Abstract.-A sensitive and specific radioimmunoassay for adenosine 3',5'-cyclic phosphate (cyclic AMP) has been developed which allows measurement of the nucleotide in extracts of 5-10 mg of tissue. The radioimmunoassay is sufficiently specific for cyclic AMP to eliminate the need for prior chromatographic separation of the cyclic nucleotide from other tissue nucleotides. The radioimmunoassay system is based upon competition of cyclic AMP with a labeled cyclic AMP derivative of high specific activity for binding sites on an antibody specific for the cyclic nucleotide. Antibody to cyclic Ai\IP was obtained by immunizing rabbits with an antigen prepared by conjugating succinyl cyclic AMP with human serum albumin. A high specific activity derivative of cyclic AAMP was prepared by synthesizing succinyl cyclic AMP tyrosine methyl ester (SCAMP-TME) and iodinating the phenolic hydroxyl group of the tyrosine moiety with "25I. Free and antibody-bound 125I-SCAMP-TME were separated by precipitation of the antibody-bound fraction with a second antibody (goat anti-rabbit gamma globulin). Displacement of 1251-SCAMP-T]NIE by unlabeled cyclic AMP when plotted as a semilogarithmic function was linear over a concentration range of 2-100 picomoles. The specificity of the antibody was tested against structurally related nucleotides, nucleosides, and purine bases. All had less than 0.005 per cent of the potency of cyclic AMP in inhibiting 125IJ SCAMIP-TME binding. The marked differences in affinity of the various cyclic nucleotides to cyclic AMP antibody would suggest that antibodies can be developed for each of the cyclic nucleotides by the principles used in this work. Adenosine 3',5'-cyclic phosphate (cyclic AMP) mediates a remarkable array of physiological phenomena' 2 and has been identified as a "secondary messenger" in the concept of hormone action proposed by Sutherland and his associates.' The methods available for the measurement of this nucleotide,3 -7 which is present in mammalian tissues in very low concentrations (0.2-1.5 m,4moles/ gm), involve a variety of complex enzymatic procedures36 and require the initial separation of cyclic AMP from other nucleotides by chromatographic techniques. We have developed a radioimmunoassay sensitive to 1-2 picomoles of cyclic AMP which permits rapid measurement of large numbers of samples using as little as 10-20 mg of tissue. The radioimmunoassay is highly specific for cyclic AMP, and therefore eliminates the need for prior chromatographic separation from other tissue nucleotides. This immunoassay is similar to the radioimmunoassay techniques developed for peptide hormones8 I and involves the competition of labeled and unlabeled cyclic AMP for antibody. In the assay to be described, free and antibody-bound cyclic AMP are then separated by precipitation of the latter with a second antibody.

ABSTRA CT The effects of extracellular nucleotides and agents which elevate intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP) concentrations on human lymphocyte metabolism have been studied. Aminophylline, isoproterenol, and prostaglandins, all of which elevate lymphocyte cyclic AMP levels, inhibited incorporation of 'H-labeled thymidine, uridine, and leucine into the DNA, RNA, and protein of phytohemagglutinin (PHA)-stimulated lymphocytes. Aminophylline inhibition was maximal only when the inhibitor was added within 1 hr after exposure of cells to PHA, suggesting that a relatively early step in the lymphocyte transformation process may be affected.The addition of various nucleotides to the culture medium also inhibited incorporation of labeled precursors. The best inhibitor, dibutyryl cyclic AMP (DU cyclic AMP), produced maximal inhibition only if present during the 1st hr after initial exposure to PHA. Among the various cyclic nucleotides derivatives of guanosine and adenine were the most effective inhibitors (substantial inhibition at 0.1 mm concentrations). However, the inhibition was not specific for nucleotides containing the cyclic phosphodiester moiety since the tri-, di-, and monophosphates of adenosine and guanosine were equally effective in diminishing thymidine uptake. The above inhibitions were not due to secondary effects of the inhibitors on the interaction of PHA with lymphocytes as judged by "25I-labeled PHA binding studies.Low concentrations (1-10 imoles/liter) of cyclic AMP produced slight stimulation of thymidine-BH uptake in resting lymphocytes (lymphocytes not stimulated with PHA). However, the effects were quite smallThese studies have been presented in part to the 53rd

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