Cyclic Nucleotide Phosphodiesterase Gene Research Articles

Identification of novel cyclic nucleotide phosphodiesterase gene cDNAs in the brain of guinea pig

Cyclic nucleotide phosphodiesterase (PDE) is a critical component of the nitric oxide (NO) signaling pathway and plays critical roles in cognition and learning, Parkinson's disease, attention deficit hyperactivity disorder, psychosis and depression. The PDEs in the brain of guinea pig have not yet been reported. The present study aimed to detect the unknown Pde cDNAs in the brain of guinea pig. Reverse transcription polymerase chain reaction (RT-PCR) and sequence comparison analysis were performed to detect the expression of Pde cDNAs and to assess the identity rates of cDNA and amino acid sequences between guinea pig and human or mouse, respectvely. The RT-PCR primers were located on the conserved region of human PDE and mouse Pde cDNAs. Eleven novel Pde cDNAs were detected in the brain of guinea pig (Cavia porcellus), including CpPde1a, CpPde1b, CpPde2a, CpPde4a, CpPde4d, CpPde5a, CpPde6c, CpPde7b, CpPde8a, CpPde9a, and CpPde10a. The identity rates of the Pde cDNA sequences between guinea pig and human ranged from 83.8% to 94.3%, and those of the amino acid sequences ranged from 91.9% to 100%. The identity rates of Pde cDNA sequences between guinea pig and mouse ranged from 84.6% to 92.1%, and those of amino acid sequences ranged from 91.2% to 99.2%. The average identity rate of the 11 Pde cDNA sequences between guinea pig and human was significantly higher (P < 0.01) than that between guinea pig and mouse. The putative partial amino acid sequences of guinea pig contained at least one of the conserved domains of human and mouse PDE proteins. These results indicate that the brain-expressed Pde genes are identified in guinea pig, which lays the foundation for further investigating the physiological roles of PDE proteins in the brain.

Identification of Novel Cyclic Nucleotide Phosphodiesterase Gene cDNAs in the Cochlea of Guinea Pig (Cavia porcellus) Through Conserved Homologous Sequences

The nitric oxide (NO) signaling pathway plays a critical role in auditory signal conversion and transduction. Cyclic nucleotide phosphodiesterase (PDE), an important component of the NO signaling pathway, has not been identified in the cochlea. Using cross-species comparison, homologous sequences of human and mouse Pde coding sequences were searched in a guinea pig genomic database and conserved homologous exons were found between human and mouse homologous sequences. Based on reverse-transcription PCR of these conserved regions, six partial Pde cDNAs were detected in the cochlea: CpPde3a, CpPde4d, CpPde8a, CpPde8b, CpPde9a, and CpPde11a. The identity rates of the six partial Pde cDNA sequences between guinea pig and human range from 83.8 to 95.5% and those of the peptide sequences range from 85.6 to 100%. The identity rates of the six Pde cDNA sequences between guinea pig and mouse range from 80.6 to 93.0% and those of peptide sequences range from 79.5 to 99.2%. The results demonstrate that multiple Pde genes are expressed in the cochlea, suggesting a NO pathway in the auditory system. Insights into this pathway will help to develop new therapeutic drugs on auditory abnormalities.

Identification and characterization of a novel cyclic nucleotide phosphodiesterase gene (PDE9A) that maps to 21q22.3: alternative splicing of mRNA transcripts, genomic structure and sequence.

Cyclic nucleotide-specific phosphodiesterases (PDEs) play an essential role in signal transduction by regulating the intracellular concentration of second messengers (cAMP and cGMP). We have identified and made an initial characterization of a full-length cDNA encoding a novel human cyclic nucleotide phosphodiesterase, PDE9A. At least four different mRNA transcripts (PDE9A1, A2, A3, A4) are produced as a result of alternative splicing of 5' exons, potentially changing the N-terminal amino acid sequences of the encoded proteins. All these predicted proteins would contain a 3',5'-cyclic nucleotide phosphodiesterase signature motif (Prosite no. PDOC00116). Northern blot analysis revealed several mRNA species of approximately 2.4 kb with varying expression patterns and intensities in most tissues examined, except blood. We have also isolated the mouse homolog of the human PDE9A2 mRNA transcript, pde9A2. The human and mouse isoforms have 93 and 83% sequence identity at the amino acid and nucleotide levels, respectively. PDE9A was mapped to 21q22.3, between TFF1 and D21S360. Comparison of the PDE9A1 cDNA with the genomic sequence from the region revealed that the gene is split into 20 exons that extend over 122 kb. Comparison of the physical map of the region and the genomic sequence further refines the mapping, with D21S113 being derived from intron 15. Several genetic disorders map to 21q22.3, including one form of bipolar affective disorder. Since functional disturbances in intraneuronal signal transmission via second messengers play an important role in the pathophysiology of affective disorders, PDE9A is a strong candidate for such a role by position and function.

Regulation of expression of the cyclic nucleotide phosphodiesterase gene in phosphodiesterase inhibitor-negative mutants of Dictyostelium discoideum.

During early development of Dictyostelium discoideum, the enzyme cyclic nucleotide phosphodiesterase (PD) is produced at a low rate during the period its specific inhibitor (PDI) is being synthesized. In addition, PD gene expression is derepressed in the aggregation-deficient (Agg-), Pdi- mutant HC35. These observations suggest that the PDI might function to regulate PD gene expression, as well as modulate its activity. To explore this idea further, five new Agg-, Pdi- mutants were isolated and analyzed. All of the mutants produced high PD activity and overexpressed PD mRNA; four exhibited elevated levels of the 2400-nucleotide aggregation transcript and one overproduced the 1900-nucleotide vegetative transcript. In contrast, PD transcripts were not elevated in two Agg-, Pdi+ mutants. To determine if PDI production regulates PD expression, HC35 cells were transformed with plasmids carrying the PDI structural gene under the control of either the vegetative or aggregative PD promoter. Neither expression of PDI by the transformants nor addition of partially purified PDI to HC35 cells affected PD transcription. These results suggest that PD overexpression in the Pdi- mutants is not a direct consequence of the inability of these cells to produce inhibitor.

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase gene is highly expressed in the limbic system of the rat brain

Cyclic AMP and cyclic GMP serve as second messengers in a variety of neural cells, modulating their metabolic and electrical activity. The cyclic GMP-stimulated cyclic nucleotide phosphodiesterase, an enzyme whose hydrolytic activity is allosterically regulated by cyclic GMP in peripheral tissues, could play an important role in the regulation of cyclic nucleotide levels in the brain. To study the presence and distribution of cyclic GMP-stimulated phosphodiesterase in the rat brain, we cloned a portion of rat liver cyclic GMP-stimulated phosphodiesterase complementary DNA by polymerase chain reaction, using degenerate phosphodiesterase-specific oligonucleotide primers. Northern blot analysis of rat tissues reveals abundant expression of cyclic GMP-stimulated phosphodiesterase messenger RNA in the brain. Northern blot analysis of brain subrogions shows especially strong expression in hippocampus and cortex, modest expression in the remainder of the forebrain and in the midbrain, and little expression in cerebellum and hindbrain. In situ hybridization studies with cyclic GMP-stimulated phosphodiesterase riboprobes confirm these northern blot results, and delineate cell groups with high levels of expression. Medial habenular nucleus is intensely labeled, as is hippocampus in the vicinity of pyramidal and granule cell bodies in areas CA1, CA2, CA3, and dentate gyrus. Other elements of the limbic system also contain cyclic GMP-stimulated phosphodiesterase messenger RNA, including olfactory and entorhinal cortices, subiculum, and amygdala. Additional cortical regions show more diffuse expression of cyclic GMP-stimulated phosphodiesterase messenger RNA, as do the basal ganglia. Cerebellum, thalamus, and hypothalamus do not show appreciable specific labeling. These studies demonstrate the presence of cyclic GMP-stimulated phosphodiesterase messenger RNA in specific regions of the rat brain, and suggest that the cyclic GMP-stimulated phosphodiesterase might modulate neuronal activity by regulating intracellular cyclic AMP levels in response to changes in intracellular cyclic GMP levels.

Characterization of a periplasmic 3':5'-cyclic nucleotide phosphodiesterase gene, cpdP, from the marine symbiotic bacterium Vibrio fischeri

Vibrio fischeri, a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids, exhibits the unusual attribute of growth on 3':5'-cyclic AMP (cAMP), apparently through the activity of a 3':5'-cyclic nucleotide phosphodiesterase (3':5'-CNP) with exceptionally high activity. The V. fischeri 3':5'-CNP is located in the periplasm, a novel cellular location for this enzyme in bacteria. To gain insight into the physiological function of this enzyme, we cloned the gene (designated cpdP) encoding it from V. fischeri MJ-1. This is the first bacterial 3':5'-CNP gene to be cloned. Sequencing and analysis of the 1.26-kb cpdP locus revealed a single open reading frame specifying a protein of 330 amino acid residues, including a 22-amino-acid leader peptide. The putative cpdP promoter contained a reasonable -10 promoter region (TATTAT) but contained no obvious -35 region; instead, a 12-bp inverted repeat (TTAAATATTTAA) occurred just upstream of this location. A possible rho-independent transcriptional terminator with a calculated free energy of -21.2 kcal.mol-1 (ca. -88.7 kJ.mol-1) followed the CpdP protein coding sequence. The predicted subunit molecular weight of 33,636 for the mature CpdP protein (36,087 less 2,451 for the leader peptide) was consistent with the molecular weight of 34,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the CpdP protein exhibited 30.3% identity with that of the low-affinity 3':5'-CNP (PDE1) of Saccharomyces cerevisiae and 33.6% identity with that of the extracellular 3':5'-CNP of Dictyostelium discoideum. The residue identities clustered in two regions, residues 100 to 146 and 238 to 269, which contained 30 of the 33 amino acids conserved in all three proteins, 4 of which were histidines. A gene replacement mutant of V. fischeri MJ-1 containing a 0.45-kb BglII deletion within the cpdP gene lacked periplasmic 3':5'-CNP activity and did not grow on cAMP, confirming for V. fischeri the relationship among cpdP, synthesis of the periplasmic 3':5'-CNP, and growth on cAMP. The mutant exhibited no obvious sensitivity to high extracellular concentrations of cAMP (5 and 10 mM), suggesting that the enzyme does not play a role in defense against extracellular cAMP.

Regulation of 2′,3′-cyclic nucleotide phosphodiesterase gene expression in experimental peripheral neuropathies

2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNPase) is an enzyme associated with central nervous system myelination. Although present in the mammalian peripheral nerve, it is not clear what its role is during myelination nor how the expression of this gene is regulated in the PNS. In this study, CNPase gene expression was studied in the crushed and permanently transected rat sciatic nerve, two models of peripheral nerve neuropathy. The Schwann cells of the crushed nerve initially demyelinate, remain in a non-myelinating condition until active regeneration induces remyelination (10–21 days after injury), whereas those of the permanently transected nerve remain in a quiescent, non-myelinating state after the initial demyelination. An increase of CNPase mRNA levels is observed during degeneration and remains high whether the peripheral nerve is regenerating or not, suggesting transcriptional activation of CNPase mRNA and/or increased CNPase mRNA stability as a response to nerve injury. In contrast, the steady state level of CNPase protein did not increase during degeneration or regeneration suggesting either negative translational regulation of CNPase gene expression or a higher turnover of this protein in the injured peripheral nerve. Furthermore, CNPase activity dropped sharply during early degeneration and remained low in the quiescent cells of the permanently transected nerve while it increased in the regenerating nerve. The results suggest that although transcriptional or post-transcriptional regulation of CNPase gene expression is not dependant on Schwann cell-axonal contact, the activity of CNPase appears to be dependent on myelination and indirectly dependent on the presence of axons in the peripheral nerve.

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.

The Cyclic Nucleotide Phosphodiesterase Gene of Dictyostelium discoideum Contains Three Promoters Specific for Growth, Aggregation, and Late Development

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) gene plays essential roles in the development of Dictyostelium discoideum during cellular aggregation and postaggregation morphogenesis. Genomic clones spanning the gene were isolated and used to determine the sequence and structure of the phosphodiesterase gene. We found an unusually complex organization for a gene of D. discoideum. Two transcripts of 2.4 and 1.9 kilobases (kb) were synthesized from start sites separated by 1.1 kb. A developmentally regulated promoter was utilized for the 2.4-kb mRNA, and a constitutive promoter regulated synthesis of the 1.9-kb transcript. The gene was found to be divided into four exons that are alternately spliced to give rise to the two mRNAs. The precursor of the 2.4-kb mRNA contained a 2.3-kb intron, whereas the precursor of the constitutive transcript was synthesized with a 1.7-kb intron. The two transcripts contained identical protein-coding regions and 400-nucleotide 3' untranslated sequences. The 2.4-kb developmentally regulated mRNA was distinguished by a long 5' untranslated leader of 666 nucleotides. The complex structure of the gene may allow multiple levels of control of the expression of the phosphodiesterase during development.

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) gene plays essential roles in the development of Dictyostelium discoideum during cellular aggregation and postaggregation morphogenesis. Genomic clones spanning the gene were isolated and used to determine the sequence and structure of the phosphodiesterase gene. We found an unusually complex organization for a gene of D. discoideum. Two transcripts of 2.4 and 1.9 kilobases (kb) were synthesized from start sites separated by 1.1 kb. A developmentally regulated promoter was utilized for the 2.4-kb mRNA, and a constitutive promoter regulated synthesis of the 1.9-kb transcript. The gene was found to be divided into four exons that are alternately spliced to give rise to the two mRNAs. The precursor of the 2.4-kb mRNA contained a 2.3-kb intron, whereas the precursor of the constitutive transcript was synthesized with a 1.7-kb intron. The two transcripts contained identical protein-coding regions and 400-nucleotide 3' untranslated sequences. The 2.4-kb developmentally regulated mRNA was distinguished by a long 5' untranslated leader of 666 nucleotides. The complex structure of the gene may allow multiple levels of control of the expression of the phosphodiesterase during development.

The expression of two transcripts of the phosphodiesterase gene during the development of Dictyostelium discoideum

One of the earliest events in the development of Dictyostelium discoideum is the induction of the cyclic nucleotide phosphodiesterase gene. During vegetative growth a small amount of secreted phosphodiesterase is synthesized. The phosphodiesterase transcript which is responsible for the vegetative enzyme has a size of 1800 nucleotides. Soon after starvation begins a more abundant mRNA with a size of 2200 nucleotides is synthesized by the developing cells. The induction of the 2200-nucleotide mRNA is dependent on protein synthesis and takes place under all regimens of growth and starvation. When growth is in axenic medium and development is in phosphate buffer, the appearance of the larger transcript is very rapid, occurring within 30 min after the onset of starvation. The initial burst of phosphodiesterase mRNA synthesis is followed by a decline in mRNA abundance unless the cells are stimulated by cAMP. When cells are grown on bacteria and development takes place on filter paper, the larger transcript appears after 4 hr, reaches a peak at 10–12 hr of development, and then slowly disappears. When prestalk and prespore cells from migrating slugs are separated, a small amount of transcript can be found only in the prestalk cells. A series of mutants blocked early in development make very little phosphodiesterase transcript or are otherwise abnormal in expression of the phosphodiesterase mRNA. Together these mutants define five independent genetic loci which affect the accumulation of the phosphodiesterase mRNA. These are the pdsA, fgdA, fgdC, fgdD, and fgdE genes.

Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum.

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum functions to maintain the responsiveness of cells to the chemoattractant cAMP during the aggregation phase of development. We have prepared a cDNA library and have isolated clones which contain a portion of the 5' untranslated region and the entire coding and 3' untranslated portions of the cyclic nucleotide phosphodiesterase gene. The primary structure of the extracellular cyclic nucleotide phosphodiesterase precursor has been deduced from the nucleotide sequence. The molecule is composed of 452 amino acids and was calculated to have a molecular mass of 51,078 daltons. Forty-nine amino-terminal residues which contain a hydrophobic leader sequence are not present in the mature extracellular enzyme. Four potential asparagine-linked glycosylation sites were found within the phosphodiesterase. An amino acid sequence homology search revealed no closely related proteins. Phosphodiesterase mRNA levels are low in growing cells and first increase soon after the onset of development. The amount of transcript then decreases before rising in abundance to maximum levels during the terminal stages of cell aggregation and apical tip formation. During formation of the fruiting body, levels of phosphodiesterase mRNA decrease. Exposure of cells to cAMP increases the amount of phosphodiesterase mRNA. Increases of mRNA abundance are correlated with increases in enzyme activity, suggesting regulation at the level of transcription.

Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli.

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.