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.     

Aminobenzoic acid derivatives as specific inhibitors of cyclic nucleotide phosphodiesterase in the rat uterus

It is shown, that p-aminobenzoic acid and its derivatives (p-acetylaminobenzoic acid and p-aminobenzoic acid hydrazide) in the concentration of 10(-6) M are the potent inhibitors (40% below the control specimens) of the phosphodiesterase activity of cyclic nucleotides in the soluble fraction of the adult rat uterus. These drugs exerted no action on the adenylate cyclase activity in membrane fractions. The inhibition is only specific to the uterus enzyme and is not revealed for other tissues. The inhibition is found to be of incompetitive character Ki for p-aminobenzoic acid hidrazide being equal to 3.2 microM.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of metabolic effects of growth hormone by various inhibitors of cyclic nucleotide phosphodiesterase

When isolated diaphragms of hypophysectomized rats were incubated with bovine growth hormone in the presence of the cyclic nucleotide inhibitors theophylline, quinine and papaverine, the stimulatory effects of the hormone on leucine incorporation into protein, α-aminoisobutyric acid and 3-O-methylglucose transport were suppressed or abolished entirely. The degree of suppression of the hormone effects appeared to correlate with the extent of glycogenolysis caused by the drugs. Thoephylline also rapidly reversed the stimulation of protein synthesis and amino acid and sugar transport produced by growth hormone. When protein synthesis and transport were stimulated by preincubation of the diaphragm with growth hormone, the subsequent addition of theophylline to the medium inhibited the hormonal effects on protein synthesis and sugar transport within 15 min and the effect on amino acid transport within 60 min. These results may mean that the rapid in vitro effects of growth hormone on protein synthesis and membrane transport in rat diaphragm muscle are mediated by a reduction in the cellular level of cyclic AMP or some other nucleotide.Attempts to block the action of growth hormone on 3-O-methylglucose transport by preincubation of the diaphragm with high concentrations (10 mM) of cyclic GMP, cyclic UMP, cyclic TMP and cyclic CMP were unsuccessful. Also an effort was made to mimic the action of growth hormone on sugar transport by incubating the diaphragm with high concentrations of imidazole and histamine, agents known to activate cyclic nucleotide phosphodiesterase. Slight stimulatory effects were obtained, but they could not be correlated with any certainty to the actions of imidazole and histamine on phosphodiesterase.Like growth hormone, insulin also stimulates protein synthesis and amino acid and sugar transport in the isolated rat diaphragm. However, the actions of insulin on these processes were not abolished by theophylline, suggesting some basic difference in the mode of action of these two hormones on protein synthesis and membrane transport in muscle.     

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.     

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.     

Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.     

Inhibition of cyclic nucleotide phosphodiesterase by carrot phytoalexin

The carrot phytoalexin, 6-methoxymellein, was isolated and purified from carrot root slices infected by the fungus Chaetomium globosum. It inhibited the basal and calmodulin-promoted activity of cyclic nucleotide phosphodiesterase. The inhibition of calmodulin-promoted diesterase activity was reduced by increasing the concentration of calmodulin or calcium while the inhibition of basal diesterase activity was reversed by the addition of magnesium to the assay mixture of the enzyme.     

Characterization of soluble uterine cyclic nucleotide phosphodiesterase.

Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.     

Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.     

Sequential changes in the cyclic nucleotide levels and cyclic nucleotide phosphodiesterase activities during development ofMyxococcus xanthus

The levels of adenosine 3′,5′-monophosphate, guanosine 3′-5′-monophosphate and the activities of their respective phosphodiesterases exhibited changes during development ofMyxococcus xanthus that are substantially consistent with role postulated for each in a previously proposed model.     

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca²⁺-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca²⁺-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca²⁺ and CDR. In subcellular fractionation experiments both the CDR and the Ca²⁺-dependent phosphodiesterase were predominantly located in the 100,000g supernatant fraction.The apparent K_m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μm, respectively, when CDR was not rate limiting. Minor increases in the apparent K_m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μm Ca²⁺ for the hydrolysis of 25 μm cGMP and by 8 μm Ca²⁺ for the hydrolysis of 25 μm cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca²⁺. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.     

Agar plate screening procedure for cyclic adenosine 3',5'-monophosphate and inhibitors of cyclic nucleotide phosphodiesterase.

A procedure is described for the semiquantitative measurement of cyclic adenosine 3',5'-monophosphate (cAMP) and detection of inhibitors of cAMP phosphodiesterase by an agar plate test. The assay organism was an adenyl cyclase-deficient mutant derived from Escherichia coli HfrH. In the presence of an acid base indicator, acid production from barbohydrate metabolism was observed as a yellow zone around filter paper disks containing cAMP. Since yellow zone formation reflects the presence of cAMP, a phosphodiesterase inhibitor can be detected indirectly by the presence of a yellow zone on assay plates from a reaction mixture of an inhibitor, phosphodiesterase, and cAMP. Three known cyclic nucleotide phosphodiesterase inhibitors were active against beef brain phosphodiesterase in this system.     

Agar plate screening procedure for cyclic adenosine 3',5'-monophosphate and inhibitors of cyclic nucleotide phosphodiesterase.

A procedure is described for the semiquantitative measurement of cyclic adenosine 3',5'-monophosphate (cAMP) and detection of inhibitors of cAMP phosphodiesterase by an agar plate test. The assay organism was an adenyl cyclase-deficient mutant derived from Escherichia coli HfrH. In the presence of an acid base indicator, acid production from barbohydrate metabolism was observed as a yellow zone around filter paper disks containing cAMP. Since yellow zone formation reflects the presence of cAMP, a phosphodiesterase inhibitor can be detected indirectly by the presence of a yellow zone on assay plates from a reaction mixture of an inhibitor, phosphodiesterase, and cAMP. Three known cyclic nucleotide phosphodiesterase inhibitors were active against beef brain phosphodiesterase in this system.     

cGMP-dependent cyclic nucleotide phosphodiesterase from human lymphoid cells

The kinetic properties of cyclic nucleotide phosphodiesterase isolated from the cytoplasmic fraction of lymphoblastoma QOS cells were studied. It was demonstrated that the enzyme can be activated in the presence of micromolar concentrations of cGMP. The kinetic properties of the enzyme are characterized by the nonlinear dependence of the cAMP hydrolysis rate on the substrate concentration. The curve becomes linear in the presence of cGMP. The molecular mass of phosphodiesterase as determined from gel filtration data is 80,0000 Da.     

The effect of proteolysis on the calmodulin activation of cyclic nucleotide phosphodiesterase

A high Km cytoplasmic cyclic nucleotide phosphodiesterase (EC 3.4.1.17) has been obtained from bovine brain. The unproteolyzed enzyme contains (63 +/- 1) X 10(3) molecular weight polypeptide chains which exhibit little if any basal catalytic activity. Complexation with calmodulin stimulates the catalytic activity nearly 2 orders of magnitude, presumably, by causing a conformational change in the enzyme which either creates or exposes the catalytic sites. Removal of about 120 amino acids from the terminal portion(s) of each polypeptide chain either by an endogenous protease or by exogenous trypsin prevents calmodulin complexation and generates a basal catalytic activity equivalent to that of the unproteolyzed enzyme-calmodulin complex. In contrast to affinity chromatography using immobilized calmodulin, blue dextran-Sepharose chromatography can be used to select for enzyme containing only unproteolyzed polypeptide chains.