Daurisoline derivatives inhibit the ability of calmodulin to stimulate cyclic nucleotide phosphodiesterase activity

Daurisoline alkaloid derivatives were found to be potent calmodulin (CaM) antagonists. The ability of daurisoline derivatives to attenuate the stimulatory effect on calmodulin activated cyclic nucleotides phosphodiesterase (CaM-PDE) was studied. These compounds did not inhibit the basal activity of this enzyme. The hydrophobicity of these compounds was related to their inhibitory potency. It is suggested that such drugs bind directly to calmodulin in a Ca2(+)-dependent fashion, as indicated by their ability to change calmodulin fluorescence.     

Role of cAMP in providing for the plastic properties of the electro-excitable membrane of neurons

In isolated snail brain, the role was studied of cyclic adenosine monophosphate (cAMP) in providing plastic properties of electro-excitable neuronal membranes of two types, habituating and non-habituating to rhythmic intracellular stimulation with depolarizing electric pulses. It has been shown that at high level of cAMP in the cell maintained with administration of dibutyryl-cAMP and (or) blockaders of phosphodiesterase in incubation medium, habituating cells lose their ability of habituation to stimulation. There is also no habituation in the presence of serotonin: serotonin effect is removed by imidazol, activator of phosphodiesterase. Imidazol promotes the development of habituation of cells, initially non-habituating to stimulation. Data are obtained on connection of Ca2+ effects and cAMP metabolism in habituating cells. On the basis of the obtained data it is suggested that the cyclase system controls plastic properties of neurones of both types, and reduction of cAMP content in the cell apparently mediates the above mentioned Ca-K-mechanism of habituation.     

Methylxanthine effects on cyclic adenosine 3':5' monophosphate phosphodiesterase activity in preparations of neonatal rat cerebellum: modification by trifluoperazine

Paradoxically, caffeine was found to stimulate the activity of 3′:5′-cyclic AMP phosphodiesterase at substrate concentrations of 14μM, in cerebellar tissue from 10-day-old rats. Pretreatment with trifluoperazine, an inhibitor of calmodulin-dependent enzyme activation, converted the stimulatory effect of caffeine to the expected inhibitory action. Trifluoperazine pretreatment also increased the inhibitory action of theophylline on the cerebellar phosphodiesterase, but had no effect on the inhibitory action of 3-isobutyl-1-methylxanthine. It is suggested that caffeine and to a lesser extent theophylline in addition to their intrinsic phosphodiesterase inhibitory activity can also cause calmodulin dependent effects on cerebellar phosphodiesterase due to calcium mobilisation.     

The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.     

Studies on griseolic acid derivatives. I. Synthesis of substituted derivatives of griseolic acid at the N1, C6, C2' or C7' position and their biological activities

Griseolic acid derivatives having a different substituent at the N1,C6,C2' or C7' position of the natural product were synthesized and their structure activity relationship to cyclic nucleotide phosphodiesterase inhibitory activity was investigated.     

Chemical constituents of plants from the genus Symplocos

The chemical constituents of Symplocos genus were reviewed with 90 structures and 28 references cited. These constituents include triterpenoids, flavonoids, lignans, phenols, steroids, alkaloids, and iridoids. Triterpenoids are the dominant constituents within the genus Symplocos, some of them exhibited antiproliferative effects. Some phenolic glycoside derivatives showed inhibitory activity against snake-venom phosphodiesterase I and human nucleotide pyrophosphatase phosphodiesterase.     

Phosphodiesterase 4 inhibitors as airways smooth muscle relaxant agents: synthesis and biological activities of triazine derivatives

A series of triazine derivatives was synthesized. The compounds were evaluated for tracheal smooth muscle relaxant and type 4 phosphodiesterase inhibitory activities. A highly significant correlation was observed between the two effects. Two compounds exhibited potent relaxant activity (EC50: 17 and 24 nM) and might be useful for the treatment of asthma.     

Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.     

Participation of the estradiol receptor in the hormonal inhibition of cyclic nucleotide phosphodiesterase in the uterus

The role of estradiol receptor was studied in the inhibitory effect of hormone on the cyclic nucleotide phosphodiesterase from immature Wistar rat uterus. It was shown that the preparative separation of the enzyme and hormone receptor by ultracentrifugation in isokinetic sucrose density gradient results in a 2.5-3-fold decrease of the estradiol effect on phosphodiesterase. This effect is completely restored after adding the separated estradiol receptor to the phosphodiesterase devoid of it. The effect of estradiol on the phosphodiesterase activity depends on a degree of receptor component aggregation: the action of estradiol on the enzyme intensities after transformation of receptor into the dissociated form (4S) and removes in the presence of the receptor component associated form (8S).     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca²⁺/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca²⁺ (50 µM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 µM); the inhibitory effect was complete at 1.0 µM. Regucalcin (1.0 µM) did not have an appreciable effect on basal activity without Ca²⁺ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160–480 U/ml). However, regucalcin (1.0 µM) did not inhibit Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 µM Ca²⁺ added. Meanwhile, Cd² (25–100 µM)-induced decrease in Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 µM). The present results suggest that regucalcin can regulate Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca²⁺ in liver cells.     

Interaction of alpha adrenergic antagonists with calmodulin

Several alpha-adrenergic antagonists inhibited the activation of calmodulin-stimulated phosphodiesterase at concentrations that had little or no effect on basal phosphodiesterase activity. The most potent of these compounds were phenoxybenzamine and dibenamine (IC50 values of about 1 microM); the amino acid ergot alkaloids ergocryptine, ergocristine, ergotamine and their dihydrogenated derivatives were less potent calmodulin-inhibitors (IC50 values of 35-80 microM). The amino ergot alkaloids ergonovine and methysergide were essentially devoid of inhibitory activity. A variety of other alpha 1-antagonists (phentolamine, tolazoline and prazosin), an alpha 2-antagonist (yohimbine), alpha-agonists (norepinephrine, phenylephrine and clonidine), beta-adrenergic antagonists (propranolol and practolol) and the beta-adrenergic agonist methoxyphenamine displayed little or no anti-calmodulin activity (IC50 values greater than 300 microM). Similarly, the alkylating agents chlorambucil and mechlorethamine also failed to inhibit calmodulin activity. Phenoxybenzamine and dibenamine inhibited calmodulin activity irreversibly, whereas the inhibition caused by other alpha adrenergic blocking agents was reversible. Phenoxybenzamine inhibited calmodulin activity by binding directly to it. This binding was calcium-dependent and irreversible. The irreversible binding and inhibition of calmodulin activity by phenoxybenzamine (or dibenamine) may serve as a useful tool for studying the sites at which drugs bind to calmodulin and may also be useful for studying the distribution and turnover of calmodulin.     

6-Nitrobenzimidazole derivatives: potential phosphodiesterase inhibitors: synthesis and structure-activity relationship

6-Nitrobenzimidazole derivatives (1-30) synthesized and their phosphodiesterase inhibitory activities determined. Out of thirty tested compounds, ten showed a varying degrees of phosphodiesterase inhibition with IC(50) values between 1.5±0.043 and 294.0±16.7 μM. Compounds 30 (IC(50)=1.5±0.043 μM), 1 (IC(50)=2.4±0.049 μM), 11 (IC(50)=5.7±0.113 μM), 13 (IC(50)=6.4±0.148 μM), 14 (IC(50)=10.5±0.51 μM), 9 (IC(50)=11.49±0.08 μM), 3 (IC(50)=63.1±1.48 μM), 10 (IC(50)=120.0±4.47 μM), and 6 (IC(50)=153.2±5.6 μM) showed excellent phosphodiesterase inhibitory activity, much superior to the standard EDTA (IC(50)=274±0.007 μM), and thus are potential molecules for the development of a new class of phosphodiesterase inhibitors. A structure-activity relationship is evaluated. All compounds are characterized by spectroscopic parameters.     

Differential Regulation of Phosphoinositide Phosphodiesterase Activity in Brain Membranes by Guanine Nucleotides and Calcium

We have shown previously that calcium and guanine nucleotides stimulate the activity of a phosphoinositide (PI) phosphodiesterase in membranes from rat cerebral cortex and that their effects are additive. To understand further guanine nucleotide- and calcium-stimulated PI phosphodiesterase activity, we have investigated the pH sensitivity and effects of inhibitors on the two modes of stimulation. NaF stimulates PI hydrolysis in brain membranes with an EC50 of 2 mM and a maximal effect at 10 mM, suggesting that a guanine nucleotide binding protein can regulate PI phosphodiesterase. Neomycin inhibited guanylylimidodiphosphate (GppNHp)-stimulated PI phosphodiesterase activity in a concentration-dependent manner, with 90% inhibition at 0.3 mM. Neomycin was not as effective at inhibiting calcium-dependent PI hydrolysis (32% inhibition at 0.3 mM). Chloroquine also had a greater inhibitory effect against GppNHp-stimulated PI phosphodiesterase activity compared to calcium-dependent activity. Guanine nucleotide- and NaF-dependent activations of PI phosphodiesterase were strongly pH-dependent, with greatest stimulation observed at pH 5-6 and inhibition at more alkaline pH. Calcium-stimulated PI hydrolysis was not as sensitive to changes in pH and had a peak of activity at pH 9. Our findings of different pH optima and differential sensitivity to inhibitors suggest that calcium and guanine nucleotides may regulate PI phosphodiesterase in rat cortical membranes through independent mechanisms.     

Differential susceptibility to biological detergents of the particulate cGMP-stimulated phosphodiesterase from rat heart: preservation of the allosteric properties of the solubilized enzyme

The effects of various biological detergents on the particulate cGMP-stimulated cAMP phosphodiesterase activity from rat heart were investigated. When added to particulate fractions, anionic and non-ionic detergents diversely increased both cAMP and cGMP phosphodiesterase activities and slightly decreased the stimulatory effect of cGMP on cAMP hydrolysis whereas cationic detergents were rather inhibitory and drastically lowered the stimulatory effect of cGMP. Among the most efficient detergents, only sodium cholate was able to solubilize phosphodiesterase activity and preserve the stimulatory effect of cGMP on cAMP hydrolysis. Furthermore, the addition of glycerol significantly improved the conservation of the allosteric properties of the enzyme. Kinetic properties of the cholate-solubilized phosphodiesterase were quite identical to those of the membrane-bound enzyme.     

大黄蒽醌衍生物与环核苷酸磷酸二酯酶、钙调素相互作用的研究

大黄蒽醌衍生物是中药大黄的主要成份。该类衍生物与钙调素(calmo-dulin,CaM)依赖的磷酸二酯酶(PDE)的相互作用表明:它们可作用子钙调素。其中,大黄酸结合CaM并抑制CaM依赖的磷酸二酯酶(CaM-PDE);而大黄素、大黄酚和芦荟大黄素既刺激CaM-PDE的活力,又刺激PDE的基础活力,其作用机制尚待阐明;当有Ca~(2+)或无Ca~(2+)条件下测定时,大黄酸对PDE基础活力均无影响。表明:象其它的CaM拮抗剂一样,大黄酸能抑制钙调素依赖的PDE的活力。     

Synthesis and anti-inflammatory activity of phthalimide derivatives,designed as new thalidomide analogues

This paper describes the synthesis and anti-inflammatory activity of new N-phenyl-phthalimide sulfonamides (3a-e) and the isosters N-phenyl-phthalimide amides (4a-e), designed as hybrids of thalidomide (1) and aryl sulfonamide phosphodiesterase inhibitor (2). In these series, compound 3e (LASSBio 468), having a sulfonyl-thiomorpholine moiety, showed potent inhibitory activity on LPS-induced neutrophil recruitment with ED(50)=2.5mg kg(-1), which was correlated with its inhibitory effect on TNF-alpha level.     

Evidence that the rate of association of adenosine 3'',5''-monophosphate to its chemotactic receptor induces phosphodiesterase activity in Dictyostelium discoideum

Adenosine 3',5'-monophosphate (cyclic AMP) mediates cell aggregation in Dictyostelium discoideum. Cell aggregation is enhanced by pulses of cyclic AMP. Application of pulses of cyclic AMP to cells that were starved only for 1 h (postvegetative cells) induces enzyme activity. One of the enzymes induced by cyclic AMP pulses is phosphodiesterase. We pulsed postvegetative cells with a set of cyclic AMP derivatives that were selected according to certain conformational and physical-chemical properties, and we measured their effect on the induction of phosphodiesterase activity. The cyclic nucleotide specificity for chemotaxis in the aggregative phase was similar to the specificity for phosphodiesterase induction in the postvegetative phase. The shape of the dose-response curves shows a paradox: the activity of a derivative, when applied at receptor-saturating concentrations, is inversely related to its affinity. These results can be explained by the assumption that the response of the chemoreceptor to different cyclic AMP derivatives is proportional to the frequency of associations (rate receptor) and not to the proportion of occupied receptors (occupation receptor). The characteristics of rate receptors and occupation receptors during chemosensory transduction will be discussed.     

Some properties of phosphodiesterase from Mycobacterium smegmatis

The phosphodiesterase of Mycobacterium smegmatis; was strongly inhibited by ATP and ADP, CTP, GTP, TTP, their corresponding deoxy derivatives and deoxy ATP were inhibitory to the enzyme while the mononucleotides AMP, CMP, GMP and TMP were slightly stimulatory. Adenosine at 2.0 mM stimulated enzyme activity of 50%.