Prostacyclin production in vitro by rabbit aortic endothelium: correction
for unstirred diffusional layers
EF Grabowski, GJ Naus and BB Weksler
The degree of mixing in fluid layers immediately adjacent to the
endothelial surface is a major variable in assessment of prostacyclin
(PGI2) production by cultured endothelial cells or intact vessel
endothelium in vitro. Lack of adequate mixing should lead to
underestimation of true production because PGI2 immediately adjacent to
endothelium would be only poorly sampled upon buffer collection. Thoracic
aortas from 38 New Zealand white rabbits were therefore excised, opened
longitudinally, and mounted endothelial side uppermost in a buffer-filled
chamber which excluded cut tissue edges from study. Production of PGI2
under unstirred and magnetically stirred conditions was measured by
radioimmunoassay (RIA) for 6-keto-PGF1 alpha. For animals pretreated with
the combination of papaverine and heparin (see below), unstimulated and
arachidonate-stimulated 6-keto-PGF1 alpha increased with stirring rate
toward limits of 2.9 and 28.5 ng/cm2/min, respectively. Unstimulated and
stimulated 6-keto PGF1 alpha measured at 650 rpm, for example, were greater
than their values at 0 rpm by factors of 3.5 (2P less than .01) and 3.7 (2P
less than .001), respectively. The process of vessel excision, however,
produces another variable: degree of injury to endothelium caused by such
factors as secondary vessel contraction and thrombin generation. Vessel
contraction and thrombin generation can be minimized, respectively, by the
use of a smooth muscle relaxant and heparin administered prior to killing
of the animals. The rabbits were, therefore, grouped according to
intravenous (IV) treatment, prior to killing, with saline, papaverine (4
mg/kg), heparin (200 U/kg) or the combination of papaverine and heparin
(same doses). As compared with pretreatment with saline, papaverine alone,
or heparin alone, pretreatment with the combination of papaverine and
saline led to increases in stimulated 6- keto-PGF1 alpha of 1.6- to
2.8-fold. By transmission electron microscopy, endothelium from animals
pretreated with saline showed ultrastructural changes, including disruption
of cytoplasm, separation without detachment of most endothelial cells from
subendothelium, and focal areas of denudation. In contrast, ultrastructural
integrity of endothelium was preserved in aortas of animals pretreated with
combined papaverine and heparin. These results support the hypothesis that
unstirred diffusional layers lead, in vitro, to underestimation of PGI2
production, especially when vessels are protected from excisional
injury.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 66,
Issue 5,
pp. 1047-1052,
11/01/1985
Copyright © 1985 by The American Society of Hematology
