Down regulation of c-myc, c-fos and erb-B during estrogen induced proliferation of the chick oviduct

Oncogenes c-myc, H-ras, c-fos and erb-B were constitutively expressed in immature chick oviduct withdrawn from estrogen administration for 2.5 weeks after 10 d of primary estrogen stimulation. Following secondary estrogen stimulation of the withdrawn chicks, synthesis of egg white proteins is rapidly induced and remaining non-functioning tubular gland cells are stimulated to proliferate with a doubling time of 24 h. During first 12 h of secondary estrogen stimulation, H-ras mRNA levels doubled and did not increase further at 24 h and 48 h. The steady state levels of c-myc, erb-B and c-fos mRNA decreased 24 h following secondary estrogen stimulation. The levels of these oncogene RNAs in oviduct were similar at 48 h following secondary estrogen stimulation to those from immature chicks administered 10 d of primary estrogen stimulation. Thus elevated expression of c-myc and c-fos mRNA does not appear to be necessary components for sustained estrogen induced cell proliferation in the chick oviduct.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.     

Changes in cyclic nucleotide levels during embryonic development of chick hearts

The effects of isoproterenol, acetylcholine (Ach), and adenosine, on cyclic AMP (cAMP) and cyclic GMP (cGMP) contents were examined in chick hearts at various stages of embryonic development. The basal cAMP content was highest (87.7 +/- 1.3 pmol/mg protein) in young (3-day) embryonic chick hearts and decreased during development (9.6 +/- 0.6 pmol/mg protein in 9-19-day-old hearts). On the other hand, the cGMP content was lowest (45.5 +/- 2.3 fmol/mg protein) in young (3-day) embryonic chick hearts and increased during development (338 +/- 15.0 fmol/mg protein in 14-19-day-old hearts). Iso increased the cAMP concentration in embryonic hearts at all ages. Ach and Ado had no effect on the cAMP content at all ages. However, the Isoproterenol-induced stimulation of cAMP was inhibited by Ach and Adenosine at all ages. In young embryonic hearts, Ach and Ado increased cGMP concentration only slightly, whereas these agents caused a substantial increase in cGMP concentration in the older hearts. Thus, there was a clear age difference in the effects of Ach and Adenosine on the cGMP and cAMP concentrations. Nitroprusside and hydrogen peroxide increased cGMP concentration in older hearts (greater than 5-day-old) but not in the 3-day-old embryonic hearts. Thus, guanylate cyclase activity may be low in young (3-day-old) hearts. It summary, the cGMP level is very low in young embryonic chick hearts, and increases markedly during development. The changes in cGMP are reciprocal to those of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

Avidin induction by dibutyryl cyclic guanosine 3',5'-monophosphate in chick oviduct organ culture

A progesterone-dependent protein, avidin, was induced by dibutyryl cGMP in a dose-dependent manner in chick oviduct organ culture. A four-fold concentration of avidin over the control samples was observed with a concentration of dibutyryl cGMP of 1 mM. The time course of avidin accumulation was essentially similar to that caused by progesterone. Dibutyryl cAMP at the same concentration had no effect on the avidin concentration in the cultured oviduct tissue or medium. These results suggest a possible role of cGMP in avidin induction or in the modulation of avidin synthesis.     

Elevated cyclic GMP concentrations in rat adrenal cortex after dexamethasone administration.

The levels of cGMP and cAMP were measured in the fasciculata-reticularis zona of the adrenal cortex in both intact and hypophysectomized young adult male rats. After administration of a single dose of dexamethasone to intact rats, cGMP levels were elevated 2–4 fold after 4hr and returned to control level after 8hr. At the same time, cAMP concentrations were moderately lowered. In hypophysectomized rats dexamethasone administration was followed by a similar increase in the cGMP level, the basal cAMP concentrations were not altered by dexamethasone. Our data suggest that dexamethasone might have a direct effect on cGMP concentration in the adrenal cortex of the rat.     

Biochemical changes in progressive muscular dystrophy. XII. Cyclic nucleotides in lymphoid and nonlymphoid organs of dystrophic mice

Concentrations of cAMP and cGMP were measured (per milligram DNA) in the lymphoid (thymus, spleen) and nonlymphoid organs (liver, brain, kidney, lungs, heart, pancreas, skeletal muscle, lens) of normal (+/+) and dystrophic (dy/dy) 129 ReJ mice aged 30, 60, and 90 days. The cAMP concentrations in the thymus did not reveal any significant differences at 30 and 60 days of dystrophy, but were considerably higher (2-fold) at 90 days. cGMP concentrations were decreased in the thymus at 30 days (0.20-fold) and markedly elevated at 60 (2-fold) and 90 days (3-fold) of the disease. The [cAMP]/[cGMP] ratio was increased (1.30-fold) at 30 days of dystrophy, and this was followed by a sharp decline at 60 days (2-fold), with a lesser decrease at 90 days (0.34-fold). In the spleen, the cAMP concentrations were augmented significantly in all stages of dystrophy (1.5- to 2.6-fold). cGMP (per milligram DNA) did not show any significant variation at 30 and 60 days of the disease but was increased (3-fold) at 90 days. The [cAMP]/[cGMP] ratio, which was enhanced in the spleen at 30 (2-fold) and 60 days (1.5-fold), demonstrated no change at 90 days of dystrophy. These results indicated significant differences in the concentration of cyclic nucleotides and their ratios in the thymus and spleen of 129 ReJ dy/dy mice. The modifications were not limited to lymphoid organs alone, having been noted in the nonlymphoid organs as well. These changes could, in turn, influence immune responsiveness and could cause immunodepression in dystrophic mice.     

Effect of lectins on the levels of cAMP and cGMP in guinea pig lymphocytes: early responses of lymph node cells to mitogenic and non-mitogenic lectins.

Guinea pig lymph node lymphocytes were reacted with two mitogenic lectins (LcL-A or PHA) and two nonmitogenic lectins (AbL or WGA)m and the levels of cAMP and cGMP were determined during 30 min of culture. Various concentrations of LcL-A and PHA caused modest (1.5 to 2-fold) rises in cAMP levels. However, the rise in cAMP did not correlate with the degree of mitogenicity. AbL caused a greater rise in cAMP than the mitogenic lectins whereas WGA had no effect on cAMP levels. None of the four lectins causes an appreciable (i.e., greater than 1.5-fold) change in cGMP levels. There does not seem to be any positive correlation between early changes of cAMP and/or cGMP and mitogenesis.     

Increased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP

The effects of cyclic nucleotides on elastin synthesis were studied in ligamentum nuchae fibroblasts by adding exogenous cyclic nucleotide derivatives or beta-adrenergic agents to cell culture medium. Elastin synthesis was enhanced (approximately 80%) by dibutyryl cGMP (Bt2cGMP) in concentrations ranging from 0.01 to 100 nM. Two other cGMP derivatives, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and 2'-deoxy-cGMP, were also potent stimulators of elastin synthesis. In the absence of calcium, basal elastin production was substantially decreased (40% of control) and cGMP analogs no longer stimulated elastin synthesis, suggesting a role for calcium in the cGMP response. Bt2cAMP had no demonstrable effect on elastin production except at high concentrations which produced a nonspecific decrease equivalent to the decrease in total protein synthesis. Similarly, elevation of endogenous cellular cAMP levels by beta-adrenergic stimulation produced no change in elastin production. When 8-Br-cGMP was added to cells together with Bt2cAMP, cGMP-dependent stimulation of elastin production was abolished by cAMP in a dose-dependent fashion. These results suggest a coordinated means by which elastin production is controlled in ligament cells, i.e. increased cGMP levels lead to a stimulation of elastin production that is reversed by cAMP.     

Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5

Using a radioimmunoassay (RIA) for the determination of adenosine 3'5' cyclic monophosphate (cAMP) and an acetylation-RIA procedure to measure guanosine 3'5' cyclic monophosphate (cGMP), we observed that cGMP levels, but not cAMP levels, were significantly elevated in murine thymocytes which had been incubated with preparations containing the thymic hormone, thymosin. Stimulation of intracellular cGMP levels was seen as early as 1 minute after incubation with thymosin fraction 5 and was maximal at approximately 10 minutes. Dose response studies indicated an optimum stimulation of cGMP with a thymosin concentration of 100 microg/ml. A control spleen fraction prepared by an identical procedure as fraction 5 did not affect the levels of either cyclic nucleotide.     

Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10^?3, 10^?4 or 10^?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [³H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of ³H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in ³H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [³H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10^?6, 10^?7 or 10^?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.     

Changes in cyclic nucleotide levels during erythroid differentiation in Friend leukemia cells

Changes in cyclic nucleotide levels were observed in Friend leukemia cells exposed to the inducers of erythroid differentiation dimethylsulfoxide (DMSO), sodium butyrate and hypoxanthine. The most extensive changes in cAMP levels occurred during the initial 24 h growth period. In uninduced cultures, cAMP concentration increased 35-fold compared with a 7-fold increase in the presence of inducer. During the same period. cGMP levels rose about 3-fold in uninduced cells compared with a 20-fold increase in induced cells.     

cGMP enhances the sonic hedgehog response in neural plate cells.

The elaboration of distinct cell types during development is dependent on a small number of inductive molecules. Among these inducers is Sonic hedgehog (Shh), which, in combination with other factors, patterns the dorsoventral (DV) axis of the nervous system. The response of a cell is dependent in part on its complement of cyclic nucleotides. cAMP antagonizes Shh signaling, and we examined the influence of cGMP on the Shh response. Cells in chick neural plate explants respond to Shh by differentiating into ventral neural-cell types. Exposure of intermediate-zone explants to cGMP analogs enhanced their response to Shh in a dose-dependent manner. The Shh response was also enhanced in dorsal-zone explants exposed to chick natriuretic peptide (chNP), which stimulates cGMP production by membrane-bound guanylate cyclase (mGC). Addition of chNP to intermediate-zone explants did not enhance the Shh response, consistent with a reported lack of mGC in this region of the neural tube. Finally, the presence of a nitric oxide (NO)-sensitive guanylate cyclase (GC) was established by demonstrating cGMP immunoreactivity in neural tissue following NO stimulation of whole chick embryos. Intracellular levels of cGMP and cAMP may thus provide a mechanism through which other factors modulate the Shh response during neural development.     

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3-5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.     

Effect of adenylyl cyclase activation on intracellular and extracellular cAMP and cGMP in preimplantation cattle blastocysts

The effects of direct and indirect activation of adenylyl cyclase on the production of intracellular and extracellular cAMP and cGMP by 13- to 16-day-old cattle embryos were determined. Embryos were incubated for 2 h in a Krebs Ringer bicarbonate medium containing the phosphodiesterase inhibitor isobutyl-methylxanthine, to which stimulating agents forskolin (100 mumol l-1), cholera toxin (2 micrograms ml-1), or both were added. Total (intra- and extracellular) basal cAMP and cGMP concentrations ranged from 6.65 +/- 0.895 to 3.4 +/- 0.708 fmol microgram-1 protein in 13-day-old embryos and from 4.05 +/- 1.151 to 0.19 +/- 0.041 fmol microgram-1 protein in 16-day-old embryos. Forskolin induced an increase (P < 0.001) in cAMP that ranged from 5.4-fold on day 13 to 2.7-fold on day 16, whereas cholera toxin induced an increase (P < 0.001) that ranged from 30-fold at day 13 to 21-fold at day 16, similar to the effect of forskolin and cholera toxin combined. Individually, forskolin and cholera toxin had no effect on cGMP concentrations, but together they induced an increase (P < 0.05). cAMP (P < 0.01) and cGMP (P < 0.001) concentrations decreased with embryo age from day 13 to day 16 for all treatments; the decrease was greater for cGMP than cAMP (5-24-fold versus 1.6-3.3-fold, respectively). It is concluded that inducible adenylyl cyclase is present in 13- to 16-day-old cattle embryos and that the embryos secrete cAMP and cGMP into the incubation medium. In addition, basal and inducible concentrations of cAMP and cGMP decrease with embryo age from day 13 to day 16. These observations indicate that cAMP and cGMP may have a role in the rapid embryonic cell proliferation that occurs at this time or in signalling to the endometrium.     

Regulation of acetylcholine receptor by cyclic AMP

In primary cultures of chick 11-day embryonic tissue a number of phosphodiesterase inhibitors were found to elevate acetylcholine receptor levels. Of these agents, Ro20-1724 was the most effective, elevating surface receptor content by 2-fold after 48 h of treatment. 8-Br-cAMP and cholera toxin, a natural activator of adenylate cyclase, mimicked the effect of Ro20-1724, while 8-Br-cGMP and dibutyryl cGMP had no effect. Cholera toxin, 8-Br-cAMP, and Ro20-1724 all increased the insertion rate of new receptor into the surface membrane without altering degradation. The enhanced insertion appears related to an actual increase in synthesis since total acetylcholine receptor was elevated by exposure to cholera toxin. In contrast, no change in creatine phosphokinase activity, myosin heavy chain content, or [35S] methionine incorporation into total cellular protein was observed during cholera toxin treatment. These results suggest that cAMP plays a role in the regulation of acetylcholine receptor.     

Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduction of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxygen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle’s metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20–22°C. Turtle brain ROS production decreased 25% within 10 min of cyanide or N₂-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of complex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner suggestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.     

Brain and ring gland cyclic AMP and cyclic GMP levels during initiation and termination of pupal diapause in flesh flies

Brain and ring gland concentrations of cyclic AMP were much higher shortly after pupariation in long day (non-diapause destined) flesh flies than in short day (destined for pupal diapause) flies. This difference was most striking in the ring gland (6 times higher in long day flies). Cholera toxin elevated brain-ring gland cAMP four-fold, thus accounting for its efficacy in averting diapause. No differences in cyclic GMP levels were detected between long and short day flies at pupariation. At diapause termination cAMP and cGMP concentrations in the brain and ring gland and cAMP in whole body homogenates changed only slightly, but whole body concentrations of cGMP rose markedly.     

Incorporation of mevalonate into dolichol and other isoprenoids during estrogen-induced chick oviduct differentiation

Incorporation of [14C]mevalonate into dolichol and other isoprenoid compounds by chick oviduct explants has been studied. A reliable assay of dolichol biosynthesis employing several chromatographic procedures, including two-dimentional TLC, was developed. Incorporation of [14C]mevalonate into dolichol by oviduct explants was linear for at least 6 h. The effect of estrogen-induced differentiation was studied by incubation of explants obtained from chicks treated for various periods of time with diethylstilbestrol. Mevalonate incorporation into dolichol, when expressed as cpm per g of tissue, was not affected by estrogen treatment, but since the oviduct increased about 100-fold in mass during differentiation, each oviduct synthesizes about 100-fold more dolichol. In most tissues, the major product of mevalonate incorporation is cholesterol. However, although approx. 90% of the non-saponifiable 14C-labeled compounds were in the so-called 'cholesterol fraction', oviduct explants from estrogenized chicks synthesized little, if any, cholesterol. A number of cholesterol biosynthetic intermediates were observed, with compounds comigrating with squalene and lanosterol accounting for about 50% of the total. Since the estrogenized chick has serum cholesterol levels in the range of 800-900 mg/dl, these results suggest that oviduct has secondary control points which allow it to inhibit cholesterol synthesis when mevalonate is used as the precursor. In support of this hypothesis is the observation that explants from untreated chicks can incorporate mevalonate into cholesterol.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes.

Ginea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E1, D1, F1 alpha, E2, D2, or F2 alpha. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2 alpha which were of low significance, but confirm earlier data. Similar results were observed with PGD1, PGE1, and PGF1 alpha with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD2 and PGE2. With PGF2 alpha, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD2 treatment.