TY - JOUR
T1 - The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes
AU - James Wedner, H.
N1 - Funding Information:
This study was supported by USPHS Program Project Grant (Core) 1 P01 AI 12450. H.J.W. is a recipient of a Research Career Development Award (5 K04 AI 00041) from NIAID, and Research Grant 1 R01 CA 24430 from the National Cancer Institute. The author would like to thank Ms. Athalia Klein and Ms. Kusarn Kukreja for excellent technical assistance and Ms. Nancy Grimshaw and Ms. Mary Anne Blue for preparing the manuscript.
PY - 1980
Y1 - 1980
N2 - The effect of diamide (diazene dicarboxylic acid bis[N,N′-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 · 10-3-1 · 10-4 M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 · 10· M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 · 10-4-1 · 10-5 M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 · 10-6 M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3-2.5 mM for cyclic AMP and 3.3-10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.
AB - The effect of diamide (diazene dicarboxylic acid bis[N,N′-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 · 10-3-1 · 10-4 M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 · 10· M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 · 10-4-1 · 10-5 M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 · 10-6 M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3-2.5 mM for cyclic AMP and 3.3-10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.
KW - (Lymphocyte)
KW - Cyclic AMP
KW - Diamide
KW - Phosphodiesterase
KW - Sulfhydryl group
UR - http://www.scopus.com/inward/record.url?scp=0018874857&partnerID=8YFLogxK
U2 - 10.1016/0304-4165(80)90390-6
DO - 10.1016/0304-4165(80)90390-6
M3 - Article
C2 - 6245710
AN - SCOPUS:0018874857
SN - 0304-4165
VL - 628
SP - 407
EP - 418
JO - BBA - General Subjects
JF - BBA - General Subjects
IS - C
ER -