Assay of Pulmonary Microvascular Endothelial Angiotensin Converting Enzyme, in Vivo: Comparison of Three Methods
We monitored the activity of pulmonary microvascular endothelial-bound angiotensin converting enzyme (ACE), in vivo, by means of multiple indicator-dilution type techniques, utilizing three different probes: the hydrolysis of two substrates, 3H-benzoyl-Phe-Ala-Pro (BPAP) and 14C-benzoyl-Ala-Gly-Pro (BAGP), and the binding of the inhibitor 3H-RAC-X-65 (RAC), all measured during a single transpulmonary passage in anesthetized rabbits, placed on total heart by-pass, so that both systemic and pulmonary circulations were fully supported by means of a two-channel extracorporeal pump. Experiments were performed at pulmonary blood flows ( \(\mathop Q\limits\) b) of 250, 400, 560 and 800 ml/min, in control or indomethacin pretreated rabbits. We also investigated the activity of pulmonary microvascular endothelial-bound 5′-nucleotidase (NCT), by measuring the dephosphorylation of its natural substrate 14C-5′-AMP. We calculated substrate utilization, mean lung transit time ( \(\mathop t\limits\) ), and volume of distribution (i.e., central blood volume) of all three substrates, as well as inhibitor binding. We also calculated Amax/Km and Bmax, the modified parameters for substrates and inhibitor, respectively. As \(\mathop Q\limits\) b increased, Amax/Km values for all three substrates and Bmax increased linearly, indicating microvascular recruitment. In experiments where either BPAP and 5′-AMP metabolism, or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between \(\mathop Q\limits\) b-induced changes in Amax/Km values of BPAP vs 5′-AMP as well as in Amax/Km of BAGP vs B. of RAC. Similarly, increasing \(\mathop Q\limits\) b increased central blood volume and decreased \(\mathop t\limits\), substrate utilization and inhibitor binding.