Assay of pulmonary microvascular endothelial angiotensin-converting enzyme in vivo: comparison of three probes.
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 bypass, 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 (Qb) of 250, 400, 560, and 800 ml/min in control or indomethacin-pretreated rabbits. ACE activity was also compared to that of pulmonary microvascular endothelial-bound 5'-nucleotidase, by measuring the dephosphorylation of its natural substrate 5'-[14C]AMP. We calculated substrate utilization, mean lung transit time (t), and volume of distribution (i.e., central blood volume) of all substrates, as well as inhibitor binding. We also calculated Amax/Km and Bmax products of enzyme mass and kinetic constants for substrates and inhibitor, respectively. As Qb increased, Amax/Km values for all three substrates and Bmax increased linearly, indicating microvascular recruitment. In experiments in which either BPAP and 5'-AMP metabolism or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between Qb-induced changes in Amax/Km values of BPAP vs 5'-AMP as well as in Amax/Km of BAGP vs Bmax of RAC. Similarly, increasing Qb increased central blood volume and decreased t. Indomethacin had no effect on most of the hemodynamic or enzyme parameters measured. We conclude that in vivo assays of ACE proceed as predicted by Michaelis-Menten kinetics and offer insights into pulmonary endothelial pathophysiology.
Orfanos
S E
SE
Department of Pharmacology & Toxicology, Medical College of Georgia, Augusta 30912.
Chen
X L
XL
Ryan
J W
JW
Chung
A Y
AY
Burch
S E
SE
Catravas
J D
JD
D000818:Animals; D004656:Enalapril / Q000031:analogs & derivatives; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D006439:Hemodynamics; D006801:Humans; D007201:Indicator Dilution Techniques; D007213:Indomethacin / Q000494:pharmacology; D008168:Lung / Q000098:blood supply* / Q000201:enzymology; D008297:Male; D008954:Models, Biological; D009842:Oligopeptides; D007703:Peptidyl-Dipeptidase A / Q000032:analysis* / Q000187:drug effects; D011817:Rabbits; D012128:Respiratory Distress Syndrome / Q000175:diagnosis; D013379:Substrate Specificity
D009842:Oligopeptides; C051316:N-(1-carboxy-3-carboxanilidopropyl)alanylproline; C085359:benzoylalanyl-glycyl-proline; C029689:benzoylphenylalanyl-alanyl-proline; D004656:Enalapril; D007703:Peptidyl-Dipeptidase A; D007213:Indomethacin