Effect Of Dibutyryl Cyclic AMP Research Articles

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.

EFFECT OF COLFORSIN ON HUMAN NEUTROPHIL SUPEROXIDE PRODUCTION AND INTRACELLULAR CALCIUM MOBILIZATION

1. The effects of dibutyryl cyclic AMP (cAMP), isoproterenol and colforsin (forskolin) were evaluated on respiratory burst in human polymorphonuclear neutrophils (PMN). 2. Dibutyryl cAMP showed a dose-dependent inhibition of n-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide production by human PMN. 3. Administration of isoproterenol induced a dose-dependent inhibition of FMLP-induced superoxide production by human PMN, and the inhibition was blocked by propranolol. 4. Administration of colforsin induced a dose-dependent inhibition of FMLP-induced superoxide production by human PMN, and the inhibition could not be blocked by propranolol. 5. Incubation with colforsin caused a significant increase in the cAMP level in human PMN. 6. Pretreatment with colforsin caused a dose-dependent inhibition in the elevation of intracellular free calcium (monitored by fura-2 fluorescence), which was observed in human PMN stimulated with FMLP. 7. These results suggest that cAMP is an inhibitory factor of superoxide production and intracellular calcium mobilization in human PMN stimulated with FMLP.

Cyclic AMPand cytochalasin B-induced arborization in cultured aortic smooth muscle cells: its cytopharmacological characterization

The present study analyzed effects of dibutyryl cyclic AMP (DB-cAMP) and cytochalasin B (CB) on the morphology of cultured aortic smooth muscle cells (SMC) from rat using phase-contrast microscopy, scanning electron microscopy, and fluorescence staining of actin filaments by the NBD-phallacidin method. The exposure of SMC to each of these agents led to rapid, extensive, and reversible (within 1-2 h of drug withdrawal) changes in their morphology including cytoplasmic arborization (stellation). The latter was preceded by (i) marginal membrane ruffles (DB-cAMP) and (ii) increased zeiotic activity (CB), which were visible within 20 min of the exposure, followed (30-90 min incubation) by a centripetal retraction of the cytoplasm and progressive development of complete or partial arborization. Further, the effects of substances interfering with the assembly-disassembly of microtubules (colchicine, taxol, lidocaine) on DB-cAMP- and CB-induced arborization were studied. None of these agents antagonized CB-induced morphological changes. Colchicine, but not lumicolchicine, taxol, or lidocaine (in a short-term study) prevented DB-cAMP-induced arborization. Taxol added to cell cultures for 24 h promoted DB-cAMP-induced arborization. Both DB-cAMP and CB resulted in the disintegration of actin filaments. The present data suggest that the arborization of cultured aortic SMC is a cytoskeleton-based process involving stabilization of microtubules and disintegration of actin filaments. Our study also suggests that the SMC arborization may represent an in vitro case of SMC stellation found in situ.

Influence of light on cyclic nucleotide metabolism in plants; effect of dibutyryl cyclic nucleotides on chloroplast components

-Dark-grown Phaseolus seedlings exposed to light for 18 hr contain more than × 3 the concentration of cyclic AMP in controls kept in an 18 hr light/6 hr dark cycle. Chloroplast cyclic AMP concentrations behaved similarly. Cyclic AMP phosphodiesterase activity of spinach chloroplasts showed a light-dependent response. The phosphodiesterase activity of chloroplasts from light-grown and dark-grown spinach seedlings exhibit similar V max but different K m values. The K m of the enzyme from light-exposed plants was more than × 30 that of the corresponding activity from dark-treated plants. The two enzymes showed different responses to the combined addition of Ca 2+ and calmodulin; a × 3 stimulation of the enzymic activity from light-treated plants was observed. The light-effects were not directly related to general photosynthetic effects, reflected in protein and chlorophyll concentrations. Light regimes also markedly modified the effects of dibutyryl cyclic AMP and dibutyryl cyclic GMP on terpenoid concentrations. Dibutyryl cyclic AMP doubled the α-tocopherol content and reduced by 40% the γ-tocopherol and chlorophyll concentrations of chloroplasts from light-grown plants; simultaneously their ubiquinone content increased by 48%. The main effect on dark-treated plants was that of dibutyryl cyclic AMP which gave a 107% increase in ubiquinone concentration. The main response to dibutyryl cyclic GMP was seen in dark-grown plants with substantial increases in the concentration of α-tocopherol, γ-tocopherol, and chlorophyll. Plants grown in darkness and then given 15 min illumination showed an 83% increase in α-tocopherol concentration and a 168% increase in ubiquinone concentration following treatment with dibutyryl cyclic GM Under these conditions the cyclic AMP analogue increased chlorophyll and ubiquinone concentrations by 57 and 78%, respectively.

Expression of tubulin, GFAP and of their encoding mRNAs during the proliferation and differentiation of cultured astrocytes

The expression of tubulin, of glial fibrillary acidic protein of their encoding mRNAs were studied in primary cultures of mouse astrocytes. The effects of dibutyryl cyclic AMP on these parameters were also analyzed. (1) The concentration of both α- and β-tubulin chains markedly increased (3-fold) between 7 and 12 days of culture and then remained at the same high value at later stages. In contrast α-tubulin mRNA concentration remained constant throughout the same culture period. (2) Glial fibrillary acidic protein concentration increased steadily reaching a 4-fold higher value at the end of the same culture period. The glial fibrillary acidic protein mRNA increased 3-fold during the first 2 weeks of culture, leveled off at day 18 and then decreased steadily returning to low values (3-fold decrease) at later stages. (3) Addition for 2 days, at day 5 or 19 of the culture, of either dibutyryl cyclic AMP or forskolin remained without effect on tubulin concentration whereas a 50% decrease in α-tubulin mRNA was observed. In the same conditions the effects of these drugs on glial fibrillary acidic protein and its mRNA were not significant. These results suggest that the increase in tubulin concentration seen between days 7 and 12, as well as that of glial fibrillary acidic protein observed during the last weeks of the culture, are not secondary to an increase either of the rate of transcription or of the stability of their encoding mRNAs.

Stimulation of aromatase activity in the fetal rat testis by cyclic AMP and FSH.

The effect of dibutyryl cyclic AMP and FSH on oestrogen biosynthesis was investigated in testes from 18- to 21-day-old fetal rats cultured in vitro in the presence of tritiated testosterone. Oestrone and oestradiol concentrations were measured by determination of constant specific activity after isotopic dilution. Dibutyryl cyclic AMP and FSH markedly stimulated the conversion of testosterone into both oestrone and oestradiol at all stages studied. Oestradiol synthesis was stimulated by two- to sevenfold, while stimulation of oestrone synthesis was even greater. The results demonstrate that the aromatase enzyme system of the fetal rat testis responds to cyclic AMP and FSH.

Effect of dibutyryl cyclic AMP on lipid synthesis in Microsporum gypseum

The influence of intracellular levels of cAMP on the lipid synthesis of Microsporum gypseum has been examined by exogenous supplementation of dibutyryl cAMP and its activators /inhibitors. Incorporation of [ 14C]acetate into various lipid fractions of M. gypseum was markedly enhanced in the presence of dibutyryl cAMP and its modulators, probably as a consequence of increased intracellular cAMP levels, which, in turn, affected the lipid biosynthesis. Increased activities of phosphatidic acid phosphatase, glycerol kinase, ethanolamine kinase and choline kinase in the presence of these additives supports the enhanced synthesis of phospholipids and suggests that lipid biosynthesis is being controlled by cAMP in M. gypseum.

The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation.

The major apoprotein of rabbit pulmonary surfactant is a developmentally and hormonally regulated sialoglycoprotein, Mr congruent to 29,000-36,000 (SP 29-36). In the present study, specific antibodies were used to isolate cloned cDNA inserts for SP 29-36 from a fetal rabbit lung cDNA library in bacteriophage lambda gt11. Two species of cDNA of 1.9 and 3.0 kilobases (kb) in size were isolated that are complementary to two species of mRNA of 2.0 and 3.0 kb which differ primarily in the lengths of their 3'-untranslated regions. The 2.0-kb species of mRNA is approximately 5 times more abundant than the 3.0-kb mRNA. The results of Southern hybridization analysis of rabbit genomic DNA cut with a number of restriction enzymes are indicative that the two mRNA species are encoded by a single gene. The two mRNA species appear to be expressed only in rabbit lung tissue and are coordinately regulated during development in vivo and in vitro; hybridizable SP 29-36 mRNA is first detectable in rabbit lung tissue on day 26 of gestation, increases to a maximum on day 31, and declines somewhat after birth. The 3.0-kb cDNA is comprised of 57 nucleotides of 5'-untranslated region, an open reading frame of 741 nucleotides, and a 3'-untranslated region of 2,165 nucleotides that contains three poly(A)-addition signals. The most 5' of the poly(A) addition signals is utilized in synthesis of the 2.0-kb SP 29-36 mRNA, while the most 3' is utilized in synthesis of the 3.0-kb mRNA. The open reading frame of the 3.0-kb cDNA encodes a protein of 247 amino acids which is highly homologous to the major apoproteins of dog and human pulmonary surfactant. The SP 29-36 cDNAs were utilized to evaluate the effects of dibutyryl cyclic AMP (Bt2cAMP) on the levels of this mRNA in fetal rabbit lung tissue in organ culture. Bt2cAMP caused an induction of SP 29-36 mRNA that was detectable as early as 2 h after its addition to the medium. This inductive effect of Bt2cAMP was blocked when cycloheximide was also present in the medium for greater than or equal to 4 h. Cycloheximide treatment also reduced the levels of SP 29-36 mRNA in control explants; this inhibitory effect on control and Bt2cAMP-treated explants was reversed within 12 h of the removal of cycloheximide from the medium. These findings suggest that a labile protein factor mediates the transcription of the SP 29-36 gene and its induction by Bt2cAMP.

Cardiovascular effects of dibutyryl cyclic AMP in the intact dog heart and the isolated cross-perfused right atrium.

This study used an isolated right atrial preparation, cross-perfused with arterial blood from a support dog. We investigated the effects of dibutyryl cyclic AMP (DBcAMP) on heart rate and arterial blood pressure of the support dog and on the sinus rate and atrial contractile force of the isolated perfused atrium. DBcAMP was injected into the external jugular vein of the support dog or into the sinus node artery of the isolated atrium. DBcAMP to the support dog induced a small increase and/or decrease in arterial blood pressure at a dose of 3 or 10 mg/Kg i.v. It produced a decrease, with or without an increase in blood pressure, at a dose of 30 mg/Kg i.v., and a dose-dependent increase in heart rate in the support dog. In the isolated atrium, positive chronotropic and inotropic effects were observed. Direct injection of DBcAMP (3-30 mg) into the sinus node artery of the isolated atrium induced positive chronotropic and inotropic effects, after initial brief negative effects, in a dose-dependent manner. DBcAMP did not change norepinephrine-induced positive chronotropic and inotropic effects in the isolated atrium. These results demonstrate that DBcAMP induces a decrease in systemic arterial blood pressure with increases in sinus rate and atrial contractile force, and acts additively with the norepinephrine-induced positive cardiac effects in the dog heart.

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.

Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells.

Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells.

Protein kinases induce isoproterenol desensitization of β-adrenoceptor-coupled adenylate cyclase system: significance of receptor occupancy

Treatment of rat reticulocytes with isopreterenol resulted in dose- and time-dependent desensitization of adenylate cyclase to β-adrenoceptor agonist stimulation. Cyclic AMP-dependent protein kinase was possibly involved in this desensitization. Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), also induced desensitization to β-adrenoceptor agonists, indicating the possible involvement of protein kinase C. The addition of a β-adrenoceptor agonist enhanced the desensitizing effect of dibutyryl cyclic AMP or TPA. T 1 2 (time for induction of half maximal desensitization) was decreased from 30 to 2.5 min in accordance with an increase in the concentration of the agonist. The extent of the desensitization was also increased by addition of the agonist. The potency with which the increase was induced was compatible with the potency for binding of the agonist to β-adrenoceptors (K D values). These results suggest that cyclic AMP- or phorbol ester-induced desensitization is enhanced by receptor occupation by β-adrenoceptor agonists.

Alpha 2-adrenergic regulation of arylalkylamine N-acetyltransferase in organ-cultured chick pineal gland: characterization with agonists and modulation of experimentally stimulated enzyme activity.

In the chicken pineal gland, norepinephrine, released at sympathetic nerve endings, plays a role in synchronizing the circadian rhythm of melatonin synthesis. This effect appears to be exerted via an adrenergic inhibition of arylalkylamine N-acetyltransferase, the melatonin rhythm-generating enzyme. The present study indicates that the nighttime peak of N-acetyltransferase activity developed by organ-cultured chick pineal glands is inhibited by adrenergic agonists with a potency order characterizing alpha 2-adrenergic receptors: UK 14,304 greater than clonidine greater than alpha-methylnorepinephrine = epinephrine greater than cirazoline greater than phenylephrine greater than isoproterenol. The mechanism of this alpha 2-adrenergic response was further analyzed in organ cultures, by studying the ability of clonidine to block the cyclic AMP-dependent and the depolarization-dependent stimulations of N-acetyltransferase activity. Clonidine prevented the rise in N-acetyltransferase activity evoked by the adenylate cyclase activators forskolin and cholera toxin or by the phosphodiesterase inhibitor Ro 20,1724. The stimulatory effect of dibutyryl cyclic AMP was also blocked by clonidine. Activation of pineal alpha 2-adrenergic receptors effectively prevented the stimulation of N-acetyltransferase by depolarizing concentrations of KCl. The possibility that the alpha 2-adrenergic effect might be exerted at a step distal to cyclic AMP production is discussed.

The effect of ionophore A23187, verapamil, and dibutyryl cyclic AMP on bile acid synthesis in isolated rat hepatocytes

The effect of the divalent cation ionophore A23187 and the calcium channel-blocker verapamil on bile acid synthesis in isolated hepatocytes in the presence and absence of dibutyryl cyclic AMP was studied. both A23187 (1μM) and verapamil (0.04 mM) caused a small (approximately 15–20%) but consistent decrease in total bile acid synthesis in the cells. When hepatocytes were incubated with dibutyryl cyclic AMP (1mM) production of total bile acid was increased by about 25%, and this effect was unchanged by A23187 but abolished by verapamil. The relative proportions of the individual bile acids prodtaced were not affected by either A23187 or verapamil. Dibutyryl cyclic AMP (1 mM) lowered the ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic + β-muricholic acid formed in the cells by about 50%. Neither A23187 nor verapamil was able to prevent this change. These results suggest that the stimulatory effect of dibutyryl cyclic AMP on total bile acid synthesis is dependent on mobilisation of calcium from intracellular stores, but its effect on the relative proportions of bile acid formed via the cholic acid versus the chenodeoxycholic acid pathway is independent of calcium movement.

Effect of Milrinone on Myocardial Mechanical Function and Cyclic AMP Content in the Fetal Rabbit

The effect of milrinone on mechanical function was studied in the isolated arterially perfused heart of the fetal (28th day of gestation) and newborn rabbits. The inotropic effect of milrinone in the fetus was significantly less than in the newborn. After milrinone infusion, myocardial cyclic AMP levels increased significantly in the two age groups and the fetal values before and after milrinone infusion were not significantly different from the newborn values. In our previous study the inotropic effects of dibutyryl cyclic AMP and high extracellular calcium in the fetus were significantly less than in the newborn. These data suggest that the diminished inotropic effect of milrinone in the fetus may be due, at least in part, to the decreased inotropism of cyclic AMP and calcium.

Regulation of nerve growth factor gene expression in primary brain monolayer cultures: effects of dibutyryl cyclic AMP and sodium butyrate

Primary brain cultures composed of a heterogeneous population of cells, most of which reacted with anti-fibronectin and only few with anti-GFAP antiserum, were found to synthesize a mRNA indistinguishable from mouse submandibular gland nerve growth factor (NGF) mRNA in hybridization characteristics and electrophoretic behavior in agarose gels. The levels of this mRNA were increased 2.5–10 fold by 1 mM dibutyryl cyclic AMP (db-cAMP) and sodium butyrate within 24 h, but not by 10 μM forskolin, and were unaffected by serum in the culture medium. These observations demonstrate that brain cells synthesize a NGF mRNA in primary culture and that the butyrate moiety of db-cAMP enhances NGF gene expression in these cells, probably by a modification of chromatin structure in and around the NGF gene.

Effects of β-adrenoceptor stimulation on intracellular Ca transients and tension in rat ventricular muscle

Effects of beta-adrenoceptor stimulation on intracellular Ca2+ transients and tension were explored in rat ventricular muscles injected with aequorin. Adrenaline (0.05-5.0 microM) and isoproterenol (0.05-1.0 microM) increased the peak of twitch tension and accelerated relaxation. The former effect depended on Ca2+ concentration in Tyrode's solution ([Ca2+]o) and the stage of the experiment. Low concentrations of these drugs added to normal Tyrode's solution containing 2 mM [Ca2+]o did not potentiate twitch tension in the early stage of the experiments. These drugs increased the peak of the aequorin light signal and slightly accelerated the falling phase of the light especially the tail. Effects of dibutyryl-cyclic AMP (DB-cAMP)(0.1-5.0 mM) and 3-isobutyl-1-methylxanthine (IBMX) (0.01-0.5 mM) were qualitatively similar to those of adrenaline and isoproterenol. Isoproterenol applied at the peak of Na-deficient contracture decreased tension without significantly changing the light signal; similar results were obtained in the presence of ryanodine (1 microM). The results were interpreted as follows: The increase of intracellular cAMP induced by beta-adrenoceptor stimulation facilitated Ca2+ uptake by sarcoplasmic reticulum (SR) and decreased Ca2+ sensitivity of contractile elements. Faster relaxation induced by cAMP was considered to be due to the decrease of Ca2+ sensitivity of contractile elements and faster Ca2+ uptake by SR. The slightly faster falling phase of light transient might be due to the faster Ca2+ uptake by SR, which predominates over the slower fall of [Ca2+]i induced by the decreased Ca2+ sensitivity of the contractile element.

Dibutyryl cyclic AMP inhibits transport dependent QO2 in cells isolated from the rabbit medullary ascending limb.

Because the medullary thick ascending limb of the loop of Henle is the target of several polypeptide hormones that stimulate adenyl cyclase in this nephron segment, we examined the effects of cyclic AMP on thick ascending limb of the loop of Henle cells isolated by enzymatic digestion and density gradient centrifugation from the outer medulla of the rabbit kidney. The functional parameter that was measured was transport dependent oxygen consumption. Oxygen consumption was measured using a polarographic oxygen electrode in a constant temperature chamber. We found that dibutyryl cyclic AMP inhibited oxygen consumption in a dose dependent way. Maximal inhibition was observed at a concentration of 10(-5) M. The effect of dibutyryl cyclic AMP was not present in the absence of either sodium, chloride or both implying that its effect is restricted to the sodium and chloride dependent oxygen consumption. The effect of dibutyryl cyclic AMP was additive to that of furosemide 10(-4) M while that of furosemide was not additive to that of cyclic AMP suggesting that the site of action of cyclic AMP is distal to that of furosemide. The effect of dibutyryl cyclic AMP was not additive to that of ouabain and was absent in cells where oxygen consumption was stimulated with amphotericin B in the absence of chloride indicating that it has no effect on Na-K-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of estrogen biosynthesis in human adipose stromal cells. Effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol esters on the synthesis of aromatase cytochrome P-450.

Using human adipose stromal cells in monolayer culture as a model system for study of the regulation of aromatase activity, as well as polyclonal antibodies raised in this laboratory against aromatase cytochrome P-450 (cytochrome P-450AROM), it was found that the rate of synthesis of cytochrome P-450AROM was stimulated by dibutyryl cyclic AMP. This stimulation was attenuated by epidermal growth factor and was potentiated by phorbol esters. These changes in cytochrome P-450AROM synthesis were associated with comparable changes in the levels of translatable cytochrome P-450AROM mRNA, as well as with changes in the activity of aromatase of these cells. By contrast, there was little change in the synthesis of the reductase component of the aromatase enzyme complex in response to these factors. The increase in mRNA was blocked by cycloheximide, indicative of a requirement for protein synthesis in mediating this inductive response. It is concluded that aromatase activity is regulated primarily by changes in the level of mRNA encoding cytochrome P-450AROM, and that such changes are likely a reflection of changes in the rate of transcription of the gene encoding this enzyme. Increases in the levels of cytochrome P-450AROM mRNA are apparently mediated by a regulatory protein(s), similar to that found for other steroidogenic forms of cytochrome P-450.

The effect of dibutyryl cyclic AMP and butyrate on F9 teratocarcinoma cellular retinoic acid-binding protein activity.

F9 teratocarcinoma cells contain a cellular retinoic acid-binding protein (CRABP) that may mediate the retinoic acid-induced differentiation of this cell line. Specific [3H]retinoic acid binding to CRABP in F9 stem cell cytosol is protein-dependent, reaches equilibrium within 4 h at 4 degrees C, and yields 643 +/- 105 fmol of [3H]retinoic acid per mg of protein with an apparent dissociation constant of 9.2 +/- 1.1 nM. When F9 stem cells are grown in the presence of either dibutyryl cyclic AMP or sodium butyrate, CRABP activity is stimulated 2-4-fold. The effect of these drugs on CRABP activity is both time and concentration-dependent, resulting in an increase in the number of binding sites for [3H]retinoic acid with no change in their affinity. The new [3H]retinoic acid-binding sites have a sedimentation coefficient of 2 S and are not displaced by excess retinol. When F9 stem cells are grown in the presence of cyclic 8-bromo-AMP or cholera toxin, no increase in CRABP activity is observed. We conclude that the stimulation of CRABP activity by dibutyryl cyclic AMP may result from the action of butyrate. In addition, the stimulation of retinoic acid-induced F9 cell differentiation by cyclic AMP analogs (Strickland, S., Smith, K.K., and Marotti, K.R. (1980) Cell 21, 347-355) and the inhibition of this differentiation by butyrate (Levine R. A., Campisi, J., Wang, S.-Y., and Gudas, L. J. (1984) Dev. Biol. 105, 443-450) are not correlated with increases or decreases, respectively, in the level of CRABP activity.