Increased gene and protein expression of the novel eNOS regulatory protein NOSTRIN and a variant in alcoholic hepatitis.
OBJECTIVE
Increased intrahepatic resistance in cirrhosis is associated with reduced endothelial NO synthase (eNOS) activity and exacerbated by superimposed inflammation. NOSTRIN induces intracellular translocation of eNOS and reduces NO generation. Our aims were to quantify and compare hepatic expression of eNOS, NOSTRIN, NOSIP, and caveolin-1 in alcoholic cirrhosis with or without superimposed alcoholic hepatitis and in normal livers.


METHODS
Biopsy specimens from 20 decompensated alcoholic cirrhotic patients with portal hypertension (10 with alcoholic hepatitis) and 6 normal livers were analyzed: real-time polymerase chain reaction for quantification of messenger RNA; Western blotting; and enzyme assays of eNOS in normal and diseased liver were performed. Localization and interaction of eNOS and NOSTRIN in liver was assessed by immunohistochemistry and co-immunoprecipitation.


RESULTS
eNOS mRNA was significantly increased and eNOS activity decreased in alcoholic hepatitis patients, despite no differences in eNOS protein expression among the patients. Patients with alcoholic hepatitis had significantly higher hepatic levels of NOSTRIN and caveolin-1 mRNA compared with cirrhosis alone or normal biopsy specimens. A NOSTRIN splice variant, not present in normal tissue, was detected on mRNA and protein levels in all alcoholic patients. Coimmunoprecipitation demonstrated association among NOSTRIN, eNOS, and caveolin-1.


CONCLUSIONS
An increase in mRNA and protein of NOSTRIN and its shortened variant in alcoholic hepatitis may partly account for the paradox of increased mRNA levels and normal protein expression but decreased enzymatic activity of eNOS in diseased liver. Such intracellular regulators of NO production may be important in the development of increased intrahepatic resistance in alcoholic hepatitis patients.
Mookerjee
Rajeshwar P
RP
Liver Failure Group, Institute of Hepatology, Division of Medicine, University College London, London, England.
Wiesenthal
Anja
A
Icking
Ann
A
Hodges
Stephen J
SJ
Davies
Nathan A
NA
Schilling
Kirstin
K
Sen
Sambit
S
Williams
Roger
R
Novelli
Marco
M
Müller-Esterl
Werner
W
Jalan
Rajiv
R
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins; D005260:Female; D015870:Gene Expression; D014644:Genetic Variation; D006519:Hepatitis, Alcoholic / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014018:Tissue Distribution; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D051242:Caveolin 1; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C470345:NOSTRIN protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D052250:Nitric Oxide Synthase Type III; C419706:NOSIP protein, human; D044767:Ubiquitin-Protein Ligases