Biphasic effects of dibutyryl cyclic adenosine 3',5'-monophosphate on synergistic stimulation of DNA synthesis by diacylglycerol, and the ionophore A23187 in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetylglycerol (OAG) and the ionophore A23187 for 8 h, [3H]-thymidine incorporation into the acid-insoluble fraction of the cells was stimulated synergistically. Further addition of dibutyryl cAMP caused a biphasic effect on the synergistic stimulation. Dibutyryl cAMP augmented the synergistic stimulation when A23187 was at the concentration of 0.075 micrograms/ml, but inhibited it when the ionophore was at 0.25 micrograms/ml. At the higher concentration of A23187, dibutyryl cAMP stimulated the [3H]thymidine incorporation when culture was for 4 h, but inhibited it when culture was for 8 h. The results were the same when 12-0-tetradecanoylphorbol-13-acetate (TPA) was used instead of OAG. Butyrate could replace dibutyryl cAMP for stimulation of [3H]thymidine incorporation in combination with TPA and A23187, but not with OAG and A23187 at the lower ionophore concentration. Dibutyryl cAMP but not butyrate stimulated ornithine decarboxylase induction caused by TPA and A23187. These results suggest that the effect of dibutyryl cAMP on DNA synthesis induced by OAG and A23187 was biphasic and depended on the concentration of A23187 and on the time of culture, and that the stimulation mechanism of butyrate is different from that of dibutyryl cAMP.     

Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells

In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.     

Differential Regulation by Butyrate and Dibutyryl Cyclic AMP of δ-Opioid, α2-Adrenergic, and Muscarinic Cholinergic Receptors in NCB-20 Cells

Long-term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the specific binding of [3H]D-Ala2,D-Leu5 enkephalin. This increase was concentration and time dependent, with an EC50 of about 480 microM and a maximal effect detected after 3-day treatment. At saturating concentration of butyrate (1 mM) the increase was three- to fourfold of the untreated control. Scatchard analysis revealed that the butyrate effect was due to an increase in the density of the opioid receptor binding sites. Butyrate also induced a smaller (about twofold) increase in the density of muscarinic cholinergic receptor binding assessed by using [3H]quinuclidinyl benzilate, whereas alpha 2-adrenergic receptor binding assessed by using [3H]clonidine was not significantly affected. The butyrate-induced opioid receptor binding could be totally abolished by the presence of cycloheximide, suggesting that the butyrate effect involves synthesis of the receptor protein. Butyrate treatment did not affect basal and prostaglandin E1-stimulated cyclic AMP levels but caused a three- to fourfold decrease in the IC50 of D-Ala2,D-Leu5 enkephalin for attenuating these cyclic AMP levels and approximately 25% increase in the maximal extent of attenuation. In contrast to the butyrate effect, long-term treatment of NCB-20 cells with 1 mM dibutyryl cyclic AMP induced an 80% decrease in the opioid and alpha 2-adrenergic receptor bindings and a 57% loss of muscarinic cholinergic receptor binding. This down-regulation of muscarinic cholinergic receptor binding sites was associated with a 35% decrease of carbachol-induced phosphoinositide breakdown, whereas the receptor up-regulation induced by butyrate was found to increase the carbachol response by about threefold. The differential regulation by butyrate and dibutyryl cyclic AMP suggests that the butyrate effect is mediated by a mechanism independent of intracellular cyclic AMP. The induction by butyrate of opioid-receptors and muscarinic cholinergic receptors in NCB-20 cells may provide a useful system for studying the regulation of gene expression of these receptor proteins.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture

Parathyroid hormone (PTH) greatly increased the level of adenosine 3', 5' cyclic monophosphate (cAMP) in rabbit costal chondrocytes in culture 2 minutes after its addition. PTH, as well as N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP) induced ornithine decarboxylase (ODC; L-ornithine carboxylyase; EC 4.1.1.17), which reached a maximum 4 hours after their addition. Neither cAMP, N6 O2' dibutyryl guanosine 3', 5' cyclic monophosphate (DBcGMP), nor sodium butyrate increased the activity of the enzyme. PTH had no effect on DNA synthesis, while DBcAMP and 8 Br-cAMP decreased DNA synthesis. Expression of the differentiated phenotype of chondrocytes in culture was also induced by PTH, DBcAMP, and 8 Br-cAMP, but not by cAMP, DBcGMP, or sodium butyrate, as judged by morphological change. Glycosaminoglycan synthesis, a characteristic of the cartilage phenotype, began to increase 8 hours after addition of PTH or DBcAMP, reaching a plateau 32 hours after their addition. These findings suggest that PTH induces increase of ODC activity and expression of the differentiated phenotype of chondrocytes through increase of cAMP and that induction of OCD is closely related to expression of the differentiated phenotype of chondrocytes.     

Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon

Bicarbonate and butyrate stimulate electroneutral Na absorption via apical membrane Na-H exchange (NHE) in rat distal colon. cAMP downregulates NHE-3 isoform and inhibits HCO3-dependent, but not butyrate-dependent, Na absorption. This study sought to determine whether 1) the apical membrane NHE-2 and NHE-3 isoforms differentially mediated HCO3- and butyrate-dependent Na absorption, and 2) cAMP had different effects on NHE-2 and NHE-3 isoforms. The effect of specific inhibitors of NHE-2 and NHE-3 isoforms (50 microM HOE 694 and 2 microM S3226, respectively) on unidirectional 22Na transepithelial fluxes performed across isolated mucosa from rat distal colon under voltage-clamp conditions was examined. HCO3 stimulation of Na absorption was inhibited by EIPA, a nonspecific inhibitor of all NHE isoforms, by S3226 and dibutyryl cAMP but not by HOE 694. In contrast, butyrate stimulation of Na absorption was not altered by dibutyryl cAMP and was not inhibited by HOE 694 in the absence of dibutyryl cAMP, but in the presence of dibutyryl cAMP was HOE694 sensitive. In contrast, S3226 inhibited butyrate-stimulated Na absorption in the absence of dibutyryl cAMP, but not in its presence. We conclude that 1) HCO3-stimulated Na absorption is mediated solely by NHE-3 isoform, whereas butyrate-stimulated Na absorption is mediated by either NHE-3 or NHE-2 isoform, and 2) dibutyryl cAMP selectively inhibits NHE-3 isoform but stimulates NHE-2 isoform. Dibutyryl cAMP does not inhibit butyrate-stimulated Na absorption as a result of its differential effects on NHE-2 and NHE-3 isoforms.     

Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.     

The effect of cyclic AMP on the growth and morphology of a normal human fibroblast parent strain and its transformed progeny line.

A normal human diploid fibroblast cell strain, Lederle 130 (Led 130), and its virus-transformed progeny line, transformed Led 130, were subjected to 0.75 and 1.5 mM concentrations of adenosine-5'-monophosphate (AMP), cyclic AMP (cAMP) and dibutyryl cyclic AMP (Bt2cAMP). While cAMP was markedly inhibitory to neoplastic cells at 1.5 mM, Bt2-cAMP was even more effective at this concentration, producing 85% inhibition by 4 days and 91% inhibition by 6 days. Bt2-cAMP was the only nucleotide to reverse morphological transformation effects in the neoplastic fibroblasts. Normal fibroblasts were inhibited in growth rate to a comparable extent by all nucleotides, and were not altered morphologically.     

Modulation of in vitro erythropoiesis: enhancement of erythroid colony growth by cyclic nucleotides.

Mammalian erythropoiesis, as assayed by erythroid colony formation in vitro, is enhanced by cyclic adenosine nucleotides and agents which are capable of raising intracellular cyclic AMP (cAMP) levels. With canine marrow cells as target, this enhancement was shown to be specific for cAMP and its mono- and dibutyryl derivatives. Adenosine and its derivatives, such as AMP, ADP and ATP, and other cyclic nucleotides, such as cGMP, dibutyryl-cGMP, cCMP and cIMP and sodium butyrate were inactive. The phosphodiesterase inhibitor, RO-20-1724, and the adenyl cyclase stimulator, cholera enterotoxin, both markedly increased colony numbers. Studies with tritiated thymidine showed that about 50% of the cells responding to either erythropoietin (ESF) or dibutyryl cAMP (db-cAMP) were in DNA synthesis. However, by unit gravity sedimentation velocity analysis, the peak of ESF-responsive colony forming cells sedimented more rapidly (8-7 +/- 0-2 mm/hr) than the peak of db-cAMP-responsive cells (7-5 +/- 0 mm/hr). These results demonstrate that adenyl cyclase-linked mechanisms influence in vitro erythropoietic proliferation and suggest that other hormones and simple molecules might interact with surface receptors and thus modulate the action of ESF at the cellular level.     

Butyrate inhibits the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.     

Cyclic AMP regulation of lactate dehydrogenase. Isoproterenol and N6,O2'-dibutyryl cyclic AMP increase the levels of lactate dehydrogenase-5 isozyme and its messenger RNA in rat C6 glioma cells

The mechanism of isoproterenol and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) induction of lactate dehydrogenase (EC 1.1.1.27) was investigated in the C6 rat glioma cell line. [3H]Leucine-labeled lactate dehydrogenase in noninduced and induced cells was quantitatively immunoprecipitated with rabbit anti-rat lactate dehydrogenase-5 antiserum. The immunoprecipitates were analyzed for 3H-labeled lactate dehydrogenase by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and isoelectrofocusing. Using this technique, it was shown that isoproterenol + 3-isobutyl-1-methylxanthine and dibutyryl cAMP cause an increase of the [3H]leucine incorporation into glioma cell lactate dehydrogenase. Analysis of the kinetics of induction and deinduction revealed no change in the rate of degradation of lactate dehydrogenase in the presence and absence of inducing agent, indicating that the induction was due to an increase in the rate of synthesis of the enzyme. The increased rate of synthesis was prevented by actinomycin D. Isoproterenol + 3-isobutyl-1-methylxanthine increased only the specific rate of synthesis of lactate dehydrogenase-5 isozyme and of the M subunit. The mechanism was further studied by assaying the level of functional mRNA coding for lactate dehydrogenase in a reticulocyte cell-free protein-synthesizing system using glioma cell poly(A)-containing RNA isolated from either isoproterenol or dibutyryl cAMP-induced cells. Analysis of the immunoprecipitated translation product by isoelectrofocusing revealed that isoproterenol or dibutyryl cAMP produced an approximately 8-fold stimulation of the poly(A) + RNA-directed synthesis of the lactate dehydrogenase M subunit. These data demonstrate that isoproterenol and dibutyryl cAMP control the level of functionally active lactate dehydrogenase mRNA in glioma cells which, in turn, determines the extent of synthesis of the lactate dehydrogenase M subunit.     

Comparison of the Effects of Forskolin and Dibutyryl Cyclic AMP in Neuroblastoma Cells: Evidence that Some of the Actions of Dibutyryl Cyclic AMP Are Mediated by Butyrate

We have compared the effects of forskolin, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP, Bt2-cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2-cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose-dependent inhibition of cell proliferation, but the effect of Bt2-cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2-cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2-cAMP than in untreated cells or in forskolin-treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2-cAMP produced histone acetylation, a well-known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2-cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1(0), a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1(0) in the cells treated with Bt2-cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2-cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.     

Alteration in gene expression at the onset of human Y-79 retinoblastoma cell differentiation

We have tested the hypothesis that differentiation and growth arrest of Y-79 human retinoblastoma cells in culture is associated with a modification of gene expression. We first examined proteins translated from mRNAs isolated from Y-79 cells growing in suspension and in attachment cultures in serum-containing medium and found them to be markedly different. This suggests that membrane-substrate interactions are of major consequence in the biochemical differentiation of these cells. Secondly, we examined the patterns of proteins translated from attached cells which had been induced to morphologically differentiate into neuronal-like and glial-like cells by serum-withdrawal and dibutyryl cAMP treatment respectively. The in vitro translatable proteins of mRNAs isolated from these cultures were found to be markedly different from those of the suspension and attachment cultures. Thirdly, we found that treatment of cells growing in attachment culture in serum-containing medium supplemented with 8-bromo cAMP, butyrate and retinoic acid as well as dibutyryl cAMP resulted in discreet alterations in proteins translated in vitro from extracted mRNAs. Although all these substances inhibit the growth of Y-79 cells, only dibutyryl cAMP and butyrate result in morphological differentiation of cells. Our results suggest that (1) attachment and morphological differentiation of Y-79 cells are both related to specific alterations in gene expression and (2) differentiation and inhibition of cell growth by various agents can be correlated with changes in translatable mRNA species although all agents do not act in the same mode.     

MODULATION OF IN VITRO ERYTHROPOIESIS: ENHANCEMENT OF ERYTHROID COLONY GROWTH BY CYCLIC NUCLEOTIDES

Mammalian erythropoiesis, as assayed by erythroid colony formation in vitro, is enhanced by cyclic adenosine nucleotides and agents which are capable of raising intracellular cyclic AMP (cAMP) levels. With canine marrow cells as target, this enhancement was shown to be specific for cAMP and its mono- and dibutyryl derivatives. Adenosine and its derivatives, such as AMP, ADP and ATP, and other cyclic nucleotides, such as cGMP, dibutyryl-cGMP, cCMP and cIMP and sodium butyrate were inactive. The phosphodiesterase inhibitor, RO-20-1724, and the adenyl cyclase stimulator, cholera enterotoxin, both markedly increased colony numbers. Studies with tritiated thymidine showed that about 50% of the cells responding to either erythropoietin (ESF) or dibutyryl cAMP (db-cAMP) were in DNA synthesis. However, by unit gravity sedimentation velocity analysis, the peak of ESF-responsive colony forming cells sedimented more rapidly (8.7 ± 0.2 mm/hr) than the peak of db-cAMP-responsive cells (7.5 ± 0 mm/hr). These results demonstrate that adenyl cyclase-linked mechanisms influence in vitro erythropoietic proliferation and suggest that other hormones and simple molecules might interact with surface receptors and thus modulate the action of ESF at the cellular level.     

Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.     

Effect of cAMP on ciliary function in rabbit tracheal epithelial cells

To study the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on respiratory ciliary activity, we measured ciliary beat frequency (CBF) of rabbit tracheal epithelium by a photoelectric method in response to cAMP analogues and agents that can increase endogenous cAMP production. Addition of 8-bromo-cAMP dose dependently enhanced CBF, with the maximal increase and the concentration necessary to produce a half-maximal response (KD) being 31.0 +/- 3.4% (SE) (P less than 0.001) and 3.2 +/- 1.5 x 10(-7) M, respectively. Other structurally dissimilar cAMP analogues dibutyryl cAMP and chlorophenylthio-cAMP likewise caused increases in CBF. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the adenylate cyclase stimulator forskolin also augmented CBF in a dose-dependent fashion and were accompanied by the increases in intracellular concentrations of cAMP. Ciliary discoordination was not observed in any of the experiments. These results suggest that cAMP may accelerate mucociliary clearance through the activation of ciliary motility and that intracellular cAMP levels appear to be an important determinant for the lung mucociliary transport functions.