Autoradiographic localization of particulate cyclic amp-dependent protein kinase in mammalian brain using[ 3H] cyclic AMP: Implications for organization of second messenger systems

Abstract

Cyclic AMP's regulatory role as an intracellular second messenger is well established. In brain and other tissues, specific proteins that bind cyclic AMP have been shown to be the regulatory subunits of cystolic and particulate cyclic AMP-dependent protein kinases. This study of the autoradiographic localization of specific [ 3H]cyclic AMP binding revealed the heterogeneous distribution of particualte cyclic AMP-dependent protein kinase in the mammalian central nervous system. Specific [ 3H]cyclic AMP binding to tissue sections was of high affinity ( K D = 60nM ) and saturable ( B max= 5pmol/mg protein ). Purine and pyrimidine nucleotide analogues demonstrated inhibition constants against [ 3H]cyclic AMP binding consistent with the specific labelling of cyclic AMP-dependent protein kinase (e.g. 8′-bromo-cyclic AMP: [ ic 50 = 130nM; inosine 3′,5′-cyclic monophosphate: ic 50 = 1μM; uridine 3′,5′-cyclic monophosphate: ic 50 = 60μM). Variations in the levels of [ 3H]cyclic AMP binding presumably reflect the presence of differing amounts of particulate cyclic AMP-dependent protein kinase in different neuronal populations. Highest densities were associated with neuronal cell layers such as the pyramidal cells of the piriform cortex and hippocampus, and granule cells of the dentate gyrus and cerebellum. High levels of binding were also found in other cortical and limbic structures, while moderate levels were found in hypothalamic, thalamic and midbrain areas. Excitotoxic lesions confirmed the localization of the enzyme in hippocampal pyramidal cells and cerebellar granule cells. Localizations reported in this study are largely consistent with results obtained using immunohistochemical methods to label cyclic AMP-dependent protein kinases. Recently, [ 3H] forskolin, a potent and selective activator of adenylate cyclase, the enzyme responsible for the formation of cyclic AMP from adenosine 5′-triphosphate, has been used to localize the activated catalytic component of this enzyme in rat brain. Regions described as being intensely labelled with [ 3H] forskolin (e.g. basal ganglia, hilus of the dentate gyrus and molecular layer of the cerebellum) were found to be associated with relatively low [ 3H] cyclic AMP binding levels. These findings suggest a marked difference between the localization of the two related enzyme entities. However, the distribution of the enzymes is indirectly correlated as high levels of particulate cyclic AMP-dependent protein kinase are present in the soma of neurons with high concentrations of adenylate cyclase in their terminals. Alternatively, it is possible that [ 3H] forskolin localizes only a subpopulation of adenylate cyclase. Interestingly, distribution studies of mRNA encoding the stimulatory form of the guanine nucleotide-binding protein, which is associated with the adenylate cyclase system, reveal that it is present in high concentrations in several cell populations containing high cyclic AMP-dependent protein kinase levels, as assessed by [ 3H]cyclic AMP binding. Future experiments utilizing in vitro autoradiography could determine regional hormone- or drug-induced changes in the amount of cyclic AMP-dependent protein kinase and may allow the localization of particulate type I and II cyclic AMP-dependent protein kinase, if sufficiently selective type I and II cyclic AMP analogues can be synthesized. These studies should help to better define the relationship between cyclic AMP binding proteins and other components of the cyclic AMP second messenger system.