Analysis of rabbit pulmonary angiotensin converting enzyme kinetics in vivo

We investigated the kinetics of pulmonary angiotensin converting enzyme (ACE) in vivo by measuring the hydrolysis of a bolus dose of a synthetic ACE substrate, benzoyl-phenylalanyl-alanyl-proline (BPAP). Estimates of apparent Km and Vmax were calculated by multiple regression analysis using a nonlinear model of enzyme kinetics. A bolus injection of indocyanine green and BPAP was given into the jugular vein of anesthetized rabbits (3-4 kg b. wt.) and serial blood samples were collected at 1-sec intervals from the carotid artery. The concentration of indocyanine green, [3H]BPAP and its metabolite in each sample was used to construct pulmonary venous outflow concentration-time curves and to assess ACE activity. Addition of 0.04 to 2.6 mumol of unlabeled BPAP per kg to the injectate reduced [3H]BPAP metabolism in a dose-dependent manner, with a calculated ID50 of 0.37 mumol/kg at the peak of the outflow curve and 0.58 mumol/kg over the integral. There was a similar correlation between the concentration of BPAP in pulmonary blood, estimated from indocyanine green outflow curves, and the metabolism of [3H] BPAP. For each animal, application of the mathematical model provided statistically significant estimates of the kinetic constants for pulmonary BPAP metabolism in vivo. The apparent Km and Vmax were 9.3 +/- 0.8 and 63.1 +/- 9.5 mumol/sec/organ, respectively; the Vmax/Km was 6.8 +/- 1.0 ml/sec/organ. BPAP did not affect any of the hemodynamic parameters measured. The technique described provides a method for detailed study of the kinetics of pulmonary ACE in vivo and effects of inhibitor drugs or other experimental treatments on the function of this enzyme in the intact animal.