Effects of angiotensinase inhibitors on plasma protein binding and IC50 determinations of renin inhibitors

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma Renin activity overestimates the potency of Renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three Renin inhibitors. All three Renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the Renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three Renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma Renin activity measured in vitro is not simply an artifact in the plasma Renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.