Exclusion of angiotensin I-converting enzyme as a candidate gene involved in exudative inflammatory resistance in F344/N rats.
BACKGROUND
Inbred LEW/N and F344/N rats respectively, are susceptible and relatively resistant to a broad range of inflammatory/autoimmune diseases. We recently identified a quantitative trait locus (QTL) on chromosome 10 that protects the F344/N rat from carrageenan-induced exudation in a dominant fashion. Angiotensin I-converting enzyme (ACE) is one of the candidate genes located in this QTL region that plays an important role in inflammation.


METHODS
RNA was extracted from both LEW/N and F344/N rat strains and used to produce full length cDNA by reverse transcription polymerase chain reaction (RT-PCR). Both strands of the PCR products were entirely sequenced to determine nucleotide differences between strains. ACE activity was measured using the synthetic substrate 3H-hippuryl-glycylglycine. ACE protein levels were determined by Western blot using a specific ACE antibody. ACE kinetic and inhibition studies were performed using specific substrates (Hip-His-Leu and Acetyl-Seryl-Aspartyl-Acetyl-Lysyl-Proline) and inhibitors (lisinopril, captopril and quinaprilat) for each C- and N-terminal active site. Finally, the dose-effects of lisinopril treatment on carrageenen-induced exudate volume and ACE activity was studied.


RESULTS
In this study, we report for the first time a missense mutation in the coding region of ACE cDNA at 5' 1021 from C to T, resulting in a Leu-341 to Phe substitution, close to the N-domain active site in the F344/N rats. Full characterization of soluble and tissue ACE in both LEW/N and F344/N rat strains showed that soluble ACE levels in serum and exudate were 1.5 fold higher in the F344/N rats than those in LEW/N rats. In addition, the soluble ACE level was inversely correlated with the exudate volume. However, the specific ACE activity and its catalytic properties were identical in both strains. Furthermore, the chronic inhibition of serum and exudate ACE levels by lisinopril treatment did not affect the exudate volume in F344/N rats, indicating that several factors besides ACE were involved in the control of carrageenan-induced exudation.


CONCLUSIONS
This report describes a complete molecular, biochemical, enzymatic and pharmacologic study of a missense mutation in the ACE cDNA in F344/N rats, that taken together, excludes ACE as a candidate gene involved with resistance to carrageenan-induced exudation in F344/N rats.
Jafarian-Tehrani
M
M
Section on Neuroendocrine Immunology and Behavior, National Institutes of Health, Bethesda, Maryland, USA.
Listwak
S
S
Barrientos
R M
RM
Michaud
A
A
Corvol
P
P
Sternberg
E M
EM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002351:Carrageenan / Q000494:pharmacology; D004252:DNA Mutational Analysis; D005122:Exudates and Transudates / Q000201:enzymology; D007249:Inflammation / Q000139:chemically induced / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D017706:Lisinopril / Q000494:pharmacology; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics; D007703:Peptidyl-Dipeptidase A / Q000097:blood / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019655:Quantitative Trait, Heritable; D051381:Rats; D011916:Rats, Inbred F344; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility
D000806:Angiotensin-Converting Enzyme Inhibitors; D002351:Carrageenan; D017706:Lisinopril; D007703:Peptidyl-Dipeptidase A