET
B receptors did not contribute to adaptive changes in ecNOS knockout mice. Furthermore, ET
B receptors are not expected to contribute to vasodilatation in ecNOS knockout mice as the vasodilatory action of ET
B receptors is mediated predominantly through ecNOS.
In the ecNOS knockout mice, the expression of ET
A receptors was down-regulated. This observation may explain the attenuated response to ET-1 in ecNOS knockout mice and makes biological sense as an adaptive mechanism. In fact, under basal conditions the portal pressure in wild type and ecNOS knockout mice is similar, suggesting that a decrease in vasoconstrictor mechanisms, in our model the decrease of the expression of ET
A receptors, compensates for a decrease in vasodilator factors, in our model the lack of ecNOS. However, this equilibrium is not maintained when a vasoactive stress, such as the perfusion of the liver with ET-1, is applied. The use of the perfused liver model allowed us to postulate that, in ecNOS knockout mice, the decreased sensitivity to ET-1 is associated to an insufficient expression of ET
A receptors. Investigation of the mechanisms by which ET
A receptors contribute to the unexpected decreased sensitivity of liver from ecNOS knockout mice to exogenous endothelin-1 was beyond the scope of this study. In a recent study, the expression of ET
A receptors was decreased in endothelium-denuded aortae of ecNOS KO mice, suggesting an adaptive mechanism similar to the one we observed. Nevertheless, in these animals, an increased expression of cyclooxygenase-2 overcame the decrease of ET
A and enabled and increased sensitivity to ET-1 [
32].
Conclusion
This study has demonstrated the capacity of adaptation of the endothelin system in a model of perfused isolated liver in ecNOS KO mice. The mechanisms involved in this adaptive response involve a decreased hepatic expression of the ET
A receptor in the absence of NO, leading to an attenuation of the vasoconstriction induced by ET-1. These data suggest that ET
A may be one major player in the regulation of hepatic vascular resistance. The expression of ET
A, which is increased in cirrhotic livers [
33], may hence represent a major pharmacological target to ameliorate portal pressure due to the imbalance between vasoconstrictive and vasodilative mechanisms. Experimental data on the use of ET
A blockers in an animal model of portal hypertension corroborate this hypothesis [
34].
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
ADG carried out parts of the animal experiments and bench work and prepared the manuscript. EB, AK, DB and HS contributed to the animal experiments and critically revised the manuscript. SS provided assistance for the endothelin expression studies and for the preparation of the manuscript. JR contributed to the design of the study, obtained funding for the study and critically revised the manuscript. All authors read and approved the final manuscript.