Inactive of atrial natriuretic factor in mice in vivo: crucial role of enkephalinase (EC 3.4.24.11)

Atrial natriuretic factor (ANF) is a hormone whose potent hemodynamic and renal actions might be beneficial in several cardiovascular disorders, but whose poor oral absorption and extremely rapid inactivation in vivo have so far prevented its therapeutic use. We have developed simple tests to study the peptides responsible for the hydrolysis of ANF in mice in vivo and to assess the effects of peptidase inhibitors. In mice injected with 125 I-ANF in low amounts the radioactivity present in kidney, a major target organ for the hormone, was analysed by HPLC, precipitation with trichloracetic acid (TCA) and in a membrane binding assay. All three parameters indicated a rapid inactivation of the hormone: 20 s after injection of 125 I-ANF the intact hormone represented less than 20% of the total kidney radioactivity. Oral pretreatment with acetorphan, a potent enkephalinase inhibitor resulted in a marked increase in the amount of intact 125 I-ANF (6-fold), TCA-precipitated (5-fold) and membrane bound radioactivity (4-fold) in the kidney; the total kidney radioactivity was enhanced by ∼2-fold. A similar protective effect was observed with other enkephalinase inhibitors, i.e. thiorphan and kelatrophan; the latter was effective at a 10-fold higher dosage. In contrast, a large variety of inhibitors of metallo-, cysteine, serine and aspartic proteinases had no or only marginal effects. Instead, captopril, an angiotensin-converting enzyme inhibitor, reduced the total and TCA-precipitable radioactivity in the kidneys. Aminopeptidase inhibitors, used either alone or in conjunction with acetorphan, displayed significant but limited protective effects. The crucial role of enkephalinase in ANF inactivation in vivo suggests that inhibitors of this peptidase could be used in a novel therapeutic approach to cardiovascular or renal diseases by protecting endogenous ANF.