Effect of high level section of the spinal cord on the cyclic AMP system in rat liver.

The effect of high level section of the spinal cord upon the hepatic cyclic AMP system was investigated in the rat. We report that transection of the spinal cord dramatically decreases the basal level of cyclic AMP from 0.88 nmol/g liver to 0.36 nmol/g at 1 h and to 0.20 nmol/g at 4 h. This was not due to increased activity of cyclic AMP phosphodiesterase or to decreased activity of basal adenylate cyclase. The sensitivity of adenylate cyclase to its usual effectors in vitro was not impaired. It is proposed that the lowering of liver cyclic AMP below its basal level after high level section of the spinal cord is due to decreased levels of hepatic catecholamines and/or plasma glucagon.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3',5'-monophosphate system to hormonal stimulation during chronic ingestion of DL-ethionine.

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3',5'-monophosphate system were examined in premalignant liver from rats chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissues quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AM content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 +/- 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 +/- 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 +/- 0.04; ethionine 0.55 +/- 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 +/- 7%; ethionine, 15 +/- 1.5%) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethionine ingestion was bilogically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (control, 185 +/- 24 pg/ml; ethionine, 1532 +/- 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14 -fold increase over basal, to 8.63 +/- 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 +/- 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of protaglandin E1 or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue. In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E₁ or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue.In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Protein kinase C activating phorbolesters enhance the cyclic AMP response to parathyroid hormone,forskolin and choleratoxin in mouse calvarial bones and rat osteosarcoma cells

The protein kinase C-(PKC) activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/l) and phorbol 12, 13-dibutyrate (PDBU; 100 nmol/l) enhanced basal cyclin AMP accumulation in cultured neonatal mouse calvaria. The cyclic AMP response to parathyroid hormone (PTH; 10 nmol/l) and the adenylate cyclase activators forskolin (1–3 mol/l) and choleratoxin (0.1 mg/ml) was potentiated in a more than additive manner by TPA and PDBU. In contrast, phorbol 13-monoacetate (phorb-13; 100 nmol/l), a related compound but inactive on PKC, had no effect on basal or stimulated cyclic AMP accumulation. In the presence of indomethacin (1mol/l), TPA and PDBU had no effect on cyclic AMP accumulation in calvarial bones per se, but were still able to cause a significant enhancement of the response to PTH, forskolin and choleratoxin. PTH-, forskolin- and choleratoxin-stimulated cyclic AMP accumulation in rat osteosarcoma cells UMR 106-01 was synergistically potentiated by TPA and PDBU, but not by phorb.-13. These data indicate that PKC enhances cyclic AMP formation and that the level of interaction may be at, or distal to, adenylate cyclase.     

Distribution of Protein I, a Synapse-Specific Phosphoprotein, and Adenylate Cyclase in the Rat Spinal Cord

The longitudinal and transverse distributions of the synapse-specific phosphoprotein Protein I and adenylate cyclase in the rat spinal cord were studied. Protein I was found to be enriched in all cervical and midlumbar (L3-L5) segments, and sparse in midthoracic and sacral segments. Adenylate cyclase activity was high in all cervical and lumbosacral segments, and low in mid-thoracic segments. Cross sectionally, both Protein I and adenylate cyclase were more enriched in the dorsal half than in the ventral half in the various segments studied. The similar topographical distributions of Protein I and adenylate cyclase in the spinal cord support the idea that adenylate cyclase may be intimately associated with Protein I in the nervous system, and could thereby regulate the state of in vivo phosphorylation of Protein I through formation of cyclic AMP.     

Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase.

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Adenylate cyclase activity and cyclic AMP levels during the development of Dictyostelium discoideum.

Adenylate cyclase activity and endogenous cyclic AMP levels were measured using a highly sensitive radioimmunoassay and protein binding assay during 24 h of development of Dictyostelium discoideum. Adenylate cyclase activity was not detected until the aggregation stage of development (10 h) when a sudden peak of activity was found. The enzyme was active at all subsequent stages, although a slow decline in activity was observed. Similarly, cyclic AMP levels were not detectable through the first 7 h of development and then showed a sudden peak at aggregation. Following aggregation the cyclic AMP levels decreased to approximately 1/2 the peak value and maintained that level throughout the remainder of the developmental cycle. Adenylate cyclase had a narrow range of substrate saturation with a maximum velocity at 1 to 4 mM ATP at both the aggregation stage (10 h) and the sorocarp stage (24 h). At levels of ATP higher than 6 mM the enzyme from both stages was strongly inhibited. No activity was observed in the absence of Mg2+ or dithiothreitol. The activity from 10-, 14-, and 20-h stages was found bound to a 25,000 x g pellet fraction. The sudden appearance of adenylate cyclase and its product cyclic AMP at aggregation provides additional evidence of a role for this nucleotide in chemotaxis, and the retention of enzyme activity and nucleotide level during the subsequent stages may reflect a further function of cyclic AMP during formation of the two cell types.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Cell cycle changes in the adenylate cyclase of C6 glioma cells

The adenylate cyclase of C6 glioma cell cultures was characterized for sensitivity to the beta-adrenergic agonist isoproterenol, as well as fluoride, and GTP as a function of the cell cycle. The mitotic phase of the cell cycle was emphasized because both the basal cellular cyclic AMP level and the intact C6 cell's capacity to accumulate cyclic AMP in response to isoproterenol decreased during mitosis. Basal and stimulated adenylate cyclase activities in mitotic cells were decreased relative to the enzyme activities in the G1, S, and G2 phases of the cell cycle. Analysis of the beta-adrenergic receptor using the radioligand(-)[3H]dihydroalprenolol showed that neither ligand affinity nor receptor density changed during the cell cycle, indicating that the reduced adenylate cyclase activity of the mitotic C6 cell was not caused by alterations in this hormone receptor. The reduction in the mitotic cell's basal adenylate cyclase activity was more prominent than the decrease in isoproterenol-, fluoride, or GTP-stimulated activities suggesting that the effectiveness of these enzymes activators (i.e., the efficiency of the coupling mechanism) was not attenuated during mitosis. These studies indicate that the intrinsic catalytic capacity (not the beta-adrenergic receptor or the coupling mechanism) of the C6 adenylate cyclase complex is reduced during mitosis and contributes to the mitotic cell's inability to accumulate and maintain the cyclic AMP concentration at the interphase level.     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

Cyclic AMP metabolism in mouse parotid glands properties of adenylate cyclase,protein kinase and phosphodiesterase

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands.Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity with a pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a K_a of 1.2·10^?7 M. Parotid cyclic AMP and cyclic GMP phosphoriesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least six peaks of enzyme activity in the pI range of 4–6.Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscles.

Cyclic AMP content, adenylate cyclase (EC 4.6.1.1) activity and phosphodiesterase I (EC 3.1.4.1) activity of the hind leg skeletal muscle and cardiac muscle in 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters were examined. In 60-day-old myopathic animals, cardiac cyclic AMP levels were higher and phosphodiesterase I activity was lower, without any changes in the basal adenylate cyclase activity, whereas in 150-day-old myopathic hamsters, cardiac cyclic AMP and basal adenylate cyclase activity were lower, without any changes in the homogenate phosphodiesterase I activity. On the other hand, basal adenylate cyclase and phosphodiesterase I activities in the skeletal muscle homogenate from 60- and 150-day-old myopathic animals were not different from the normal values but the skeletal muscle cyclic AMP levels were significantly less in 60-day-old myopathic hamsters only. The plasma cyclic AMP levels in 60-day-old myopathic hamsters, unlike 150-day-old myopathic animals, were higher than the normal. Although these results reveal differences in myopathic cardiac and skeletal muscles, it is concluded that changes in adenylate cyclase-cyclic AMP system in myopathy are dependent upon the degree of disease.     

Adenosine 3',5'-monophosphate formation by preparations of rat liver soluble guanylate cyclase activated with nitric oxide, nitrosyl ferroheme, S-nitrosothiols, and other nitroso compounds

Cyclic AMP formation from ATP was stimulated by unpurified and partially purified soluble hepatic guanylate cyclase in the presence of nitric oxide (NO) or compounds containing a nitroso moiety such as nitroprusside, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), nitrosyl ferroheme, and S-nitrosothiols. Cyclic AMP formation was undetectable in the absence of NO or nitroso compounds and was not stimulated by fluoride or glucagon, indicating the absence of adenylate cyclase activity. The nitroso compounds failed to activate, whereas fluoride or glucagon activated, adenylate cyclase in washed rat liver membrane fractions. Cyclic GMP formation from GTP was markedly stimulated by the soluble hepatic fraction in the presence of NO or nitroso compounds. Cyclic AMP formation by partially purified guanylate cyclase was competitively inhibited by GTP and cyclic GMP formation is well-known to be competitively inhibited by ATP. Therefore, it appears that activated guanylate cyclase, rather than adenylate cyclase, was responsible for the formation of cyclic AMP from ATP. Formation of cyclic AMP of cyclic GMP was enhanced by thiols, inhibited by hemoproteins and oxidants, and required the addition of either Mg2+ or Mn2+. Further, several nitrosyl ferroheme compounds and S-nitrosothiols stimulated the formation of both cyclic AMP and cyclic GMP by the soluble hepatic fraction. These observations support the view that soluble guanylate cyclase is capable, under certain well-defined conditions, of catalyzing the conversion of ATP to cyclic AMP.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

Short-term changes in levels of cyclic AMP, adenylate cyclase, and phosphodiesterase during the initiation of sperm motility in rainbow trout

In order to clarify the role of the system that generates and degrades cyclic AMP during the initiation of motility of trout sperm, short-term changes in levels of intraspermatozoal cyclic AMP, adenylate cyclase, and phosphodiesterase were measured. Levels of cyclic AMP and the activity of adenylate cyclase increased and reached a maximum level 1 sec after transfer of sperm to K+-free medium, where they became motile, and then decreased rapidly. However, there were no changes in either parameter in sperm which remained immotile in K+-rich medium. In addition, an increase in the activity of phosphodiesterase was observed 4 sec later than the increase in levels of cyclic AMP and adenylate cyclase. These findings suggest that a very rapid change in the level of intracellular cyclic AMP occurs within 1 sec, at the moment of spawning, by the activation of adenylate cyclase and phosphodiesterase, and regulates the initiation of trout sperm motility.     

Adenylate Cyclase Activity in the Superior Cervical Ganglion of the Rat

Abstract: Adenylate cyclase activity in cell-free homogenates of the rat superior cervical ganglion (SCG) was assayed under a variety of experimental conditions. Adenylate cyclase activity was decreased by approximately one-half when 1 m M EGTA was included in the homogenization buffer and assay mixture, indicating the presence of a Ca²⁺-sensitive adenylate cyclase in the ganglion. In the presence of EGTA, basal adenylate cyclase activity in homogenates of the SCG was 12.9 ± 0.6 pmol cyclic AMP/ganglion/10 min. Enzyme activity was stimulated three- to fourfold by 10 m M NaF or 10 m M MnCl₂, Both GTP and its nonhydrolyzable analog guanylylimidodiphosphate (GppNHp) stimulated adenylate cyclase in a concentration-dependent manner over the range of 0.1–10.0 μ M . Stimulation by GppNHp was five to six times greater than that produced by GTP at all concentrations tested. Decentralization of the ganglion had no effect on basal or stimulated adenylate cyclase activity. Receptor-linked stimulation of adenylate cyclase was not obtained with any of the following: isoproterenol, epi-nephrine, histamine, dopamine, prostaglandin E₂, or va-soactive intestinal peptide. Thus the receptor-linked regulation of adenylate cyclase activity appears to be lost in homogenates of the ganglion.     

Amitriptyline-Mediated Inhibition of Neurite Outgrowth from Chick Embryonic Cerebral Explants Involves a Reduction in Adenylate Cyclase Activity

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline-mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin-stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP-dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline-mediated inhibition of neurite outgrowth.     

The periodic change in adenosine 3',5'-monophosphate concentration in sea-urchin eggs

The level of adenosine 3',5'-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0 X 10(-7)M and 1.5 X 10(-6)M, respectively. The activity of adenylate cyclase in a 105 000 X g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl2 increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5 X 10(-6)M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

Lack of correlation between hormonal effects on cyclic AMP and glycogenolysis in rat liver.

Portions of liver were obtained by biopsy from rats infused with various concentrations of glucagon or epinephrine and analyzed for cyclic AMP, glycogen, phosphorylase activity, and glycogen synthetase I activity. The response of tissue cyclic AMP to glucagon or epinephrine was far less sensitive than other metabolic parameters; at certain lower doses of glucagon or epinephrine, glycogen decomposed without a simultaneous increase in the hepatic level of cyclic AMP. It is probable that hormonal activation of adenylate cyclase results in an increase of cyclic AMP only in its small “active” pool without detectable changes in its much larger inactive or bound pool. Though the active cyclic AMP is expected to be released into the circulation or to be labeled with [³H]adenine in preference to the inactive nucleotide, neither the increase of cyclic AMP in the vena cava in vivo nor the incorporation of [³H]adenine into tissue cyclic AMP in liver slices in vitro exhibited more sensitivity to glucagon than the hepatic level of cyclic AMP as a whole. Thus, it remains to be settled whether cyclic AMP is compartmentalized in the cell or plays no essential role in the stimulation of hepatic glycogenolysis induced by small doses of hormones.