Cyclic AMP Content Of Cells Research Articles

Action of 50 Hz magnetic fields on cyclic AMP and intercellular communication in monolayers and spheroids of mammalian cells.

To investigate the influence of physiological parameters such as cell density and three-dimensional cell contact on the biological action of a 2 mT/50 Hz magnetic field, mouse fibroblasts were exposed as monolayers and as multicellular spheroids. Changes in cyclic AMP content of cells and alterations in gap junction-mediated intercellular communication were measured immediately after 5 min of exposure to the field. In monolayers of intermediate cell density (1 x 10(5) cells/cm2), the field treatment caused an increase in cAMP to 121% of the control level, whereas, at 3 x 10(5) cells/cm2 (near confluence), a decrease to 88% of the unexposed cells was observed. Furthermore, field exposure stimulated gap-junction communication to 160% of the control level as determined by Lucifer yellow dye exchange. In spheroids, alterations in the radial profile of cellular cAMP were observed that were due both to field-induced local cAMP changes and to increased gap-junction permeability for this second messenger, the latter causing radial cAMP gradients to be flattened. The results indicate a strong dependence of field action on physiological parameters of the system exposed.

Glucagon-like peptide-1 binding to rat skeletal muscle

We have found [ 125I]glucagon-like peptide-1(7–36)-amide-specific binding activity in rat skeletal muscle plasma membranes, with an estimated M r of 63,000 by cross-linking and SDS-PAGE. The specific binding was time and membrane protein concentration dependent, and displaceable by unlabeled GLP-1(7–36)-amide with an ID 50 of 3 × 10 −9 M of the peptide; GLP-1(1–36)-amide also competed, whereas glucagon and insulin did not. GLP-1(7–36)-amide did not modify the basal adenylate cyclase activity in skeletal muscle plasma membranes. These data, together with our previous finding of a potent glycogenic effect of GLP-1(7–36)-amide in rat soleus muscle, and also in isolated hepatocytes, which was not accompanied by a rise in the cell cyclic AMP content, lead us to believe that the insulin-like effects of this peptide on glucose metabolism in the muscle could be mediated by a type of receptor somehow different to that described for GLP-1 in pancreatic B cells, where GLP-1 action is mediated by the cyclic AMP-adenylate cyclase system.

Control of histamine induced inositol phospholipid hydrolysis in cultured human tracheal smooth muscle cells

[ 3H]Inositol phosphate responses to histamine and a range of other agonists were studied in cultured human tracheal smooth muscle cells. Histamine (EC 50 6.5 μM), bradykinin (EC 50 9.7 nM), carbachol (EC 50 10μM), substance P and NaF all produced concentration dependent [ 3H]inositol phosphate formation in these cells. The response to histamine was inhibited by mepyramine (K A 4.3 × 10 9 M −1), indicating the involvement of the histamine H 1 receptor in this response. The inositol phosphate response to histamine was apparently desensitized following prolonged agonist exposure. The response to histamine was inhibited by phorbol dibutyrate (IC 50 6 nM), and this inhibitory effect was reversed by staurosporine (150 nM). Isoprenaline (1 μM), rolipram (0.1–100 μM) and 3-isobutyl-1-methylxanthine (0.1 mM) all produced small inhibitory effects upon histamine induced inositol phosphate formation. These results demonstrate that cultured human tracheal smooth muscle cells express histamine H 1 receptors coupled to phosphoinositidase C and suggest that the inositol phosphate response induced by stimulation of this receptor subtype is inhibited by activation of protein kinase C and, to a lesser extent, by elevation of cell cyclic AMP content.

Inhibition of gastric acid secretion by epidermal growth factor. Effects on cyclic AMP and on prostaglandin production in rat isolated parietal cells

Histamine (0.5 mM) stimulated the cyclic AMP content of cell suspensions containing greater than 80% parietal cells. Epidermal growth factor (EGF) inhibited this stimulatory effect of histamine, but had no effect on basal cyclic AMP content. The half-maximally effective concentration of EGF for inhibition of histamine-stimulated cyclic AMP was 3.9 nM. The equivalent measurement for the inhibition of histamine-stimulated aminopyrine accumulation was 3.0 nM. Aminopyrine accumulation was measured because it provides an index of the secretory activity of the cell. The cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibitory effect of EGF on cyclic AMP content. This effect of IBMX was not caused by its ability to raise cellular cyclic AMP content in the presence of histamine. Prevention by IBMX of the inhibitory action of EGF on histamine-stimulated aminopyrine accumulation had been shown previously [Shaw, Hatt, Anderson & Hanson (1987) Biochem. J. 244, 699-704]. EGF stimulated prostaglandin E2 (PGE2) production in the cell fraction containing greater than 80% parietal cells, with the half-maximally effective concentration being 7.5 nM. EGF was ineffective in stimulating PGE2 production if the cell fraction was depleted of parietal cells (12%), or if 0.5 mM-histamine was added to the enriched parietal-cell fraction. In conclusion, EGF may inhibit histamine-stimulated acid secretion by decreasing the cyclic AMP content of parietal cells. This effect could be mediated by an increase in cyclic AMP phosphodiesterase activity, but it is unlikely to involve an effect of EGF on parietal-cell prostaglandin production.

Chronic exposure to opiate agonists increases proenkephalin biosynthesis in NG108 cells

The neuroblastoma-glioma NG108 cell line has been shown to contain both a δ-opiate receptor and enkephalin peptides. In this paper, the presence of authentic proenkephalin mRNA and proenkephalin-derived peptides are demonstrated. Growth of the cells in the presence of etorphine for 5–7 days resulted in a 3-fold increase of proenkephalin mRNA, which was accompanied by comparable increases in proenkephalin peptides and free enkephalin. The effect was mimicked by either morphine or [ d-Ala 2, d-Met 5]enkephalinamide, and was blocked by naloxone. The EC 50 for the effect of etorphine was 10 −9 M. The cyclic AMP content of cells grown for 5 days in the presence of etorphine was the same as that of control cells. Forskolin treatment also increased the proenkephalin mRNA content of the cells: the effect was not additive with that of etorphine, suggesting that the effect of opiate agonists was not occurring through their inhibition of adenylate cyclase. The results suggest that proenkephalin synthesis in NG108 cells can be regulated by two different mechanisms, one involving cyclic AMP while the other, regulated by the opiate receptor, is yet to be determined.

Inhibition of mast cell adenosine responsiveness by chronic exposure to adenosine receptor agonists

Mast cell adenosine receptors are up-regulated functionally and numerically by chronic exposure to receptor antagonists, but their response to long-term treatment with receptor agonists has not been studied. To address this issue cultured mouse bone marrow-derived mast cells were exposed to N-ethylcarboxamide adenosine (NECA), an adenosine receptor agonist that augments stimulated mast cell mediator release. Cells grown for 3 days in 1 nM NECA responded normally to A23187 or antigen in releasing β-hexosaminidase, but the ability of exogenous adenosine to potentiate this mediator release was attenuated markedly. This inhibition of adenosine responsiveness was partially present after 10 min of 1 μM NECA exposure and complete after 4 hr. The inhibitory effects could be reversed by washing NECA-exposed cells and returning them to culture for more than 4 hr. The adenosine present in the fetal calf serum coupled with deoxycoformycin attenuated mast cell adenosine responsiveness. The NECA-treated cells also exhibited a hyporesponsiveness to adenosine's augmentation of cell cyclic AMP content. This hyporesponsiveness was specific for adenosine receptors in that exogenous isoproterenol was able to increase cyclic AMP levels to a similar degree in both control and NECA-treated cells. Thus, chronic NECA exposure induces a homologous desensitization of mast cell adenosine receptors.

Adenosine 3',5'-cyclic monophosphate-dependent release of prolactin from GH3 pituitary tumour cells. A quantitative analysis.

The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.

Cyclic AMP levels during stimulation and inhibition of histamine release from rat mast cells by the calmodulin inhibitor trifluoperazine.

The calmodulin inhibitor trifluoperazine represses compound 48/80-induced histamine release. The effects appear to take place at a step distal to the entrance of calcium into the cell. Trifluoperazine in itself induces histamine release from rat peritoneal mast cells. This release is totally independent of extracellular calcium and at high concentrations of the drug (greater than or equal to 20 microM), it is due to lysis of the cells. Trifluoperazine reduces the compound 48/80-induced decrease in mast cell cyclic AMP content, probably by inhibiting a cyclic nucleotide phosphodiesterase. The trifluoperazine-induced repression of histamine release does not seem to be correlated to the ability of the drug to alter the compound 48/80 induced changes in cyclic AMP.

Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK1 cells.

We have previously demonstrated that a cultured porcine kidney cell, LLC-PK(1), maintains the characteristics of a polar renal epithelial cell in culture, and responds to salmon calcitonin and [arginine]vasopressin by increasing cyclic AMP content. To demonstrate the usefulness of this cell line as a model for the study of the biochemical events distal to cyclic AMP production, the activation of cyclic AMP-dependent protein kinase was examined. Intact cells in monolayer demonstrated progressive increases in cyclic AMP content and activation of protein kinase in response to [arginine]vasopressin (2-200nm) and salmon calcitonin (0.03-30nm) with both hormones fully activating the enzyme at a cell cyclic AMP content of 35pmol/mg of protein. Of the total cyclic AMP-dependent protein kinase activity, 80% was found in the 27000g supernatant fraction of sonicated cell material, and this soluble protein kinase could be fully activated by hormone. Conversely, the 27000g pellet contained a significant proportion of cyclic AMP-independent protein kinase and only 20% of total cell cyclic AMP-dependent protein kinase; the latter showed little response to hormone. On the basis of DEAE-cellulose chromatography, type II protein kinase was the predominant isoenzyme in both soluble and particulate fractions of the LLC-PK(1) cells and the soluble fractions of rat and guinea-pig renal medulla. Thus, the LLC-PK(1) cell line can serve as a model for hormonal modulation of protein kinase and as a potential source for defining the endogenous substrates for these enzymes.

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a 32P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE 1, PGE 2 and to a less extent by PGF 2α and PGA 2. In the case of PGE 2, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE 1, PGE 2 and PGF 2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE 2. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.

Effects of epinephrine, adrenocorticotrophic hormone, and theophylline on adenosine 3', 5'-monophosphate phosphodiesterase activity in fat cells.

When rat fat cells were incubated with ACTH, epinephrine, or theophylline for 2 to 10 min, cyclic AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, E.C. 3.1.4.17) activity (K(m) about 0.39 muM) in the 100,000 x g sediment fraction of homogenates was increased 35 to 50%. The effects of epinephrine and ACTH were concentration dependent and maximal increases were produced with concentrations similar to those that maximally stimulate lipolysis. Theophylline (0.5 mM) similarly increased phosphodiesterase activity but did not enhance the effects of maximally effective concentrations of the hormones. The changes in phosphodiesterase activity following addition of ACTH or theophylline paralleled changes in cell cyclic AMP content; both reached a maximum within 5 min and then declined, approaching basal levels after 20 or 30 min. The increased phosphodiesterase activity in cells incubated for 5 min with epinephrine reverted to basal levels within 2.5 min after the addition of propranolol. Our data are consistent with the view that there is a component of the fat-cell phosphodiesterase, perhaps localized in the plasma membrane, whose activity can be acutely modified by the concentration of its substrate, cyclic AMP. As previously reported (J. Biol. Chem.248, 7164-7170, 1973), exposure of fat cells to insulin increases the activity of a low K(m) phosphodiesterase also localized in the 100,000 x g sediment fraction of fat cell homogenates. In the presence of insulin, however, phosphodiesterase remained elevated for at least 40 min and there was no significant change in fat-cell cyclic AMP content. When phosphodiesterase activity was elevated and cyclic AMP content maintained at a high level by incubation of cells with ACTH plus theophylline, insulin produced a further increase in enzyme activity. Whether or not insulin and ACTH (or epinephrine or theophylline) affect the same phosphodiesterase, there seems little doubt that the underlying mechanisms are different.

Prostaglandin E 1 effects on adenosine 3':5'-cyclic monophosphate concentration and phosphodiesterase activity in fibroblasts (mouse L cells-tissue culture-enzyme kinetics-prostaglandin homologues).

Incubation of L-929 and L-2071 fibroblasts with prostaglandin E(1) (PGE(1)) caused a rapid increase in the cyclic AMP content of these cells. A maximal effect was produced with 0.2 mug PGE(1) per ml. At a concentration of 4 mug/ml, PGE(2) was almost equally effective, but PGF(2alpha) and PGA(2) were much less so. 2.6 muM epinephrine, 0.4 mM serotonin, and 0.2% ethanol were without effect. In L-929 cells, cyclic AMP concentrations remained elevated for 2-5 hr, and then declined, although even after a 24-hr incubation the medium contained PGE(1) in a concentration sufficient to increase maximally the cyclic AMP content of cells not previously exposed to this compound. A second addition of PGE(1) after 5 or 24 hr did not produce another increase in the concentration of cyclic AMP. After incubation with PGE(1) for 24 hr, cyclic AMP phosphodiesterase activity, assayed with 0.56 muM substrate, was increased 30-100%; the activity rose further between 24 and 48 hr. It is suggested that the increase in phosphodiesterase activity that appears to be a consequence of prolonged elevation of cyclic AMP concentration may account at least in part for the apparent "refractoriness" to PGE(1) that develops after incubation for several hours with this compound.