Radioimmunoassay of Cyclic Nucleotides

Abstract

Adenosine 3′,5′-cyclic monophosphate (cAMP) mediates a remarkable array of physiologic responses, fulfilling the role of a second messenger in virtually every cell type and tissue through its ability to stimulate protein kinases (Robison et al. 1968). Cyclic 3′,5′-guanosine monophosphate (cGMP), like cAMP, is very widely distributed, although its physiologic function remains uncertain despite clear-cut changes in a few tissues in response to physiologic stimuli. Both of these cyclic nucleotides are present in mammalian tissues at extremely low concentrations (cAMP 0.2–1.5 nmol/kg; cGMP 0.008–0.06 μimol/kg) and their measurement has therefore been very difficult (Steiner et al. 1970). In the mid-1960s, a number of enzymatic procedures were developed for measuring cyclic nucleotides, all of which required large amounts of tissue and extensive chromatographic purification before analysis, making it impossible to conduct measurements in large numbers of tissue samples. In the late 1960s, competitive binding assays were developed for the measurement of cAMP, involving either the use of anti-2′-0-suc- cinyl cAMP antibodies (Steiner et al. 1969,1972) or naturally occurring cAMP- binding proteins (Gilman 1970). The radioimmunoassay was both highly specific for cAMP and sensitive to 1–2 pmol, permitting rapid measurements of large numbers of samples in as little as 10 mg tissue (Steiner et al. 1969, 1972).