Extraction of cyclic amp for the determination in the competitive protein binding assay (original) (raw)
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Biochimica et Biophysica Acta (BBA) - General Subjects, 1980
The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 • 10-3--1 • 10 -4 M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 • 10 -4 M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 • 10-4--1 • 10 -5 M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 • 10 -6 M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a g i of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels.
Effect of Serum on Isoproterenol-induced Cyclic AMP Accumulation in Human Lymphocytes
Acta pharmacologica et toxicologica, 1985
The effects of autologous serum on basal and isoproterenol (JPR) or prostaglandin E, (PGE,) stimulated adenosine 3',5' cyclic monophosphate (cAMP) levels were investigated in human lymphocytes. For all blood donors. serum (25% (vfv)) lowered the basal cAMP content. In contrast, the responsiveness of the lymphocyte cAMP accumulation to (-)-IPR was increased. This effect was most clearly demonstrable in bicarbonate buffered incubation medium (40-50% increase of maximal response), but was also seen in phosphate buffered medium (10-20% increase). Serum did not alter the sensitivity of the lymphocytes to IPR. The response to PGE, , which was a considerable more effective stimulator of cAMP accumulation than IPR. was not affected in any consistent way by serum. The results indicate that serum influences the regulation of lymphocyte cAMP and that this effect may partly be exerted at the level of the f:l-adrenoceptors.
British journal of pharmacology, 1994
1. Glucocorticoids and elevations of intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) may affect lymphocyte activation, proliferation and effector functions in similar ways. Therefore, we have investigated the effects of the glucocorticoid, dexamethasone, on human lymphocyte cyclic AMP formation. 2. Treatment of resting human lymphocytes with the glucocorticoid, dexamethasone, sensitized prostaglandin E2-stimulated cyclic AMP accumulation in a time- and concentration-dependent manner. 3. In membranes of lymphocytes treated for 24 h with 100 nM dexamethasone, maximal adenylyl cyclase activity stimulated by prostaglandin E2, isoprenaline, guanosine 5'-triphosphate (GTP), forskolin and MnCl2 was significantly enhanced; the EC50 for these agents was not significantly altered. 4. beta 2-Adrenoceptor density, immunodetectable alpha-subunits of the G-proteins Gs and Gi, and pertussis toxin-substrates were not significantly altered by dexamethasone treatment. 5. I...
Basal cyclic AMP levels in human blood mononuclear cells
European Journal of Clinical Investigation, 1984
Basal cyclic AMP levels in blood mononuclear cells from healthy human donors has been studied. A great variation in cyclic AMP content ranging from below 2 to 20 pmol cells was seen. Incubation of the cells at 37°C mostly showed a time-dependent decrease in basal cyclic AMP, levelling off after 16 h. When treated synchronously, cells from blood from the first sample of blood sampling contained more cyclic AMP than that of later samples. Presence of adenosine deaminase, theophylline or indomethacin influenced cyclic AMP concentrations in a non-consistent manner. Variations in cyclic AMP content may in some individuals be ascribed to early increase of cyclic AMP, in others to a progressive reduction in basal cyclic AMP during blood sampling, cell preparation and incubation.
Calcitonin and calcium ionophores: cyclic AMP responses in cells of a human lymphoid line
Proceedings of the National Academy of Sciences, 1978
Receptors for calcitonin, as assayed by the specific binding of 125I-labeled salmon calcitonin and stimulation of cyclic AMP formation, were found in 8866 cells derived from a human lymphoid line. The affinity of calcitonin from different species and of various analogues of human calcitonin for the binding sites and their ability to stimulate cyclic AMP formation were closely related to their hypocalcemic activity and presumably reflected biological properties of the hormones. Besides calcitonin, prostaglandin E1 and beta-adrenergic catecholamines stimulated cyclic AMP formation in these cells. The calcium ionophores, A23187 and Br-X-573A, did not influence the specific binding of 125I-labeled salmon calcitonin. A23187, however, suppressed basal and calcitonin-stimulated formation of cyclic AMP in the presence of at least 0.6 mM calcium in the incubation medium. Br-X-537A did not require extracellular calcium to suppress basal and calcitonin-stimulated formation of cyclic AMP, sugge...
British Journal of Clinical Pharmacology, 1981
1 The influence of acebutolol, atenolol, pindolol and timolol on human lymphocyte cyclic AMP (cAMP) and its stimulation by isoprenaline in vitro has been studied. 2 Acebutolol and atenolol (10-8-10-6M) had no significant influence on lymphocyte cAMP levels or on isoprenaline-stimulated increase in cAMP. 3 Pindolol and timolol significantly antagonised the effect of isoprenaline, and pA2 values were calculated to be 8.12 and 8.04 respectively. This suggests that 832-adrenoceptors are involved in this phenomenon. 4 Only pindolol produced a significant increase in lymphocyte cAMP, which is consistent with its partial agonist activity.
Immunopharmacology, 1999
The role of the cAMP pathway as an immunomodulatory system has been an area of intensive research. Pharmacological elevation of the cAMP pathway inhibits T lymphocyte proliferation and production of Th1-type cytokines. The effects of Ž. cAMP are thought to be mediated via activation of the intracellular receptor, protein kinase A PKA. We investigated the inhibitory effects of cAMP elevation on human lymphocyte proliferation and function by utilising a range of selective inhibitors of PKA. Elevation of cAMP activity by dbcAMP, Sp-cAMPS and forskolin induced significant decreases of Con A stimulated PBMC proliferation. Co-incubation with the selective PKA inhibitors HA1004, KT5720 and Rp-cAMPS showed these antiproliferative effects to persist, despite measurable PKA activity being inhibited to that of untreated cells or less. IL-2 production was also inhibited by dbcAMP in the presence of HA1004 and Rp-cAMPS. It has been demonstrated that the inhibitory effects of pharmacological elevations in cAMP on human T cell proliferation and IL-2 production do not require PKA activity. These observations indicate that control of lymphocyte proliferation and functional status by cAMP proceeds through PKA-independent events. Identification of the underlying mechanisms behind these effects would increase our understanding of the cAMP cascade and may provide a potentially novel target for immunomodulation.
The Biochemical journal, 1977
Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the...