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Query: UNIPROT:P06889 (Mol)
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The dunce (dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility. The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases or base-pairs). To characterize these RNAs and to understand their sequence heterogeneity, we isolated and analyzed 29 new and independent cDNA clones representing the dnc RNAs. Restriction mapping, hybridization analysis and sequence determination of these cDNA clones and the corresponding genomic exons resolved these into six different classes. Exons defined by the cDNA clones are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites: two of these were mapped by parallel S1-nuclease and primer extension experiments. In addition, some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence. Altogether, the results indicate that the size and sequence heterogeneity of dnc transcripts results from transcription initiation at multiple sites, alternative splicing, and processes which generate different 3' ends. The existence of multiple protein products is suggested by the alternative use of exons which code for portions of the open reading frame. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons and internal differences arising from the optional inclusion of a 39 base-pair exon and from the alternative use of two 3' splice sites separated by six base-pairs. Expression of a cDNA clone in yeast containing a large portion of the open reading frame produced cAMP PDEase activity identical in properties to the Drosophila enzyme affected by the dnc mutation. The results suggest that the remarkable structural complexity of dnc may reflect an intricate control of the spatial and/or temporal expression of various isoforms of cAMP PDEase.
J Mol Biol 1991 Dec 05
PMID:Characterization of the memory gene dunce of Drosophila melanogaster. 166 Sep 26

Three phosphodiesterase (PDE) type III inhibitors were tested and found to inhibit Xenopus oocyte maturation induced by insulin with apparent IC50 values of 2.2 +/- 0.2 microM Cl-930, 25 +/- 3 microM imazodan (Cl-914), and 786 +/- 237 microM piroximone (MDL 19,205). The same rank order of potencies was observed for inhibition of insulin-like growth factor-I (IGF-I)-induced oocyte maturation, with IC50 values of 5.5 +/- 0.9 microM Cl-930, 54 +/- 4 microM imazodan, and 1190 +/- 395 microM piroximone. Oocyte maturation induced by microinjection of Ha p21ras was also inhibited by pretreatment of oocytes with Cl-930 or imazodan, with IC50 values of 4.3 +/- 1.2 and 59 +/- 4 microM, respectively. Progesterone-induced maturation was not affected by PDE III inhibitor action; and, neither type IV PDE inhibitors (Ro 20, 1724 or rolipram) nor dipyridamole (a type V PDE inhibitor) inhibited cell division induced by IGF-I or microinjected Ha p21ras. In addition, while insulin-stimulated oocyte PDE activity measured in vivo after microinjection of 200 microM [3H] cAMP was inhibited by nonselective and type III-specific drugs (with IC50 values of 4.2 +/- 1.8 microM Cl-930 and 26 +/- 6 microM imazodan), type IV and type V inhibitors did not inhibit hormone-stimulated enzyme activity. This pharmacological evidence demonstrates a necessary role for PDE III in insulin-, IGF-I-, and p21ras-induced meiotic cell division in Xenopus laevis oocytes.
Mol Endocrinol 1991 Dec
PMID:Type III phosphodiesterase plays a necessary role in the growth-promoting actions of insulin, insulin-like growth factor-I, and Ha p21ras in Xenopus laevis oocytes. 166 4

Treatment of quiescent MG-63 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF) stimulates the rapid accumulation of c-myc RNA. We have now determined that a similar effect can be induced by cAMP. Treatment with forskolin (an activator of adenylate cyclase), IBMX (a phosphodiesterase inhibitor), PGE1, and isoproterenol stimulated accumulation of both cAMP and c-myc RNA, but no increase in either cAMP or c-myc RNA was seen with the inactive forskolin analog 1,9-dideoxyforskolin. Forskolin and IBMX acted synergistically in stimulating accumulation of both cAMP and c-myc RNA. However, three lines of evidence indicated that PDGF action is not mediated by cAMP. First, PDGF treatment caused no elevation of cAMP within 1 h, even in the presence of IBMX. Second, the kinetics of c-myc RNA elevation after treatment with PDGF or forskolin were similar, ruling out delayed onset of cAMP stimulation. Finally, simultaneous treatment with forskolin and the calcium ionophore A23187 enhanced the elevation of c-myc RNA levels; no such effect was seen with PDGF. We had previously shown that PDGF action is not affected by prior treatment of MG-63 cells with TPA, a treatment which desensitizes the c-myc response to TPA. Similarly, TPA pretreatment had minimal effect on forskolin or IBMX-induced c-myc expression. These data suggest that cAMP, phorbol esters, and PDGF act independently to stimulate c-myc RNA expression in MG-63 cells. However, nuclear runoff experiments and RNA half-life measurements demonstrated that PDGF, phorbol ester, and cAMP all act to increase the transcription of the MYC gene.
Mol Cell Biol 1990 Jan
PMID:Platelet-derived growth factor-stimulated c-myc RNA accumulation in MG-63 human osteosarcoma cells is independent of both protein kinase A and protein kinase C. 168 64

The voltage-gated Ca2+ current (ICa) in cardiac myocytes is regulated by cAMP-dependent phosphorylation. Although the regulation of ICa via mechanisms involving modulation of cAMP synthesis is well understood, the regulation of cAMP degradation has been less thoroughly investigated. The goal of the present study was to investigate the participation of different subclasses of cAMP phosphodiesterase (PDE) in regulating cAMP-dependent phosphorylation of Ca2+ channels in frog ventricular myocytes. Cardiomyocytes were isolated enzymatically and mechanically and were patch-clamped using the whole-cell configuration of the patch-clamp technique. The effects of various low-Km cAMP PDE inhibitors on ICa were examined. None of the inhibitors tested [milrinone, indolidan, 1-methyl 3-isobutyl xanthine (MIX), rolipram, or Ro 20-1724] were able to elevate ICa in the absence of elevated cAMP, although they all increased ICa in the presence of submaximal levels of cAMP. This result suggests that these compounds do not act directly on Ca2+ channels but rather modulate cAMP degradation. Half-maximal effects were observed with 1.4 microM milrinone and 3.4 microM MIX. Milrinone was effective when applied from either the extracellular or intracellular surface, whereas MIX was effective only when applied from the extracellular solution. In the presence of internal cGMP, which stimulates the cGMP-stimulated PDE, the low-Km cAMP PDE inhibitors had no effect on ICa, whereas high concentrations of MIX, which inhibit the cGMP-stimulated PDE, increased ICa. This would support the hypothesis that cGMP-stimulated PDE either has a much stronger capacity to hydrolyze cAMP or is more efficiently coupled to Ca2+ channels than the low-Km cAMP PDEs.
Mol Pharmacol 1990 Sep
PMID:Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells. 169 53

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.
Am J Respir Cell Mol Biol 1990 Oct
PMID:Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers. 169 99

The interaction of hormones acting via the mobilization of calcium and stimulation of cAMP levels in cells was examined by determining the effects of carbachol and forskolin on cAMP and cGMP accumulation in mouse parotid gland. Treatment of isolated acini with either carbachol (0.01 to 20 microM) or forskolin (1 microM) alone produced little or no increase in cAMP levels; carbachol, however, augmented the effect of forskolin on cAMP accumulation approximately 3- to 4-fold. The effects of carbachol on forskolin-stimulated cAMP levels were further augmented approximately 10-fold in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (MIX) but not in the presence of "low Km" cGMP-inhibited phosphodiesterase inhibitor milrinone. Augmentation of cAMP levels also occurred in the presence of carbachol plus the beta-adrenergic agonist isoproterenol (0.01 microM). In either the presence or absence of forskolin, carbachol increased cGMP levels independently of the inclusion of MIX and in a fashion parallel to that observed for cAMP accumulation. In the presence of forskolin (1 microM), the concentration of carbachol that produced half-maximal effects on cAMP and cGMP levels was 0.62 and 0.72 microM, respectively. Similar values were obtained in the presence of MIX. Cyclic GMP levels were also enhanced by carbachol plus isoproterenol. Hydroxylamine, as well as dibutyryl-cGMP and 8-bromo-cGMP in combination with forskolin, mimicked the effects of carbachol plus forskolin on cAMP levels. LY83583 (6-anillino-5,8-quinolinedione), an agent that lowers cGMP by inhibiting guanylate cyclase, reduced basal levels of cGMP and also completely prevented the increase in cGMP caused by carbachol plus forskolin. In these experiments, however, the augmentation of forskolin-stimulated cAMP levels by carbachol was reduced by approximately 50%. Additional studies suggest that calcium is also required for carbachol augmentation of forskolin-stimulated cAMP accumulation by effects on the adenylate cyclase complex. Augmentation of cAMP levels by carbachol did not involve effects on cAMP degradation. The results suggest that, when cAMP synthesis is stimulated by forskolin or isoproterenol, the muscarinic agonist carbachol augments cAMP accumulation by mechanisms involving cGMP and calcium in mouse parotid gland.
Mol Pharmacol 1990 Oct
PMID:Regulation of cAMP metabolism in mouse parotid gland by cGMP and calcium. 170 Feb 70

The effect of exogenous prostaglandin E2 (PGE2) on hormone-dependent adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated by microradioimmunoassay in collecting tubules microdissected from the cortex (CCT) or outer medulla (MCT) of the rat kidney. Two phosphodiesterase inhibitors were used: either a xanthine derivative (isobutyl-methylxanthine (IBMX, 1 mM] active on all forms of phosphodiesterase or Ro 20-1724 (50 microM) active on the phosphodiesterase type III. A prostaglandin synthesis inhibitor was added to all media. In the presence of IBMX, 0.3 microM PGE2 inhibited by 39.1% the response induced in the CCT by the beta-adrenergic agonist isoproterenol (1 microM). Under the same experimental conditions, arginine vasopressin (AVP)-stimulated cAMP accumulation in CCT or MCT was not affected by PGE2. In the presence of Ro 20-1724, 0.3 microM PGE2 did not modify the response to 1 nM AVP in CCT but inhibited this response in MCT samples (mean inhibition: 52.7%). The inhibition by PGE2 was dose dependent with a maximum at 0.3 microM, observed for all concentrations of AVP tested (from 50 pM to 1 nM) and did not affect the concentration of AVP inducing half-maximal cAMP accumulation. In a second experimental series performed in the presence of adenosine deaminase, an A1-adenosine agonist [theta)-N6-(R-phenylisopropyl)adenosine (PIA, 0.1 microM] also decreased the response to 1 nM AVP in the MCT. The addition of an A1-adenosine antagonist relieved the effect of PIA but did not modify the inhibition observed with PGE2. Thus PGE2 decreased the synthesis of cAMP in beta-adrenergic sensitive cells in rat CCT and might affect the catabolism of AVP-dependent cAMP level rather than its synthesis in rat MCT.
Mol Cell Endocrinol 1990 Oct 22
PMID:Two mechanisms of inhibition by prostaglandin E2 of hormone-dependent cell cAMP in the rat collecting tubule. 170 42

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.
Mol Cell Biol 1991 Dec
PMID:tpr-met oncogene product induces maturation-producing factor activation in Xenopus oocytes. 171 75

The regulation by FSH (follitropin; follicle-stimulating hormone) of FSH receptor mRNA and protein (FSH binding) was studied using cultured Sertoli cells isolated from 21-day-old rats. FSH induced a dose-dependent and almost complete down-regulation of receptor mRNA at 4 h after addition of the hormone. At subsequent time points (16 h and later) the FSH receptor mRNA levels had returned close to control values. The effect of FSH was mimicked by dibutyryl cyclic AMP (dbcAMP) and forskolin, and the phosphodiesterase inhibitor methyl-isobutylxanthine (MIX) prolonged the FSH action. These findings indicate that the effect of FSH on its receptor mRNA was mediated by cAMP. A down-regulatory effect of FSH and dbcAMP on FSH receptor mRNA was also observed in the presence of the protein synthesis inhibitor cycloheximide, suggesting a direct effect of FSH/dbcAMP on the expression of the FSH receptor gene. Transcriptional run-on experiments revealed that FSH did not inhibit initiation of the FSH receptor gene; hence a post-transcriptional mechanism is involved. Binding of 125I-FSH to the cultured Sertoli cells was rapidly (4 h) decreased when the cells were incubated with FSH or FSH in combination with MIX. This effect can be explained by ligand-induced receptor sequestration. In contrast, incubation of Sertoli cells with dbcAMP had no effect on binding of 125I-FSH after 4 h, but resulted in a 60% loss of FSH binding sites after 24 h, probably caused by decreased mRNA expression. In conclusion, FSH receptor down-regulation in Sertoli cells is effected not only by the well-documented ligand-induced loss of receptors from the plasma membrane, but also involves a cAMP-mediated decrease of FSH receptor mRNA through a post-transcriptional mechanism.
Mol Cell Endocrinol 1991 Jul
PMID:Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression. 172 86

Insulin-like growth factor-I (IGF-I) stimulated Xenopus laevis oocyte ribosomal S6 kinase activity 5- to 10-fold, with an apparent EC50 of 0.8 +/- 0.1 nM after 90 min of hormone treatment. IGF-I-stimulated enzyme activity was inhibited by treatment of oocytes with nonselective phosphodiesterase (PDE) inhibitors, with apparent IC50 values of 2 +/- 1 microM papaverine, 20 +/- 2 microM isobutylmethylxanthine, and 128 +/- 16 microM theophylline. Type III PDE inhibitors also inhibited IGF-I-stimulated S6 kinase activity with IC50 values of 9.7 +/- 0.3 microM Cl-930 and 84 +/- 23 microM imazodan (Cl-914). These drugs apparently affected an intracellular molecular event leading to activation of S6 kinase, since Cl-930 prevented IGF-I-stimulation of S6 kinase, but had no direct inhibitory effect when added to the S6 kinase enzyme assay mixture. While hormone-stimulated S6 kinase activity was inhibited by isobutylmethylxanthine (nonselective PDE inhibitor) and Cl-930 (PDE III inhibitor), Ro 20, 1724 and rolipram (PDE IV inhibitors) and dipyridamole (PDE V inhibitor) had no significant effect on activated enzyme levels. The time course for IGF-I stimulation of oocyte S6 kinase displayed a small early peak of activity approximately 0.15-0.4 time required for 50% of cell population to display white spots (GVBD50) and a second major increase in activity at 0.6-0.7 GVBD50 that was sustained until meiotic maturation was complete. The second wave of enzyme activation was inhibited by Cl-930, but the early increase was not.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1991 Dec
PMID:Inhibitors of phosphodiesterase III block stimulation of Xenopus laevis oocyte ribosomal S6 kinase activity by insulin-like growth factor-I. 172 88


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