Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Interralation of prostaglandin endoperoxide (prostaglandin G2) and cyclic 3′,5′-adenosine monophosphate in human blood platelets

The prostaglandin endoperoxide, prostaglandin G₂, in platelet-rich plasma may produce reversible platelet aggregation without secretion, irreversible aggregation with secretion of platelet constituents inhibited by indomethacin, or the latter effects despite indomethacin, depending on the concentration of the endoperoxide. Irreversible aggregation and platelet secretion induced by prostaglandin G₂ apparently result from the action of ADP, since these responses are inhibited by 2-n-amylthio-5′-AMP (an inhibitor of the actions of ADP on platelets) and they do not occur in heparinized platelet-rich plasma. Prostaglandin G₂ lowers the platelet level of cyclic 3′,5′-AMP. Its actions are inhibited by elevation of cyclic AMP levels by prostaglandin E₁ or dibutyryl cyclic AMP or adenosine. Like malondialdehyde production induced by thrombin, ADP, or arachidonic acid, prostaglandin G₂-induced malondialdehyde production is reduced by dibutyryl cyclic AMP and prosraglandin E₁. Platelet activation by prostaglandin G₂ is enhanced by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)-adenine.The action of prostaglandin G₂ on platelets is more complex then previously reported.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

Thromboxane A₂ plays and important role in arachidonic acid- and prostaglandin H₂-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I₂, prostaglandin I₁, and prostaglandin E₁) and dibutyryl cyclic AMP inhibit both thromboxane A₂ formation and arachidonate-induced aggregation platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A₂ is still formed. Prostaglandin H₂-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A₂ production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cycyooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate mode for studying relationship between the platelet cyclooxygenase and platelet function.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Stimulation of palatal glycosaminoglycan synthesis by cyclic AMP

Intracellular levels of cyclic AMP in primary cultures of mouse embryo palate mesenchyme cells were elevated by exogenous administration of dibutyryl cAMP, 8Br-cAMP, prostaglandin E₂ or prostacyclin. Glycosaminoglycan synthesis was stimulated in a dose-dependent manner. Qualitative analysis by DEAE anion exchange chromatography and sensitivity to hyaluronidase digestion indicated preferential stimulation of hyaluronic acid synthesis. Cyclic AMP may thus play a role in regulating the synthesis of palatal glycosaminoglycans known to be requisite for normal development of the palate.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Opposing actions of methylxanthines and dibutyryl cyclic AMP on 1,25 dihydroxyvitamin D3 production and calcium fluxes in isolated chick renal tubules

In contrast to dibuturyl cyclic AMP, the methylxanthine phosphodiesterase inhibitors theophylline and caffeine were found to inhibit the conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃ in isolated renal tubules from vitamin D deficient chicks. This inhibition occurred at concentrations of methylxanthines which were shown to increase renal tubule cyclic AMP levels. No effect of theophylline or caffeine on 25 hydroxyvitamin D₃ metabolism in isolated chick renal mitochondria was detected. Because of a demonstrated inhibitory action of calcium (10 and 20 μmol/l) on renal mitochondrial conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃, the effect of theophylline and dibutyryl cyclic AMP on cellular calcium-45 efflux and total renal tubule calcium content was estimated. Theophylline 10 mmol/l was found to inhibit renal tubular calcium efflux and to increase total cellular calcium content, while dibutyryl cyclic AMP 1 mmol/l had the reverse effect on both parameters. Divergent actions of the methylxanthines and dibutyryl cyclic AMP on the formation of 1,25 dihydroxyvitamin D₃ and renal tubule calcium efflux and content support the hypothesis that intracellular calcium is an important regulator of renal vitamin D metabolism. The results indicate that observed actions of methylxanthines cannot always be ascribed to cyclic AMP accumulation.     

Effect of Noradrenaline and Prostaglandin E<Subscript>1</Subscript> on Adenosine 3′,5′;-Monophosphate Formation in Isolated Pericardial Fat Cells of Man

NORADRENALINE increases the intracellular concentration of adenosine 3′,5′-monophosphate (cyclic AMP)^1,2 which, in turn, enhances glycogenosis³ and lipolysis^4,5 in adipose tissue by increasing Phosphorylase and lipase activities. Prostaglandin E₁ (PGE₁) antagonizes the induced increases in Phosphorylase activity^6,7 and glycerol release in human adipose tissues^8,9 and isolated adipocytes⁷. The finding that the stimulatory effects of the cyclic AMP analogue N⁶—O² dibutyryl cyclic AMP, which mimics the hormonal effect of noradrenaline in human fat cells, are not blocked by PGE₁⁷ suggests that noradrenaline and PGE₁ alter fat cell metabolism by acting on the adenyl cyclase system¹⁰. Whether noradrenaline and PGE₁ alter concentrations of cyclic AMP in human fat cells, however, has not been reported.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

Studies of effects of cyclic adenosine 3′,5′-monophosphate in regulation of human hemopoiesis in vitro

Summary Prostaglandin E₁ (PGE₁), high concentrations of dibutyryl cyclic AMP (dbcAMP), and theophylline were strikingly inhibitory both to tritiated thymidine ([³H]TdR) incorporation into bone marrow deoxyribonucleic acid (DNA) in vitro and to granulocytic colony growth. Autoradiography revealed that lower concentrations of dbcAMP were stimulatory to red blood cell precursors. This study was supported in part by United States Public Health Service Grant AM15163, by Health Research Council of the City of New York Career Scientist Award I-683, and by a Veterans Administration Medical Investigatorship to V. H.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Correlation between changes in intracellular level of cyclic AMP, activation of cyclic AMP-dependent protein kinase, and the morphology of Chinese hamster ovary cells in culture.

The morphological conversion of Chinese hamster ovary cells induced by treatment with dibutyryl cyclic AMP is correlated with increases in the intracellular level of cyclic AMP and the activation of cyclic AMP-dependent protein kinase. When cholera toxin is used to induce the increase in intracellular cyclic AMP, a similar correlation is obtained. Treatment of cells with prostaglandin E₁, which causes a transient increase in intracellular cyclic AMP and a transient activation of protein kinase activity, does not result in the morphology change. From these studies we conclude that a stable activation of the cyclic AMP-dependent protein kinase, which results from an increase in intracellular cyclic AMP, induces the morphological conversion of Chinese hamster ovary cells through phosphorylation of one or more cellular components.     

Similar and opposite effects of dibutyryl cyclic AMP and insulin on glucose metabolism in adipose tissue

The effects of N⁶-2′-O-dibutyryl cyclic AMP on glucose metabolism and lipolysis in fragments of rat epididymal adipose tissue were studied. Measurements were made of glucose uptake, conversion of glucose carbon to CO₂ and tissue fatty acids and glyceride-glycerol, lactate production, and glycerol release. Low concentrations of dibutyryl cyclic AMP (0.1–0.5 mM) increased all parameters of glucose metabolism and inhibited glycerol release in tissue from both normally fed and fasted rats. Higher concentrations of dibutyryl cyclic AMP (3–5 mM) diminished glucose utilization and greatly accelerated lipolysis. Insulin, 50 μunits/ml, accelerated glucose metabolism in the presence of either low or high concentrations of dibutyryl cyclic AMP though the effect of insulin was greatly reduced by 3 mM dibutyryl cyclic AMP. Tissue exposed to concentrations of dibutyryl cyclic AMP which inhibited glucose metabolism (5 mM), then rinsed and reincubated without dibutyryl cyclic AMP, displayed increased glucose utilization. The results of these experiments emphasize the need for caution in interpretation of the effects of dibutyryl cyclic AMP on adipose tissue metabolism and the need for further research to elucidate the role of cyclic AMP in the regulation of glucose metabolism.     

Lipoprotein lipase activity in cultured macrophage cell line J7742 and its increase in variants deficient in adenylate cyclase and cyclic AMP-dependent protein kinase

Three macrophage cell lines, J774₂, CT₂ and J7H₁ were compared with respect to synthesis and secretion of lipoprotein lipase. The enzyme activity measured was characterized as lipoprotein lipase on the basis of serum dependence and inhibition by 1 M NaCl. Enzyme activity in all three lines increased with time in culture and the highest activity was found in the medium of the CT₂ line which is adenylate cyclase deficient while that in the J7H₁ line, cyclic AMP-dependent protein kinase deficient, was intermediate. The half life of the enzyme activity in conditioned medium from all three lines was 30–40 min, suggesting that the different levels of activity observed do represent different levels of enzyme production by the cells. About 80% of the lipoprotein lipase activity from all three lines was present in the medium and 50–70% of cellular activity could be released into the medium by a 3-min exposure to heparin. In addition, 24 h incubation with heparin enhanced enzyme secretion in all three lines. To determine the role of cyclic AMP in the regulation of lipoprotein lipase activity use was made of dibutyryl cAMP, methyl isobutylxanthine (IBMX) and cholera toxin. These agents strikingly depressed lipoprotein lipase activity in the J774₂ line but only dibutyryl cAMP was active in the CT₂ line (adenylate cyclase deficient). In the J7H₁ (protein kinase deficient) line there was no response to dibutyryl cAMP or IBMX over the first 4 h of incubation. Addition of these agents did not affect total cell protein synthesis. The present findings indicate that in the intact cells changes in cyclic AMP levels are associated with a change in the activity of lipoprotein lipase.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element     

Changes in enzyme patterns produced by high potassium concentration and dibutyryl cyclic AMP in organ cultures of sympathetic ganglia

—Preliminary experiments had shown that acetylcholine, the putative mediator of trans-synaptic induction of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in vivo, did not lead to an increase in these enzyme activities in mouse superior cervical ganglia kept in organ culture. It was the aim of the present study to evaluate whether increases in tyrosine hydroxylase and dopamine β-hydroxylase evoked by other stimuli such as potassium or dibutyryl cyclic AMP in such an in vitro system are representative for in vivo trans-synaptic induction where changes in the levels of enzymes involved in norepinephrine synthesis or degradation are strictly confined to TH and DBH. In the presence of elevated concentrations of potassium or 5 mm dibutyryl cyclic AMP under organ culture conditions TH and DBH as well as DOPA decarboxylase and monoamine oxidase were significantly (P < 0.025) increased. The increase in total activities of TH and DBH were completely, those of DOPA decarboxylase and monoamine oxidase partially, inhibited by cycloheximide. In the presence of high concentrations of potassium, the total protein content of the ganglia was 28 per cent higher than in culture controls while dibutyryl cyclic AMP had no significant effect. Cycloheximide alone caused the protein content to fall to 70 per cent of that in control cultures. The loss of protein in the presence of cycloheximide was not accompanied by a simultaneous loss of TH, DOPA decarboxylase or monoamine oxidase, but DBH was decreased. Potassium was shown to increase the incorporation of [³H]leucine into TCA-insoluble protein during an early culture period but dibutyryl cyclic AMP showed no such effect. An increase in the rate of incorporation of [³H]leucine into protein was seen in both the control and elevated potassium cultures after 48 h. This increase did not occur in the presence of dbcAMP. The difference in enzyme patterns under conditions of elevated potassium and dibutyryl cyclic AMP and the fact that no changes in the levels of endogenous cyclic AMP were observed during exposure to 54 mm -potassium for a time period sufficient to initiate changes ultimately leading to elevated TH levels argues against the mediation of the potassium-induced enzyme increases by cAMP. Since changes in enzyme patterns caused by potassium and dbcAMP were not similar to patterns seen in vivo under conditions of trans-synaptic induction we conclude that use of this system as an in vitro model for in vivo trans-synaptic induction necessitates great caution.