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 th at protects the F344/N rat from carrageenan-induced exudation in a dominant fashion. Angiotensin I-converting enzyme (ACE) is one of the candidate gen es located in this QTL region that plays an important role in inflammation. Materials and Methods: RNA was extracted from both LEW/N and F344/N rat str ains and used to produce full length cDNA by reverse transcription polymera se 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 H-3-hippuryl-glycylglycine. ACE protein levels were determined by Western blot using a specific ACE antibo dy. ACE kinetic and inhibition studies were performed using specific substr ates (Hip-His-Leu and Acetyl-Seryl-Aspartyl-Acetyl-Lysyl-Proline) and inhib itors (lisinopril, captopril and quinaprilat) for each C- and N-terminal ac tive 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-3 41 to Phe substitution, close to the N-domain active site in the F344/N rat s. 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 f old higher in the F344/N rats than those in LEW/N rats. In addition, the so luble ACE level was inversely correlated with the exudate volume. However, the specific ACE activity and its catalytic properties were identical in bo th strains. Furthermore, the chronic inhibition of serum and exudate ACE le vels by lisinopril treatment did not affect the exudate volume in F344/N ra ts, indicating that several factors besides ACE were involved in the contro l of carrageenan-induced exudation. Conclusions: This report describes a complete molecular, biochemical, enzym atic 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 wit h resistance to carrageenan-induced exudation in F344/N rats.