A new competition binding assay for determination of the cAMP content of human leukocytes.
We developed a competition binding assay for measurement of the cAMP content of human cells based on specific binding sites for this messenger in a crude bovine adrenocortical preparation. The mean cAMP content of human granulocytes and lymphocytes was 1.13 +/- 0.32 microM and 1.81 +/- 0.23 microM, respectively. Stimulation of granulocytes with formyl-methionyl-leucyl-phenylalanine (f-MLP) induced a 2-3 fold increase in cAMP between 10 and 45 sec, which returned to resting values after 1 min. Lymphocytes did not react to f-MLP with a transient rise in intracellular cAMP content. This competition binding assay for cAMP is in essence similar to that for Ins(1,4,5)P3 (7), but conditions for the simultaneous assessment of both messengers could not be found. The present assay is rapid, easy to perform, sensitive and considerably cheaper than commercial kits for assessment of the cAMP content of cells.
Zomerdijk
T P
TP
Dept. of Infectious Diseases, University Hospital, Leiden, The Netherlands.
Nibbering
P H
PH
Bezemer
A C
AC
Löwik
C W
CW
van Furth
R
R
D000302:Adrenal Cortex / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D000242:Cyclic AMP / Q000032:analysis / Q000097:blood*; D006098:Granulocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008214:Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D011865:Radioisotope Dilution Technique; D014316:Tritium
D014316:Tritium; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP