Effects of cyclic AMP-elevating hormones and autacoids on LPS-activated rat peritoneal, bronchoalveolar and hepatic (Kupffer) macrophages

Peritoneal, bronchoalveolar and hepatic (Kupffer) macrophages activated in vitro by endotoxin, exhibit alterations in nitric oxide production when certain hormones or other biologically active agents (autacoids) are present in the culture medium. They also show changes in acid beta-glucuronidase activities and morphological changes concerning cell size and general appearance. Agents known to elevate the intracellular levels of cyclic AMP, e.g. adrenalin, prostaglandin E2 and dopamine, increase the nitric oxide production in all three types of macrophage. The addition of H-89, an inhibitor of protein kinase A, abolishes the increase in nitric oxide production. Adrenalin also increases the extracellular activity of beta-glucuronidase. The results of this work suggest that cyclic AMP-elevating hormones and autacoids affect the functions of endotoxin-activated macrophages, such as the production of nitric oxide and the activity of acid beta-glucuronidase.     

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.     

The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils. The inhibition of neutrophil movement and the elevation of cyclic AMP levels by catecholamines, prostaglandins, theophylline and cholera toxin.

Agents known to affect intracellular levels of cyclic AMP in many diverse systems have been tested for their effect on the chemotaxis induced by Escherichia coli culture filtrates, spontaneous motility and cyclic AMP levels of rabbit peritoneal neutrophils. Prostaglandin E1 and A1 but not prostaglandin F2alpha increased neutrophil cyclic AMP levels and, correspondingly, only the former two prostaglandins inhibited chemotaxis. Nevertheless, a quantitative relationship between prostaglandin stimulation of cyclic AMP and inhibition of chemotaxis could not be found. Epinephrine, isoproterenol, and, to a much lesser extent, norepinephrine increased neutrophil cyclic AMP through beta adrenergic stimulation. Only epinephrine and isoproterenol inhibited chemotaxis, but the inhibition was variable and not related to the ability of these catecholamines to increase intracellular cyclic AMP. Cholera toxin increased neutrophil cyclic AMP after a 30-min lag period which paralled its inhibitory effect on chemotaxis and spontaneous motility. However, the effect on chemotaxis require 50 ng/ml of toxin whereas the effect on cyclic AMP was manifested at 2 ng/ml of toxin. Prior to 30-min preincubation there was no effect of even 1250 ng/ml of toxin on either cyclic AMP or chemotaxis. Choleragenoid prevented the effects of toxin on both cyclic AMP and chemotaxis. The bacterial chemotactic factor obtained from E. coli culture filtrates did not effect a measurable change in levels of neutrophil cyclic AMP. The data indicate that even though cyclic AMP is not, in the main sequence of events, triggering the chemotactic response, increases in neutrophil cyclic AMP may modulate the movement and thus the chemotactic responsiveness of the neutrophil.     

Cyclic AMP-dependent control of the rat hepatic glutathione disulfide-sulfhydryl ratio.

The disulfide-sulfhydryl ratio of rat hepatic tissue has been found to vary diurnally lowest in the early morning and highest in the early evening (Isaacs, J. (1976) Fed. Proc. 35, 1472, and Isaacs, J. and Binkley, F. (1977) Biochim. Biophys. Acta 497, 192-204). Intraperitoneal injections of dibutyryl cyclic AMP induces an increase in hepatic glutathione protein mixed disulfides (GSSProt) combined with a corresponding decrease in reduced glutathione (GSH) and protein sulfhydryl (ProtSH). Also, dibutyryl cyclic AMP caused hepatic catalase activity to decrease and to increase hepatic production of peroxide molecules. A decrease in catalase activity directs more of the increased peroxide into the glutathione peroxidase pathway. This leads to increased amounts of oxidized glutathione (GSSG) which ultimately results in increased levels of GSSProt. Therefore cyclic AMP may mediate its effect on the disulfide-sulfhydryl ratio via control over catalase and peroxide generation. Support for this idea is provided by the close temporal correlation between the diurnal variations in cyclic AMP, hepatic catalase, peroxide generation and GSSProt-GSH levels.     

Does the circadian pacemaker act through cyclic AMP to drive the melatonin rhythm in chick pineal cells?

Cyclic AMP is a key regulator of melatonin production in the chick pineal gland. Agents that raise cyclic AMP levels (such as forskolin), or cyclic AMP analogues (such as 8-bromocyclic AMP), increase melatonin synthesis and release, whereas agents that lower cyclic AMP levels (including light) decrease melatonin synthesis and release. A circadian oscillator in these cells also raises and lowers melatonin output. We have been investigating the relationships between cyclic AMP and the circadian pacemaker in the regulation of melatonin production. In the chick pineal (unlike certain neuronal systems), the weight of the evidence indicates that cyclic AMP is not on an entrainment pathway to the circadian pacemaker. Instead, cyclic AMP appears to act downstream from the pacemaker. The pacemaker might itself act directly through cyclic AMP, regulating melatonin content by raising and lowering cyclic AMP levels. If this were the case, and if the effects of cyclic AMP levels on melatonin output are saturable (as they must be), then, in the face of such saturating levels of cyclic AMP, the pacemaker should no longer raise or lower melatonin output. To test this prediction, maximally effective concentrations of forskolin and 8-bromocyclic AMP were determined. Both agents markedly increased melatonin output. After 36 hr, cells were refractory to additional stimulation of melatonin output by addition of both agents together, or by higher concentrations of forskolin (although cyclic AMP levels could still be raised further). Nonetheless, the circadian pacemaker continued to raise and lower melatonin output: The rhythm persisted in the face of saturating levels of cyclic AMP. It is therefore suggested that the circadian pacemaker in chick pineal cells acts with, not through, cyclic AMP to regulate melatonin synthesis. Cyclic AMP and the pacemaker act synergistically to regulate serotonin N-acetyltransferase activity and the melatonin rhythm, with cyclic AMP mediating acute effects and amplitude regulation.     

Regulation of adenosine 3' :5'-monophosphate efflux from rat glioma cells in culture*.

Rat glioma cells grown in culture secrete cyclic adenosine 3':5'-monophosphate (cyclic AMP) into the culture medium following stimulation by beta-agonistic catecholamines. Agents which reduced cellular ATP levels such as valinomycin, oligomycin, and uncouplers of oxidative phosphorylation, inhibited cyclic AMP efflux. Secretion of cyclic AMP was also prevented by prostaglandin A-1 and pharmacological agents including probenecid and papaverine. Of the latter agents, only papaverine reduced ATP levels. These results suggest that the transport of cyclic AMP across animal cell membranes is energy-dependent and subject to regulation.     

Involvement of cyclic AMP in the production of the acid protease Acp1 by Sclerotinia sclerotiorum

The acid protease Acp1 is produced by Sclerotinia sclerotiorum during plant infection. We explored the mechanism involved in the triggering of that production and found that cyclic AMP played a positive role. Acp1 could be produced in the sole presence of exogenous cyclic AMP. The use of molecules known to increase or decrease the intracellular cyclic AMP levels confirmed the impact of this nucleotide on the protease production and suggested its endogenous site of action. Further pharmacological studies showed the specific effect of cyclic AMP on Acp1 production and suggested that protein kinase A would be its likely target. Together, these results provide the first indication that the production of a pathogenesis-related fungal protease could depend on a cyclic AMP/Protein kinase A signalling pathway.     

Cyclic AMP-dependent control of the rat hepatic glutathione disulfide-sulfhydryl ratio

The disulfide-sulfhydryl ratio of rat hepatic tissue has been found to vary diurnally lowest in the early morning and highest in the early evening (Isaacs, J. (1976) Fed. Proc. 35, 1472, and Isaacs J. and Binkley, F. (1977) Biochim. Biophys. Acta 497, 192–204). Intraperitoneal injections of dibutyryl cyclic AMP induces an increase in hepatic glutathione protein mixed disulfides (GSSProt) combined with a corresponding decrease in reduced glutathione (GSH) and proteon sulfhydryl (ProtSH). Also, dibutyryl cyclic AMP caused hepatic catalase activity to decrease and to increase hepatic production of peroxide molecules. A decrease in catalase activity directs more of the increased peroxide into the glutathione peroxidase pathway. This leads to increased amounts of oxidized glutathione (GSSG) which ultimately results in increased levels of GSSProt. Therefore cyclic AMP may mediate its effect on the disulfide-sulfhydryl ratio via control over catalse and peroxidase generation. Support for this idea is provided by the close temporal correlation between the diurnal variations in cyclic AMP, hepatic catalase, peroxidase generation and GSSProt-GSH levels.     

3',5'-cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na+-channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine-induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP.     

The cardiac sarcoplasmic reticulum - glycogenolytic complex, an internal beta adrenergic receptor.

Agents which stimulate the formation of cyclic AMP are well known to cause positive inotropic effects on the heart. This review suggests that the myocardial cell is multicompartmented and has several effector sites for cyclic AMP-mediated events and therefore several possible receptor sites for cyclic AMP-mediated agents. It is possible that some of the complex relationships between cyclic AMP-mediated agents and cardiac physiologic functions result from differences among agonist receptors and enzymes in the intracellular compartments. Examination of the characteristics of intracellular drug receptors may yield a new frontier for cardiac pharmacology.     

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

Thromboxane A2 plays an 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 in 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 cyclooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate model for studying the relationship between the platelet cyclooxygenase and platelet function.     

Production of cyclic AMP and adenosine by the brain and prothoracic glands of Manduca sexta

Relatively large amounts of cyclic AMP are produced by the prothoracic glands (source of the insect moulting hormone or moulting hormone percursor) of the tobacco hornworm, Manduca sexta. Pharate pupal glands produce more cyclic AMP than early fifth instar larval glands, and the addition of aminophylline enhances cyclic AMP accumulation. The much lower cyclic AMP level in the absence of aminophylline indicates the presence of potent cyclic AMP phosphodiesterase activity. Brains (sources of the prothoracicotropic hormone) also produce cyclic AMP but at a lower rate. Brains efficiently produce adenosine from ATP while β-ecdysone inhibits adenosine formation in early fifth instar larval brains. β-Ecdysone stimulates adenyl cyclase in brains of both stages when aminophylline and fluoride are present but has no effect on cyclic AMP accumulation in prothoracic glands. The absence of fluoride greatly reduces the amount of cyclic AMP produced by prothoracic glands when aminophylline is present. No cyclic AMP is accumulated in prothoracic glands when both fluoride and aminophylline are absent or in brains when fluoride is absent, notwithstanding the presence of aminophylline. Other insect tissues were also analysed for cyclic AMP production and none showed levels nearly as high as the prothoracic glands, suggesting a close relationship between cyclic AMP production and the function of the gland.     

Enzymatic radioiodination of succinyl cyclic AMP tyrosine methyl ester by lactoperoxidase and radioimmunoassay for cyclic AMP.

A highly specific and simple radioimmunoassay for cyclic AMP with a sensitivity of 0.04 picomoles/tube has been developed according to the method of Steiner et al., using 125I-succinyl cyclic AMP tyrosine methyl ester as a tracer. The tracer with higher immunoactivities could be simply and constantly prepared by an enzymatic iodination procedure utilizing lactoperoxidase, radioactive iodide and hydrogen peroxide generated by glucose-glucose oxidase system, rather than by chloramine-T procedure.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

3′,5′‐Cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na⁺‐channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine‐induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 281–288, 2001     

Effect of resection of small intestine on the interaction of vasoactive intestinal peptide with rat colonic epithelial cells

Vasoactive intestinal peptide (VIP) receptors and VIP-dependent cyclic AMP production were studied in rat colonic epithelial cells 3 days after a 60% resection of the small intestine. Basal cyclic AMP levels were similar in both control and resected animals. The potency, but not the efficiency, of the peptide on the stimulation of cyclic AMP production was diminished in cells from resected rats. Accordingly, the affinity of VIP receptors, but not the binding capacity, decreased as a consequence of the loss of a part of the small intestinal mucosa. These observations are consistent with the known inhibitory role of cyclic AMP on cell proliferation in colonic epithelium and other tissues and suggest a participation of VIP acting through the cyclic nucleotide in the compensatory hyperproliferative response of the colon following massive resection of the small intestine.     

Guanine nucleotide binding in human placental syncytiotrophoblast membranes and comparative regulation of adenylate cyclase in syncytiotrophoblast, turkey erythrocyte and bovine calf testes membranes by guanosine-5'-triphosphate

Guanosine-5'-triphosphate (GTP) binds specifically to syncytiotrophoblast plasma membranes and increases the production of cyclic AMP in these membranes. 1. In syncytiotrophoblast membranes, GTP alone caused a significant increase in the basal levels of cyclic AMP in a dose dependent manner. 2. GTP alone did not significantly stimulate cyclic AMP production in turkey erythrocyte or bovine calf testes membranes. 3. GTP decreased Gpp(NH)p-mediated cyclic AMP production while increasing NaF-mediated cyclic AMP production in placental, erythrocyte and testes membranes. 4. Since cyclic AMP has been reported to regulate the levels of placental hormones, and it is shown in this study that GTP increases cyclic AMP production in the placenta, this study suggests: (A) placental GTP levels may indirectly regulate placental hormone production, (B) placental beta adrenergic (BA) mediated adenylate cyclase activity may not be regulated in the same manner as the BA system of avian erythrocytes.     

Effect of cyclic AMP on the intracellular degradation of newly synthesized collagen

Prostaglandin E1 and cholera toxin increased the intracellular levels of cyclic AMP of human lung fibroblasts. With prostaglandin E1, the increase in cyclic AMP occurred within 10 min followed by a decline to less than one-half of peak values in 6 h. With cholera toxin, the increase occurred within 60 min but the level of cyclic AMP remained increased for 6 h. Both agents caused a decrease in collagen production as expressed as the proportion of newly synthesized protein represented by collagen. The increase in cyclic AMP levels was accompanied by a marked increase in the proportion of newly synthesized collagen which was degraded intracellularly prior to secretion. Analysis of the degraded collagen showed it to be predominantly less than 1000 daltons in molecular mass, but still in peptide linkage. The data are consistent with the hypothesis that cyclic AMP levels in diploid fibroblasts regulate the amount of collagen produced by fibroblasts, at least in part, by modulating the level of intracellular collagen degradation.     

The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.     

Cyclic AMP induces synthesis of prostaglandin E1 in platelets

Although platelets are known to synthesize small amounts of prostaglandin E1 the control of the formation of this prostanoid has not been investigated. Incubation of human platelet-rich plasma with various compounds which are known to increase cyclic AMP concentration in platelets and inhibit platelet aggregation also increased intracellular prostaglandin E1 synthesis. The prostaglandin E1 was isolated by high pressure liquid chromatography and definitively identified by negative and positive ionization mass spectroscopy. The amounts of prostaglandin E1 formed were proportional to the concentration of cyclic AMP in platelets. Prostacyclin (10 nM) which is the most potent stimulator of cyclic AMP formation increased intracellular cyclic AMP by 4.6 fold and prostaglandin E1 level by 3 fold over the basal levels. Addition of theophylline, a cyclic AMP phosphodiesterase inhibitor, together with prostacyclin increased cyclic AMP concentration 8.7-fold and prostaglandin E1 level 12-fold compared to basal concentrations. Dibutyryl cyclic AMP (2 mM) and 8-bromo cyclic AMP (0.1 mM) increased prostaglandin E1 levels by 3 fold and 2 fold over the basal level, respectively. Prostaglandin D2 (3 microM) when added to platelet-rich plasma increased the cyclic nucleotide levels by 2 fold concomitant with 2 fold increase in prostaglandin E1 concentration. In contrast prostaglandin E2 or prostaglandin F2 alpha which had no effect on cyclic AMP level did not affect the prostaglandin E1 synthesis. Addition of 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, to platelet-rich plasma inhibited both the increase of intracellular prostaglandin E1 and cyclic AMP levels induced by prostacyclin.