cAMP analogs and selective inhibitors used to study low Km Mucor rouxii cAMP phosphodiesterase.

1. The sensitivity of partially purified low Km phosphodiesterase (PDE) from Mucor rouxii to pharmacological agents and cAMP analogs was studied. The IC50 obtained were compared with those reported for PDEs from higher eukaryotes. 2. The best inhibitors of the hydrolysis of 1 microM cAMP were SQ 65.442 (IC50 c 10 microM), dipyridamol and CI 930. cGMP was not an inhibitor (IC50 greater than 1000 microM). 3. The cAMP analogs were tested as inhibitors of the hydrolysis of 0.1 microM cAMP. 8-Aminohexylamino cAMP was the best inhibitor with an IC50 of c 1 microM. 4. A sedimentation profile of Mucor PDE was assayed in the presence of several pharmacological inhibitors and cAMP analogs. No isoforms with different sensitivity towards the inhibitors were detected. Forms with slightly different behaviour towards some cAMP analogs were observed.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Cyclic AMP-dependent Cl Secretion Is Regulated by Multiple Phosphodiesterase Subtypes in Human Colonic Epithelial Cells

The role of phosphodiesterase (PDE) isoforms in regulation of transepithelial Cl secretion was investigated using cultured monolayers of T84 cells grown on membrane filters. Identification of the major PDE isoforms present in these cells was determined using ion exchange chromatography in combination with biochemical assays for cGMP and cAMP hydrolysis. The most abundant PDE isoform in these cells was PDE4 accounting for 70-80% of the total cAMP hydrolysis within the cytosolic and membrane fractions from these cells. The PDE3 isoform was also identified in both cytosolic and membrane fractions accounting for 20% of the total cAMP hydrolysis in the cytosolic fraction and 15-30% of the total cAMP hydrolysis observed in the membrane fraction. A large portion of the total cGMP hydrolysis detected in cytosolic and membrane fractions of T84 cells was mediated by PDE5 (50-75%). Treatment of confluent monolayers of T84 cells with various PDE inhibitors produced significant increases in short-circuit current (Isc). The PDE3-selective inhibitors terqinsin, milrinone and cilostamide produced increases in Isc with EC50 values of 0.6 nM, 8.0 nM and 0.5 microM respectively. These values were in close agreement with the IC50 values for cAMP hydrolysis. The effects of the PDE1-(8-MM-IBMX) and PDE4-(RP-73401) selective inhibitors on Isc were significantly less potent than PDE3 inhibitors with EC50 values of >7 microM and >50 microM respectively. However, the effects of 8-MM-IBMX and terqinsin on Cl secretion were additive, suggesting that inhibition of PDE1 also increases Cl secretion. The effect of PDE inhibitors on Isc were significantly blocked by apical treatment with glibenclamide (an inhibitor of the CFTR Cl channel) and by basolateral bumetanide, an inhibitor of Na-K-2Cl cotransport activity. These results indicate that inhibition of PDE activity in T84 cells stimulates transepithelial Cl secretion and that PDE1 and PDE3 are involved in regulating the rate of secretion.     

cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties]

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.     

Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum

Inhibition of cGMP-specific phosphodiesterase type V (PDE5) has been shown to improve penile erection in patients with erectile dysfunction. We report here the cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had been isolated from nonpenile tissues. The third isoform was novel and hence called PDE5A3. The deduced amino acid sequence of PDE5A3 was the same as the C-terminal 823-residue sequence of PDE5A1 and PDE5A2. While PDE5A1 and A2 isoforms were expressed in all tissues examined, the A3 isoform was confined to tissues with a smooth muscle or cardiac muscle component. When expressed in COS-7 cells, PDE5A1, A2, and A3 isoforms had similar cGMP-catalytic activities with K(m) of 6.2, 5.75, and 6.06 microM, respectively. Their cGMP-catalytic activities were inhibited by zaprinast with IC(50) values of 3.2 microM, 1.3 microM, and 1.6 microM, respectively, and by sildenafil with IC(50) of 28, 14, and 13 nM, respectively.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

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.

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.     

Isolation and characterization of PDE10A, a novel human 3', 5'-cyclic nucleotide phosphodiesterase.

A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized. PDE10A1 encodes a protein that is 779 amino acids in length. An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated. The proteins encoded by the two variants share 766 amino acids in common. This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs. Northern analysis revealed that PDE10A is widely expressed. The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27. A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast. It hydrolyzed both cAMP and cGMP. Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX.     

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.     

Identification of substrate specificity determinants in human cAMP-specific phosphodiesterase 4A by single-point mutagenesis

To identify amino acids that might be involved in discriminating guanosine-3',5'-cyclic phosphate (cGMP) towards adenosine-3',5'-cyclic phosphate (cAMP) binding in the cAMP-specific phosphodiesterases, alignments of different human cyclic nucleotide phosphodiesterases (PDEs) were performed. Eight amino acid residues that are highly conserved in the cAMP-hydrolysing phosphodiesterases (PDE1, PDE3, PDE4, PDE7, PDE8) and that did not show any homologies to the cGMP-specific phosphodiesterases (PDE5, PDE6, PDE9) were selected from these alignments. Using the technique of site-directed mutagenesis, derivatives of PDE4A carrying single mutations at these conserved residues (amino acid positions are given according to the human PDE4A isoform HSPDE4A4B; accession number L20965) were generated and expressed in COS1 cells. The expression products were characterised with regard to cAMP and cGMP hydrolysis and sensitivity towards type-specific inhibitors. The mutation of Phe484 toward Tyr, Ala590 toward Cys, Leu391 and Val501 towards Ala had no significant influence on substrate affinity or specificity. However, the exchange of Trp375 and Trp605 for aliphatic residues abolished catalytic activity and the exchange of Pro595 for Ile led to sevenfold decrease of substrate affinity and an 14-fold decrease of the affinity towards the PDE4-specific inhibitor 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram). Both effects may provide evidence for a structural importance of Trp375, Trp605 and Pro595 for PDE function. By exchanging the aspartate residue for asparagine or alanine at position 440 of the human PDE4A4B isoform, the substrate specificity was altered from the highly specific cAMP hydrolysis to an equally efficient cAMP and cGMP binding and hydrolysis. In addition, the IC(50) values for common PDE4-specific inhibitors like rolipram, N-(3,5-dichlorpyrid-4-yl)-3-cyclopentyl-oxy-4-methoxy-benzamide (RPR-73401) and 8-methoxy-5-N-propyl-3-methyl-1-ethyl-imidazo[1,5-a]-pyrido[3,2-e]-pyrazinone (D-22888) were dramatically increased. These results demonstrate an important role of the aspartate at position 440 in determining substrate specificity and inhibitor susceptibility of PDE4A. The strong conservation of this residue suggests that Asp440 may play a similar role in other cAMP-PDEs.     

Inhibition of cyclic GMP hydrolysis in human corpus cavernosum smooth muscle cells by vardenafil, a novel, selective phosphodiesterase type 5 inhibitor.

One of the key mediators of penile erectile function is nitric oxide (NO), which activates soluble guanylyl cyclase within the smooth muscle of erectile tissue and stimulates the production of cGMP. In addition to synthesis by cyclases, intracellular cGMP concentrations are tightly regulated by phosphodiesterases, which hydrolyze and inactivate cyclic nucleotides. In this study, we compared the inhibition of cGMP hydrolysis by vardenafil and sildenafil; two inhibitors selective for phosphodiesterase type 5 (PDE5). Vardenafil is a novel, high affinity PDE5 inhibitor currently under clinical development. In soluble extracts of human corpus cavernosum smooth muscle cells, vardenafil and sildenafil effectively inhibited cGMP hydrolysis at substrate concentrations of 1, 5 and 10 microM cGMP. The IC50 values for vardenafil were approximately 5-fold lower than for sildenafil at the substrate concentrations tested. Dixon plot analyses of the inhibition data demonstrated that vardenafil had a smaller inhibition constant (Ki = 4.5 nM) than sildenafil (Ki = 14.7 nM) in the same cellular extracts. In intact cells, 10 microM of the nitric oxide donor sodium nitroprusside resulted in a minimal (17%) increase in cGMP, relative to basal levels (321 +/- 65 fmol/mg prot). Treatment of cells with 10, 50 or 100 nM vardenafil, in the presence of 10 microM sodium nitroprusside, elevated cGMP levels in a dose dependent fashion, from 63% to 137% of basal levels. Equimolar concentrations of sildenafil also caused dose dependent increases in intracellular cGMP, but to a lesser extent (27-60%). These observations suggest that vardenafil is a more potent PDE5 inhibitor, than sildenafil in vitro. The more pronounced increase of cGMP in the presence of NO in intact cells suggests that vardenafil will be effective at lower doses than sildenafil under clinical conditions.     

Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.     

Expression and characterization of deletion recombinants of two cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE-3)

cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed inEscherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (<1 μM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with [³²P]-cGMP. The IC₅₀ values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP. These authors contributed equally to this work.     

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.     

The calcium/calmodulin-dependent phosphodiesterase PDE1C down-regulates glucose-induced insulin secretion.

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion.