Potency and mechanism of action of E4021, a type 5 phosphodiesterase isozyme-selective inhibitor, on the photoreceptor phosphodiesterase depend on the state of activation of the enzyme.
The ability of inhibitors selective for the type 5 phosphodiesterase isozyme (PDE5) to act on the photoreceptor PDE isozyme (PDE6, the central effector enzyme for visual transduction) is poorly understood. Because PDE5 inhibitors are currently used as therapeutic agents, it is important to assess the potency and mechanism of action of this class of PDE inhibitor on PDE6. We show that E4021 (sodium 1-[6-chloro-4-(3, 4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-ca rboxylate sesquihydrate) inhibits activated PDE6 (KI = 1.7 nM) as potently as PDE5. This makes E4021 the most potent inhibitor of PDE6 discovered to date. The effectiveness of E4021 to inhibit nonactivated PDE6 (with bound inhibitory gamma subunits) is reduced 40-fold compared with the activated enzyme. Furthermore, at intermediate E4021 concentrations and high cGMP concentrations, nonactivated PDE undergoes activation of cGMP hydrolysis rather than inhibition. We demonstrate direct competition of E4021 and the gamma subunits for binding to the catalytic site. Measurements of cGMP binding to noncatalytic regulatory sites on the catalytic subunits of PDE6 rule out an allosteric effect of E4021 by direct binding to these noncatalytic sites. We conclude that E4021 is a competitive inhibitor of cGMP hydrolysis and that the gamma subunit also competes with both E4021 and substrate for catalytic site binding. An understanding of the effects of PDE5-targeted drugs on retinal PDE6 requires a knowledge of the complex interactions among substrate, drug, and inhibitory gamma subunit at the catalytic site of both nonactivated and activated forms of PDE6. (+info)
cAMP-dependent induction of PDE5 expression in murine neuroblastoma cell differentiation.
The present study demonstrates, in both hybrid NG108-15 and mouse neuroblastoma N18TG2 cells, the presence and regulation of PDE5 mRNA during cell differentiation. PDE5 cDNA probes in Northern blot analysis recognize a approximately 9 kb transcript in bovine lung as well as in mouse neuroblastoma cells. Hybridization on total RNA extracted from dibutyryl-cAMP-treated NG108-15 cells shows a 5-fold increase of PDE5 9 kb mRNA: such an increase is not observed in N18TG2 although we observed a similar increase in the enzymatic activity of both cell lines. Our data demonstrate that PDE5 gene expression can be regulated by cAMP and suggest the existence of a complex regulatory system for PDE5 activity. (+info)
Inhibition of cyclic GMP-binding cyclic GMP-specific phosphodiesterase (Type 5) by sildenafil and related compounds.
The cGMP-binding cGMP-specific phosphodiesterase (PDE5) degrades cGMP and regulates the intracellular level of cGMP in many tissues, including the smooth muscle of the corpus cavernosum of the penis. Sildenafil (Viagra), a specific PDE5 inhibitor, promotes penile erection by blocking the activity of PDE5, which causes cGMP to accumulate in the corpus cavernosum. In the present study, sildenafil, like other PDE5 inhibitors, stimulates cGMP binding to the allosteric sites of PDE5 by interacting at the catalytic site of this enzyme, but the drug does not compete with cGMP for binding at the allosteric sites. Both sildenafil and zaprinast are competitive inhibitors of PDE5, and double-inhibition analysis shows that these two inhibitors added together interact with the catalytic site of PDE5 in a mutually exclusive manner. After site-directed mutagenesis of each of 23 conserved amino acid residues in the catalytic domain of PDE5, the pattern of changes in the IC50 values for sildenafil or UK-122764 is similar to that found for zaprinast. However, among the three inhibitors, sildenafil exhibits the most similar pattern of changes in the IC50 to that found for the affinity of cGMP, implying similar interactions with the catalytic domain. This may explain in part the stronger inhibitory potency of sildenafil for wild-type PDE5 compared with the other inhibitors [sildenafil (Ki = 1 nM) > UK-122764 (Ki = 5 nM) > zaprinast (Ki = 130 nM)]. The affinity of each of these inhibitors for PDE5 is much higher than that of cGMP itself (Km = 2000 nM). It is concluded that residues such as Tyr602, His607, His643, and Asp754 may form important interactions for sildenafil in PDE5, but because these amino acids are conserved in all mammalian PDEs, the selectivity and potency of sildenafil is likely to be provided by a nonconserved residue or residues in the PDE5 catalytic domain. (+info)
Improvement of mortality by long-term E4010 treatment in monocrotaline-induced pulmonary hypertensive rats.
We investigated the effects of long-term treatment with a selective phosphodiesterase 5 inhibitor E4010, 4-(3-chloro-4methoxybenzyl)amino-1-(4-hydroxypiperidino)-6-phth alazin ecarbonitrile monohydrochloride, on the survival rate of rats with pulmonary hypertension induced by monocrotaline (MCT). After an s.c. injection of 40 mg/kg MCT (day 0), male Wistar rats of 4 weeks of age were divided into four groups. Vehicle-treated rats (control, n = 8) and MCT-treated rats (n = 32) were fed a commercial diet. E4010-treated rats were given a commercial diet containing 0.01% (E4010 0.01%, n = 32) and 0.1% (E4010 0.1%, n = 32) of E4010, respectively. At day 23, all rats in the control group and 28.1% of those in the MCT group (P <.01 versus control) were alive. Although the survival rate of E4010 0.01%-treated rats was not improved (50%) compared with MCT, those at 0.1% showed a significant difference (84. 4%, P <.01 versus MCT). For MCT rats (n = 9), right ventricle weight and the levels of plasma atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cGMP, and cyclic AMP were higher compared with control (n = 8). In E4010 0.1%-treated rats (n = 27), the right ventricular hypertrophy was suppressed, and the increase in plasma cGMP level was amplified compared with MCT without any effects on plasma ANP, BNP, and cyclic AMP levels. Accordingly, we consider that the mechanism of action of E4010 may be related to the decreased pulmonary arterial pressure caused by the augmentation of pulmonary arterial relaxation through an ANP and/or BNP-cGMP system. These results suggest that E4010 will be useful for the treatment of pulmonary hypertension. (+info)
Genomic origin and transcriptional regulation of two variants of cGMP-binding cGMP-specific phosphodiesterases.
We have reported alternative splice variants of cGMP-binding cGMP-specific phosphodiesterases (PDE5A), i.e. rat PDE5A2, human PDE5A1, canine PDE5A1 and PDE5A2, which possess distinct N-terminal sequences. In this study, the DNA sequences corresponding to the unique N-terminal portions of PDE5A1 and PDE5A2 were shown to be tandemly located upstream of exons encoding the common region of PDE5A in both human and rat PDE5A genes. The presence of human PDE5A2 and rat PDE5A1 transcripts in lung was confirmed by reverse transcriptase-PCR. These results indicated that two variant forms of PDE5A exist in humans, canines and rats. We examined the tissue distribution of the two variants of human PDE5A in adult and fetal humans. The patterns of expression of the two alternatively spliced transcripts of human PDE5A in human tissues differed. Many putative regulatory elements including cAMP response elements were observed in the 5'-untranslated region and intron of the PDE5A gene. The levels of the PDE5A transcripts, especially the PDE5A2 transcripts, were increased by a cAMP analogue in cultured rat vascular smooth muscle cells, indicating that the PDE5A2 is an inducible variant of PDE5A in rats. (+info)
Studies of the molecular mechanism of discrimination between cGMP and cAMP in the allosteric sites of the cGMP-binding cGMP-specific phosphodiesterase (PDE5).
The regulatory domain of the cGMP-binding cGMP-specific 3':5'-cyclic nucleotide phosphodiesterase (PDE5) contains two homologous segments of amino acid sequence that encode allosteric cyclic nucleotide-binding sites, referred to as site a and site b, which are highly selective for cGMP over cAMP. The possibility that the state of protonation in these sites contributes to cyclic nucleotide selectivity was investigated. The binding of cGMP or cAMP was determined using saturation and competition kinetics at pH values between 5.2 and 9.5. The total cGMP binding by PDE5 was unchanged by variation in pH, but the relative affinity for cGMP versus cAMP progressively decreased as the pH was lowered. Using site-directed mutagenesis, a conserved residue, Asp-289, in site a of PDE5 has been identified as being important for cyclic nucleotide discrimination in this site. It is proposed that deprotonation of Asp-289 enhances the number and strength of bonds formed with cGMP, while concomitantly decreasing the interactions with cAMP. (+info)
Autoregulation of nitric oxide-soluble guanylate cyclase-cyclic GMP signalling in mouse thoracic aorta.
1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity. (+info)
Impaired cerebral vasodilator responses to NO and PDE V inhibition after subarachnoid hemorrhage.
Subarachnoid hemorrhage (SAH) is associated with impaired nitric oxide (NO)-mediated cerebral vasodilatation. We tested the hypothesis that SAH causes alterations in the production of, hydrolysis of, or responsiveness to cGMP in the rat basilar artery in vivo. Rats were injected with saline or autologous blood into the cisterna magna. Two days later, effects of vasoactive drugs on basilar artery diameter were examined using a cranial window preparation. Vasodilator responses to ACh, sodium nitroprusside (SNP), and low concentrations (+info)