Cyclic Nucleotide-dependent Protein Kinases: VIII. AN ASSAY METHOD FOR THE MEASUREMENT OF ADENOSINE 3',5'-MONOPHOSPHATE IN VARIOUS TISSUES AND A STUDY OF AGENTS INFLUENCING ITS LEVEL IN ADIPOSE CELLS

Abstract

Abstract An assay method has been developed for the measurement of tissue levels of adenosine 3',5'-monophosphate (cyclic AMP) based upon the ability of the cyclic nucleotide to activate cyclic AMP-dependent protein kinase purified from bovine heart, kidney cortex, or brain. The method has been used to measure cyclic AMP levels in mouse brain and in rat brain, liver, fat pads, and isolated adipose cells under various conditions. The results obtained with the three different sources of enzyme agreed well, both with each other and with published values. The limit of sensitivity of the assay method for cyclic AMP was about 2 x 10-9 m. In isolated adipose cells, Filipin, a polyene antibiotic with antilipolytic properties, decreased the intracellular cyclic AMP level, apparently by causing its leakage from the cells into the incubation medium. Vitamin K5, also an antilipolytic agent, unexpectedly potentiated the action of norepinephrine and corticotropin in elevating intracellular cyclic AMP levels in adipose cells. The data also show a differential inhibition by Ca++, EDTA, and dl-β-hydroxy-N-tert-butyl-2,4-dichlorophenethylamine of the action of various lipolytic hormones in elevating cyclic AMP levels in adipose cells. It is suggested that there occur at least three distinct types of hormone receptor in adipose cells, each interacting specifically with one of three catagories of hormone, namely, (a) norepinephrine, (b) glucagon, and (c) corticotropin and thyroid-stimulating hormone.