Abstract-Contraction and relaxation of vascular smooth muscle and cardiac myocytes are key physiological events in the cardiovascular system. These events are regulated by second messengers, cAMP and cGMP, in response to extracellular stimulants. The strength of signal transduction is controlled by intracellular cyclic nucleotide concentrations, which are determined by a balance in production and degradation of cAMP and cGMP. Degradation of cyclic nucleotides is catalyzed by 3Ј,5Ј-cyclic nucleotide phosphodiesterases (PDEs), and therefore regulation of PDEs hydrolytic activity is important for modulation of cellular functions. Mammalian PDEs are composed of 21 genes and are categorized into 11 families based on sequence homology, enzymatic properties, and sensitivity to inhibitors. PDE families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, enzymatic regulation by phosphorylation and regulatory proteins, subcellular localization, and interaction with association proteins. Each unique variant is closely related to the regulation of a specific cellular signaling. Thus, multiple PDEs function as a particular modulator of each cardiovascular function and regulate physiological homeostasis. (Circ Res. 2007;100:309-327.)

cDNA encoding a novel phosphodiesterase (PDE) was isolated from a human fetal lung cDNA library and designated PDE10A. The deduced amino acid sequence contains 779 amino acids, including a putative cGMP binding sequence in the amino-terminal portion of the molecule and a catalytic domain that is 16 -47% identical in amino acid sequence to those of other PDE families. Recombinant PDE10A transfected and expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 0.26 and 7.2 M, respectively, and V max with cGMP was almost twice that with cAMP. Of the PDE inhibitors tested, dipyridamole was most effective, with IC 50 values of 1.2 and 0.45 M for inhibition of cAMP and cGMP hydrolysis, respectively. cGMP inhibited hydrolysis of cAMP, and cAMP inhibited cGMP hydrolysis with IC 50 values of 14 and 0.39 M, respectively. Thus, PDE10A exhibited properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. PDE10A transcripts were particularly abundant in the putamen and caudate nucleus regions of brain and in thyroid and testis, and in much lower amounts in other tissues. The PDE10A gene was located on chromosome 6q26 by fluorescent in situ hybridization analysis. PDE10A represents a new member of the PDE superfamily, exhibiting unique kinetic properties and inhibitor sensitivity.Cyclic nucleotides cAMP and cGMP are well known as second messengers and regulate many functions in various tissues (1-4). Intracellular cAMP and cGMP concentrations are controlled via stimulation of adenyl and guanyl cyclases in response to extracellular signaling and their degradation by cyclic nucleotide phosphodiesterases (PDEs), 1 respectively. Many kinds of PDEs are involved in the metabolism of cyclic nucleotides. Based on their amino acid sequence homology, biochemical properties, and inhibitor profiles, seven PDE families have been recognized in mammalian tissues (5, 6). PDE1 is Ca 2ϩ / calmodulin-dependent, hydrolyzing both cAMP and cGMP. PDE2 is stimulated by cGMP and hydrolyzes cAMP and cGMP.PDE3 is cGMP-inhibited. PDE4 is cAMP-specific and rolipramsensitive. PDE5 is cGMP-specific. PDE6 is a photoreceptor cGMP PDE. PDE7 is cAMP-specific and rolipram-insensitive. Very recently, cDNAs encoding two kinds of novel PDEs were isolated from humans and mice (7-11). One is cAMP-specific (PDE8), and the other is cGMP-specific (PDE9). PDE7 and the two latter PDEs are insensitive to the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). cDNA cloning of these novel PDEs was done by an approach using bioinformatics. A search of data bases of expressed sequence tags (ESTs) was performed using parts of PDE sequences such as the catalytic domain. The approach was shown to be effective for the isolation of novel PDE cDNAs.Genome sequencing projects are progressing in many organisms, providing us information of a variety of pathways involved in the cyclic nucleotide metabolism necessary to maintain life. In Caenorhabditis elegans, which is a small soil nematode found in temperate regions, there are five pairs of autosomal chromosomes and a...

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the Nterminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP-and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 3.0 and 1.4 M, respectively, and the V max value with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K m values as PDE11A4, the relative V max values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMPand cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products. Cyclic nucleotide phosphodiesterases (PDEs)1 metabolize cAMP and cGMP, which are second messengers regulating many functions in various cells and tissues. Based on their amino acid sequence homology, biochemical properties, and inhibitor profiles, many kinds of PDEs have been identified in mammalian tissues (1-3). The PDE1 family is Ca 2ϩ /calmodulindependent and hydrolyzes both cAMP and cGMP. PDE2 is stimulated by cGMP and hydrolyzes cAMP and cGMP, while PDE3 is cGMP-inhibited. The cAMP-specific and rolipram-sensitive PDEs belong to the PDE4 family. PDE5 is a cGMPbinding, cGMP-specific PDE. The photoreceptor cGMP PDEs are in the PDE6 family. PDE7 is cAMP-specific and rolipraminsensitive. PDE8 is a cAMP-specific PDE, and PDE9 is a cGMP-specific PDE (3-8). Recently, we revealed a new member of the PDE group, PDE10A, which hydrolyzes both cAMP and cGMP (9). Some of these PDEs constitute subfamilies encoded by distinct genes. In each PDE family, alternative splice variants have been reported (1, 10, 11). In many cases, different gene products and alternative splice variants in each PDE family show different expression patterns in tissues and different subcellular localization (1,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). PDEs encoded by alternatively spliced mRNAs have been reported to differ in their regulation by some kinases including cAMP-dependent protein kinase (cAK) and cGMP-dependent kinase (cGK) and associated proteins (19, 23). Thus, cyclic nucleotide levels are controlled by a complex system. Each ...

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.

cGMP‐binding, cGMP‐specific phosphodiesterase which is encoded by the PDE5A gene plays important roles in cardiovascular system, and is a significant target molecule of therapeutic agents. However, little is known about molecular characteristics of the human PDE5A gene. The 4.4‐kb cDNA encoding human PDE5A was isolated from lung and placenta cDNA libraries. The deduced amino acid sequence analysis demonstrated that N‐terminal amino acid sequence is dissimilar to that of rat PDE5A [Kotera, J., Yanaka, N., Fujishige, K., Imai, Y., Akatsuka, H., Ishizuka, T., Kawashima, K. & Omori, K. (1997) Eur. J. Biochem. 249, 434−442]. Human PDE5A mRNA is produced in high amounts in various tissues such as pancreas, skeletal muscle, placenta, heart, thyroid, adrenal cortex, testis, small intestine and stomach. In addition, the megakaryocyte‐like cell line Dami cells and two types of human vascular smooth muscle cells also produce the mRNA. Over 100‐kb chromosomal DNA corresponding to the human PDE5A gene was isolated and analyzed. The human PDE5A gene was revealed to contain 21 exons. Comparison of genomic organization with the rod photoreceptor phosphodiesterase β‐subunit gene (PDE6B), which is another kind of cGMP‐specific phosphodiesterase, has shown that the PDE5A and PDE6B genes are very similar in their relative exon−intron organization. In particular, the evolutionary relatedness of these genes was suggested in the catalytic domain. Furthermore, chromosomal location of the PDE5A gene was defined as being chromosome 4q26 by fluorescent in situ hybridization analysis.