Cyclic nucleotide phosphodiesterase PDE1C1 in human cardiac myocytes

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.     

Isoforms of cyclic nucleotide phosphodiesterase PDE3 and their contribution to cAMP hydrolytic activity in subcellular fractions of human myocardium

Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.     

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) splice variants from bovine cardiac muscle

Calmodulin-dependent cyclic nucleotide phopshodiesterase (PDE1) has been extensively characterized and is a key enzyme involved in the complex interaction between cyclic nucleotide and Ca(2+) second-messenger systems. It is well established that PDE1 exists in different isozymes. For example, bovine brain tissue has two PDE1 isozymes (PDE1A2 and PDE1B1) whereas only one form (PDE1A1) is reported in bovine cardiac tissue. In this study, we report the cloning of two cDNA splice variants of PDE1: PDE1-small and PDE1-large, from bovine cardiac tissue. Their amino acid sequence similarity to PDE1 sequences from other mammalian species showed that all are very conserved, suggesting their importance in cellular functions. Interestingly, compared to other mammalian species, bovine PDE1A, PDE-small and PDE-large show a deletion at the C-terminal end of the catalytic domain of the gene. Although the significance of this deletion at this crucial location of the gene is not known, we have successfully over-expressed both PDE1-small and PDE1-large splice variants in E. coli and these splice variants are characterized in terms of Western blot, biotinylated calmodulin overlay and peptide mass fingerprinting. Results from these studies suggested that these two splice variants belong to the PDE1 superfamily. To our knowledge, this is the first report on cloning and characterization of these cDNA variants from bovine cardiac tissue. Since there are at least two isoforms of PDE1 in bovine heart tissue, this merits further in-depth study.     

Functions of the N-terminal region of cyclic nucleotide phosphodiesterase 3 (PDE 3) isoforms

The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain ( approximately 50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-Delta189, MPDE3B, and M3B-Delta196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta321 and M3B-Delta302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-Delta397 and H3A-Delta457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-Delta510 and M3B-Delta604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-Delta196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-Delta607, H3A-Delta721, and M3B-Delta604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.     

Plasma membrane cyclic nucleotide phosphodiesterase 3B (PDE3B) is associated with caveolae in primary adipocytes

Caveolae, plasma membrane invaginations particularly abundant in adipocytes, have been suggested to be important in organizing insulin signalling. Insulin-induced activation of the membrane bound cAMP degrading enzyme, phosphodiesterase 3B (PDE3B) is a key step in insulin-mediated inhibition of lipolysis and is also involved in the regulation of insulin-mediated glucose uptake and lipogenesis in adipocytes. The aim of this work was to evaluate whether PDE3B is associated with caveolae. Subcellular fractionation of primary rat and mouse adipocytes demonstrated the presence of PDE3B in endoplasmic reticulum and plasma membrane fractions. The plasma membrane PDE3B was further analyzed by detergent treatment at 4 degrees C, which did not solubilize PDE3B, indicating an association of PDE3B with lipid rafts. Detergent-treated plasma membranes were studied using Superose-6 chromatography which demonstrated co-elution of PDE3B with caveolae and lipid raft markers (caveolin-1, flotillin-1 and cholesterol) at a Mw of >4000 kDa. On sucrose density gradient centrifugation of sonicated plasma membranes, a method known to enrich caveolae, PDE3B co-migrated with the caveolae markers. Immunoprecipitation of caveolin-1 using anti caveolin-1 antibodies co-immunoprecipitated PDE3B and immunoprecipitation of flag-PDE3B from adipocytes infected with a flag-PDE3B adenovirus resulted in co-immunoprecipitation of caveolin-1. Studies on adipocytes with disrupted caveolae, using either caveolin-1 deficient mice or treatment of adipocytes with methyl-beta-cyclodextrin, reduced the membrane associated PDE3B activity. Furthermore, inhibition of PDE3 in primary rat adipocytes resulted in reduced insulin stimulated glucose transporter-4 translocation to caveolae, isolated by immunoprecipitation using caveolin-1 antibodies. Thus, PDE3B, a key enzyme in insulin signalling, appears to be associated with caveolae in adipocytes and this localization seems to be functionally important.     

Regulation by insulin of cyclic nucleotide phosphodiesterase (PDE) from rat luteal cells

The effect of insulin on cyclic nucleotide phosphodiesterase (PDE) in rat luteal cells was studied. Cells were obtained from PMSG/hCG primed rats and further incubated or not with insulin. The hormone produced an increase of enzyme activity after a 10 min incubation of intact cells. Maximal stimulation was achieved at 0.2 nM of insulin. Two peaks of cyclic nucleotide phosphodiesterase activity were resolved after chromatography of cell cytosolic extracts on DEAE-cellulose. These peaks (I and II) were active with cAMP as substrate but only peak I was active with cGMP. The enzyme activity of both peaks was increased in cells treated with insulin. Phosphodiesterase activity in the two peaks show two kinetic components for cAMP hydrolysis, one of high affinity (Km 2-4 microM) and the other of low affinity (47-56 microM). Treatment of the cells with insulin produced a 2 to 8 fold increase of the Vmax of these peaks. In addition after stimulation with insulin, the activation of peak I phosphodiesterase by calmodulin was less effective.     

Calmodulin-dependent cyclic nucleotide phosphodiesterase in an experimental rat model of cardiac ischemia-reperfusion

In the present study, we investigated the activity and expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) and the effects of calpains in rat heart after ischemia and reperfusion. Immunohistochemical studies indicated that CaMPDE in normal heart is localized in myocardial cells. Rat ischemic heart showed a decrease in CaMPDE activity in the presence of Ca2+ and calmodulin; however, in ischemic-reperfusion tissue a progressive increase in Ca2+ and calmodulin-independent cyclic nucleotide phosphodiesterase (CaM-independent PDE) activity was observed. Perfusion of hearts with cell-permeable calpain inhibitor suppressed the increase of Ca2+ and CaM-independent PDE activity. Protein expression of CaMPDE was uneffected by hypoxic injury to rat myocardium. The purified heart CaMPDE was proteolyzed by calpains into a 45 kDa immunoreactive fragment in vitro. Based on these results, we propose that hypoxic injury to rat myocardium results in the generation of CaM-independent PDE by calpain mediated proteolysis, allowing the maintenance of cAMP concentrations within the physiological range.     

Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei

The identity and location of nuclear cyclic nucleotide phosphodiesterases (PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fractions from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensitive to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the cardiac nuclear envelopes (NE) were similar to those measured in the corresponding intact nuclei, thus suggesting that most PDE activity is associated with the nuclear membrane. Moreover, the main hydrolytic activities in cardiac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC(50), K(m) and K(i) values was typical of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-stimulated PDE) represented a minor component (8-9%) of total hydrolytic activity. Solubilization of nuclear envelopes and HPLC separation also yielded rolipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the presence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enriched, P1 pellet. In addition, Western blot analysis demonstrated the presence of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. However, in the same preparations, the presence of PDE4A could not be ascertained. Altogether, these results suggest an intrinsic and predominant association of these nuclear PDEs with the NE and much likely with nucleopore complexes.     

Identification of rat cyclic nucleotide phosphodiesterase 11A (PDE11A): comparison of rat and human PDE11A splicing variants.

We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual-substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino-acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue-specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS-7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3-1.6 and 2.1-3.9 microM, respectively), the Vmax values differed significantly (RNPDE11A4 > RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 > HSPDE11A2 > HSPDE11A3 > HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants.     

A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP

The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis.     

A FYVE-containing unusual cyclic nucleotide phosphodiesterase from Trypanosoma cruzi

Cyclic-nucleotide-specific phosphodiesterases (PDEs) are key players in the intracellular signaling pathways of the important human pathogen Trypanosoma cruzi. We report herein the identification of an unusual PDE from this protozoal organism. This enzyme, TcrPDEC, is a member of the class I PDEs, as determined from the presence of a characteristic signature sequence and from the conservation of a number of functionally important amino acid residues within its catalytic domain. Class I PDEs include a large number of PDEs from eukaryotes, among them all 11 human PDE families. Unusually for an enzyme of this class, TcrPDEC contains a FYVE-type domain in its N-terminal region, followed by two closely spaced coiled-coil domains. Its catalytic domain is located in the middle of the polypeptide chain, whereas all other class I enzymes contain their catalytic domains in their C-terminal parts. TcrPDEC can complement a PDE-deficient yeast strain. Unexpectedly for a kinetoplastid PDE, TcrPDEC is a dual-specificity PDE that accepts both cAMP and cGMP as its substrates.     

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.     

Visual detection of AMP and real-time monitoring of cyclic nucleotide phosphodiesterase (PDE) activity in neutral aqueous solution. Chemosensor-coupled assay of PDE and PDE inhibitors

A phosphate chemosensor ensemble, [Zn(H-bpmp)(pyrocatechol violet)](+) is useful for a real-time assay of phosphodiesterases (PDEs) in a neutral aqueous solution. In addition, a simple and convenient screen and assay procedures for inhibitors of PDEs have been demonstrated using IBMX, a nonselective PDE inhibitor as an example.     

Proteolytic activation of calmodulin-dependent cyclic nucleotide phosphodiesterase

Purified calmodulin-stimulated cyclic nucleotide phosphodiesterase from brain, a homodimer of 59-kDa subunits, was activated by limited proteolysis with trypsin, alpha-chymotrypsin, Pronase, or papain and could not be further stimulated by addition of Ca2+ and calmodulin. Proteolysis increased Vmax and had little effect on the Km for cGMP. Treatment with alpha-chymotrypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) produced, sequentially, 57- and 45-kDa peptides from the bovine and 55-, 53-, and 38-kDa peptides from the ovine enzyme. This protease-treated phosphodiesterase exhibited a Stokes radius of 3.9 nm and an S20,w value of 4.55; comparison with the hydrodynamic properties observed for native enzyme (4.3 nm, 5.95 S) strongly suggests a dimeric protein of Mr approximately 80,000-90,000. The proteolyzed species does not interact significantly with calmodulin immobilized on agarose, nor does it show complex formation with 2-dimethylaminonaphthalene-1-sulfonyl-calmodulin even at micromolar concentrations of protein. Proteolysis, in the presence of calmodulin plus Ca2+, fully activated phosphodiesterase, producing the same intermediate peptides; however, final peptides from the bovine and ovine enzymes were 47 and 42 kDa, respectively, indicating a new, specific conformation of the enzyme. When EGTA was added to such incubations, these peptides were cleaved to those of the size seen when proteolysis was carried out entirely in the presence of EGTA. The initial rate of activation was increased by the presence of Ca2+ and calmodulin, suggesting that, in complex, phosphodiesterase exhibits a site with increased susceptibility to proteolysis. Since calmodulin can still interact with a fully activated form of the enzyme, it appears that retention of calmodulin binding can occur concomitantly with damage to that portion of the phosphodiesterase molecule responsible for suppression of its basal catalytic activity.     

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.     

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.     

Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca²⁺-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.