Effect Of Dibutyryl Cyclic AMP Research Articles

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.

Effect of isoproterenol on myocardial mechanical function and cyclic AMP content in the fetal rabbit

The effect of isoproterenol on mechanical function was studied in the isolated arterially perfused heart of the fetal (21st and 28th day of gestation) and newborn rabbits. The inotropic effect of isoproterenol in the fetus was less than in the newborn. In contrast, myocardial cyclic AMP levels after isoproterenol infusion in the fetus were greater than in the newborn. The inotropic effects of dibutyryl cyclic AMP and of calcium in the fetus were less than in the newborn. These data suggest that the process from beta-receptor to cyclic AMP in the fetus was equally or even more responsive to isoproterenol than in the newborn. The diminished inotropic effect of isoproterenol in the fetus may be due, at least in part, to the decreased inotropic effect of calcium.

Ｄｉｂｕｔｙｒｙｌ ｃｙｃｌｉｃ ＡＭＰの電気生理学的効果

Dibutyryl cyclic AMP (DBcAMP) は, 重症心不全に対しその有効性が注目されているが, その臨床電気薬理学的性質についてはほとんど検討されていない.そこで洞不全症候群 (SSS) 4例, 房室ブロック3例, 心室性不整脈2例の計9例, 男7例, 女2例, 年齢22～81歳 (平均58±24歳) を対象とし, DBcAMPO.05～0.1mg/kg/minを30分間点滴静注し, その電気生理学的効果を検討した.DBcAMPは洞周期, 房室結節, 心房, 心室の不応期を有意に短縮させたが, AH時間, HV時間には有意な変化を及ぼさなかった.また2対1房室ブロックを示した例ではDBcAMP後1対1伝導に改善した.以上よりDBcAMPは房室伝導障害, SSSに対し効果的と考えられた.

Adenine nucleotides and ventricular fibrillation

The isolated perfused rat heart was used to study the influence of adenine nucleotides and their metabolites on vulnerability to ventricular fibrillation. In this model the incidence of ventricular arrhythmias after coronary artery ligation is determined by the extracellular K+ concentration; with perfusate K+ of 2.0 and 3.0 mmol/l hearts develop a high incidence of ventricular arrhythmias and fibrillation while arrhythmias are not encountered with perfusate K+ of 9.0 mmol/l. Assay of adenine nucleotides in uninvolved and ischaemic myocardium of these hearts showed a direct relationship between incidence of ventricular fibrillation and tissue levels of cyclic AMP but not tissue levels of lactate, high energy phosphates, adenosine, inosine and hypoxanthine/xanthine. Administration of dibutyryl cyclic AMP to isolated rat hearts reduced the ventricular fibrillation threshold; this action of cyclic AMP was effectively antagonized by adenosine and its N-ethylcarboxamido analogue but not by 2-chloroadenosine, phenylisopropyladenosine, cyclohexyladenosine and the adenosine deaminase inhibitor, EHNA. 2-Chloroadenosine, like adenosine, inhibited the increase in heart rate caused by DBcAMP. All the adenosine analogues had antiarrhythmic activity against spontaneously occurring ventricular arrhythmias during coronary artery occlusion. Adenosine analogues also antagonized the effect of dibutyryl cyclic AMP whereby it prolongs the QT interval. Adenosine, by as yet incompletely defined mechanisms, may act as an antagonist to the cyclic AMP mediated increase in vulnerability which contributes to the genesis of ventricular fibrillation in the early phase of myocardial ischaemia.

Positive inotropic effects of dibutyryl cyclic AMP in isolated perfused rat hearts

Positive inotropic effects of dibutyryl cyclic AMP in isolated perfused rat hearts

Dibutyryl-cyclic AMP inhibits cholesterol esterification in J 774 monocyte-like cells

The effect of dibutyryl-cyclic AMP (dbcAMP) and theophylline was investigated on oleic acid incorporation into cholesteryl esters and triacylglycerols in the mouse monocyte-macrophage cell line J 774. 24h pretreatment of macrophages with dbcAMP decreased cholesteryl ester formation in a dose-dependent manner (about 4 fold reduction for dbcAMP 10 −4M + theophylline 10 −3M), while oleic acid incorporation into triacylglycerols was markedly (2 to 3 fold) enhanced. The catabolism of acetylated LDL was only slightly affected (about 15–20% reduction with dbcAMP 5×10 −4M + theophylline 10 −3M). Acyl Coenzyme A:cholesterol-O-acyl-transferase activity, measured in vitro on cell homogenates, was reduced in dbcAMP-treated cells, whereas diacylglycerol acyltransferase activity was increased. These results suggest that cyclic AMP can modulate cholesteryl ester and triacylglycerol formation in macrophages, and that these metabolisms are inversely regulated. Agents which increase cyclic AMP intracellular level could be of interest for reducing cholesteryl ester accumulation in macrophages.

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 m M dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.

The effect of dibutyryl cyclic AMP on the excretion of taurocholic acid from isolated rat liver cells

Dibutyryl cyclic AMP (50–1000 μM) was found to increase the initial rate of efflux of taurocholic acid from isolated rat hepatocytes. Efflux of the bile acid was inhibited by sodium, and in the absence of sodium dibutyryl cyclic AMP failed to stimulate the rate. Increasing the concentration of calcium from 0 to 1.2 mM had no effect on the initial rate of taurocholic acid efflux from the cells, but the absence of calcium markedly reduced the effect of dibutyryl cyclic AMP. The results suggest that changes in the fluxes of sodium and calcium are involved in the effect of the cyclic nucleotide on taurocholic acid efflux from the cells.

Anti-immunoglobulin and phorbol ester induce phosphorylation of proteins associated with the plasma membrane and cytoskeleton in murine B lymphocytes.

Protein phosphorylations are rapidly induced in intact B cells by antibodies to surface immunoglobulin (anti-IgM) and by phorbol 12-myristate 13-acetate (PMA). A comparison of the molecular weight, isoelectric points, phosphopeptides, and phosphoamino acids of the phosphoproteins induced by anti-IgM and by PMA suggests that anti-IgM acts through the activation of protein kinase C. This conclusion is strengthened by the observation that prolonged treatment with PMA ablates the ability of anti-IgM to induce phosphorylation, presumably by depleting cellular protein kinase C. Furthermore, the effects of dibutyryl cyclic AMP on protein phosphorylation are quite distinct from the effects of anti-IgM. The six most prominent phosphoproteins induced by PMA, with approximate Mr values of 47, 55, 62, 68, 68, and 65-70 X 10(3), are associated with the plasma membrane. Of these, four are apparently associated with the cytoskeleton, suggesting that the phosphorylation of cytoskeletal proteins may be important events early in B cell activation. Examination of protein phosphorylation in cell lines derived from different tissues has identified one major B cell phosphoprotein (Mr 65-70 X 10(3), which is absent in T cells, and two phosphoproteins (Mr 55 and 68 X 10(3), which are observed in cells of hematopoietic origin but which are absent or uncommon in other cell types.

Effect of cyclic AMP on lipid accumulation and metabolism in human atherosclerotic aortic cells

The effects of dibutyryl cyclic AMP (db cAMP), cholera toxin, and methylisobutylxanthine on the content and metabolism of lipids in smooth muscle cells cultured from normal and atherosclerotic intima of human aorta have been studied. Db cAMP (0.1 mM) decreased the levels of triglycerides and esterified sterols 1.5-3-fold in cells cultured from fatty streaks and atherosclerotic plaques. Cholera toxin (100 μg/ml) and methylisobutylxanthine (0.1 mM) stimulated by 2-fold the cyclic AMP level in aortic smooth muscle cells and decreased the content of triglycerides and esterified sterols by 2–3-fold. Prolonged treatment with db cAMP and methylisobutylxanthine resulted in a fall in- the intracellular phospholipids and free sterols. Using the labelled precursors, [3H]acetate and [ 14C]oleate, it was established that the agents increasing the intracellular cyclic AMP level inhibit the formation of sterols and fatty acids, impede the synthesis of phospholipids, triglycerides and esterified sterols, and stimulate their hydrolysis. The data demonstrate that cyclic AMP facilitates the regression of cellular lipoidosis by altering the intracellular lipid metabolism.

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.

Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue.

Antibodies directed against the major apoprotein of rabbit lung surfactant, a 29-36-kDa glycoprotein, were used to study changes in the levels of translatable surfactant apoprotein mRNA in rabbit lung tissue during development, as well as the effects of cortisol and cyclic AMP analogues on the levels of surfactant apoprotein and its mRNA in fetal rabbit lung tissue in organ culture. The major surfactant apoprotein and its mRNA were undetectable in lung tissues of 21-day gestational age fetal rabbits. Translatable mRNA specific for the major surfactant apoprotein was first detectable in lung tissues of 26-day fetuses, increased 25-fold on day 28, reached peak levels at day 31, and declined after birth. Incubation of 21-day fetal rabbit lung explants with cortisol in serum-free medium resulted in an increase in the specific content of the 29-36-kDa apoprotein. Cyclic AMP analogues and forskolin, an activator of adenylate cyclase, also caused a marked increase in the accumulation of surfactant apoprotein. When fetal lung explants were incubated with cortisol and dibutyryl cyclic AMP in combination, the specific content of the surfactant apoprotein was increased to levels greater than that of explants treated with either cortisol or dibutyryl cyclic AMP alone. These effects of dibutyryl cyclic AMP and cortisol on surfactant apoprotein accumulation were associated with comparable changes in the levels of translatable surfactant apoprotein mRNA. Thus, we have shown for the first time that the induction of pulmonary surfactant apoprotein synthesis during differentiation in vitro and in vivo is associated with an increase in the level of translatable mRNA and that cortisol and cyclic AMP increase both the accumulation of the major surfactant apoprotein and the corresponding mRNA in fetal rabbit lung tissue in vitro.

Effects of dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparation

In order to elucidate the role of cyclic nucleotides in the suprachiasmatic nucleus, we examined the effect of dibutyryl cyclic AMP (c-AMP) and dibutyryl cyclic GMP (c-GMP) on single neuronal activity of the suprachiasmatic nucleus of the rat. A hypothalamic slice about 300 μm thick, including the suprachiasmatic nucleus, was cut coronally using a vibratome. By means of bath- as well as iontophoretic application, c-AMP and c-GMP caused mainly an inhibition and facilitation in suprachiasmatic neuronal activity, respectively. The present results suggest that c-AMP and c-GMP play an important role in regulation of suprachiasmatic neuronal activity as a second messenger and that these two nucleotides may function as a reciprocal messenger.

Effects of cyclic nucleotides, betazole hydrochloride and acetylcholine on pepsinogen secretion from isolated rabbit gastric mucosa.

The effects of dibutyryl cyclic AMP (db-cAMP), dibutyryl cyclic GMP (db-cGMP), betazole hydrochloride (betazole) and acetylcholine (ach.) on pepsinogen secretion from isolated rabbit gastric mucosa were studied using an organ culture system. The 10(-3) M db-cAMP, but not db-cGMP of any concentration, produced a significant enhancement of pepsinogen secretion into the culture medium. In the presence of aminophylline (phosphodiesterase inhibitor), betazole stimulated pepsinogen secretion at concentrations of 10(-8), 10(-6) and 10(-4) M. The 10(-4) M betazole stimulated pepsinogen secretion most strongly (208% of control) and 10(-6) M betazole induced submaximal secretion (137% of control). Ach. stimulated pepsinogen secretion at 10(-8), 10(-6), 10(-4) and 10(-2) M. The peak secretion occurred at 10(-4) M ach. (303% of control). Betazole (with aminophylline) against a background of 10(-6) M ach. (submaximal stimulation dose), produced an intense stimulation of pepsinogen secretion at the concentrations of 10(-8), 10(-6) and 10(-4) M, and the secretion rate expressed as percent of control were 277, 350 and 329%, respectively. These responses were not considered the additive action by betazole and ach. but the potential interaction between the two agents in pepsinogen secretion. From these findings, we conclude that betazole-ach. interdependency exists in in vitro pepsinogen secretion.

Effect of dibutyryl cyclic AMP on morphologic features and biologic markers of a human salivary gland adenocarcinoma cell line in culture.

Dibutyryl cyclic AMP (dB-cAMP) has marked effects on the growth, morphologic features, and biologic markers of a human salivary gland adenocarcinoma cell line in culture. A cell line with ultrastructure and biologic markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of dB-cAMP. Major alterations, such as process formation and expression of myofilaments and oxytocin receptor in addition to myosin and S-100 protein, were observed in those cells with a phenotype similar to myoepithelial cells. Both the anchorage-independent and anchorage-dependent growths were markedly suppressed in the presence of dB-cAMP. After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Effects of dibutyryl cyclic AMP and theophylline on in-vitro development of the secondary palate in the embryonic chick

The avian secondary palate is normally cleft and does not show a rise in intracellular cyclic AMP like that of the fused mammalian palate. The secondary palate of the embryonic chick (HH stages 25–34) was exposed in vitro to dibutyryl cyclic AMP and theophylline to determine whether raising intracellular cyclic-AMP levels would alter medial-epithelial development. Differentiation of the medial epithelium was not altered but there was mucous-cell hyperplasia in nasal epithelium and inhibition of membrane-bone formation in the mesenchyme. Thus the developmental factors that cause the avian palate to be cleft and the mammalian palate to be fused are more complex than differences in intracellular cyclic-AMP levels.

Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function.

Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)

Renin release from kidney cortical slices in response to isoproterenol and glucagon is decreased in vitamin E-deficient rats

The effects of vitamin E (VE)-deficiency on renin release by various agents were examined using rat kidney cortical slices. Isoproterenol and glucagon in the presence or absence of theophylline increased renin release in the control group, while their stimulatory effects were attenuated by VE-deficiency. These decreased responses of renin release to isoproterenol and glucagon due to VE-deficiency were restored to the control level by dietary supplementation of dl-α-tocopheryl acetate or N, N′-diphenyl-p-phenylenediamine. The stimulatory effect of dibutyryl cyclic AMP or theophylline on renin release was not affected by VE-deficiency. These results suggest that in case of VE-deficiency, the response of renin release to stimuli is decreased via cyclic-AMP production.

Dibutyryl-cyclic GMP stimulation of Ca2+ -ATPase activity in rat brain synaptic membranes.

The effects of dibutyryl cyclic AMP (db-cAMP) and dibutyryl cyclic GMP (db-cGMP) were tested on Ca2+-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities in lysed synaptosomes prepared from whole rat brains (minus cerebellum). At concentrations from 0.1 to 2.0 mM, db-cGMP produced a selective, concentration-dependent increase in Ca2+-ATPase activity. Both db-cGMP and db-cAMP slightly reduced Mg2+-ATPase activity, whereas neither compound had concentration-dependent effects on (Ca2+ + Mg2+)-ATPase activity. These findings suggest that the Mg2+-independent, Ca2+-ATPase activity in rat brain is regulated by a cyclic GMP-dependent process. Further, the data provide evidence that the Ca2+-ATPase activity in lysed synaptosomal membranes represents an enzyme that is distinguishable from both the Mg2+ -and (Ca2+ + Mg2+)-ATPase.

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis.

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.