Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis

doi:10.1053/gast.2000.17831

Gastroenterology

Volume 119, Issue 3, September 2000, Pages 794-805

https://doi.org/10.1053/gast.2000.17831 Get rights and content

Abstract

Background & Aims: The leukotrienes C₄/D₄/E₄ (cysteinyl-LTs) are 5-lipoxygenase (5-LO)-derived eicosanoids with potent vasoconstrictor, proliferative, and profibrogenic properties that may participate in key pathophysiologic events in liver cirrhosis. We examined the cysteinyl-LT biosynthetic pathway in liver tissue and purified liver cells isolated from rats with carbon tetrachloride–induced cirrhosis, and assessed the vasoactive properties of LTD₄ in hepatic stellate cells (HSCs) and anesthetized rats. Methods & Results: Liver homogenates from cirrhotic rats had increased 5-LO mRNA and cysteinyl-LT content, as determined by Northern blot and enzyme immunoassay, respectively. In isolated rat liver cells, 5-LO mRNA expression was found to be restricted to Kupffer cells. However, among the liver cells (i.e., hepatocytes, Kupffer cells, HSCs, and sinusoidal endothelial cells), hepatocytes exhibited the highest ability to generate cysteinyl-LTs from the unstable intermediate LTA₄. Hepatocytes from cirrhotic rats showed an enhanced baseline generation of cysteinyl-LTs, but their ability to synthesize cysteinyl-LTs from exogenous LTA₄ was found to be similar to that of hepatocytes from normal animals. Both LTD₄ and hepatocyte-conditioned medium increased intracellular Ca²⁺ concentration and induced contraction in HSCs, suggesting that hepatocyte-derived cysteinyl-LTs could act in a paracrine fashion on nearby nonparenchymal liver cells. The relevance of these in vitro findings was further established in vivo by the observation that LTD₄ significantly increased portal pressure in anesthetized rats. Conclusions: These data suggest a role for hepatocyte-derived cysteinyl-LTs in mediating hepatic vascular tone abnormalities in cirrhosis.

GASTROENTEROLOGY 2000;119:794-805

Section snippets

Induction of cirrhosis in rats

Cirrhosis was induced by inhalation of CCl₄ (twice weekly, Monday and Friday)¹⁶ in 15 male adult Wistar rats (Criffa S.A., Santa Perpètua de Mogoda, Spain). Both cirrhotic and control (n = 20) rats were fed ad libitum with standard chow and distilled water containing phenobarbital (0.3 g/L) as drinking fluid.

Liver tissue collection

Livers were collected from rats anesthetized with an intramuscular injection of ketamine (50 mg/kg body wt) and rinsed with ice-cold Dulbecco's phosphate-buffered saline (PBS) with calcium

Results

The enzyme 5-LO catalyzes the oxygenation of arachidonic acid to the epoxide intermediate LTA₄, which in turn is conjugated with glutathione by the enzyme LTC₄ synthase to form the cysteinyl-LT. Thus, we first examined the expression of these key enzymes in liver tissue by Northern and Western blot analyses. As shown in Figure 1A, liver tissue from control animals did not show 5-LO mRNA expression.

. (Upper panel) Representative Northern blot analysis of 5-LO mRNA expression in liver tissue from 2

Discussion

We assessed hepatic cysteinyl-LT production in experimental cirrhosis. The results clearly indicate that the 5-LO metabolic pathway is markedly enhanced in livers from rats with CCl₄-induced cirrhosis. Particularly, we found a prominent expression of 5-LO mRNA associated with a parallel increase in cysteinyl-LT levels in liver homogenates from cirrhotic rats. Of note, we were unable to detect 5-LO mRNA expression by Northern blot analysis in liver tissue from control rats, but obtained the

Acknowledgements

The authors thank Drs. Marcelina Párrizas and Josep Maria Nicolas for expert assistance, Montse Bernat for technical support, and Dr. J. Evans (Merck Frosst Canada Inc) for providing the specific polyclonal antibody to LTC₄ synthase.

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^☆: Address requests for reprints to: Joan Clària, Ph.D., DNA Unit, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. e-mail: [email protected]; fax: (34) 93-4515272.

^☆☆: Supported in part by Comisión Interministerial de Ciencia y Tecnología (grant SAF 97/0120) and Dirección General de Investigación Científica y Técnica (PB93-1018) y Fondo de Investigaciones Sanitarias (FIS 00/0616). E.T. and M.B.-M. are recipients of fellowships from Ministerio de Sanidad y Consumo (BEFI 98/9314) and Fundació Privada Clínic per la Recerca Biomèdica, respectively.

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Liver, Pancreas, and Biliary TractHepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis☆,☆☆

Abstract

Section snippets

Induction of cirrhosis in rats

Liver tissue collection

Results

Discussion

Acknowledgements

Prostaglandins

Biochim Biophys Acta

J Biol Chem

Eur J Pharmacol

J Biol Chem

J Hepatol

Gastroenterology

Biochem Biophys Res Commun

J Hepatol

J Biol Chem

Arch Biochem Biophys

Cell Biol Int

Hepatology

Eur J Med Chem

Pharm Res

Leukotrienes and lipoxins: structures, biosynthesis, and biological effects

Science

The eicosanoids: prostaglandins, thromboxanes, leukotrienes and related compounds

Leukotrienes and other products of the 5-lipoxygenase pathway. Biochemistry and relation to pathobiology in human diseases

N Engl J Med

Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin

J Immunol

On the mechanism of induction of DNA synthesis in cultured arterial smooth muscle cells by leukotrienes. Possible role of protaglandin endoperoxide synthase products and platelet-derived growth factor

J Cell Sci

Liver, Pancreas, and Biliary Tract
Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis☆,☆☆