Cyclic nucleotides in neuronal and glial-enriched fractions from the nerve cord of Manduca sexta.

Neuronal and glial fractions were isolated from the CNS of Manduca sexta. The addition of serotonin caused a significant increase in the cyclic AMP levels in both fractions. A lesser increase in cyclic AMP occurred in the presence of norepinephrine. None of the neurotransmitters examined had any appreciable effect on the levels of cyclic GMP.     

Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids

—Guanosine 3′,5’cyclic monophosphate (cyclic GMP) levels in incubated slices of mouse cerebellum are increased 10-fold by glutamate and two-to three-fold by glycine or γ-aminobutyric acid (GABA). Glutamate also produces a 10-fold increase in adenosine 3′,5’cyclic monophosphate (cyclic AMP) in the same tissue. However, GABA decreases cyclic AMP levels 30-40 per cent, and glycine produces only a transient 50 per cent accumulation of this cyclic nucleotide. Theophylline slightly augments the accumulation of cyclic GMP produced by all three amino acids but markedly attenuates the accumulation of cyclic AMP produced by glutamate. In the absence of Ca²⁺, none of the three amino acids has any effect on cyclic GMP levels, and glutamate produces only a 50 per cent rise in cyclic AMP levels. The decrease of cyclic AMP levels produced by GABA is not affected by theophylline or by the absence of Ca²⁺. These data suggest an involvement of both cyclic GMP and cyclic AMP in the neurochemical actions of glutamate, GABA and glycine.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta . Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M co-eluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [³H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.     

Cyclic AMP and the control of aggregative phase gene expression in Dictyostelium discoideum.

The induction of aggregative phase functions and the acceleration of the onset of aggregation competence by nanomolar pulses of cyclic AMP can be mimicked by exposing developing cells to a high extracellular concentration of either cyclic AMP or cyclic GMP (5 × 10^?4M) during the first 1–2 hr of development. Pulses of cyclic AMP have previously been shown to result in oscillations of intracellular cyclic AMP concentration; we show that high extracellular concentrations of cyclic AMP and cyclic GMP cause intracellular cyclic AMP levels to increase. We describe a mutant, HM11, which has elevated levels of intracellular cyclic AMP from the beginning of development and which begins to accumulate cell-associated phosphodiesterase, an aggregative phase enzyme, within an hour of starvation. Our data suggest that the expression of aggregative phase functions is controlled by an elevation of intracellular cyclic AMP which may be either continuous or periodic.     

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine.A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor.The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

Variation of the levels of cyclic AMP and cyclic GMP during development of the insect Ceratitis capitata.

Changes in the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) during development were studied in the Dipterous Ceratitis capitata. The developmental patterns were different to each other. Cyclic AMP showed a sharp maximum in the larval stage to decrease afterwards during adult development. Changes of cyclic GMP exhibited an opposite pattern, although its levels were always higher than those of cyclic AMP.     

Inhibition of platelet aggregation by 6-keto-PGE1; lack of an effect on cyclic GMP levels

The effects of 6-keto-PGE₁ on aggregatory responses to arachidonic acid (AA), adenosine diphosphate (ADP) and collagen were studied in human platelet-rich plasma (PRP). In addition, experiments were carried out to determine if these effects correlate with changes in platelet cyclic AMP and cyclic GMP levels. 6-Keto-PGE₁ incubated in PRP produced dose-related increases in platelet cyclic AMP levels whereas platelet cyclic GMP levels were unchanged. Control aggregations induced by AA and ADP did not alter cyclic AMP and cyclic GMP levels whereas control aggregations induced by collagen elevated cyclic GMP levels while cyclic AMP levels were unchanged. 6-Keto-PGE₁ produced a dose-dependent inhibition of platelet aggregation induced by AA, ADP and collagen and this inhibition correlated with a dose-related increase in cyclic AMP levels. Since 6-keto-PGE₁ does not consistently alter cyclic GMP levels in human PRP, the present data support previous studies suggesting that 6-keto-PGE₁ produces inhibition of platelet aggregation through the stimulation of cyclic AMP accumulation.     

Oppositional effects of acetylcholine and isoproterenol on isometric tension and cyclic nucleotide concentrations in rabbit atria.

The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.     

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.     

Control of juvenile hormone production: the relationship between precursor supply and hormone synthesis in the tobacco hornworm,Manduca sexta

Tissue culture conditions for the production of juvenile hormone by excised corpora allata from the tobacco hornworm, Manduca sexta, were optimized and hormone output was monitored by incorporation of [¹⁴C-methyl]-methionine. Production was moderate in early fifth instar but undetectable in late fifth instar larvae. It was low in pupae, unmeasurable in adult males and high in adult females. Hormone production by glands of adult females was unaffected by the presence in the medium of juvenile hormone III, the hormone analogue, Methoprene, dibutyrylcyclic AMP, dibutyryl cyclic GMP and 25-hydroxycholesterol, an inhibitor of cholesterol synthesis in mammals.The assay of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in homogenates of corpora allata from M. sexta was optimized and activity in homogenates from animals at various life stages was determined. The correlation between the activity of the enzyme and hormone production by intact glands was excellent in all cases examined except for adult males, which have very high HMG-CoA reductase levels, but produce no hormone.Unlike the HMG-CoA reductase of mammalian tissue, the activity of corpus allatum enzyme is unaffected by Mg²⁺ ATP or fluoride ion, an inhibitor of phosphoprotein phosphatase. The activity is also unaffected by the presence of cAMP, cGMP, IBMX or 25-hydroxycholesterol.     

Cyclic nucleotide phosphodiesterase activities from isolated fat cells: correlation of subcellular distribution with effects of nucleotides and insulin

Cyclic nucleotide phosphodiesterasc activities were determined in fractions of fat cell homogenates, prepared either by differential centrifugation or by centrifugation on discontinuous sucrose gradients.In the supernatant fraction (150,000g supernatant in 0.25 m sucrose, or 92,000g supernatant in 0.32m sucrose): (a) there was 70% of the cyclic AMP phosphodiesterase activity of the whole homogenate, and over 90% of the cyclic GMP phosphodiesterase activity; (b) double reciprocal kinetic plots were nonlinear for both substrates; (c) cyclic (GMP, 0.02-2 μm, activated hydrolysis of 10 μm cyclic AMP; (d) 25 or 50 μm cyclic GMP noncompetitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 38 μm); (e) cyclic AMP, 0.1 μm, slightly activated hydrolysis of 10 μm cyclic GMP; (f) 10 or 20 μm cyclic AMP competitively inhibited hydrolysis of 5–20 μm cyclic GMP (K_i = 18 μm).In the particle fraction (1000g, 1000-16,000g, and 16,000–150,000g pellets in 0.25m sucrose, or 0.8-1.2m sucrose interface at 92,000g): (a) there was 30% of the cyclic AMP phosphodiesterase activity of whole homogenate, but less than 5% of the cyclic GMP phosphodiesterase; (b) the double reciprocal kinetic plot of hydrolysis of cyclic AMP was nonlinear; (c) cyclic GMP, 0.02-2μm, did not affect hydrolysis of 10 μm cyclic AMP; (d) 5 or 10 μm cyclic GMP competitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 1.9 μm).Incubation of fat cells with insulin, 40 ng/ml, increased the maximum velocity of particulate high-affinity cyclic AMP phosphodiesterase, but did not affect the supernatant activity. Addition of insulin after homogenization of the cells had no effect on any phosphodiestesterase activity.     

Regulation of cyclic GMP elevation in the developing antennal lobe of the sphinx moth,Manduca sexta

In the moth, Manduca sexta, 3′,5′‐guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20‐hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity‐dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal‐lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal‐lobe neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 359–375, 1999     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: concentrations in Morris hepatomas of different growth rates

Cyclic AMP and cyclic GMP levels were examined in Morris hepatoma explants in vivo. All eight tumor lines examined had significantly elevated cyclic AMP and cyclic GMP levels when compared to normal liver from tumor-bearing rats. No apparent correlation was observed between the rates of tumor growth and cyclic nucleotide levels; however, two tumor lines (3924A and 7288ctc) had very high levels of cyclic GMP.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Cyclic AMP phosphodiesterase activity in the midgut of Manduca sexta

Midgut preparations from Manduca sexta larvae exhibit potent cyclic AMP phosphodiesterase activity. The enzyme exhibits a pH optimum at pH 8·8 and the assay reaction is linear for at least 30 min. Enzyme activity is greatest in larvae during the second day of the fourth and fifth instars and decreases at the end of the instar. Midgut cyclic AMP phosphodiesterase activity rises again in the pharate pupa and during pharate adult development. Whether these alterations in emzyme activity reflect humorally controlled morphogenetic changes in the midgut or are involved in the physiological functions of the midgut is as yet not known.     

Activity of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by bovine heart and rat liver cyclic nucleotide phosphodiesterases.

The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10^?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10^?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10^?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca²⁺. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP.