RELATIONSHIP BETWEEN VOLUME OF BATHING MEDIUM AND ECTOENZYME ACTIVITY IN
MONOLAYERS OF CULTURED BOVINE PULMONARY ARTERIAL ENDOTHELIAL CELLS.
Papapetropoulos,Andreas, Lynn A. Elmore and John D. Catravas.
Vascular Biology Center and Department of Pharmacology & Toxicology
Medical College of Georgia, Augusta, GA 30912
APStracts 3:0070L, 1996.
It is unclear whether all or a fraction of the capillary plasma volume (V pc )
serves as the reaction volume (V r ) for pulmonary capillary endothelial
ectoenzymes, in vivo . Cultured endothelial cell monolayers (EC) provide a
convenient model for studying EC-bound enzyme - V r relationships. Since the
Michaelis-Menten parameter V max (=E*k cat /V r , where E is enzyme mass and k
cat is the constant of product formation) is inversely proportional to V r ,
we hypothesized that increasing the volume of medium (V m ) bathing EC
monolayers would proportionally reduce the calculated V max (or V max /K m )
values of an ectoenzyme reacting with a substrate only if, and as long as, V m
= V r . To test this hypothesis, studies were performed in bovine pulmonary
arterial EC grown to confluence. Activities of angiotensin converting enzyme
(ACE) and 5'nucleotidase (NCT) were assayed in Earle's balanced salts solution
utilizing [ 3 H]-BPAP and [ 14 C]-5'AMP as substrates, respectively. Under
first order reaction conditions and at constant substrate concentrations
([BPAP]=15nM, [AMP]=1[mu]M), V max /K m ratios of ACE and NCT declined to 20%
of their original values, as V m increased from 0.6ml to 2ml. ACE activity was
also studied at constant substrate mass (BPAP=7pmol) under first order
reaction conditions; again, enzyme activity (V max /K m ) declined
proportionally to increasing V m . Under zero order reaction conditions
([BPAP]=250[mu]M), ACE activity (V max ) was similarly related to V m . Linear
regression analyses revealed that ACE or NCT would recognize up to at least
3ml V m , a volume vastly exceeding that of V pc in a section of the capillary
bed composed of an equivalent number of endothelial cells, and thus suggesting
that V pc could serve as the reaction volume for pulmonary capillary EC
ectoenzymes in vivo .
Received 30 October 1995; accepted in final form 13 May 1996.
APS Manuscript Number L312-5.
Article publication pending Am. J. Physiol. (Lung, Cell. Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 28 May 96