Direct measurement of nitric oxide release from vascular
endothelial cells.
Guo, Jin-Ping, Toyoaki Murohara, Michael Buerke, Rosario Scalia, and
Allan M. Lefer.
Department of Physiology, Jefferson Medical College, Thomas
Jefferson University, Philadelphia, PA 19107
APStracts 3:0169A, 1996.
Measurement of nitric oxide (NO) concentrations in endothelial
preparations is vital for our understanding of the role of NO in
vascular biology. A NO-selective electrode was used to directly
measure NO release from isolated rat aortic endothelium, cultured rat
aortic endothelial cells (RAECs) and from organic NO donors. Basal
release of NO was detectable in confluent RAEC with a range of 140 -
504 picomoles per 1x105 cells (n = 10). This basal release of NO was
significantly attenuated by treatment with a competitive NO synthase
(NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1 mM) to
42+/-14 picomoles per 1x105 cells (p<0.01). The basal release of
NO was also significantly inhibited by a calmodulin antagonist W-7
(N-(6-aminoheyxl)-5-chloro-1-napthalenesulfonamide hydrochloride; 15
[mu]M) to 28+/-14 picomoles per 1x105 cells (p<0.01). The
substrate for NO synthesis, L-arginine (1 mM), significantly
stimulated NO release (p<0.05 vs. control basal release).
Furthermore, stimulation of cultured RAECs with two endothelium
-dependent vasodilators, acetylcholine (ACh; 100 nM) and A23187 (1
[mu]M), resulted in a significant increase in NO release [574+/-112
picomoles per 1x105 cells (n=5) in ACh- and 658+/-119 picomoles per
1x105 cells (n=5) in A23187-stimulated RAECs]. Treatment of RAECs
with the calmodulin inhibitor W-7 (15 [mu]M) for 30 minutes resulted
in a significant reduction in stimulated NO release by L-arginine,
ACh, or A23187, indicating that NO detected in the present system is
mostly derived from the calmodulin-dependent constitutive form of NO
synthase (ecNOS). Basal release of NO was also detectable in isolated
rat aortic rings with intact endothelium. NO release was
significantly attenuated by L-NAME and augmented by human superoxide
dismutase. These data indicate the usefulness of the amperometric
measurement of NO employing a NO-specific electrode in biological
systems and this method is also useful in demonstrating the
effectiveness of NO donors in vitro.
Received 19 September 1995; accepted in final form 14 March 1996.
APS Manuscript Number A1017-5.
Article publication pending Journal of Applied Physiology.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 1 April 96