The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding. Consequences for cAMP signaling pathway and characterization of a phosphatidic acid binding site.

Hormones and growth factors induce in many cell types the production of phosphatidic acid (PA), which has been proposed to play a role as a second messenger. We have previously shown in an acellular system that PA selectively stimulates certain isoforms of type 4 cAMP-phosphodiesterases (PDE4). Here we studied the effect of endogenous PA on PDE activity of transiently transfected MA10 cells overexpressing the PA-sensitive isoform PDE4D3. Cell treatment with inhibitors of PA degradation, including propranolol, induced an accumulation of endogenous PA accompanied by a stimulation of PDE activity and a significant decrease in both cAMP levels and protein kinase A activity. Furthermore, in FRTL5 cells, which natively express PDE4D3, pretreatment with compounds inducing PA accumulation prevented both cAMP increase and cAMP-responsive element-binding protein phosphorylation triggered by thyroid-stimulating hormone. To determine the mechanism of PDE stimulation by PA, endogenous phospholipids were labeled by preincubating MA10 cells overexpressing PDE4D3 with [(32)P]orthophosphate. Immuno- precipitation experiments showed that PA was specifically bound to PDE4D3, supporting the hypothesis that PDE4D3 activation occurs through direct binding of PA to the protein. PA binding site on PDE4D3 was characterized by engineering deletions of selected regions in the N-terminal regulatory domain of the enzyme. Deletion of amino acid residues 31-59 suppressed both PA-activating effect and PA binding, suggesting that this region rich in basic and hydrophobic residues contains the PA binding site. These observations strongly suggest that endogenous PA can modulate cAMP levels in intact cells, through a direct activation of PDE4D3.

Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

Relationships between phosphatidic acid and cyclic nucleotide phosphodiesterases in activated human blood mononuclear cells.

We have previously shown that mitogenic activation of human PBMC rapidly increases both the intracellular phosphatidic acid (PA) level and cyclic nucleotide phosphodiesterase (PDE) activity, with time-course responses, suggesting a causative relationship between the two events. PA also directly stimulated cAMP-PDE activity in acellular systems. Thus the mitogenic properties of PA night be due to its ability to lower the level of cAMP, a negative effector of lymphocyte activation, through PDE activation. In this study, human PBMC were stimulated either with the mitogenic lectin ConA, the anti-CD3 mAb OKT3, or the phorbol ester TPA. All three agonists increased the radiolabeled PA level and the PA mass in treated cells and simultaneously increased cytosolic and particulate cAMP- and cGMP-PDE activities, with significant positive correlations between PA accumulation and PDE activities. Furthermore, the ConA-induced PDE activation was dose-dependently reduced by treatment of PBMC with the diacylglycerol-kinase inhibitor R59022. This compound also dose-dependently lowered the PA level and inhibited the proliferative response to ConA. In addition, TPA-induced PDE activation was totally abolished by ethanol, which strongly reduced PA accumulation in response to the phorbol ester. These data suggest that PA increase may be linked to mitogen-induced PDE activation. Experiments performed in the presence of rolipram indicated that ConA and TPA stimulated both the rolipram-sensitive PDE4 and the rolipram-insensitive PDE activities, OKT3 being more active on PDE4. All three agonists stimulated the cGMP-specific PDE5. These results suggest that PA is an important component of the mechanisms that maintain a low level of cyclic nucleotides, which is a prerequisite for an optimal lymphoproliferative response.

Protective effect of docosahexaenoic acid against hydrogen peroxide-induced oxidative stress in human lymphocytes.

Oxidatively stressed lymphocytes exhibit decreased proliferative response to mitogenic stimulation. Although several sensitive targets involved in lymphocyte suppression have already been identified, little is known about the influence of oxidative stress on cyclic nucleotide phosphodiesterases (PDE) (EC 3.1.4.17), thought to play a major role in the control of cyclic AMP (cAMP) level, a well-recognized negative effector of lymphoproliferation. Although the polyunsaturated fatty acid content of membrane phospholipids is thought to be directly related to the extent of oxidant-induced lipid peroxidation, some n-3 fatty acids also seem to have antioxidant effects, depending on the concentration used and the overall redox status of the cells in question. Results of the present study showed that human peripheral blood mononuclear cells (PBMC) as well as rat thymocytes were relatively resistant to a short-term exposure (10 min) to hydrogen peroxide (H2O2). Indeed, H2O2-induced lipid peroxidation, estimated by malondialdehyde (MDA) production, was only 2-fold increased by H2O2 concentrations lower than 2 mM, whereas a larger increase (10-fold) could be observed in PBMC at the highest dose (5 mM). Previous enrichment of PBMC with 5 microM docosahexaenoic acid (22:6n-3), brought to the cells as a fatty acid-albumin complex (ratio 1), significantly reduced MDA production induced by low doses of H2O2, the protective effect no longer being observed at the highest doses. In contrast, eicosapentaenoic acid (20:5n-3) did not have any protective effect. Cytosolic PDE activities of both human PBMC and rat thymocytes were significantly inhibited (40-50%) after H2O2 treatment of the cells, whereas particulate PDE activities were not modified. Different responses of PDE activities to H2O2 treatment were observed when PBMC were first enriched with 22:6n-3 prior to H2O2 addition. In 22:6n-3-treated cells, the H2O2-induced inhibition of both cAMP- and cGMP-PDE cytosolic activities was abolished, whereas the particulate activities were increased by the highest H2O2 concentration used (5 mM). At the same time, the glutathione peroxidase (glutathione: oxidoreductase, EC 1.11.1.9) (GSH-Px) activity of PBMC and thymocytes was only marginally inhibited by H2O2 addition (20%), and pretreatment of the cells with 22:6n-3 did not modify the slight inhibitory effect of H2O2. Collectively, these results suggest that lymphocytes are relatively resistant to H2O2-induced lipid peroxidation due to their high GSH-Px content, and that low doses of 22:6n-3 are able to prevent some of the H2O2-induced alterations such as lipid peroxidation and PDE inhibition. Docosahexaenoic acid might thus offer some protection against oxidant-induced lymphocyte suppression.

Regulation of PDE-4 cAMP phosphodiesterases by phosphatidic acid.

Phosphatidic acid (PA) has been previously shown to activate specifically some of the isoforms of type 4 cylic nucleotide phosphodiesterases (PDE-4) in an acellular system. In the present work, we have investigated the mechanism of PA-activating effect by using a recombinant PA-sensitive isoform, PDE-4D3. The enzyme was specifically activated by acidic phospholipids, but not by zwitterionic phospholipids or anionic detergents. The importance of the role of PA acidic groups in the activation process was confirmed by studying the influence of pH and ionic strength on activation. Crosslinking experiments suggested that PA might influence the ability of PDE-4D3 to form dimers. Binding studies performed with radiolabeled PA showed that PA binds to a PDE-4D3 preparation in a saturable manner. Specifically bound PA was displaced by anionic, but not by zwitterionic phospholipids. With a preparation of PDE-4B2, a PDE-4 isoform insensitive to PA activation, PA binding was only displaced by high concentrations of unlabeled PA, suggesting that high-affinity PA binding sites are only present on PDE-4D3. These data support the hypothesis that PA-activating effect depends on direct binding of the effector on specific sites carried by the PDE-4D3 protein.

Docosahexaenoic acid lowers phosphatidate level in human activated lymphocytes despite phospholipase D activation.

N-3 polyunsaturated fatty acids from marine oil have been shown to decrease T cell-mediated immune function both in animals and humans, and to inhibit the mitogen-induced lymphoproliferative response when added to lymphocyte culture medium. As phosphatidic acid (PA) is a key mediator of the mitogenic process, the present study aims to investigate whether docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, the main n-3 fatty acids from fish oil, are able to alter the mitogen-induced synthesis of PA, when added to the culture medium of human peripheral blood mononuclear cells (PBMC). Incubation of PBMC in a medium containing 5 microM DHA bound to 5 microM human delipidated serum albumin induced a 2-fold increase in the basal PA mass whereas incubation with EPA, in the same conditions, had no effect. In contrast, both fatty acids markedly reduced the concanavalin A (ConA)-induced production of PA as compared with untreated cells. Paradoxically, phospholipase D (PLD) activity, evidenced by the synthesis of phosphatidylbutanol, was only detected in DHA-treated cells further stimulated by ConA, indicating that both DHA and ConA are required for PLD activation. Similarly, an increased diacylglycerol (DAG) mass was only observed in DHA-treated cells stimulated by ConA, whereas no modification occurred in control or EPA-treated cells stimulated or not by ConA. Furthermore, 1-butanol suppressed the ConA-induced increase of DAG mass observed in DHA-treated cells, indicating that phosphatidate was the source of the newly synthesized diacylglycerol. Altogether, these results show that, in concanavalin A-activated human peripheral blood mononuclear cells, docosahexaenoate stimulates both phospholipase D and phosphatidate phosphohydrolase activities, which ultimately results in an increased diacylglycerol production at the expense of phosphatidate.

Selective activation of rolipram-sensitive, cAMP-specific phosphodiesterase isoforms by phosphatidic acid.

In rat thymic lymphocytes, accumulation of phosphatidic acid (PA) occurs at the same time as decrease in cAMP levels and activation of a cAMP-specific phosphodiesterase (PDE) [type 4, EC 3.1.4.17 (PDE4)]. We investigated the nature of the PDE activated by PA and the mechanism of activation by using recombinant cAMP-specific PDE4 isoforms derived from three different genes (PDE4A, PDE4B, and PDE4D). The "long" variants expressed from each gene (PDE4A5, PDE4B1, and PDE4D3) were activated by PA, whereas the "short" variants (PDE4A1, PDE4B2, PDE4D1, and PDE4D2) were not. Phosphatidylserine was an activator that was as effective as PA, whereas phosphatidylcholine was ineffective, indicating that activation was restricted to anionic phospholipids. PA caused an increase in the Vmax value of PDE4D3 without affecting the Km value of the enzyme for the cAMP substrate. PA also caused a change in the Mg2+ requirement for hydrolysis. Half-maximal stimulation of the PDE was obtained with approximately 10 microg/ml PA. Although protein kinase A-mediated phosphorylation of PDE4D3 produces effects similar to those elicited by PA, the mechanism of PA-induced activation was not found to involve a phosphorylation. Instead, several observations suggest that PA may directly interact with the enzyme. The stimulation of cAMP PDEs by PA and other acidic phospholipids may be a mechanism by which growth factors and hormones modulate the cAMP-dependent signal transduction pathway during cell stimulation.

Modulation of rat thymocyte proliferative response through the inhibition of different cyclic nucleotide phosphodiesterase isoforms by means of selective inhibitors and cGMP-elevating agents.

We have investigated the role played by cyclic nucleotide phosphodiesterases (EC 3.1.4.17) in the control of T-lymphocyte response to mitogenic agents by their ability to influence the cellular level of cAMP. The importance of this messenger as a negative regulator in this cell type is well established. Multiple isoenzymes of phosphodiesterase were fractionated from the cytosol of rat thymic lymphocytes by high performance liquid chromatography on an anion exchange column. In addition to the type II, III, IV isoforms that we have already described [Valette et al., Biochem. Biophys. Res. Commun. 169:864-872 (1990)], a phosphodiesterase fraction sharing several of the characteristics of type V, cGMP-binding phosphodiesterase, was detected. Non-isoform-selective inhibitors of phosphodiesterase such as dipyridamole, papaverine, and methyl-isobutylxanthine were able to totally prevent the proliferative response of thymocytes to stimulation by the mitogenic lectin concanavalin A. In contrast, the selective inhibitor of type IV phosphodiesterases rolipram induced a rather moderate inhibition of proliferation, not exceeding 60%; and the selective inhibitors of type III and type V phosphodiesterases, milrinone and M&B 22,948, respectively, displayed only marginal inhibitory effects. The association of the type III and IV phosphodiesterase inhibitors produced synergistic inhibition of proliferation, which could then be almost totally suppressed. These inhibitory effects on cell multiplication were reflected at the level of the cell cAMP content; only rolipram was able to induce a significant (approximately 50%) increase in cAMP, and this increase was potentiated by the presence of milrinone, reaching almost 100%. The type V phosphodiesterase selective inhibitor M&B 22,948 displayed similar properties to those of milrinone, which suggests that it indirectly inhibited the type III, cGMP-inhibitable isoenzyme, by inducing a cGMP rise. This hypothesis was supported by evidence of a significant raising effect of M&B 22,948 on cGMP level, and by the ability of a cGMP-elevating agent, sodium nitroprusside, to mimic the synergistic effects of milrinone associated with rolipram. Furthermore, 8-bromo-cGMP, a potent activator of cGMP-dependent protein kinase, which showed only weak inhibitory effects on thymic type III phosphodiesterase, failed to alter the effects of rolipram on the cell proliferation. These results allow us to delineate a role for types III, IV, and V phosphodiesterase in the control of cAMP level during the proliferative response of thymic lymphocytes. They also suggest that endogenously formed cGMP might participate in the regulation of cAMP level in the cells by means of the inhibition of the type III phosphodiesterase.(ABSTRACT TRUNCATED AT 400 WORDS)

Impaired G-proteins and cyclic nucleotide phosphodiesterase activity in T-lymphocytes from patients with sarcoidosis.

Among the various immune abnormalities which characterize active sarcoidosis, a low proliferative response of peripheral blood lymphocytes to mitogenic lectins has long been observed. Since membrane-associated G-proteins are very likely to be crucial elements in lectin signal transduction, we investigated the binding of 5'-guanylylimidodiphosphate (GppNHp), a non hydrolyzable GTP analogue, to blood total lymphocyte membranes and to blood T-lymphocyte membranes from patients with active sarcoidosis, and from healthy control subjects. GppNHp binding was markedly decreased in peripheral cells from patients with sarcoidosis as compared to controls, suggesting the occurrence of a defect at the level of G-protein(s). A further characterization of G-proteins in these cells by means of ADP-ribose-labelling in the presence of bacterial toxins brought forward a significant decrease in the labelling of a 40 kDa protein, the major pertussis toxin substrate, in membranes from sarcoid patients, while the labelling of the major 44 kDa cholera toxin substrate proved to be unchanged with respect to control membranes. It is hypothesized that, in sarcoid lymphocytes, a defect in the negative control of adenylate cyclase mediated by the inhibitory G-protein Gi, prevents the lowering of cAMP necessary to normal mitogenic response of blood lymphocytes to stimulation. cAMP degradation by the specialized enzyme phosphodiesterase constitutes another critical step in the control of cAMP levels. Both cAMP and cGMP phosphodiesterase activities were found decreased in blood total lymphocyte preparations from sarcoid patients. With purified T-cells, although the mean cAMP and cGMP phosphodiesterase activities from sarcoid patients were found more markedly decreased with respect to healthy donors, only the decrease in cGMP phosphodiesterase was found statistically significant. The role these defects in cyclic nucleotide degradation potentially play in the disturbance of blood lymphocytes response associated with sarcoidosis is discussed.

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.

Decreased cyclic nucleotide phosphodiesterase activity in human peripheral blood mononuclear cells from elderly women.

1. Both adenosine 3':5'-cyclic monophosphate and guanosine 3':5'-cyclic monophosphate phosphodiesterase activities of peripheral blood mononuclear cells were markedly decreased in elderly women as compared with young control women. 2. In contrast, the ability of these cells to bind guanosine 5'-[beta, gamma-imido]triphosphate, a non-hydrolysable analogue of guanosine 5'-triphosphate, was the same in both groups. 3. These findings are discussed in the context of the decline in immune function which occurs with increasing age.

Concanavalin A stimulates the Rolipram-sensitive isoforms of cyclic nucleotide phosphodiesterase in rat thymic lymphocytes.

Pretreatment of rat thymic lymphocytes with Concanavalin A induced a very early (30 min) and substantial increase (+90%) of the soluble cAMP phosphodiesterase activity. The crude cytosolic phosphodiesterase activity of rat thymocytes could reproducively be resolved by Mono-Q ion exchange high performance liquid chromatography into four separate phosphodiesterase peaks: a cGMP-stimulated, two cAMP-specific Rolipram-sensitive and a cGMP-inhibited cardiotrope-sensitive peaks. Concanavalin A stimulated very specifically the activity of the two predominant cAMP-specific Rolipram sensitive peaks whereas it only slightly modified the cGMP-stimulated and the cGMP-inhibited forms. The present results strongly suggest that the Rolipram-sensitive cAMP PDE activity may play a key role in the control and regulation of mitogen-induced thymocyte proliferation.

Cyclic nucleotide phosphodiesterases and methyltransferases in purified lymphocytes, monocytes and polymorphonuclear leucocytes from healthy donors and asthmatic patients.

Cyclic nucleotide phosphodiesterase and phospholipid N-methyl-transferase activities were simultaneously measured in purified polymorphonuclear cell-, mononuclear cell-, lymphocyte- and monocyte-homogenates from control subjects, from patients with atopic asthma and from patients with non-atopic asthma. Whereas cyclic AMP and cyclic GMP phosphodiesterase activities were found to be about 10-fold lower in polymorphonuclear than in mononuclear cells, phospholipid N-methyltransferase proved to be rather similar in each cell type from control donors. Cyclic AMP phosphodiesterase and phospholipid N-methyltransferase were significantly decreased in polymorphonuclear cells and monocytes from asthmatic patients compared with the control group while cyclic GMP phosphodiesterase was significantly impaired only in the monocyte subpopulation.

Differential susceptibility to biological detergents of the particulate cGMP-stimulated phosphodiesterase from rat heart: preservation of the allosteric properties of the solubilized enzyme.

The effects of various biological detergents on the particulate cGMP-stimulated cAMP phosphodiesterase activity from rat heart were investigated. When added to particulate fractions, anionic and non-ionic detergents diversely increased both cAMP and cGMP phosphodiesterase activities and slightly decreased the stimulatory effect of cGMP on cAMP hydrolysis whereas cationic detergents were rather inhibitory and drastically lowered the stimulatory effect of cGMP. Among the most efficient detergents, only sodium cholate was able to solubilize phosphodiesterase activity and preserve the stimulatory effect of cGMP on cAMP hydrolysis. Furthermore, the addition of glycerol significantly improved the conservation of the allosteric properties of the enzyme. Kinetic properties of the cholate-solubilized phosphodiesterase were quite identical to those of the membrane-bound enzyme.

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta. Separation and inhibition by selective reference phosphodiesterase inhibitors.

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.

Effect of estrogen on adenosine 3'5',cyclic monophosphate in quail oviduct: possible involvement in estradiol-activated growth.

Previous studies have shown that estradiol indirectly stimulated the proliferation of oviduct epithelial cells in the quail. The present study was undertaken to investigate the effects of estradiol and other steroid hormones on the cAMP concentration. The ability of forskolin, a specific activator of the catalytic subunit of adenylate cyclase, to induce oviduct cell proliferation and specific protein synthesis (progesterone receptor) in the absence of estrogen was also tested. Administration of estradiol benzoate (EB) to immature female quails produced a transient surge in oviduct cAMP concentration. After EB injection, cAMP concentration increased by 36.7% after 6 h and returned to control values after 12 h. This rise in oviduct cAMP concentration preceded the beginning of DNA synthesis. The same effect was observed even in the absence of increased plasma estradiol, when the hormone was perfused through the hepatic portal vein. Estriol, estrone, and testosterone failed to elevate cAMP concentrations. After repeated EB injections, the oviduct cAMP concentration declined below the control value (-66% after 72 h). A similar drop in the cAMP concentration was observed in developing quails during the proliferative phase of the luminal epithelial and glandular cells. Administration of forskolin to immature female quail pretreated with EB rapidly increased the oviduct cAMP concentration, induced a burst of DNA synthesis, and shortened the prereplicative period. In addition, forskolin administration did not increase the progesterone receptor concentration. These results demonstrate that cAMP is involved in the mechanism by which estradiol indirectly stimulates oviduct epithelial cell proliferation in the quail. The events that may take place during the prereplicative period and the antiproliferative effect of progesterone through a sustained increase in the cAMP concentration in the oviduct are discussed.

Cyclic nucleotide phosphodiesterase activity in the quail oviduct: hormonal regulation and involvement in estrogen-induced growth.

A previous study has shown that cAMP was involved in estrogen-activated growth in the quail oviduct. The present study was undertaken to investigate the hormonal regulation of 3',5'-cyclic nucleotide phosphodiesterase activity in the oviduct. Tamoxifen, an antiestrogen compound, and 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase, were also used to determine the relationship between estradiol-induced cell proliferation and cAMP phosphodiesterase activity. Cyclic nucleotide phosphodiesterase was almost completely restricted to the cytosolic fraction (108,000 X g supernatant) of the quail oviduct homogenate. By affinity chromatography on immobilized calmodulin, we separated and partially characterized three different forms of the enzyme. They differed in their cyclic nucleotide specificities, kinetics, and sensitivity to calmodulin. In vivo, estradiol benzoate (EB) modulated crude cytosolic phosphodiesterase activity. cAMP and cyclic-GMP hydrolyzing activities increased between 12 and 48 h after a single injection of EB and then declined to return to control value by 96 h. Estrone, 17 alpha-estradiol, progesterone, and testosterone were ineffective, while estriol slightly increased cyclic-GMP hydrolyzing activity. When administered with EB, tamoxifen drastically increased oviduct cAMP concentration while it completely inhibited oviduct growth and the activation of cAMP phosphodiesterase induced by EB alone. Moreover, 3-isobutyl-1-methylxanthine produced a dose-dependent inhibition of oviduct cell proliferation when given with EB. These results demonstrate that the activation of cAMP phosphodiesterase after an injection of EB and the subsequent decrease in oviduct cAMP concentration are necessary for the epithelial cells to achieve their proliferative cycle.