Inhibitory effects of dibutyryl cyclic AMP and theophylline on the melanosome transformation in the embryonic chick pigmented retina cultured in vitro.

When the retinal pigment epithelial cells of chick embryo are cultured in monolayer conditions, the pigment granules are lost from the cytoplasm. The first structural change in depigmentation is the transformation of pigment granules into the degradative organelles designated as the dense body and melanosome complex. The cells are grown in medium containing DBcAMP of various doses from 10^?5 to 10^?2M. Cell proliferation is retarded by treatment with DBcAMP (10^?3M). The transformation of pigment granules is almost completely prevented in all 1-day cultured cells. In 5-day cultured cells continuously treated with more than 10^?4M, the transformation is not only prevented, but the synthesis of pigment granules is stimulated. A similar result is obtained by the administration of 10^?3M theophylline. 5′-AMP does not prevent the transformation of pigment granules but seems to stimulate the synthesis of pigment granules. On the other hand, cGMP is ineffective both on prevention of transformation and on synthesis of pigment granules. The mechanisms of the transformation of pigment granules are discussed.     

Compartive behavioral effects of dibutyryl cyclic AMP and prostaglandin E1 in mice.

Three behavioral tests, spontaneous locomotor activity (SLMA), exploratory behavior (EB) and rotarod performance (RP), a measure of neuromuscular coordination, were used to stuey the interaction of PGE1 (1 mg/kg i.p., 10 min. pretreatment) with DBcAMP (25 mg/kg i.p., 25 min. pretreatment) in mice. A dose-response relationship of PGE1 (0.01-5.0 mg/kg) to SLMA was determined, with a significant decrease in SLMA produced by a dose of 0.1 mg/kg. decreases in SLMA were produced by PGE1 (79%), DBcAMP (41%) and DBcAMP-PGE1 combination (71%). Similar decreases in EB were observed. Although no significant difference between controls and DBcAMP was observed in RP, 52% of mice tested were RP failures following PGE1 and a 100% failure rate was induced by the combination. Mice were treated with a second injection of DBcAMP or PGE1 or the combination 24 hr following the first injection. Behavioral activity of these mice was observed 25 min (DBcAMP) or 10 min (PGE1) after the second dose was administered. A second injection of DBcAMP failed to decrease SLMA and EB from controls; moreover, SLMA began to return towards control levels as early as 2 hr between injections. The second injection of PGE1 or DBcAMP+PGE1 produced the same behavior as that produced by the first injection. On the basis of these results, the relationship of cyclic nucleotides and PGs to behavioral activity is discussed.     

Trophoblast estrogen synthetase stimulation by dibutyryl cyclic AMP and theophylline: increase in cytochrome P-450 content.

The inclusion of both dibutyryl cyclic AMP and theophylline in the culture medium of human malignant trophoblast cells (JAr line) for 72 hours results in an enhanced estrogen secretion through the increased specific activity of estrogen synthetase (aromatase), a cytochrome P-450 mono-oxygenase enzyme system. The data described here suggest that this increased aromatase activity is due to an increase in the concentration of only one component of the mono-oxygenase system, cytochrome P-450.     

Effect of dibutyryl cyclic AMP and theophylline on lipoprotein lipase secretion by human monocyte-derived macrophages

The effect of dibutyryl cyclic AMP and theophylline on lipoprotein lipase secretion was investigated after a 24 h pretreatment of human monocyte-derived macrophages. Both the effectors decreased in a dose-dependent manner the enzyme activity recovered in the culture medium. The decrease in lipoprotein lipase activity appeared to be related to reduced enzyme synthesis without apparent modification of its stability and half-life and was conversely associated with an increase of lysosomal acid hydrolase activities. This effect was reversible on removal of the nucleotide. The present findings suggest that cyclic AMP may play a role in lipoprotein lipase expression in human macrophages and therefore may participate in the regulation of lipoprotein uptake by these cells, which are strongly implicated in the atherogenic process.     

Glucose synthesis by isolated guinea-pig hepatocytes. Effect of cyclic AMP and dibutyryl cyclic AMP.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Differential effects of testosterone and dibutyryl cyclic AMP on the meiotic maturation of mouse oocytes in vitro

Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Inhibition by cyclic AMP and dibutyryl cyclic AMP of transport of organic acids in kidney cortex.

1. Cyclic adenosine 3',5'-monophosphate and N-6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate decrease the initial entry rate and the steady-state uptake of p-aminohippurate and uric acid by rabbit kidney cortex slices. 2. N-6-2'-O-Dibutyryl adenosine 3'-5'-monophosphate inhibits the tubular transport of p-aminohippurate competitively. 3. Isoproterenol, known to increase cyclic nucleotide concentration of the cortical tubules by activation of adenyl cyclase, decreases p-aminohippurate transport. Antidiuretic hormone which is known to stimulate only medullary adenyl cyclase has no effect on p-amino-hippurate uptake by cortical slices. 4. Theophylline, which inhibits cyclic nucleotide phosphodiesterase and, therefore, enhances the cellular accumulation of endogenous cyclic nucleotide, depresses p-aminohippurate transport.     

Effect in vivo of beta-adrenergic stimulation, angiotensin II, dibutyryl cyclic AMP, and theophylline on tonin concentration in rat saliva and submaxillary gland.

Tonin (an enzyme present in rat submaxillary gland and saliva) has previously been shown to be able, unlike renin and reninlike substances, to release angiotensin II either directly by acting on an appropriate substrate or from angiotensin I. The administration of a beta-adrenergic drug, isoproterenol, produces a rise of tonin concentration in saliva without affecting its concentration in the submaxillary gland. Prior administration of a beta blocker, propranolol, partially prevents this effect. The administration of theophylline increases the tonin concentration in both saliva and the submaxillary gland, whereas dibutyryl cyclic AMP increases tonin concentration in the former. These results suggest that beta-adrenergic stimulation enhances both tonin release into the saliva and tonin synthesis in the submaxillary gland, and that these effects might be mediated by cyclic AMP. Infusion of angiotensin II blocked the stimulatory effect of isoproterenol on salivary tonin. 1Sar-8Ile-angiotensin II is both a weak antagonist of angiotensin II in this respect and a strong agonist in terms of blocking the effect of isoproterenol another role mirrored in other physiological mechanisms of derivatives of angiotensin II.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

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.