Cyclic AMP-induced cytolysis in S49 cells: selection of an unresponsive "deathless" mutant.

Wild-type S49 lymphoma cells respond to cyclic adenosine 3', 5'-monophosphate (cAMP) by inducing cAMP phosphodiesterase, halting growth in the G1 phase of the cell cycle and subsequently dying. By using a counter selection procedure, we have isolated a new class of mutants of S49 cells termed "deathless" that are resistant to cytolysis, but otherwise respond like the wild-type cells to cAMP. Upon removal of the cyclic nucleotide, D-cells resume their normal growth. Unlike all other cAMP-resistant mutants of S49 cells isolated until now, the D- mutant has a functionally normal cAMP-dependent protein kinase and retains normal ability to induce phosphodiesterase and arrest cell growth in G1. It is probable that the altered gene product of the D- mutant is distal to protein kinase and in a biochemical pathway separate from that of cAMP induction of phosphodiesterase or growth arrest. The D- mutant may facilitate studies of the mechanism of cAMP-induced cytolysis and growth regulation in S49 cells.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

Voltage sensor movement and cAMP binding allosterically regulate an inherently voltage-independent closed-open transition in HCN channels

The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4-S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed-open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.     

Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms

It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.     

Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels.

The activation of cyclic nucleotide-gated (CNG) channels is a complex process comprising the initial ligand binding and a consecutive allosteric transition from a closed to an open configuration. The cone and olfactory CNG channels differ considerably in cyclic nucleotide affinity and efficacy. In each channel, the cyclic nucleotide-binding site is connected to the last transmembrane segment of the channel by a linker peptide (C-linker) of approximately 90 amino acids. Here we report that replacement of three amino acids in the cone C-linker by the corresponding amino acids of the olfactory channel (I439V, D481A and D494S) profoundly enhanced the cAMP efficacy and increased the affinities for cAMP and cGMP. Unlike the wild-type cone channel, the mutated channel exhibited similar single-channel kinetics for both cGMP and cAMP, explaining the increase in cAMP efficacy. We thus conclude that the identified amino acids are major determinants of channel gating.     

Elevation of cyclic AMP activates an actin-dependent contraction in teleost retinal rods

Agents which elevate cyclic AMP (cAMP) cause teleost retinal rods to contract. We have characterized this cAMP effect and have evaluated the role of the cytoskeleton in cyclic nucleotide-induced contraction, using actin and microtubule inhibitors. The necklike myoid region of the rod contracts in the dark and elongates in the light. If long, light-adapted rods are cultured with cAMP analogs and IBMX, rods contract to their short dark-adapted position. Cyclic nucleotide- induced rod contraction occurs in constant light, requires a phosphodiesterase inhibitor, and is specific to cAMP (db cyclic GMP, 8- bromocyclic GMP, 5'AMP, and adenosine have no effect on rod myoid length). Cyclic AMP effects on rod length are consistent with observations from several species that cAMP levels are higher in dark- adapted than in light-adapted retinas. Since rod myoids contain paraxially aligned actin filaments and microtubules, we have used the motility inhibitors cytochalasin D and cold and nocodazole to investigate the roles of these cytoskeletal elements in rod contraction. Cyclic nucleotide-induced contraction is not inhibited when myoid microtubules are disrupted with cold and nocodazole treatments, but contraction is blocked if myoid actin filaments are disrupted with cytochalasin D. Thus, we conclude that actin filaments, but not microtubules, are required for rod contraction. We propose that rod contraction in vivo is triggered by a rise of cytoplasmic cAMP at onset of darkness and that this contraction is mediated by an actin- dependent mechanism.     

In vitro transformation of androgen receptor from murine skeletal muscle by cAMP

Sedimentation constants and DNA-cellulose-binding of cytosolic androgen receptor from murine skeletal muscle were determined in presence of cyclic nucleotides. Without cAMP, two testosterone-binding fractions of similar amount at 4-5S and 8-9S were obtained. With 3 microM cAMP the receptor sedimented predominantly at 4-5S. Binding of testosterone-receptor-complexes to DNA-cellulose was enhanced by increasing cAMP-concentrations and reached a maximum at 20-90 nM cAMP depending on the DNA-concentrations in the test. A similar DNA-binding characteristic was obtained after partial purification of the receptor by affinity chromatography. cGMP had no effect. We conclude that the androgen receptor is transformed in vitro by cAMP.     

A mechanistic and kinetic analysis of the interactions of the diastereoisomers of adenosine 3'',5''-(cyclic)phosphorothioate with purified cyclic AMP-dependent protein kinase.

The binding affinities of the diastereoisomers of adenosine 3',5'-(cyclic)phosphorothioate, Sp-cAMP[S] and Rp-cAMP[S], for the cyclic AMP- (cAMP-)binding sites on purified and reconstituted pig heart type II cAMP-dependent protein kinase holoenzyme were determined by measuring the ability of these compounds to displace [3H]cAMP from this enzyme. Sp-cAMP[S], a cAMP agonist, displaced 50% of the [3H]cAMP bound to the holoenzyme at a concentration 10-fold higher than that of cAMP; Rp-cAMP[S], a cAMP antagonist, required a 100-fold higher concentration relative to cAMP. Activation of the isolated holoenzyme, determined as phosphotransferase activity, was measured in the presence of the agonist and in the absence and in the presence of increasing concentrations of the antagonist. The results of fitting the activation data to sigmoid curves with a non-linear-regression program and to Hill plots by using a linear-regression program showed that Rp-cAMP[S] had no effect on Vmax, increased the EC50 values for agonist activation and had no effect on the co-operativity of activation (h). A Ki value of 11 microM was determined for Rp-cAMP[S] inhibition of cAMP-induced activation of purified type II cAMP-dependent protein kinase. Electrophoresis of the holoenzyme on polyacrylamide gels under non-denaturing conditions in the presence of saturating concentrations of the diastereoisomers resulted in 100% dissociation of the subunits with Sp-cAMP[S] and 0% dissociation with Rp-cAMP[S]. Sp-cAMP[S], the isomer with an axial exocyclic sulphur atom, binds to the holoenzyme, releases the catalytic subunit and activates the phosphotransferase activity. Rp-cAMP[S], the isomer with an equatorial exocyclic sulphur atom, binds to the holoenzyme but does not result in dissociation, and thus acts as a competitive inhibitor of phosphotransferase activity.     

Expression of early developmental genes in Dictyostelium discoideum is initiated during exponential growth by an autocrine-dependent mechanism.

Throughout growth, Dictyostelium cells continuously produce an autocrine factor, PSF, that accumulates in proportion to cell density. Production of PSF declines rapidly when cells are shifted to starvation conditions, and the properties of PSF are distinct from those of regulatory factors produced by starving cells. During late exponential growth, PSF induces expression of several early developmental genes, including those for proteins important in cAMP signaling and cell aggregation. Examples are the aggregation stage cAMP receptor (cAR1), the aggregation-specific form of cyclic nucleotide phosphodiesterase, and gp24 (contact sites B). Through PSF, growing cells detect environmental conditions (cell number high, food approaching depletion) that are appropriate for production of the gene products needed to initiate aggregation and development.     

Interleukin 1 and cyclic AMP induce kappa immunoglobulin light-chain expression via activation of an NF-kappa B-like DNA-binding protein.

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.     

The annexin 2-S100A10 complex and its association with TRPV6 is regulated by cAMP/PKA/CnA in airway and gut epithelia

The formation of a heterotetrameric complex between annexin 2 (anx 2) and S100A10 plays an important role in regulating the cellular distribution and biochemical properties of anx 2. A major distinction between the anx 2-S100A10 complex and other annexin-S100 complexes is that S100A10 binding to anx 2 occurs independently of calcium. Here we describe a cyclic 3',5'-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA, EC 2.7.1.37)-dependent mechanism regulating anx 2-S100A10 complex formation and its interaction with the transient receptor potential vanilloid type 6 channel (TRPV6) in airway and gut epithelia. In both 16HBE14o- and Caco-2 cells, forskolin (FSK) stimulated increased anx 2-S100A10 complex formation, which was attenuated by either PKA inhibitors or calcineurin A (CnA) inhibitors. The anx 2-S100A10 complex association with TRPV6 was dependent on FSK-induced CnA-dependent dephosphorylation of anx 2. Analysis of the significance of the cAMP/PKA/CnA pathway on calcium influx showed that both PKA and CnA inhibitors attenuated Ca(45) uptake in Caco-2, but not 16HBE14o-, cells. Thus, the cAMP/PKA/CnA-induced anx 2-S100A10/TRPV6 complex may require additional factors for calcium influx or play a role independent of calcium influx in airway epithelia. In conclusion, our data demonstrates that cAMP/PKA/CnA signalling is important for anx 2-S100A10 complex formation and interaction with target molecules in both absorptive and secretory epithelia.     

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.     

A variant of S49 mouse lymphoma cells with enhanced secretion of cyclic AMP.

A novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP-dependent protein kinase. The variant is also resistant to N6-monobutyryl cAMP but is sensitive to killing by 8-bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2'-butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.     

Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor.

Activating mutations of the thyroid-stimulating hormone receptor (TSHr) have been identified as a cause of toxic adenomas. Germline-inactivating TSHr mutations have been described as a cause of congenital hypothyroidism. The effects of combining activating and inactivating mutations within a single receptor was studied. The double mutant T477I/P639S contained an activating TSHr mutation (P639S) together with an inactivating one (T477I). The other one (I486M/P639S) contained two activating mutations. Constructs were expressed in COS-7 cells and basal and TSH-stimulated cyclic AMP (cAMP) accumulation and inositol phosphate (IP) production were determined. The expression at the cell surface was studied both with binding and fluorescence-activated cell scanning analysis. Our results show that the effect of combining the two activating mutations is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha. Surprisingly the double mutant containing the T477I behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways. These data show that an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the membrane surface when combined with an activated form of the receptor.     

Metabotropic Glutamate Receptor (mGluR)-Mediated Potentiation of Cyclic AMP Responses Does Not Require Phosphoinositide Hydrolysis: Mediation by a Group II-Like mGluR

Abstract: Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to G_s. It has been suggested that mGluR-mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR-mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1 S ,3 R -ACPD-stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1 S ,3 R -ACPD induced a cAMP response that was greater than additive. Finally, ( RS )-3,5-dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2 S ,1' R ,2' R ,3' R )-2-(2,3-dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR-mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.     

PKA from Saccharomyces cerevisiae can be activated by cyclic AMP and cyclic GMP.

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP in 3 x 10(-6) M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.     

Reduction of adenine nucleotide content of clone 4 MDCK cells: effects on multiplication, protein synthesis, and morphology

The antitumor agent hadacidin (N-formyl-hydroxyamino-acetic acid), at 4 mM, inhibited the multiplication of clone 4 Madin Darby canine kidney (MDCK) cells within 24 hr. Growth resumed rapidly upon replacement of hadacidin with aspartate, an observation consistent with the drug's action as a competitive inhibitor of adenylosuccinate synthetase, an enzyme in adenine nucleotide biosynthesis. Data indicate that the drug-treated cells were arrested in S phase of the cell cycle. Accompanying inhibition of multiplication was a 16-fold increase in the area occupied by the cells and a refractoriness to release by treatment with trypsin. None of these changes occurred when 0.5 mM adenosine was included in the incubation mixture containing the inhibitor. Hadacidin decreased the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) content of the cells as well as the rate at which 3H-leucine was incorporated into protein. In the presence of 1 mM dibutyryl cAMP and theophylline, the drug had no effect on cell division and protein synthesis. The data suggest that, in clone 4 MDCK cells, the effects of hadacidin are mediated by diminishing the level of cAMP.