Enhanced synthesis and reduced metabolism of endothelin-1 (ET-1) by hepatocytes - an important mechanism of increased endogenous levels of ET-1 in liver cirrhosis

doi:10.1016/S0168-8278(00)80302-5

Journal of Hepatology

Volume 33, Issue 5, November 2000, Pages 725-732

https://doi.org/10.1016/S0168-8278(00)80302-5 Get rights and content

Abstract

Background/Aims: Hepatic concentration of endothelin-1 (ET-1) is increased in human and experimental liver cirrhosis. Because of its potent actions in the liver, ET-1 has been suggested to play an important role in the pathophysiology of cirrhosis. Since hepatocytes are the major cell type to metabolize ET-1, we investigated whether their reduced capacity to degrade ET-1 is a mechanism of its elevated levels in cirrhosis.

Methods: The expression of ET-1 receptors, ET-1 and endothelin converting enzyme (ECE), and metabolism of ET-1 and ECE activity were compared in hepatocytes isolated from control and carbon tetrachloride-induced cirrhotic rats.

Results: ET-1 receptor density and receptor-mediated internalization of ET-1 were significantly increased in cirrhotic hepatocytes relative to the control cells. However, compared to control hepatocytes, metabolism of ET-1 by the cirrhotic cells was reduced significantly. Interestingly, hepatocytes were found to contain preproET-1 mRNA, ECE-1 mRNA and ET-1. PreproET-1 mRNA and ET-1 levels were increased in cirrhotic hepatocytes but their ECE mRNA and ECE activity were not altered.

Conclusions: These results provide the first evidence that hepatocytes have the ability to synthesize ET-1 and demonstrate that decreased metabolism and enhanced synthesis of ET-1 in hepatocytes are an important mechanism of its elevated levels in cirrhosis.

Section snippets

Materials and Methods

Rat (1–39) big ET-1 (Sigma, St Louis, MO, USA); collagenase CLS1 (Worthington, Freehold, NJ, USA); William's medium E, penicillin G, streptomycin and fetal bovine serum (GibcoBRL, Grand Island, NY, USA); endothelin-1, sarafotoxin S6c and BQ-123 (American Peptide, Sunnyvale, CA, USA); [¹²⁵I] endothelin-1 (2200 Ci/mmol) (DuPont-NEN, Boston, MA, USA) were purchased from the indicated sources.

Morphology of cultured hepatocytes

The size of cirrhotic hepatocytes was significantly smaller than that of control cells (21±1.2 μM versus 28±1.6 μM; p<0.01) (Fig. 2). Interestingly, unlike the control cells that formed well-organized plates, cirrhotic hepatocytes tended to aggregate during culture.

ET-1 and ECE in the liver and hepatocytes

The concentrations of ET-1 in plasma and livers of cirrhotic rats were respectively 1.5- and 3-fold higher than in control rats (Table 1). The increase in hepatic ET-1 concentration was consistent with an increase in preproET-1 mRNA

Discussion

Hepatic ET-1 levels increase in cirrhotic rats 3., 21. and humans 4., 20.. Enhanced hepatic synthesis of ET-1 seems to be its main cause, as indicated by higher concentration of hepatic venous ET-1 than portal venous ET-1 in cirrhotic patients (20). This investigation has demonstrated that enhanced synthesis and reduced metabolism of ET-1 in hepatocytes can be an important mechanism of elevated endogenous and circulating ET-1 in cirrhosis. Previously, preproET-1 mRNA transcript could not be

Acknowledgements

Supported by a VA Merit award and a grant DK 54411–01 from the NIH to CRG. We thank Ms. Lori Perez and Mr. McKinley Blair for excellent technical assistance and Dr. Xuedong Chi for Western blot analysis.

References (49)

M Pinzani et al.
Endothelin 1 is overexpressed in human cirrhotic liver and exerts effects on activated hepatic stellate cells
Gastroenterology
(1996)
CR Gandhi et al.
Hepatic effects of endothelin: metabolism of [¹²⁵I]endothelin-1 by liver-derived cells
Arch Biochem Biophys
(1993)
A Gabriel et al.
Superoxide-induced changes in endothelin (ET) receptors in hepatic stellate cells
J Hepatol
(1998)
K Stephenson et al.
Endothelin association with cultured rat Kupffer cells: characterization and regulation
Hepatology
(1995)
CR Gandhi et al.
Endothelin, a potent peptide agonist in the liver
J Biol Chem
(1990)
TA Tran-Thi et al.
Regulation of endothelin-1 action on the perfused rat liver
FEBS Lett
(1993)
SB Mustafa et al.
Endothelin stimulates platelet activating factor synthesis by cultured rat Kupffer cells
Hepatology
(1995)
K Ohnaka et al.
Purification and characterization of a phosphoramidon-sensitive endothelin-converting enzyme in porcine aortic endothelium
J Biol Chem
(1993)
D Xu et al.
ECE-1 a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1
Cell
(1994)
M Ihara et al.
Biological profiles of highly potent novel endothelin antagonists selective for the ET_A receptor
Life Sci
(1992)

DL Williams et al.

Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes

Biochem Biophys Res Commun

(1991)

A Gabriel et al.

Down-regulation of endothelin receptors by transforming growth factor β1 in hepatic stellate cells

J Hepatol

(1999)

T Sakurai et al.

cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA

Biochem Biophys Res Commun

(1991)

K Shimada et al.

Cloning and functional expression of endothelin converting enzyme from rat endothelial cells

J Biol Chem

(1994)

H Yoshimi et al.

Regional distribution of immunoreactive endothelin in rats

Peptides

(1989)

K Kitamura et al.

Regional distribution of immunoreactive endothelin in porcine tissue abundance in inner medulla of kidney

Biochem Biophys Res Commun

(1989)

A Hemsen et al.

Presence of endothelin-1 and endothelin-3 in peripheral tissues and central nervous system of the pig

Reg Peptides

(1991)

R Shao et al.

Regulation of endothelin-1 synthesis by endothelin-converting enzyme-1 during wound healing

J Biol Chem

(1999)

H Rieder et al.

Sinusoidal endothelial liver cells in vitro release endothelin-augmentation by transforming growth factor β and Kupffer cell-conditioned media

Klin Wochenschr

(1991)

C Housset et al.

Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1

Proc Natl Acad Sci USA

(1993)

CR Gandhi et al.

Increased hepatic endothelin-1 levels and endothelin receptor density in cirrhotic rats

Life Sci

(1996)

A Caligiuri et al.

Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ET_A receptors on large cholangiocytes

Am J Physiol

(1998)

F Laura et al.

Endothelin-1 is synthesized and inhibits cyclic adenosine monophosphate-dependent anion secretion by an autocrine/paracrine mechanism in gallbladder epithelial cells

J Clin Invest

(1998)

CR Gandhi et al.

Hepatic effects of endothelin: receptor characterization and endothelin-induced signal transduction in hepatocytes

Biochem J

(1992)

Cited by (51)

Aldosterone alters the chromatin structure of the murine endothelin-1 gene
2016, Life Sciences
Citation Excerpt :
It should be noted that under non-pathological conditions the ET-1 protein is expressed at low levels in hepatocytes. Indeed, until 2000 it was thought that hepatocytes did not produce any ET-1 and were only involved in the metabolism of the protein [14]. This cell line does show responsiveness to aldosterone treatment for other well-known aldosterone early response genes, Sgk-1 and Scnn1a (Fig. 2A).
Aldosterone increases sodium reabsorption in the renal collecting duct and systemic blood pressure. Paradoxically, aldosterone also induces transcription of the endothelin-1 (Edn1) gene to increase protein (ET-1) levels, which inhibits sodium reabsorption.
Here we investigated changes in the chromatin structure of the Edn1 gene of collecting duct cell lines in response to aldosterone treatment. The Edn1 gene has a CpG island that encompasses the transcription start site and four sites in the 5′ regulatory region previously linked to transcriptional regulation.
The chromatin structure of the Edn1 gene was investigated using a quantitative PCR-based DNaseI hypersensitivity assay in murine hepatocyte (AML12), renal cortical collecting duct (mpkCCD_C14), outer medullary collecting duct1 (OMCD1), and inner medullary collecting duct-3 (IMCD-3) cell lines.
The CpG island was uniformly accessible. One calcium-responsive NFAT element remained at low chromatin accessibility in all cell lines under all conditions tested. However, the second calcium responsive NFAT element located at − 1563 bp upstream became markedly more accessible in IMCD-3 cells exposed to aldosterone. Importantly, one established aldosterone hormone response element HRE at − 671 bp relative to the transcription start site was highly accessible, and another HRE (− 551 bp) became more accessible in aldosterone-treated IMCD-3 and OMCD1 cells.
The evidence supports a model in which aldosterone activation of the mineralocorticoid receptor (MR) results in the MR-hormone complex binding at HRE at − 671 bp to open chromatin structure around other regulatory elements in the Edn1 gene.
Regulation of the Norepinephrine Transporter by Endothelins: A potential therapeutic target
2015, Vitamins and Hormones
Citation Excerpt :
ET-1 gene expression is enhanced by stress, and it plays a relevant role in diverse systemic disorders associated with endothelium injury like hypertension, uremic hemolytic syndrome, thrombocytopenic purpura, and heart failure (Hynynen & Khalil, 2006; Itoh et al., 1988). Immunoreactive ET-1 and mRNA ET-1 were reported in the blood vessels, heart, liver, kidney, peripheral nervous system, and CNS (Giaid et al., 1989; Kuddus, Nalesnik, Subbotin, Rao, & Gandhi, 2000; Naicker & Bhoola, 2001; Naidoo, Mahabeer, & Raidoo, 2001; Naidoo, Naidoo, Mahabeer, & Raidoo, 2004; Rubanyi & Botelho, 1991; Zhan & Rockey, 2011). ET-1 is the peptide with most potent vasoconstrictor property known (Yanagisawa, Inoue, et al., 1988; Yanagisawa, Kurihara, et al., 1988).
Neuronal norepinephrine (NE) uptake is a crucial step in noradrenergic neurotransmission that regulates NE concentration in the synaptic cleft. It is a key mechanism mediated by the NE transporter (NET) which takes the neurotransmitter into the presynaptic neuron terminal or the adrenal medulla chromaffin cell. The activity of NET is short and long terms modulated by phosphorylation mediated by protein kinases A, C, and G and calcium–calmodulin-dependent protein kinase, whereas the transporter availability at the cell surface is regulated by glycosylation. Several neuropeptides like angiotensins II, III, and 1–7, bradykinin, natriuretic peptides, as well as endothelins (ETs) regulate a wide variety of biological effects, including noradrenergic transmission and in particular neuronal NE uptake. Diverse reports, including studies from our laboratory, show that ETs differentially modulate the activity and expression of NET not only in normal conditions but also in diverse cardiovascular diseases such as congestive heart failure and hypertension. Current literature supports a key role for the interaction between ETs and NE in maintaining neurotransmission homeostasis and further suggests that this interaction may represent a potential therapeutic target for various diseases, particularly hypertension.
Endothelin-1 and its role in the pathogenesis of infectious diseases
2014, Life Sciences
Citation Excerpt :
It is formed by conversion of pre-pro-ET-1 into the intermediate precursor big ET-1, which is then cleaved by the ET converting enzyme (ECE) to form the active 21-amino acid peptide. ET-1 is synthesized by a variety of cells including endothelial cells, macrophages, cardiomyocytes, and neurons (Table 1) (Schinelli, 2002; Vignon-Zellweger et al., 2012; Kedzierski and Yanagisawa, 2001; van Harmelen et al., 2008; MacCumber et al., 1990; Nakagomi et al., 2000; Tonnessen et al., 2000; Yanagisawa et al., 1988; Kuddus et al., 2000; Jamal and Schneider, 2002; Ehrenreich et al., 1992; Matsushima et al., 2004; Giaid et al., 1989; Pomonis et al., 2001; Yu et al., 1995). ET-1 is constitutively synthesized and acts in an autocrine and paracrine manner in tissues throughout the body; its physiological plasma concentration is ~ 1 pM (Kedzierski and Yanagisawa, 2001).
Endothelins are potent regulators of vascular tone, which also have mitogenic, apoptotic, and immunomodulatory properties (Rubanyi and Polokoff, 1994; Kedzierski and Yanagisawa, 2001; Bagnato et al., 2011). Three isoforms of endothelin have been identified to date, with endothelin-1 (ET-1) being the best studied. ET-1 is classically considered a potent vasoconstrictor. However, in addition to the effects of ET-1 on vascular smooth muscle cells, the peptide is increasingly recognized as a pro-inflammatory cytokine (Teder and Noble, 2000; Sessa et al., 1991). ET-1 causes platelet aggregation and plays a role in the increased expression of leukocyte adhesion molecules, the synthesis of inflammatory mediators contributing to vascular dysfunction. High levels of ET-1 are found in alveolar macrophages, leukocytes (Sessa et al., 1991) and fibroblasts (Gu et al., 1991). Clinical and experimental data indicate that ET-1 is involved in the pathogenesis of sepsis (Tschaikowsky et al., 2000; Goto et al., 2012), viral and bacterial pneumonia (Schuetz et al., 2008; Samransamruajkit et al., 2002), Rickettsia conorii infections (Davi et al., 1995), Chagas disease (Petkova et al., 2000, 2001), and severe malaria (Dai et al., 2012; Machado et al., 2006; Wenisch et al., 1996a; Dietmann et al., 2008). In this minireview, we will discuss the role of endothelin in the pathogenesis of infectious processes.
Augmenter of liver regeneration: An important intracellular survival factor for hepatocytes
2008, Journal of Hepatology
Citation Excerpt :
Transfection of primary hepatocytes with antisense oligonucleotide for ALR mRNA (ALR-AS) led to mitochondrial and cellular depletion of ALR, profound loss of ATP, mitochondrial release of cytochrome c, cellular release of LDH and apoptotic/necrotic death of hepatocytes. Hepatocytes were prepared as described previously [23], and suspended (0.25 × 106 cells/ml) in William’s medium E supplemented with 2 mM l-glutamine, penicillin/streptomycin, 10% FBS, and 10−6 M insulin. Cells were plated at a density of 0.063 × 106/cm2, the medium renewed 3 h later, and the cells used following overnight incubation.
Augmenter of liver regeneration (ALR), a protein synthesized and stored in hepatocytes, is associated with mitochondria, and possesses sulfhydryl oxidase and cytochrome c reductase activities. We sought to determine the effects of ALR depletion in hepatocytes by antisense oligonucleotide transfection.
Rat hepatocytes in primary culture were transfected with antisense oligonucleotide for ALR mRNA (ALR-AS) or scrambled oligonucleotide. Various analyses were performed at times up to 24 h after transfection.
Treatment with ALR-AS caused a decrease in ALR mRNA, cellular depletion of ALR protein primarily from mitochondria, and decreased viability. Flow cytometric analysis of ALR-AS-transfected hepatocytes stained with annexin-V_cy3 and 7-aminoactinomycin D revealed apoptosis as the predominant cause of death up to 6 h; incubation beyond this time resulted in necrosis in addition to apoptosis. ALR-AS-transfection caused release of mitochondrial cytochrome c, activation of caspase-3, profound reduction in the ATP content, and cellular release of LDH. Inhibition of caspase-3 inhibited the early phase of ALR-AS-induced death but not the late phase that included ALR and LDH release.
These results suggest that ALR is critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis.
The future treatment of portal hypertension
2007, Best Practice and Research in Clinical Gastroenterology
Citation Excerpt :
In human cirrhosis, endothelin upregulation parallels loss of hepatic function.63,64 Also in humans, increased endothelin levels are due both to increased synthesis and to decreased metabolism.65 In two rat models of portal hypertension—the bile-duct-ligated rat and partial portal vein ligation—only endothelin receptor subtype B (ETB) receptors appeared to be upregulated.66,67
Increased understanding of the hyperdynamic circulation syndrome has resulted in novel therapeutic approaches, some of which have already reached clinical practice. Central to the hyperdynamic circulation syndrome is an imbalance between the increase in different vasodilators (foremost among which is nitric oxide) and the compensatory increase in vasoconstrictors—usually accompanied by a blunted response. This chapter discusses the role of endothelin in the pathogenesis of the syndrome and in future treatment approaches. A relatively new area of research in this field is the role of infection and inflammation in the initiation and maintenance of the hyperdynamic circulation syndrome. The use of antibiotics in the setting of acute variceal bleeding is standard practice. Studies have suggested that chronic manipulation of the intestinal flora could have beneficial effects in the treatment of portal hypertension. The bile salts are another novel and interesting target. Although their vasoactive properties have been known for some time, recent data demonstrate that their effects could be central in the pathogenesis of the hyperdynamic circulation syndrome, and that manipulation of the composition of the bile acid pool could be a therapeutic approach to portal hypertension. Finally, hypoxia and angiogenesis play a role in the development of portal hypertension and the formation of collaterals. This role needs to be further defined but it appears likely that this phenomenon is yet another target for therapeutic intervention.
Lanreotide on collateral response to endothelin-1 and vasopressin in cirrhotic rats
2006, Journal of the Chinese Medical Association
Portosystemic collaterals in cirrhosis are often complicated with hemorrhage. Endothelin-1 (ET-1) and argi-nine vasopressin (AVP) have been shown to directly constrict collaterals of portal hypertensive rats. Furthermore, octreotide, a long-acting somatostatin analog, enhances ET-1-related collateral vasoconstriction if administered in the perfusate before ET-1 incubation. However, long-term effects of somatostatin analogs on collateral response to ET-1 and AVP remain unclarified. This study investigated the effects of lanreotide (a longer-acting somatostatin analog when compared with octreotide) on the portosystemic collateral vascular reactiveness to ET-1 and AVP in cirrhotic rats.
Liver cirrhosis was induced in Sprague-Dawley rats by common bile duct ligation (BDL). On the 33^rd day after BDL, rats received 1 intramuscular injection with lanreotide (10 mg/kg) or distilled water. On the 43^rd day after BDL, systemic, and portal hemodynamics were evaluated. By an in situ perfusion model, ET-1 (10⁻¹⁰ −10⁻⁷ mol/L) and AVP (10⁻¹⁰ −3 × 10⁻⁷ mol/L) were applied to assess the collateral responses.
Heart rate, mean arterial pressure, and portal pressure were not modified by lanreotide. Lanreotide pretreat-ment enhanced collateral vascular response to ET-1 and reached statistical significance at 10⁻⁷ mol/L without inducing changes to AVP.
Lanreotide augmented the collateral vascular responsiveness to ET-1 but not to AVP in cirrhotic rats.

View all citing articles on Scopus

View full text

Enhanced synthesis and reduced metabolism of endothelin-1 (ET-1) by hepatocytes - an important mechanism of increased endogenous levels of ET-1 in liver cirrhosis

Abstract

Section snippets

Materials and Methods

Morphology of cultured hepatocytes

ET-1 and ECE in the liver and hepatocytes

Discussion

Acknowledgements

Gastroenterology

Arch Biochem Biophys

J Hepatol

Hepatology

J Biol Chem

FEBS Lett

Hepatology

J Biol Chem

Cell

Life Sci

Biochem Biophys Res Commun

J Hepatol

Biochem Biophys Res Commun

J Biol Chem

Peptides

Biochem Biophys Res Commun

Reg Peptides

J Biol Chem

Sinusoidal endothelial liver cells in vitro release endothelin-augmentation by transforming growth factor β and Kupffer cell-conditioned media

Klin Wochenschr

Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1

Proc Natl Acad Sci USA

Increased hepatic endothelin-1 levels and endothelin receptor density in cirrhotic rats

Life Sci

Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes

Am J Physiol

Endothelin-1 is synthesized and inhibits cyclic adenosine monophosphate-dependent anion secretion by an autocrine/paracrine mechanism in gallbladder epithelial cells

J Clin Invest

Hepatic effects of endothelin: receptor characterization and endothelin-induced signal transduction in hepatocytes

Biochem J

Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ET_A receptors on large cholangiocytes