Radioimmunoassay for Cyclic Nucleotides

Abstract

Abstract Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) were measured in mammalian tissues and fluids by radioimmunoassay in a variety of physiological and pharmacological conditions. Basal concentrations and responses to hormonal stimuli were in close agreement with results from previous studies using other assay techniques. Cyclic nucleotide phosphodiesterase treatment of urine and trichloroacetic acid extracts of various tissues completely destroyed the immunoreactive material in each assay except for a tissue blank in kidney, liver, and skeletal muscle of 24%, 40%, and 15%, respectively, in the cGMP assay. The nature of this cross-reacting substance has not been identified. Hormonal stimuli causing marked increases in cAMP in vivo or in vitro did not affect the level of cGMP, indicating that the concentration of each of these nucleotides is controlled independently. Adrenalectomy caused an increase in cGMP in skeletal muscle, but did not affect cAMP in this or other tissues. The concentration of cGMP in skeletal muscle from adrenalectomized rats reverted toward normal after treatment with dexamethasone. Adenylate cyclase and guanylate cyclase activities in subcellular fractions were estimated by immunoassay of the amount of cAMP and cGMP formed from ATP and GTP, respectively. The high specificity and sensitivity of the cAMP and cGMP immunoassays allowed detection of cyclic nucleotide in the supernatant of the reaction mixture without prior concentration or separation procedures. No difference in adenylate cyclase activity in liver plasma membranes was observed between this technique and that of measuring conversion of [α-32P]ATP to [32P]cAMP. Guanylate cyclase activity was detectable in soluble fractions from liver and renal cortex and was not affected by glucagon, dexamethasone, thyroxine, or sodium fluoride added in vitro.