The relation of endogenous adenosine cyclic 3':5'-monophosphate to the antagonistic effects of adenosine and colchicine on cell shape.

Adenosine and colchicine have antagonistic effects on cell shape. When Chinese Hamster lung fibroblasts (CHE36-6) or SV40 transformed 3T3 (SV3T3) cells are incubated with colchicine (1 muM) for one hour at 37 degrees C, they round up into spheres with short spikes. Cells treated with adenosine (1 muM-minus 4 mM) for one hour become refractile and develop spindly processes. However, when the two compounds are added simultaneously, the characteristic responses to either drug are abolished and the cells appear normal. The counteraction is specific for adenine and its derivatives, adenosine being the most effective of the compounds we tested. Accumulation of colchicine or adenosine is not altered significantly by the presence of the other drug, ruling out decreases in uptake as the basis of the mutual antagonism. The morphological changes can be observed under conditions where there are no changes in intracellular cAMP levels (such as incubation with low concentrations of adenosine or cordycepin, an adenosine analog that cannot be directly converted to cAMP). Colchicine does not alter cAMP content of control or adenosine-treated cells. These data show that adenine compounds have potent effects on cell shape, and the antagonistic effects of adenosine and colchicine on cell shape are not mediated through changes in intracellular cAMP levels.     

Dual effects of dopamine in rat adrenal glomerulosa cells

The effects of dopamine (DA) on cAMP production and aldosterone secretion were compared in freshly isolated cells and in primary cultures of rat adrenal glomerulosa cells. Under isolated conditions, glomerulosa cells exhibited dopamine receptors from DA-1 and DA-2 subclass, whereas in cultured conditions, where cells are very sensitive to their known stimuli, cells only exhibited dopamine receptors from the DA-1 subclass. Moreover, unlike freshly isolated cells, dopamine stimulated both cAMP production and aldosterone secretion in 3-day cultured preparations. These effects were receptor specific since they were completely suppressed by Scherring 23390 (a specific DA-1 antagonist) and were unaffected by a beta-adrenergic antagonist. As in vivo rat adrenal cortex contains DA, we discuss a possible involvement of this neurotransmitter in the regulation of aldosterone secretion.     

Role of cyclic AMP and ammonia in induction and maintenance of post-aggregative differentiation in a suspension culture of Dictyostelium discoideum

The effects of ammonia and cAMP on prespore and prestalk differentiation of Dictyostelium discoideum were investigated by monitoring eight developmentally regulated proteins as differentiation markers under the shake culture conditions in glucose/albumin medium. In the medium containing cAMP, cells form small agglomerates and undergo prespore differentiation [19]. Under the conditions where agglomeration was prevented, ammonia induced four marker proteins out of eight tested in the presence of cAMP, which included not only a prespore specific enzyme but also cell-type non-specific proteins. No inhibitory effect of ammonia was observed in presumptive cell differentiation. These results suggest that ammonia is an inducer of differentiation at the protein level as well as the mRNA level as found previously [24]. The effects of cAMP were examined with special attention to the difference between induction of differentiation and maintenance of differentiated state in this specific medium. The induction of differentiation from early aggregative cells was cAMP-dependent with all the marker proteins tested. This agrees with the observations so far obtained in other culture systems. However, when already differentiated cell masses (slugs) were dissociated and shaken in this specific medium, only two enzymes required cAMP to maintain the activity while five out of eight kinds of the proteins continued to be expressed as in undisturbed slugs even without cAMP. This suggests that for the maintenance of the differentiated state after slug disaggregation cAMP may not be required with respect to the majority of proteins, if cells are provided with some favorable conditions such as glucose/albumin medium.     

Interactions between insulin and the cyclic AMP system of Cloudman S91 mouse melanoma cells

Insulin inhibits the proliferation of wild-type Cloudman S91 mouse melanoma cells. The effects, which are mediated through specific, high-affinity receptors for insulin, appear to involve interactions with the cAMP system. Our evidence is as follows: (1) Cloudman cells have a cAMP requirement for proliferation and pigmentation. Exposure of cells to insulin results in a lowering of intracellular cAMP levels and inhibition of both cell division and pigment formation. (2) The effects of insulin are reversed by agents which raise cAMP levels, or by the cAMP analogue dibutyryl cAMP. (3) A mutant cell line with a temperature-dependent requirement for cAMP is most sensitive to the growth inhibitory effects of insulin when its requirements for cAMP are maximal. (4) Mutants selected only for alterations in their response to Insulin frequently have concomitant alterations in their cAMP systems. (5) The melanotropin-responsive adenylate cyclase system is stimulated following prolonged exposure of cells in culture to insulin. Although we do not know the mechanism(s) for the interactions between the insulin and the cAMP system, our initial findings suggest that protein phosphorylation/dephosphorylation reactions are involved.     

Frequency encoding of pulsatile signals of cAMP based on receptor desensitization in Dictyostelium cells

Dictyostelium discoideum amoebae represent a prototype for the study of periodic signaling in intercellular communication. These cells synthesize cAMP in response to cAMP pulses. Cell responsiveness in Dictyostelium can be characterized by the capability to generate a large number of significant responses to cAMP signals in a given amount of time. The existence of a frequency of pulsatile cAMP signals yielding maximum responsiveness is demonstrated by analysis of a realistic model for cAMP synthesis, based on receptor desensitization. The optimal frequency of stimulation closely depends on the kinetics of receptor desensitization and resensitization in target cells. Synthesis of cAMP is determined both in conditions where cells are not excitable and in conditions where they relay suprathreshold pulses of cAMP. Moreover, the effect of the stimulus waveform is investigated, and several measures of cell responsiveness are compared. The results provide an explanation for the effectiveness of cAMP pulses delivered at 5 min intervals, and for the failure of pulses delivered at 2 min intervals, in inducing slime mold development. Besides applying to intercellular communication in Dictyostelium, the present analysis bears on patterns of pulsatile signaling observed for hormones and growth factors. In all these cases, it appears that pulsatile signals can be encoded in terms of their frequency on the basis of desensitization in target cells.     

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.     

Anti-proliferative effects of 8-chloro-cAMP and other cAMP analogs are unrelated to their effects on protein kinase A regulatory subunit expression

Conflicting reports have attributed 8-chloro-cAMP (Cl-cAMP)-mediated inhibition of tumor cell growth to either a toxic 8-chloro-adenosine (Cl-AdR) breakdown product or a Cl-cAMP-mediated decrease in ratio of Type I to Type II regulatory (R) subunits of protein kinase A (PKA). Using the MCF-7 human breast cancer and S49 mouse lymphoma cell lines as models, we show that the effects of Cl-cAMP and other cAMP analogs on growth and R subunit expression are unrelated. MCF-7 cell growth was insensitive to most analogs and inducers of cAMP, but was potently inhibited by Cl-cAMP acting through uptake and phosphorylation of its Cl-AdR breakdown product. Possible roles of adenosine receptors or P(2) purinoceptors in these Cl-cAMP-mediated growth effects were ruled out by studies with agonists and antagonists. Cholera toxin markedly decreased the ratio of Type I to Type II R subunits in MCF-7 cells without affecting growth, while growth inhibitory concentrations of Cl-cAMP or Cl-AdR had insignificant effects on this ratio. In S49 cells, where PKA activation is known to inhibit cell growth, PKA-deficient mutants retained sensitivity to both Cl-cAMP and the related 8-bromo-cAMP. Adenosine kinase (AK)-deficient S49 cells were inhibited only by higher concentrations of these 8-halogenated cAMP analogs. Of the commonly used cAMP analogs, only 8-(4-chlorophenylthio)-cAMP acted purely as a cyclic nucleotide-having no effect on PKA-deficient cells, but strongly inhibiting both wild-type and AK-deficient cells. Where growth inhibitory concentrations of most cAMP analogs reduced RI expression in the AK-deficient mutant, a functionally equivalent concentration of (N(6), O(2'))dibutyryl-cAMP maintained or increased this expression.     

cAMP-induced modulation of the growth yield of Saccharomyces cerevisiae during respiratory and respiro-fermentative metabolism

The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism.     

cAMP-induced modulation of the growth yield of Saccharomyces cerevisiae during respiratory and respiro-fermentative metabolism

The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism.     

Ca++ antagonists distinguish different requirements for cAMP-mediated gene expression in the cellular slime mold, Dictyostelium discoideum

Cyclic AMP is essential for the accumulation of many prespore mRNAs and can advance the time of appearance of mRNAs specifically enriched in prestalk cells. Additionally, when late-developing cells are washed free of cAMP, a number of growth phase mRNAs reaccumulate. This reaccumulation can be suppressed by cAMP. These effects of cAMP are all mediated through the cell surface cAMP receptor and can occur under conditions where the receptor-associated adenylate cyclase is inactive, indicating that the initial intracellular transduction event necessary for expression of these mRNAs does not depend upon cAMP synthesis. The dihydropyridine derivatives, nifedipine and nitrendipine, are highly specific Ca++ channel blockers. They are shown here to prevent the influx of Ca++ from the external medium that occurs in response to cAMP binding to the cell surface receptor during development. These two compounds as well as another Ca++ antagonist, 8-N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate (TMB-8) and a calmodulin inhibitor, N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W7), all specifically decrease cAMP-mediated prespore mRNA accumulation in a dose-dependent manner. They also prevent cAMP from suppressing the expression of the growth phase genes. The growth phase mRNAs reaccumulate in cAMP-treated cells in the presence of increasing concentrations of these drugs. By contrast, cAMP induction of the pre-stalk-enriched mRNA is not as significantly affected by these agents. These results raise the possibility that the cell surface cAMP receptor can couple to different signal transduction systems and thereby induce or suppress the expression of different sets of cAMP-regulated genes during development.     

New roles for DIF? Effects on early development in Dictyostelium

The DIFs are unusual, chlorinated molecules which induce stalk cell differentiation during the later, multicellular phase of Dictyostelium development. Here we provide evidence that one or more DIFs have a role during early development, when small amounts are known to be made. Initial indications came from an optical technique which detects changes in shape or cohesion of cells in suspension (Gerisch and Hess, PNAS 71, 2118, 1974). After a period of optical inactivity at the start of development, cell suspensions normally produce spontaneous spike-shaped light-scattering oscillations synchronised by oscillations in extracellular cAMP levels, followed by sinusoidal oscillations where the synchroniser is not known. DIFs 1 and 2 produce optical responses from cells at all these early stages of development. The phase of both spiked and sinusoidal oscillations can be shifted, indicating an effect on the oscillator in each case. We find further: (1) cAMP oscillations and cAMP relay during spiked oscillations are transiently inhibited by DIF-1. (2) DIF-1 causes a transient decrease in cellular cGMP levels in cells taken before oscillations commence and likewise inhibits the cGMP response to a cAMP stimulus in cells taken later in development. Cytoskeletal organization and hence cell shape might be affected by DIF-1 by this indirect route. (3) The effects of DIF-1 are transient, even though it is essentially stable in the cell suspension. Cells somehow adapt to DIF-1. (4) The effects are chemically specific: DIF-1 and DIF-2 are active at 10(-7) to 10(-8) M, with DIF-2 being the more active, whereas related compounds have little or no activity at 10(-6) M. These results indicate that cells are responsive to DIFs 1 and 2 from the start of development and suggest a wider role for the DIFs. This role might involve effects on cAMP signalling and on intracellular second messengers.     

cAMP has distinct acute and chronic effects on aquaporin-5 in lung epithelial cells

Aquaporin-5 (AQP5) is present on the apical membrane of epithelial cells in various secretory glands as well as on the apical membrane of the airway epithelium, airway submucosal glands, and type 1 pneumocytes, where it can participate in respiratory tract water homeostasis. We examined the effects of cAMP on AQP5 distribution and abundance. When AQP5-expressing mouse lung epithelial cells were treated with cAMP or the beta-adrenergic agonist terbutaline, a biphasic AQP5 response was observed. Short term (minutes) exposure to cAMP produced internalization of AQP5 off of the membrane and a decrease in protein abundance. Both of these responses were blocked by inhibition of protein kinase A and the decrease in abundance was blocked by chloroquine, indicating lysosome-mediated degradation. Sustained cAMP exposure (hours) produced an increase in membrane localization and increased abundance; these effects were also blocked by protein kinase A inhibition. The beta-adrenergic agonist terbutaline produced changes in AQP5 abundance in mouse trachea and lung, consistent with our findings in cultured epithelial cells. Purified AQP5 protein was phosphorylated by protein kinase A but not protein kinase C or casein kinase II, and aquaporin-5 was phosphorylated in cultured cells after long term (but not short term) exposure to cAMP. These studies indicate that cAMP and beta-adrenergic agonists produce distinct short and long term effects on AQP5 distribution and abundance that may contribute to regulation of lung water homeostasis.     

Inhibition of choriogonadotropin-activated steroidogenesis in cultured Leydig tumor cells by the Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.     

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.     

Induction of metabolic changes and down regulation of bovine parathyroid hormone-responsive adenylate cyclase are dissociable in isolated osteoclastic and osteoblastic bone cells.

Bovine parathyroid hormone (PTH), dibutyryl cAMP, and calcium each induce similar metabolic changes in isolated bone cells. PTH and calcium, but not dibutyryl cAMP, result in desensitization of osteoclastic and osteoblastic bone cells to PTH. In osteoblastic cells, calcium effects are specific for PTH receptor.adenylate cyclase complexes and responsiveness to other hormones is not reduced while in osteoclastic cells, small effects of high calcium on prostaglandin E1- and epinephrine-inducible cAMP accompany the large decreases seen in cAMP response to PTH. The membrane effects of calcium and of PTH appear to be independently regulated as PTH-induced desensitization can be initiated in the absence of calcium. In addition, calcium effects on PTH-sensitive adenylate cyclase follow a different calcium dose-response than PTH-like metabolic changes. These results suggest that the effect of calcium on the membrane is not directly related to its induction of PTH-like metabolic changes. A possible role of calcium as an in vivo regulator of bone cell sensitivity to PTH is discussed.     

Protein Kinase A-Dependent and -Independent Signaling Pathways Contribute to Cyclic AMP-Stimulated Proliferation

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.     

The role of butyrate in the reverse transformation reaction in mammalian cells.

The reverse transformation reaction of Chinese hamster ovary cells from compact, epithelial-like, randomly growing, heavily knobbed, lectin reactive cells into stretched, tighly adherent, smooth-surfaced, lectin resistant, fibroblast-like cells normally elicited by dibutyryl cAMP can be produced to its complete extent by N6-monobutyryl cAMP or 8-bromo-cAMP, O2'-monobutyryl cAMP is ineffective as is cAMP itself in the absence of an inhibitor of phosphodiesterase activity. In the presence of a phosphodiesterase inhibitor, cAMP is fully effective. These results indicate that the role of the butyryl groups of dibutyryl cAMP and, especially, the N6-butyryl, in the reverse transformation reaction is protection of the cAMP analogue from degradation. Butyrate at concentrations of about 1 mM does produce a response which to some extent mimics that of cAMP analogues. The cells, however, fail to assume a fibroblastic-like shape, but rather become flattened. The butyrate effect is much slower and less readily reversible than that evoked by cAMP analogues. Butyrate produces an approximately 2-fold increase in intracellular cAMP levels. These results are consistent with the hypothesis that butyrate effects, in part, are mediated by AMP.