Cyclic Nucleotide-dependent Protein Kinases

Abstract

Abstract An assay method has been developed for the measurement of tissue levels of guanosine 3',5'-monophosphate (cyclic GMP) based upon the ability of the cyclic nucleotide to activate cyclic GMP-dependent protein kinase prepared from either lobster tail muscle or silkmoth pupal fat body. The method has been used to measure cyclic GMP levels in various biological tissues and human urine, and in slices of heart and brain incubated under various conditions. The results obtained with this new method agree well with published values of tissue levels of cyclic GMP. The limit of the sensitivity of the assay method is about 0.5 pmole of cyclic GMP in an incubation volume of 0.2 ml (2.5 x 10-9m). Acetylcholine caused a rapid (within 0.5 min) increase of 5- to 10-fold in the cyclic GMP level in rat ventricular slices. The cyclic GMP level returned to the basal value within 5 min. Acetylcholine also effectively lowered the increase in myocardial adenosine 3',5'-monophosphate (cyclic AMP) caused by isoproterenol or glucagon. Isoproterenol or glucagon, in reverse fashion, effectively lowered the increase in myocardial cyclic GMP level caused by acetylcholine. The basal level of myocardial cyclic AMP was not significantly influenced by acetylcholine, nor the basal cyclic GMP level by isoproterenol or glucagon alone. Acetylcholine increased cyclic GMP levels and decreased cyclic AMP levels in rabbit cerebellar slices. In cerebral cortex slices, acetylcholine increased cyclic GMP levels without affecting cyclic AMP levels. Norepinephrine, on the other hand, lowered the cyclic GMP levels while greatly increasing the cyclic AMP levels of cerebellum. Histamine was without effect on cyclic GMP levels in cerebellum but increased cyclic AMP levels. In cerebral cortex, histamine caused a slight increase in cyclic GMP levels accompanied by a great elevation in cyclic AMP levels. These data support the concept that the levels of cyclic GMP and cyclic AMP are under separate regulatory control.