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Query: UNIPROT:P06889 (Mol)
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Phosphodiesterase type-5 (PDE-5) inhibitors including sildenafil, vardenafil and tadalafil are a new class of vasoactive drugs that have been developed for treatment of erectile dysfunction in patients. A growing number of studies in recent years suggest that sildenafil may be used clinically for treatment of pulmonary hypertension and endothelial dysfunction. In addition, recent studies primarily from our laboratory suggested that sildenafil has preconditioning-like powerful cardioprotective effect in the animal models of ischemia-reperfusion injury. Sildenafil has been found to exert cardioprotection through nitric oxide generated from endothelial and/or inducible nitric oxide synthases and opening of mitochondrial ATP-sensitive potassium channels. Future demonstration of the cardioprotective effect in patients with the relatively safe and effective FDA-approved PDE-5 inhibitors, such as sildenafil, could have an enormous impact on bringing the long-studied phenomena of ischemic and pharmacologic preconditioning to the clinical forefront.
J Mol Cell Cardiol 2004 Feb
PMID:Cardioprotection with phosphodiesterase-5 inhibition--a novel preconditioning strategy. 1487 43

Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation.
Mol Reprod Dev 2004 Apr
PMID:Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation. 1499 41

Phosphodiesterase catalyzes the hydrolysis of the intracellular second messenger 3',5'-cyclic AMP (cAMP) into the corresponding 5'-nucleotide. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and COPD. We have determined crystal structures of the catalytic domain of PDE4B complexed with AMP (2.0 A), 8-Br-AMP (2.13 A) and the potent inhibitor rolipram (2.0 A). All the ligands bind in the same hydrophobic pocket and can interact directly with the active site metal ions. The identity of these metal ions was examined using X-ray anomalous difference data. The structure of the AMP complex confirms the location of the catalytic site and allowed us to speculate about the detailed mechanism of catalysis. The high-resolution structures provided the experimental insight into the nucleotide selectivity of phosphodiesterase. 8-Br-AMP binds in the syn conformation to the enzyme and demonstrates an alternative nucleotide-binding mode. Rolipram occupies much of the AMP-binding site and forms two hydrogen bonds with Gln443 similar to the nucleotides.
J Mol Biol 2004 Mar 19
PMID:Crystal structures of the catalytic domain of phosphodiesterase 4B complexed with AMP, 8-Br-AMP, and rolipram. 1500 52

Plasma membranes were isolated from 3T3-L1 adipocytes. Plasma membrane phosphodiesterase (PM-PDE) was measured in the presence of 5 microM cilostamide. Time course and cAMP dose response ranging from 0 to 2 microM were measured. PM-PDE remained linear up to 20 min. Non-linear curve fitting analysis showed that the low Km cAMP dose data fit a two component curve significantly better than a one component curve, indicating that there are two iso-forms of PDE in the plasma membrane of 3T3-L1 adipocytes, similar to swine adipocytes. The Km and Vmax values for this two component curve were Km1=0.12 microM, Vmax1=3.08 pmol min(-1) mg(-1) protein, and Km2=3.67 microM, Vmax2=83.8 pmol min(-1) mg(-1) protein. Inhibitors of PDE1, PDE2 and PDE5 failed to inhibit PM-PDE, as observed in swine adipocyte plasma membranes. However, PDE4 inhibitors were three-fold more effective at inhibiting PDE in 3T3-L1 PM compared to swine adipocyte PM. One mM 1, 3-dipropyl-8-p-sulfophenylxanthine (DPSPX) inhibited PM-PDE by approximately 75% in both preparations. These data demonstrate that PM-PDE is distinct from microsomal membrane PDE and may be responsible for extracellular cAMP metabolism to AMP in 3T3-L1 adipocytes.
Comp Biochem Physiol B Biochem Mol Biol 2004 Mar
PMID:Plasma membrane-bound cyclic AMP phosphodiesterase activity in 3T3-L1 adipocytes. 1505 May 18

Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs.
Mol Carcinog 2004 Jun
PMID:Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-kappaB in mouse epidermal cl41 cells. 1517 Aug 15

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC --> TA, GC --> AT, etc.) principally via its major adduct [+ta]-B[a]P-N2-dG. Recent findings suggest that different lesion bypass DNA polymerases may be involved in different mutagenic pathways, which is the subject of this report. [+ta]-B[a]P-N2-dG built into a plasmid in a 5'-TGT sequence gives approximately equal numbers of G --> T and G --> A mutations when host E. coli are UV irradiated prior to transformation, so this sequence context was chosen to investigate what DNA polymerases are involved in G --> T versus G --> A mutations. G --> T mutations decline (>10-fold) if E. coli either are not UV-irradiated or are deficient in DNA polymerase V ((delta)umuD/C), demonstrating a role for damage-inducible DNA Pol V in a G --> T pathway. G --> T mutations are not affected by transformation into E. coli deficient in either DNA polymerases II or IV. While the work herein was in progress, Lenne-Samuel et al. [Mol. Microbiol. 38 (2000) 299] built the same adduct into a plasmid in a 5'-GGA sequence, and showed that the frequency of G --> T mutations was similar in UV-irradiated and unirradiated host E. coli cells, suggesting no involvement by damage-inducible, lesion bypass DNA polymerases (i.e., not II, IV or V); furthermore, a role for DNA Pol V was explicitly ruled out. The easiest way to reconcile the findings of Lenne-Samuel et al. with the findings herein is if two G --> T mutagenic pathways exist for [+ta]-B[a]P-N2-dG, where sequence context dictates which pathway is followed. In contrast to the G --> T mutations, herein G --> A mutations from [+ta]-B[a]P-N2-dG in the 5'-TGT sequence context are shown not to be affected by UV-irradiation of host E. coli, and are not dependent on DNA Pol V, or Pol II, Pol IV, or the damage-inducible, but SOS-independent UVM system. Published studies, however, have shown that G --> A mutations are usually enhanced by UV-irradiation of host E. coli prior to the introduction of plasmids either site-specifically modified with [+ta]-B[a]P-N2-dG or randomly adducted with (+)-anti-B[a]PDE; both findings imply the involvement of a lesion-bypass DNA polymerase. These disparate results suggest the existence of two G --> A mutagenic pathways for [+ta]-B[a]P-N2-dG as well, although confirmation of this awaits further study. In conclusion, a comparison between the evidence presented herein and published findings suggests the existence of two distinct mutagenic pathways for both G --> T and G --> A mutations from [+ta]-B[a]P-N2-dG, where in each case one pathway is not damage-inducible and not dependent on a lesion-bypass DNA polymerase, while the second pathway is damage-inducible and dependent on a lesion-bypass DNA polymerase. Furthermore, DNA sequence context appears to dictate which pathway (as defined by the involvement of different DNA polymerases) is followed in each case.
 ...
PMID:A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in E. coli. 1517 47

The molecular mechanisms underlying the regulation of vas deferens (VD) motility and semen emission are still poorly understood. We now report evidence on VD expression of phosphodiesterase type 5 (PDE5), which regulates nitric oxide (NO)-induced relaxation and cGMP breakdown in smooth muscle cells. In human VD, the PDE5 abundance was relatively high (>3 x 10(6) molecules/microg total RNA), although 10-fold lower than in corpora cavernosa (CC). Also cGMP metabolising activity was higher in CC than in VD. However, both tissues share the same sensitivity to a broad panel of cGMP-related PDE inhibitors: sildenafil, tadalafil, dipyridamole, zaprinast, vinpocetine, EHNA and cilostamide. Based on the rank order of potency of these PDE inhibitors, we found that the cGMP metabolizing activity in human VD mostly corresponds to PDE5. PDE5 was immunolocalized in all the muscular layers of human and rabbit VD and was found to be negatively involved in regulating NO-induced relaxation. In addition, by using a rabbit model of hypogonadotropic hypogonadism, we found that PDE5 gene expression and activity are androgen-dependent in VD, as previously demonstrated in CC. In fact, the sensitivity to a NO-donor (NCX4040), its enhancement by PDE5 inhibitors and the PDE5-related cGMP breakdown were all affected by androgen manipulation. Our results provide a hypothesis explaining the beneficial effects of PDE inhibitors in patients with rapid ejaculation.
Mol Hum Reprod 2005 Feb
PMID:Expression and functional activity of phosphodiesterase type 5 in human and rabbit vas deferens. 1564 Apr 38

It is well known that CpG dinucleotide steps in DNA, which are highly methylated at the 5-position of cytosine (meC) in human tissues, exhibit a disproportionate number of mutations within certain codons of the p53 gene. There is ample published evidence indicating that the reactivity of guanine with anti-B[a]PDE (a metabolite of the environmental carcinogen benzo[a]pyrene) at CpG mutation hot spots is enhanced by the methylation of the cytosine residue flanking the target guanine residue on the 5'-side. In this work we demonstrate that such a methylation can also dramatically affect the conformational characteristics of an adduct derived from the reaction of one of the two enantiomers of anti-B[a]PDE with the exocyclic amino group of guanine ([BP]G adduct). A detailed NMR study indicates that the 10R (-)-trans-anti-[BP]G adduct undergoes a transition from a minor groove-binding alignment of the aromatic BP ring system in the unmethylated C-[BP]G sequence context, to an intercalative BP alignment with a concomitant displacement of the modified guanine residue into the minor groove in the methylated meC-[BP]G sequence context. By contrast, a minor groove-binding alignment was observed for the stereoisomeric 10S (+)-trans-anti-[BP]G adduct in both the C-[BP]G and meC-[BP]G sequence contexts. This remarkable conformational switch resulting from the presence of a single methyl group at the 5-position of the cytosine residue flanking the lesion on the 5'-side, is attributed to the hydrophobic effect of the methyl group that can stabilize intercalated adduct conformations in an adduct stereochemistry-dependent manner. Such conformational differences in methylated and unmethylated CpG sequences may be significant because of potential alterations in the cellular processing of the [BP]G adducts by DNA transcription, replication, and repair enzymes.
J Mol Biol 2005 Mar 04
PMID:Methylation of cytosine at C5 in a CpG sequence context causes a conformational switch of a benzo[a]pyrene diol epoxide-N2-guanine adduct in DNA from a minor groove alignment to intercalation with base displacement. 1570 9

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), a member of the 2H phosphoesterase superfamily, is firmly bound to brain white matter and found mainly in the central nervous system of vertebrates, and it catalyzes the hydrolysis of 2',3'-cyclic nucleotide to produce 2'-nucleotide. Recent studies on CNP-knockout mice have revealed that the absence of CNP causes axonal swelling and neuronal degeneration. Here, the crystal structure of the catalytic fragment (CF) of human CNP (hCNP-CF) is solved at 1.8A resolution. It is an alpha+beta type structure consisting of three alpha-helices and nine beta-strands. The structural core of the molecule is comprised of two topologically equivalent three-stranded antiparallel beta-sheets that are related by a pseudo 2-fold symmetry. Each beta-sheet contains an H-X-T-X motif, which is strictly conserved among members of the 2H phosphoesterase superfamily. The phosphate ion is bound to the side-chains of His and Thr from each of the two motifs. Structural comparison of hCNP-CF with plant 1'',2''-cyclic nucleotide phosphodiesterase (CPDase) and bacterial 2'-5' RNA ligase reveals that the H-X-T-X motifs are structurally conserved among these enzymes, but the surface properties of the active site are quite different among the enzymes, reflecting the differences in their substrates. On the basis of the present crystal structure of the hCNP-CF/phosphate complex, the available structure of the CPDase/cyclic-nucleotide analogue complex, and the recent functional studies of rat CNP-CF, we propose a possible substrate-binding mode and catalytic mechanism of CNP, which employs the nucleophilic water molecule activated by His310. The proposed mechanism is basically equivalent to the second step of the well-accepted reaction mechanism of RNase A. Since the overall structure of hCNP-CF differs considerably from that of RNase A, it is likely that the similar active sites with two catalytic histidine residues in these enzymes arose through convergent evolution.
J Mol Biol 2005 Feb 25
PMID:Crystal structure of the catalytic fragment of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase. 1571 63

Sustained increases in intracellular cGMP concentrations ([cGMP]i) inhibit cell growth and induce apoptosis. We now report that a cGMP-specific phosphodiesterase, PDE5, plays a dominant role in regulating [cGMP]i transitions that inhibit cell growth and control susceptibility to apoptosis in pulmonary endothelium. Atrial natriuretic peptide (ANP) activates guanylyl cyclase A/B and induces a rapid [cGMP]i rise 2-5 min after its application, in both pulmonary arterial endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs). However, increased [cGMP]i in PAECs is transient and decays within 10 min due to cytosolic PDE5 hydrolytic activity. Increased [cGMP]i in PMVECs is sustained for >3 h due to the absence of PDE5. Indeed, at any ANP concentration, the sustained (30 min) [cGMP]i rise is greater in PMVECs than in PAECs, unless PAECs are also treated with the PDE5 inhibitor zaprinast. Using RT-PCR, Western blot analysis, immunoprecipitation, and DEAE chromatography, we resolved the expression and activity of PDE 5A1/A2 only in PAECs. Similarly, PDE5 expression was restricted to extra-alveolar endothelium in vivo. ANP induced growth inhibition and apoptosis in PMVECs, but similar effects were not seen in PAECs unless ANP treatment was combined with zaprinast. ANP blocked the VEGF-induced proliferation and migration in PMVECs. Collectively, these data suggest that PDE5-regulated [cGMP]i controls endothelial cell growth and apoptosis, representing a mechanism of heterogeneity between two endothelial phenotypes.
Am J Physiol Lung Cell Mol Physiol 2005 Aug
PMID:Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells. 1579 63


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