Differential long-term effects of tannic acid on adenyl cyclase activity and lipolysis in rat adipocytes

Plants elaborate a variety of secondary metabolites such as hydrolysable tannins which are relatively abundant in fruits, vegetables and beverages in the human diet. We have studied the in vivo long-term effect consumption of tannic acid-supplemented drinking water (0.05%, w/v) on the rat adipocyte adenyl cyclase system and on lipolysis. We found that 14-day tannic acid supplementation did not significantly affect either body growth or food consumption, while fat pads weight was higher than that of the control, although the difference was not significant. On the other hand, tannic acid supplementation decreased both basal and isoproterenol-stimulated lipolysis significantly whereas cyclic AMP production as well as adenyl cyclase activity increased significantly. These results are at a first glance contradictory as cyclic AMP accumulation and lipolysis are positively correlated in rat adipocytes. They suggest at least that the tannic acid diet led to an inhibition of cyclic AMP-dependent protein kinase activity followed by a decrease in lipolysis in rat adipocytes, and to an increased activity of the type VI adenyl cyclase subunit of rat fat cells. This subunit is known to be negatively regulated under phosphorylation by cyclic AMP-dependent protein kinase. More in-depth studies are required to examine whether tannic acid could at least modify the expression of the catalytic subunit of adenyl cyclase, G-proteins and cyclic AMP-dependent protein kinase and/or alter their activities.     

Cyclic AMP-Dependent Protein Phosphorylation in Chemosensory Neurons: Identification of Cyclic Nucleotide-Regulated Phosphoproteins in Olfactory Cilia

Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). In control nonsensory (respiratory) cilia, the cyclic AMP-dependent protein kinase is practically GTP gamma S-insensitive. GTP gamma S activation of the olfactory enzyme appears to be mediated by a stimulatory GTP-binding protein (G-protein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [gamma-32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide-dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 microM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP-dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.     

Opposite Regulation of Adenylyl Cyclase by Protein Kinase C in Astrocyte and Microglia Cultures

Abstract: We studied the regulation of cyclic AMP responses by protein kinase C (PKC) in purified astrocyte and microglia cultures obtained from the neonatal rat brain. In astrocytes, a 10-min treatment with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4β-phorbol 12,13-didecanoate (4β-PDD) (but not with 4α-PDD) or with diacylglycerol, which activate PKC, dose-dependently enhanced cyclic AMP accumulation induced by the β-adrenergic agonist isoproterenol and the adenylyl cyclase activator forskolin. Such enhancement was prevented by the PKC inhibitors staurosporine and calphostin-C and by down-regulation of PKC and was not related to activation of membrane receptors or G_s proteins or to inhibition of G_i proteins or phosphodiesterases. Instead, the activity of adenylyl cyclase doubled in PMA-treated astrocytes. In microglia, a 10-min treatment with PMA or PKC inhibitors did not affect cyclic AMP accumulation, whereas longer treatments with PMA or 4β-PDD (but not 4α-PDD) inhibited the cyclic AMP response in a time- and dose-dependent manner. Such inhibition was mimicked by staurosporine and calphostin-C. Also, in the case of microglia, the modulation of cyclic AMP responses appeared to occur at the level of adenylyl cyclase, and not elsewhere in the cyclic AMP cascade. The inhibition of microglial adenylyl cyclase was apparently not due to aspecific cytotoxicity. A differential regulation of adenylyl cyclase by PKC in astrocytes and microglia may help to explain qualitative and quantitative differences in the response of these cells to various physiological and pathological stimuli.     

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans.

Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.     

Metabolism of Adenosine 3′,5′-Cyclic Monophosphate and Induction of Fruiting Bodies in Coprinus macrorhizus

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.     

Effects of ethanol on gastric acid secretion and gastric mucosal cyclic AMP in the rat.

The effect of ethanol on the secretion of gastric acid and the content of cyclic AMP of the gastric mucosa was studied in rats. Intravenously, ethanol (10 to 800 mg/kg) had no effect on the output of acid. Upon local application into the stomach, ethanol (1 to 10%) caused a concentration-dependent inhibition of the output of gastric acid. The effect was evident within 5 min. At the concentration of 1 %,ethanol decreased the rate of acid secretion maximally by about 30%. At the concentration of 3 %, the maximal inhibition was about 70 %. At the concentration of 10 %, ethanol caused a total cessation of the output of acid within 20 to 60 min.Five and 25 min after the administration of 10 % ethanol into the stomach, the gastric mucosal content of cyclic AMP was decreased by approximately 50 %. Also in vitro, the mucosal content of cyclic AMP was decreased by ethanol within 5 min. The decrease was about 30 % with 2.5 % ethanol, approximately 60 % with 10 % ethanol, and approximately 45 % with 20 % ethanol. Alcohol inhibited the activity of the cyclic AMP phosphodiesterase of the gastric mucosa in a competitive manner. The K_i-value was 0.16 M which would correspond to an alcohol concentration of 9.1 % (v/v). Ethanol caused a concentration-dependent inhibition of the activity of the gastric mucosal adenyl cyclase. By 0.166 M (9.4 %) alcohol the inhibition was nearly 100 %.It is concluded that the ethanol-induced decrease of cyclic AMP in the gastric mucosa is due to a decreased formation of the nucleotide. The accompanying inhibition of the output of acid by ethanol is consistent with the view that cyclic AMP is an intracellular regulator of the gastric acid secretion. In view of the role of cyclic AMP in the control of the integrity of the cells, it is suggested that the ethanol-induced damage of gastric mucosa might also be, at least partly, due to the decreased mucosal content of cyclic AMP.     

Enzymatic regulation of mast cell activation and secretion by adenylate cyclase and cyclic AMP-dependent protein kinases

This review details the biochemical events that follow IgE dimerization by antigen and cross-linking of receptors and are linked with the early rise in cyclic AMP. That the monophasic rise in cyclic AMP at 15 s is essential to the degranulation process is evident by pharmacological manipulation of adenylate cyclase, using specific activators and inhibitors to achieve potentiation and inhibition of immunologic release, respectively. Although only a small percentage of membrane adenylate cyclase is transmembrane linked to IgE-Fc perturbation, its product, cyclic AMP, is elevated during activation and is responsible for the activation of two protein kinase isoenzymes at 30-60 s. This sequence appears to be essential for secretion to occur, as evidenced by dose-related inhibition of both beta-hexosaminidase release and protein kinase activation by adenylate cyclase inhibitors. Competitive activation of cyclic AMP-dependent protein kinase activity by a phosphodiesterase inhibitor leads to inhibition of mediator release by diverting an essential enzyme or recruiting an inhibitory sequence. The precise functional role of the mast cell cyclic AMP-dependent protein kinases has not yet been identified, but there is much evidence in other cell types that protein phosphorylation is an essential accompaniment to cellular regulation. Although other apparently essential biochemical steps are noted, such as uncovering a serine esterase, methylation of membrane phospholipid, and increased Ca2+ influx, only a portion of the activation-secretion response is presented here as a sequence, namely, the IgE-Fc receptor-initiated, transmembrane-coupled activation of adenylate cyclase and the subsequent cytoplasmic cyclic AMP-dependent activation of types I and II protein kinases.     

Inhibition of plasminogen activator secretion by cyclic AMP in a macrophage-like cell line.

The continuous cell line, J774.2, exhibits many macrophage-like functions such as latex and Fc-mediated phagocytosis, antibody mediated phagocytosis, antibody mediated cytotoxicity, chemotaxis, and lysozyme secretion. Cyclic AMP stimulates Fc-mediated phagocytosis and inhibits the growth of J774.2. To further evaluate the relationship between cyclic AMP and the specialized functions exhibited by these cells. Variants deficient in phagocytosis, adenylate cyclase and cyclic AMP-dependent protein kinase were derived. We have now shown that J774.2 also secretes plasminogen activator and that this secretion is rapidly and specifically inhibited by 8-bromoadenosine 3':5'-cyclic monophosphoric acid (8 Br--cAMP) or cholera toxin under conditions where lysozyme secretion is unaltered. Utilizing protein kinase-deficient variants, the ability of cyclic AMP to inhibit plasminogen activator secretion was shown to be mediated by a cyclic AMP-dependent protein kinase. We conclude that cyclic AMP has diametrically opposing effects on two macrophage-like functions: Fc-mediated phagocytosis and plasminogen activator secretion.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.     

Lymphocyte protein kinase activity in cells from young and elderly men and women

Protein kinase activity of lymphocytes isolated from human subjects was assayed using histone as substrate. The activity was stimulated about twofold by cyclic AMP and total enzyme activity, determined in the presence of cyclic AMP, was inhibited by 65% by the specific heat-stable inhibitor of cyclic AMP-dependent protein kinase. Histone phosphorylation was not stimulated by cyclic GMP in the presence of the inhibitor. Cyclic AMP-dependent protein kinase could be activated in vitro by incubating intact cells with isoproterenol or with forskolin and was reflected by a significant (P less than 0.05) increase in the protein kinase activity ratio. In contrast to these well-characterized adenylate cyclase activators, incubating cells for up to 2 hr in vitro in the presence of the specific beta-blocker propranolol had no significant effect on the amount of cyclic AMP-dependent protein kinase that was in the activated state. When compared in subjects between the ages of 21 and 74 years, lymphocyte protein kinase activity was unaltered by age or gender. These results indicate that cyclic nucleotide-dependent protein kinase is of the cyclic AMP-dependent variety in the human lymphocyte. A low amount of the cyclic AMP-dependent activity (about 15%) is in the already activated state in freshly isolated cells, and this is not further reduced by incubation in vitro or by beta-blockade. In contrast to previously reported changes in the capacity to synthesize cyclic AMP, lymphocyte protein kinase is unaltered by gender or age in human subjects.     

Effects of vanadate on protein kinases in rat hepatocytes.

In rat hepatocytes, vanadate modifies neither the intracellular concentration of cyclic AMP nor the --cyclic AMP/+cyclic AMP activity ratio for cyclic AMP-dependent protein kinase. Vanadate can, however, counteract the increase in cyclic AMP and the increase in the --cyclic AMP/+cyclic AMP activity ratio of cyclic AMP-dependent protein kinase induced by glucagon. On the other hand, vanadate treatment of hepatocytes can produce a time- and concentration-dependent increase in cyclic AMP- and Ca2+-independent casein kinase activity. Maximal activation at the optimal time with 5 mM-vanadate was about 70% over control. A clear relationship was observed between the activation of casein kinase and the inactivation of glycogen synthase after vanadate treatment. These results suggest that casein kinase activity may be involved in vanadate actions in rat hepatocytes.     

Platelet aggregation. II. Adenyl cyclase, prostaglandin E1, and calcium

In exploration of the proposal that prostaglandin E1 (PGE1) inhibits platelet aggregation via stimulation of adenyl cyclase, the temporal relationship of adenosine cyclic 3',5' monophosphate (cyclic AMP) synthesis and inhibition of ADP-induced aggregation in response to PGE1 was studied. The requirement for calcium in aggregation led to the investigation of the effects of calcium ions on platelet adenyl cyclase activity. PGE1 stimulated the synthesis of cyclic AMP from adenosine-5'-triphosphate-8-14-C by platelet membrane fractions and also increased cyclic AMP synthesis in intact platelets previously incubated for 2 hours with adenosine-14-C. The accumulation of cyclic AMP increased signficiantly at low concentrations of PGE1 and reached a maximum at about 1 mug. Regardless of the inducing agent, calcium ions are an absolute requirement for the aggregation of platelets.     

THE EFFECT OF NEONATAL THYROIDECTOMY ON THE DEVELOPMENT OF THE ADENOSINE 3'',5''-MONOPHOSPHATE SYSTEM IN THE RAT BRAIN

Abstract— The effect of neonatal thyroidectomy on the cyclic AMP system in the developing rat brain was examined. Administration of ¹³¹I at birth led to a 16 per cent reduction in brain weight and a 70 per cent reduction in body weight by 40 days of age. The level of cyclic AMP in the brain increased 5-fold between birth and 40 days of age and this increase was partially reduced by early thyroidectomy. A similar increase in the activity of adenyl cyclase and phosphodiesterase was observed during development, but thyroidectomy produced no detectable changes in the activity of either enzyme. The activity of the cyclic AMP-dependent protein kinase was already maximal at birth and also was unaffected by thyroidectomy.
Norepinephrine increased levels of cyclic AMP 4- to 5-fold in brain slices prepared from adult rats, but was without effect on slices prepared from newborn or 3-day-old rats. The response to norepinephrine in thyroidectomized rats did not differ from that in control rats at any of the ages examined. Our findings indicate that neonatal hypothyroidism does not deleteriously affect the development of the cyclic AMP system in the rat brain.     

Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase.

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.     

5-hydroxytryptamine-induced relaxation of neonatal porcine vena cava in vitro

5-hydroxytryptamine (5-HT) caused concentration-dependent relaxation of isolated rings from porcine vena cava contracted with alpha-methyl 5-HT or prostaglandin F2 alpha. Relaxation was not blocked by propranolol (1 micron), atropine (1 micron), indomethacin (3 microns), mepyramine (1 micron), cimetidine (1 micron), or cocaine (10 microns). Further receptor analysis could not be performed by antagonism of the relaxant response but was possible using 5-HT induced increases in cyclic AMP. Methysergide (1 micron) but not cyproheptadine (0.1 micron), specifically antagonised the 5-HT induced increase in cyclic AMP with an estimated pA2 of 7.19. The alpha-methyl analogue of 5-HT, a potent agonist at M and D receptors, did not cause relaxation or elevate cyclic AMP. These results suggest that the 5-HT receptor described here is not of the classical M or D type and unlike that described thus far in the vasculature. This receptor shares some similarities with brain 5-HT1 receptors since both may be linked with adenylate cyclase.     

Lanthanum: inhibition of ACTH-stimulated cyclic AMP and corticosterone synthesis in isolated rat adrenocortical cells

Lanthanum (La+++) is a well-known Ca++ antagonist in a number of biological systems. It was used in the present study to examine the role of Ca++ in the regulation of adenyl cyclase of the adrenal cortex by ACTH. In micromolar concentrations, .La+++ inhibited both cyclic AMP and corticosterone response of isolated adrenal cortex cells to ACTH. However, a number of intracellular processes were not affected by La+++. These include the stimulation of steroidogenesis by dibutyryl cyclic AMP, conversion of several steroid precursors into corticosterone, and stimulation of the latter by glucose. Thus, inhibition of steroidogenesis by La+++ appears to be solely due to an inhibition of ACTH-stimulated cyclic AMP formation. Electron microscope examination showed that La+++ was localized on plasma membrane of the cells and did not appear to penetrate beyond this region. Since La+++ is believed to replace Ca++ at superficial binding sites on the cell membrane, it is proposed that Ca++ at these sites plays an important role in the regulation of adenyl cyclase by ACTH. Similarities in the role of Ca++ in "excitation-contraction" coupling and in the ACTH-adenyl cyclase system raise the possibility that a contractile protein may be involved in the regulation of adenyl cyclase by those hormones which are known to require Ca++ in the process.     

Adenosine 3',5' cyclic monophosphate in Euglena gracilis

$\text{Euglena gracilis}$ contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. $\text{Euglena}$ also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.     

Properties and cellular distribution of cyclic AMP-dependent protein kinase from thymus

A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10^?6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP. The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.     

Mastoparan inhibits beta-adrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts

Mastoparan, a wasp venom toxin, has various pharmacological activities, the mechanisms of which are still unknown. To clarify the action of mastoparan on G protein-coupled receptor-mediated signaling, we previously examined the effect of mastoparan on G(q)-mediated signaling and demonstrated that mastoparan binds to gangliosides causing a decrease in Galpha(q/11) content in lipid rafts, and resulting in the inhibition of G(q)-mediated phosphoinositide hydrolysis (Sugama et al., Mol. Pharmacol., 68, 1466, 2005). In the present study, we examined the effect of mastoparan on beta-adrenoceptor-G(s) signaling in 1321N1 human astrocytoma cells. Mastoparan inhibited isoproterenol-induced elevation of cyclic AMP in a concentration-dependent manner. Although mastoparan is known to be an activator of G(i), pertussis toxin only slightly attenuated mastoparan-induced inhibition of cyclic AMP elevation, suggesting that a major part of the inhibition of cyclic AMP elevation induced by mastoparan is not mediated by Galpha(i). By contrast, mastoparan-induced inhibition of cyclic AMP elevation was clearly attenuated by preincubation of the cells with ganglioside mixtures. Moreover, mastoparan changed the localization of Galpha(s) in lipid rafts without disrupting the structure of lipid rafts. Fluorescent staining analysis showed that mastoparan released GFP-Galpha(s) from plasma membranes into the cytosol. These results suggest that the mastoparan-induced suppression of cyclic AMP elevation is mainly caused by changing the localization of Galpha(s) in lipid rafts into a compartment in the cellular interior where it is not available to activate adenylyl cyclase.     

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.