Effect of delta 9-tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetrahymena

Cyclic nucleotide levels were determined in division-synchronized Tetrahymena and the effect of delta 9-tetrahydrocannabinol (THC) on the cyclic nucleotide levels was studied. In non-drug-treated division-synchronized cells, there was no statistically significant variation in the level of cAMP and cGMP during the G2 period, preceding the first division. During the free running cell cycle (the interval of time between the first and second synchronous division) the twofold increase in the level of cAMP was statistically significant; however the variation in the level of cGMP was not statistically significant. THC caused a lowering of cAMP and cGMP levels throughout the 4-experimental treatment. The suppression of cAMP and cGMP levels altered the cyclic nucleotide pattern of the cell cycle. The cAMP pattern was changed particularly in the G2 period preceding the first synchronous division, and immediately after division during the free running cell cycle. THC treatment caused division delays of approximately 8-15 min in the onset of the first and second synchronous division. However, the duration of the free running cell cycle (110-120 min) was unchanged. The suppression of cyclic nucleotide levels resulting from THC treatment is discussed in relation to delays in the division schedule.     

Post-translational modifications of the regulatory subunits of cAMP-dependent protein kinases during G1/S progression

During the G1/S transition of the cell cycle variations in the labelling by 8-N3-[32P]cAMP of the protein kinase A regulatory subunits RI and RII, used as a probe to monitor post-translational modifications that may regulate cAMP binding, were observed in synchronized HeLa cells. A decrease in 8-N3-[32P]cAMP labelling of RI, RII and RII phosphorylated by the catalytic subunit of PKA was correlated with the increased percentage of cells in phases G1. An increase in 8-N3-[32P]cAMP incorporated into the 54-kDa RII subunit during progression from G1 to S was correlated with an increase in intracellular cAMP. A transient increase in Mn-SOD activity was detected in cells arrested at the G1/S transition using two different techniques, suggesting that oxidative modulation of regulatory subunits by free radicals may modify cAMP binding sites during the cell cycle. Decreased photoaffinity labelling by 8-N3-[32P]cAMP of RI, RII and autophosphorylated RII subunits was found to be an inherent characteristic of PKA in the G1/S transition.     

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.     

Adenosine 3'':5''-monophosphate content and actions in the division cycle of synchronized HeLa cells

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.     

Phosphorylation of ribosomal proteins during the cell cycle of Physarum polycephalum

Physarum polycephalum has been used as a model system to study the phosphorylation of ribosomal proteins during the cell cycle. The results showed that the phosphate content of S3, the major ribosomal phosphoprotein in this organism, was constant during all phases of the cell cycle. No additional ribosomal phosphoproteins were observed. These results differ significantly from those reported earlier by Rupp, R.G., Humphrey, R.M. and Shaeffer, J.R. (Biochim. Biophys. Acta (1976) 418, 81-92) and suggest that the use of thymidine or hydroxyurea to synchronize cell population may affect the phosphorylation of ribosomal proteins. The results are discussed in relation to protein synthesis and cAMP level during the cell cycle.     

The effect of trifluoroperazine on cAMP levels in the rat myocardium during various phases of heart cycle

Trifluoperazine, a calmodulin inhibitor, has been studied for its effect on cAMP content in rat heart tissue at various cardiac cycle stages with beta-adrenoreceptors stimulation by izadrine. When trifluoperazine is absent, the cAMP level is found to be decreased at the beginning of the cardiac cycle in comparison with the middle of the cardiac cycle. Trifluoperazine (6 X 10(-6) M) induces an increase of the content of cyclic nucleotides at the beginning of the cardiac cycle and has no effect on its level in the middle of the cardiac cycle. This leads to a decrease of the difference between cAMP concentrations in the defined cardiac cycle regions. The calmodulin-dependent reactions might be expected to play an important role in the regulation of the myocardium cAMP concentration change during the cardiac cycle.     

Circadian clock regulation of the bimodal rhythm of cyclic AMP in wild-type Euglena

Cell-cycle traverse is associated with fluctuations in the cellular content of cAMP; artificial alterations of these levels phase-shift cell division in free-running cultures of achlorophyllous Euglena maintained in constant darkness (DD). The phase shifts observed, however, are only transient: the cell division rhythm rephases to that of unperturbed controls. This implies that the second messenger functions downstream of the circadian oscillator. Further, the level of cAMP is known to indicate carbon nutrient status and the competency of cells to traverse various restriction points in the cell cycle of other eukaryotes. We wished to determine the profile of cAMP content in free-running, dividing and non-dividing cultures of green, wild-type cells, which survive well during prolonged growth arrest. We monitored cAMP content in photoautothropic cultures of E. gracilis (strain Z) at 25 degrees C under either an entraining light-dark cycle comprising 12 h of light and 12 h of darkness (LD:12,12) or free-running (LD:1/2,1/2) regimes. cAMP content in rhythmically dividing, light-phased or free-running cells exhibited bimodality [peaks at CT (circadian time) 9-14 and CT 19-22). Expression of cAMP content on a per milligram total cellular protein basis caused the day trough (CT 1-3) to be even more distinct. Non-dividing, free-running, photoautotrophic cultures displayed a similarly phased bimodality in cAMP content. These findings in wild-type Euglena confirm that the bimodal rhythm of cAMP content is regulated by the circadian oscillator that underlies division rhythmicity but is not dependent on the cell division cycle. We will now determine the effect of the fluctuating cAMP levels on the phosphorylation status and activity of cell-cycle regulatory proteins.     

Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain uncompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPlb expression. Moreover, although these agents used alone did not affect GPllb/llla expression, they markedly reversed phorbol ester-induced GPllb/llla expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPlb and GPllb/llla expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis. © 1995 Wiley-Liss, Inc.     

Cross-talk between the autocrine (mitogenic) pheromone loop of the ciliate Euplotes raikovi and the intracellular cyclic AMP concentration

Cell type-specific protein signals, called pheromones, are constitutively secreted by Euplotes raikovi and bound back in autocrine fashion, with a positive effect on the vegetative (mitotic) cell growth. In cells growing suspended with their secreted pheromone, it was found that any interruption of this autocrine signaling loop was immediately followed by an effective enhancement of the basal intracellular cyclic AMP (cAMP) level. To establish a cause-effect relationship between these pheromone-induced variations in the cytoplasmic cAMP level and cell growth, cells ready to pass from a resting stage to a new growth cycle were conditioned either to incorporate a cAMP analog resistant to phosphodiesterase degradation, or to utilize cAMP released (following cell irradiation) from incorporated "caged" cAMP. Cells responded at every induced increase in their basal cAMP level by markedly decreasing their commitment to start a new growth cycle. It was deduced that the autocrine signaling of E. raikovi pheromones involves cAMP as inhibitor of its mitogenic activity.     

Anoxia-induced changes in cAMP contents in the cat cerebral cortex]

Effect of the cessation of oxygen supply on cAMP content and neuronal spike activity (NSA) in the cortex brain was studied. The interruption of oxygen supply during in first decades of seconds evoked changes in the pattern of NSA, followed with the decrease of cAMP content (to 56 +/- 10%). Then the phase of neuronal hyperactivity and increase of cAMP level (to 198 +/- 26%) took place. The content of cAMP approximated the basal one in 2.5 min anoxia. Anoxia during 5 min resulted in direct opposite shifts of cAMP content in two groups of cats (an increase up to 223 +/- 11%, and decrease up to 75 +/- 8%, respectively, which correlated with individual features of NSA recovery in postanoxic period and values of cAMP basal level in the cortex of different animals. Upon 30 min reoxygenation after 2.5 min anoxia a decline of the content of cAMP (to 63 +/- 12%) accompanied enhance of NSA. This period of reoxygenation after 5 min anoxia demonstrated two types of reactions, observed in different groups of cats: the first type--NSA tended to normalization with the level of cAMP 44 +/- 8% below basal level, and the second type--insufficient recovery of NSA attended by value of cAMP 90 +/- 13% above basal level.     

Changes in the composition of membrane phospholipids during the cell cycle of Escherichia coli

Phospholipid concentrations have been estimated throughout the successive cell cycle in synchronously growing culture of E. coli B/r. Total phospholipid phosphorus was shown to be doubled in the period of time between two cell divisions, whereas during the division itself it did not change. Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) exhibit a stepwise increase during the cell cycle. It should be noted that the phase of accumulation of these lipids could shift depending on the duration of the cell cycle. The fall in level of PE was followed by a short-term increase (5-10 min). At the same time the level of cardiolipin was observed to be significantly increased.     

Inhibition by vanadate of cyclic AMP production in rat corpora lutea incubated in vitro

Vanadate, a normal constituent of cells, has been reported to affect a variety of enzymes involved in phosphate transfer; the findings regarding adenylate cycle vary with the tissue and experimental system. In the corpus luteum, cyclic AMP (cAMP) stimulates steroidogenesis; and prostaglandin F2 alpha, which induces luteal regression, inhibits luteinizing hormone (LH)-induced cAMP accumulation. We examined the influence of orthovanadate on cAMP concentration in isolated corpora lutea from pseudopregnant rats. With 2 mM vanadate, basal cAMP level was unaffected, but LH-induced cAMP accumulation was inhibited by 45-68%. Lower doses of vanadate (0.2-1 mM) were almost as effective. When added simultaneously with LH, vanadate was inhibitory within 25 min, but no inhibition occurred when vanadate was added for 30 min to tissue pretreated with LH for 60 min. The decrease in cAMP accumulation was observed also when corpora lutea were exposed to vanadate in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM), indicating that vanadate inhibits cAMP synthesis. Vanadate may increase cytosolic calcium by inhibiting ion pumps in cell membranes. Thus, we examined the effect of vanadate in corpora lutea incubated in calcium-depleted medium and found that vanadate still inhibited cAMP formation. Vanadyl sulfate (0.4 and 2 mM) reduced the LH-induced cAMP accumulation as effectively as vanadate. Thus, the use of vanadate as a tool for exploring physiological regulators of luteal adenylate cyclase should be considered.     

Rebound increase of basal cAMP level in NG108-15 cells during chronic morphine treatment: effects of naloxone and chloramphenicol

The effects of morphine on the basal cAMP level in the neuroblastoma X glioma NG108-15 hybrid cell line have been studied. Morphine (10 microM) added to the incubation media at hr 0 caused a rapid and significant decrease in the cAMP level up to hr 1; the level then slowly returned to the control at hr 6, and gradually increased to its peak at hr 36, returning to the control at hr 60. These results provide the first evidence for a delayed rebound increase of cAMP during morphine treatment. Naloxone (10 microM) added at hr 0 concomitantly with morphine blocked the morphine-induced decrease in cAMP level at hr 1 and attenuated its increase at hr 36. However, when naloxone was added at hr 5.5, the cAMP level significantly increased at hr 6, and at hr 36 the cAMP level increase was the same as in the case of morphine alone. Furthermore when naloxone was added 0.5 hr prior to harvesting the cells at hr 6, 12, 24 and 36, the cAMP level showed an immediate increase at each time point up to about the same level as observed with morphine alone at hr 36. Chloramphenicol, a protein synthesis inhibitor (100 microM) itself caused little or no change in the cAMP level. Added 30 min before morphine, chloramphenicol decreased the morphine-induced rebound increase at hr 36 in a concentration-dependent manner without any significant effect on cAMP decrease at hr 1. However when chloramphenicol was added at hr 5.5, the morphine-induced rebound increase at hr 36 was also attenuated, thereby suggesting an involvement of macromolecular synthesis in the rebound increase of cAMP which may be used as a model for the development of morphine dependence.     

The relationship of cAMP levels and the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides to the HeLa cell cycle.

Cellular cAMP levels as well as the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides were determined in synchronized cultures of HeLa cells after pulse-labelling with [¹⁴C]adenine. The cAMP levels were found to be maximal in G 1 and minimal in G 2 and mitosis, as previously reported by others. The rate of labelling of the cAMP pools, however, was found to be maximal in G 2 and decreased to a minimum in G 1. This suggests that the rate of cAMP synthesis is highest when pool level is lowest and vice versa. A comparison of cAMP levels and the rate of 5′AMP pool labelling throughout the HeLa cell cycle indicated an inverse relationship. Such a relationship emphasizes the role of the cyclic 3′,5′-phosphodiesterase activity during the cell cycle. The kinetics of pool labelling of IMP, ATP, and hypoxanthine throughout the cell cycle suggested that the adenylate energy charge fluctuated as a function of the cell cycle. The apparent activation of the adenylate cyclase during G 2 and mitosis as reflected by the increased rate of cAMP pool labelling suggests that the super phosphorylation of H 1 histone during G 2-mitotic transition may be mediated by cAMP-dependent phosphokinases.     

The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in human endometrial glandular epithelial cells

The effects of relaxin (RLX), forskolin (Fk), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro, a phophodeisterase inhibitor) on the accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in human endometrial glandular epithelial cells were studied. Epithelial glands were isolated from the endometrium by digesting the viable tissue fragments with collagenase. The epithelial glands were incubated with Ro, RLX, and Fk separately or in combination. The amount of cAMP was determined at the end of incubation. A moderate increase in cAMP content was observed in epithelial glands incubated with Ro alone. Accumulation of cAMP after incubation with RLX was observed only in the presence of Ro. Increase of cAMP content in response to RLX and Ro was time- and dose-dependent. The accumulation of cAMP was apparent in 5 min, reached the maximum after 15 min, and remained elevated for 17 h incubation. One nanogram per millileter RLX was effective to increase the cAMP content, with a maximal response at 100 ng/ml. The effect of Ro and the combined effect of Ro and RLX on cAMP accumulation were studied in epithelial glands of 20 endometrial specimens obtained during different stages of the menstrual cycle. When epithelial glands were incubated with Ro alone, the cAMP concentration in glands from proliferative endometria was 120 +/- 67 pmol/mg protein (n = 6, means +/- SD), significantly higher than that of secretory endometria, 42 +/- 37 (n = 14, p = 0.007). RLX and Ro caused an additional increase of cAMP accumulation, 2- to greater than 10-fold increase over the sample incubated with Ro alone. There was no significant difference between proliferative and secretory phases (500 +/- 410, n = 6, and 470 +/- 300, n = 14, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP regulates vegetative growth and cell cycle in <Emphasis Type="Italic">Candida albicans</Emphasis>

We demonstrate here the regulatory role of cAMP in cell cycle of Candida albicans. cAMP was found to be a positive signal for growth and morphogenesis. Phosphodiesterase inhibitor aminophylline exhibited significant effects, i.e., increased growth, as well as induced morphogenesis. Atropine and trifluoperazine negatively regulated (inhibited) growth and did not induce morphogenesis. These changes were attributed to increase in cAMP levels and protein kinase A (PKA) activity in presence of aminophylline, while reduction was observed in atropine and trifluoperazine (TFP) grown cells. Alteration in cAMP signaling pathway affected the cell cycle progression in Candida albicans. Increased cAMP levels in aminophylline grown cells reduced the duration of cell cycle by inciting the cell cycle-specific expression of G1 cyclins (CLN1 and CLN2). However atropine and trifluoperazine delayed the expression of G1 cyclins and hence prolonged the cell cycle. Implication of cAMP signaling pathway in both the cell cycle and morphogenesis further opened the channels to explore the potential of this pathway to serve as a target for development of new antifungal drugs.     

Cell cycle control by Ca2+ in Saccharomyces cerevisiae

We established an experimental system suitable for study of cell cycle regulation by Ca2+ in the yeast Saccharomyces cerevisiae. Systematic cell cycle analysis using media containing various concentrations of Ca2+, a Ca2(+)-ionophore (A23187), and a Ca2(+)-chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) revealed that simultaneous addition of 10 microM A23187 and 10 mM EGTA to cells growing in a Ca2(+)-deficient medium at 22 degrees C caused rapid decrease in intracellular Ca content and resulted in transient G1 arrest followed by block mostly at G2/M, as revealed by flow cytometry. Recovery from G1 arrest was not due to coordinated initiation of DNA synthesis and bud emergence: unbudded cells with S or G2/M DNA were observed. Examination of terminal phenotype suggested that Ca2+ was required at all the stages of the cell cycle except for the initiation of DNA synthesis. The intracellular cAMP level decreased within 10 min of addition of A23187 and EGTA. No significant transient G1 arrest was observed in cells incubated with 8-Br-cAMP, or RAS2val19 and delta bcy1 mutants, which produce a high level of cAMP and have constitutively activated cAMP-dependent protein kinase, respectively. These results indicate that Ca2+ is essential for cell cycle progression and suggest that Ca2+ may regulate the cAMP level. This system will be useful for genetic and molecular studies on cell cycle events regulated by Ca2+.     

The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue.

The intracellular level of cGMP was independent of the rate of cell division in cells derived from virally infected brain tissue. The phosphodiesterase inhibitor R07-2956 (4-dimethoxybenzyl-2-imidazolidinone) increased the intracellular level of cGMP in virally infected brain cells, but it did not effect the level of cAMP. There was no correction between the increase in cGMP levels following addition of R07-2956 and changes in mitotic activity in the brain cell cultures. Experimental manipulations which increased the cAMP level were accompanied by a decreased mitotic rate indicating there was a correlation between mitotic activity and the level of cAMP in the same cells. Raising the intracellular level of cAMP by exogenous db-cAMP or cAMP or the use of other phosphodiesterase inhibitors routinely increased the level of cGMP as well. Conversely increasing the intracellular cGMP level by adding the exogenous cGMP increased the level of both cGMP and cAMP.A tissue culture system was used with the cell line derived from viral infected human brain tissue originally obtained from a patient with subacute sclerosing panencephalitis (SSPE). The intracellular levels of cAMP and cGMP were monitored by radioimmunoassay following manipulation of the system by addition of exogenous cGMP (0.05 mM), addition of exogenous db-cAMP (0.5 mM), or cAMP (0.5 mM) and the use of phosphodiesterase inhibitors: theophylline (1.0 mM), papaverine (50 μg/ml), 4-3-butoxy-4-methoxy benzyl-2-imidozalidinone (R020-1724) and R07-2956. Cell division was monitored in treated and non-treated cultures at 24 h intervals by analyzing the cell number and mitotic index.High levels of cGMP were found in cells which were not actively dividing but high levels were just as apt to be present in dividing cells. There was an inverse relationship between cell division and the level of cAMP.     

Regulation of Melatonin Production by Pineal Photoreceptor Cells: Role of Cyclic Nucleotides in the Trout (Oncorhynchus mykiss)

Abstract: The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N -acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.     

Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.