Cyclic Nucleotide Phosphodiesterase Activity Research Articles

Pharmacological and biochemical effects of the cardiotonic agent Org10325 in isolated cardiac and vascular tissue preparations

1. The pharmacological and biochemical effects of a novel cardiotonic agent, Org10325 have been studied in isolated cardiac and vascular tissue preparations. 2. Org10325 produced concentration-dependent (0.15-4.8 mM) positive inotropic, positive chronotropic and vascular relaxant responses in rabbit isolated papillary, atrial and aortic preparations, respectively. The maximal chronotropic effect (45%) was significantly less than the isoprenaline maximum. The inotropic effects of Org10325 were not modified by alpha- or beta-adrenoceptor blockade or by pretreatment with reserpine. Org10325 was at least 23 times more potent at relaxing aortic strips pre-contracted with phenylephrine than with KCl. 3. Org10325 (74 microM) potentiated (10-14 fold) the positive inotropic effects of isoprenaline in rabbit isolated papillary muscles. Carbachol inhibited the positive inotropic effect of Org10325. Both the positive inotropic and vasorelaxant effects of Org10325 were accompanied by increases in cyclic AMP but not cyclic GMP. 4. In rat perfused heart preparation Org10325 increased phosphorylase a, cyclic AMP-dependent protein kinase activities and stimulated phosphorylation of contractile proteins (troponin-I and C-protein). 5. Org10325 selectively inhibited the cyclic AMP hydrolytic activity of cyclic AMP high affinity cyclic nucleotide phosphodiesterase (PDE) isoenzymes, PDE III (IC50 65 microM) and PDE IV (IC50 71 microM), from rabbit cardiac ventricle. Weak inhibition (IC50 greater than 250 microM) of PDE I and PDE II was observed. 6. The results show that the cardiac and vascular effects of Org10325 are mediated by an increase in cellular cyclic AMP due to inhibition of PDE III and PDE IV activities. However, in contrast to other PDE-inhibitors OrglO325 produced a marked increase in relaxation time of isolated papillary muscle suggesting the involvement of additional cyclic AMP-independent mechanisms of action.

Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney

Homogenization of rat kidney under isotonic conditions and in the presence of protease inhibitors showed that some 92% of the cyclic AMP phosphodiesterase activity and some 83% of the cyclic GMP phosphodiesterase activity was released into the soluble fraction. Analysis of soluble phosphodiesterase activity by FPLC on a Mono-Q column resolved four distinct fractions expressing cyclic nucleotide phosphodiesterase activity. Lineweaver-Burk plots for the hydrolysis of both cyclic GMP and cyclic AMP yielded linear results. The first two peaks (KPDE-MQ-II, KPDE-MQ-III) showed higher activities towards cyclic GMP than cyclic AMP with the ratio of their V max values for the hydrolysis of cyclic AMP/cyclic GMP being 0.66 and 0.16, respectively. For the second two peaks (KPDE-MQ-IV, KPDE-MQ-V) the V max ratios for the hydrolysis of cyclic AMP/cyclic GMP were 6.4 and 16.7, respectively. All enzymes exhibited similar low K m values for both cyclic AMP and cyclic GMP but had very different V max values. KPDE-MQ-II was activated by Ca 2+/calmodulin. The cyclic AMP phosphodiesterase activity of KPDE-MQ-III was augmented by the presence of low concentrations of cyclic GMP. Thermal denaturation studies showed that the phosphodiesterase activity of each fraction decayed as a single exponential indicating that each phosphodiesterase fraction contained but a single phosphodiesterase activity. The inhibitors IBMX, zaprinast, milrinone, amrinone, buquineran, carbazeran, ICI 118233, ICI 63197 exerted selective effects on the activities of these enzymes. We compared the action of these compounds on cyclic GMP phosphodiesterases from bovine retina. Over the concentration ranges used, the bovine retinal enzyme was only inhibited by IBMX, zaprinast and carbazeran. The cytosolic isoenzymes of cyclic AMP phosphodiesterases play a much more important role in metabolizing cyclic AMP in kidney compared with liver, where the activity of membrane-bound isoenzymes predominate.

Phospholipid metabolism modulates cyclic nucleotide phosphodiesterase activity in rat heart microsomes

Cyclic nucleotide phosphodiesterase activity in rat heart microsomes is attributable to several isoenzymatic forms: a cyclic AMP-specific, a cyclic GMP-specific, and a cyclic GMP-stimulated enzyme. Incubation of microsomes with an exogenous phospholipase C (C. welchii) induced a marked stimulation (+126%) of cyclic AMP phosphodiesterase and a moderate stimulation (+49%) of cyclic GMP-phosphodiesterase in the membrane-bound fraction. Besides, a notable fraction of activity was solubilized by the treatment. A parallel decrease in the activating effect of cyclic GMP on the hydrolysis of cyclic AMP was observed in the membranes (down to 18% of the control effect). It resulted from a marked stimulation of the basal activity, while the activated level was unaffected. The treatment by an exogenous phospholipase D induced more moderate modifications. The addition to microsomes of oleyl,acetyl-glycerol, but not of long chain-diacylglycerols, partly reproduced the phospholipase C effect. Phosphatidate also induced variations in phosphodiesterase activity, and could thus participate in the phospholipase effects. These results suggest that endogenous phospholipases, the activity of which is modulated by hormonal stimuli, might influence phosphodiesterase activity in cardiac membranes by producing phospholipid metabolites, with potential consequences on heart contractility.

Cyclic nucleotide phosphodiesterase and glutathione peroxydase activities in heart of rats fed diets containing different oils

Cyclic nucleotide phosphodiesterase and glutathione peroxydase activities in heart of rats fed diets containing different oils

Prostaglandin E2 inhibition of growth in a colony-stimulating factor 1-dependent macrophage cell line.

The effects of prostaglandin E2 (PGE2) were examined in a murine macrophage cell line (BAC1.2F5) that was completely dependent on colony-stimulating factor-1 (CSF-1) for both growth and survival. The addition of PGE2 to cultures of BAC1.2F5 cells resulted in the inhibition of CSF-1-induced [3H]thymidine incorporation and cell proliferation. The inhibitory effects of PGE2 were mimicked by the addition of dibutyryl-cyclic AMP, and the effectiveness of PGE2 was markedly potentiated by 1-methyl-3-isobutylxanthine, a potent inhibitor of cyclic nucleotide phosphodiesterase activity. PGE2 caused a 10-fold elevation of the intracellular cyclic AMP concentration, whereas CSF-1 neither increased cyclic AMP levels nor attenuated the rise in cyclic AMP promoted by PGE2. However, CSF-1 may indirectly regulate cyclic AMP levels since in the absence of CSF-1, BAC1.2F5 cells actively synthesized PGE2, whereas PGE2 production was abruptly terminated by the addition of CSF-1. In BAC1.2F5 cells, PGE2 increases the intracellular cyclic AMP concentration, thereby blocking cell proliferation, but does not down-regulate the CSF-1 receptor or abrogate the functions of CSF-1 necessary for cell survival.

Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases. Use of phosphodiesterase inhibitors to evaluate their roles in regulating cyclic nucleotide levels in intact cells

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.

Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells

Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 μM), the major hydrolytic activity was for cAMP while at a high concentration (20 μM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.

The 3′,5′-cyclic nucleotide phosphodiesterases of meristematic and differentiated tissues of pea roots

Cyclic nucleotide phosphodiesterase activity was separately extracted from meristematic and differentiated regions of pea root tips. Meristematic tissue yielded two main cyclic nucleotide phosphodiesterase activities essentially similar to their counterparts in the differentiated tissue. One of the activities (50–75% satn ammonium sulphate fraction) hydrolysed cyclic AMP, cyclic GMP and cyclic CMP, with a preference for the latter. The other activity (75–100% satn ammonium sulphate fraction) was more specific; it showed a preference for cyclic CMP but was devoid of activity towards cyclic GMP. Partial purification of the first enzyme was achieved by gel-filtration chromatography on Sephadex G-200. The second enzymic activity was separated by affinity chromatography on Affi-Gel Blue into two fractions, a non-retained peak (A) and a retained peak (B). Peak B was × 10 more active towards cyclic CMP than was peak A and there was insufficient of the latter activity for further investigation. The peak B enzyme exhibited low storage stability; its K m and V max were, respectively, 0.5 mM and 122 nmol min −1 mg −1 protein. Preincubation with Fe 3+, Mg 2+ and Ca 2+ gave small but reproducible stimulation. EDTA inactivated it but the effect could be alleviated synergistically by Ca 2+ and Fe 3+. Together, these two cations stimulated the non-inhibited enzyme by 73%. It was not stimulated by calmodulin in the presence of Ca 2+, nor was it inhibited by calmidazolium. The results indicate that of the two main enzymic activities found in pea root tips, one is similar to the multifunctional cyclic nueleotide phosphodiesterase of pig liver, and the other, which fails to hydrolyse cyclic GMP, is unlike any previously found in plant or animal tissues. Theophylline (2 mM) inhibited the peak B enzyme by 88% and root growth by 60%; cytokinins also inhibited both the enzyme and the growth of pea roots.

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 Ca 2+-dependent fashion, as indicated by their ability to change calmodulin fluorescence.

Cyclic AMP links amino acid chemoreceptors to ion channels in olfactory cilia

Amino acids, olfactory stimuli for the channel catfish (Ictalurus punctatus), were tested for the ability to affect cyclic AMP metabolism in isolated olfactory cilia. Ten stimuli, representative of each receptor subtype and covering a wide range of electrophysiological potency, elicited similar increases in guanine nucleotide-dependent cyclic AMP formation. Stimulus-dependent activation of adenylate cyclase was observed only when receptor occupancy approached or exceeded 50%. Stimulus activation of adenylate cyclase was additive with that obtained with guanine nucleotide alone, was independent of receptor specificity, and poorly correlated with neural potency. Stimuli did not affect cyclic nucleotide phosphodiesterase activity in isolated cilia preparations. Both cyclic AMP and cyclic GMP reversibly increased membrane conductance in isolated ciliary membranes incorporated in artificial phospholipid bilayers. The cyclic nucleotide-gated conductance was activated by either nucleotide with equal potency, did not require exogenous ATP or GTP, and was mediated by 44 pS non-selective cation channels. Taken together, these results suggest that, under appropriate conditions of receptor occupancy, cyclic AMP links amino acid chemoreceptor binding to membrane depolarization by directly gating cation channels in olfactory cilia.

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.

Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes, from liver and hepatocytes, identifies a novel IBMX-insensitive form

DEAE chromatography of a high speed supernatant fraction from a homogenate of rat liver, prepared under isotonic conditions in the presence of protease inhibitors, yielded three peaks of cyclic nucleotide phosphodiesterase activity (PDE activity). The first peak could be resolved on Affi-gel Blue chromatography to yield a Ca 2+/calmodulin stimulated cyclic GMP specific PDE and a cyclic AMP and cyclic GMP hydrolysing PDE whose activity was insensitive to Ca 2+/calmodulin. These two activities could also be clearly resolved by Mono-Q chromatography of soluble extracts from both liver and hepatocytes. These had different molecular weights, kinetics of substrate utilization, thermostabilities, dependence on Mg 2+ and inhibitor sensitivities. The cyclic AMP and cyclic GMP utilizing PDE resolved in these procedures appears to be a novel enzyme form (PDE-MQ-I) which is insensitive to inhibition by the so-called non-selective PDE inhibitor IBMX and displays catalytic activity in the absence of Mg 2+. None of the inhibitors tested were capable of inhibiting this form showing that the catalytic activity of this species could be distinguished from all the other soluble activities. This novel enzyme hydrolysed both cyclic AMP and cyclic GMP with K m values of 25 μM and 237 μM, respectively. The V max ratio of hydrolysis of cyclic GMP/cyclic AMP was above unity (1.4). It accounted for 30% of the soluble cyclic AMP PDE activity and 10% of the cyclic GMP PDE activity assessed at 1 μM substrate. Gel filtration of PDE-MQ-I indicated a size of 33,150 Da, in contrast to the size of 237,500 Da observed for the Ca 2+/calmodulin PDE-MQ-II. Thermal inactivation of PDE-MQ-I and PDE-MQ-II yielded single exponential decays with t 1 2 values of 6.33 min and 0.7 min at 60° respectively. In the presence of saturating Ca 2+, PDE-MQ-II was activated by calmodulin with an EC 50 of ca. 30 ng/ml. In the presence of calmodulin, PDE-MQ-II was activated by Ca 2+ with an EC 50 of ca. 20 μM. Chromatography of homogenates on Mono-Q also identified a cyclic GMP-activated cyclic nucleotide PDE (PDE-MQ-III) and two cyclic AMP specific activities (PDE-MQ-IV and PDE-MQ-V). These exhibited very different inhibitor sensitivities and could be readily distinguished using the compound Ro-20-1724 which yielded IC 50 values for inhibition of >500 μM, 13 μM and 1.5 μM, respectively, for the hepatocyte enzymes. PDE-MQ-III to -V exhibited K m values for the hydrolysis of cyclic AMP of 37.7 μM, 0.62 μM and 0.62 μM and Hill coefficients of 1.62, 0.69 and 1.01, respectively. Rolipram was found to potently inhibit both the cyclic AMP specific forms. The compound ICI 118233 failed to inhibit any of the isolated soluble phosphodiesterases. A similar multiplicity of phosphodiesterase species was observed in the soluble fraction from both rat liver homogenates and isolated hepatocytes. We suggest that care be taken in interpreting experiments performed to assess adenylate cyclase activity in intact cells by monitoring intracellular cyclic AMP accumulation in the presence of IBMX due to the possible presence of an IBMX-insensitive phosphodiesterase.

Ribonuclease in tomato vacuoles: high-performance liquid chromatographic analysis of ribonucleolytic activities and base specificity

We have developed isocratic high-performance liquid chromatographic techniques for assaying monomeric products of action of ribonucleolytic enzymes using RNA and diribonucleoside monophosphates as substrates. The methods allow detection of phosphodiesterase, phosphotransferase, cyclic nucleotide phosphodiesterase and nucleotidase activities. In addition to a classification of ribonucleases under study, quantitative measurements can be used to assess enzyme specificity with respect to bases adjacent to the cleavage site. The methods have been used to characterize a tomato vacuolar ribonuclease. The enzyme splits phosphoester bonds by a phosphotransferase reaction yielding 2′,3′-cyclic nucleotide monophosphates. Only the respective purine nucleotides were further decyclized to the 3′-nucleoside monophosphates with 2′,3′-cGMP as the best substrate. Analysis of products of yeast RNA hydrolysis revealed that the enzyme splits preferentially adjacent to purine residues, in particular to guanine ( G≫A=U>C). The same order of base preference was essentially found using diribonucleoside monophosphates as substrates ( G≫A>U>C and G>U=C>A for bases in 5′ and 3′ position, respectively).

Effect of emoxypin on basal cyclic nucleotide phosphodiesterase activity and late receptor potential of the isolated retina

Effect of emoxypin on basal cyclic nucleotide phosphodiesterase activity and late receptor potential of the isolated retina

Effect of non-ionic detergent on the activity of dog heart cyclic-nucleotide phosphodiesterases

Effect of non-ionic detergent on the activity of dog heart cyclic-nucleotide phosphodiesterases

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low K m cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,l-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC 50 = 17 μM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.

Quantitation by fast‐atom bombardment/mass‐analysed ion kinetic energy spectrometry: Kinetic analysis of cyclic nucleotide phosphodiesterase activity

Quantitation of cyclic nucleotide phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Characteristic peaks of the substrate, cyclic AMP, and product, AMP, were identified in positive-ion FAB mass spectra and MIKE scans of the protonated molecules. By spiking enzyme incubates with known quantities of cyclic AMP and AMP and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the concentrations of cyclic AMP and AMP in solution at the end of a series of enzyme incubations have been estimated. From the data obtained the Km and Vmax of the enzymes were calculated as 181 microM and 28.6 nmol/min respectively, showing excellent agreement with values of the Michaelis constant, Km = 205 microM and the maximum velocity Vmax = 33.2 nmol/min obtained by radioactive assay.

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin

Triacontanol (TRIA) treatment of plasma membrane‐enriched vesicles from barley (Hordeum vulgare L., cv. Conquest) roots resulted in stimulation of membrane‐associated, divalent cation‐dependent ATPase activity (EC 3.6.1.3). The stimulation at physiologically active concentrations of TRIA (10−11–10−9M) occurred only when the vesicles were treated with TRIA in the presence of calmodulin. Octacosanol, the C28‐analogue of TRIA, had no effect on divalent cation‐dependent ATPase activity. Consistent with in vivo studies, simultaneous treatment of vesicles with weight equivalents of TRIA and octacosanol reduced the stimulation of ATPase activity. The effect of calmodulin on the stimulation of ATPase activity was diminished by calmidazolium, a specific inhibitor of calmodulin. Circular dichroism studies did not show a change in the α‐helix content of calmodulin in the presence of TRIA. TRIA also had no apparent effect on soluble calcium‐calmodulin 3′,5′‐cyclic nucleotide phosphodiesterase activity. Removal of excess TRIA from the medium after treatment still resulted in stimulation of divalent cation‐dependent ATPase activity in the presence of calmodulin was comparable to treated vesicles from which excess TRIA had not been removed. These data further support the contention that TRIA affects membrane structure and function.

Effect of phospholipase C treatment on the activity of the particulate cyclic nucleotide phosphodiesterase of rat brain

1. 1. The activity of cAMP phosphodiesterase (PDE) was studied in a 10,000 g particulate fraction prepared from rat brain. 2. 2. Phospholipase C such as sphingomyelin choline phosphodiesterase (SMase), phosphatidylinositol phosphodiesterase (PIase) and phosphatidylcholine phosphohydrolase (PCase) were used to deplete phospholipid(s) from the particulate fraction and their effects on PDE activity were investigated. 3. 3. Treatment with SMase or Plase did not affect PDE activity whereas treatment with PCase resulted in inhibition. 4. 4. It was also found that the PCase used not only hydrolysed phosphatidylcholine but also other phospholipids such as phosphatidylethanolamine, phosphatidylserine and sphingomyelin.

Benzylisoquinoline compounds inhibit the ability of calmodulin to activate cyclic nucleotide phosphodiesterase

Benzylisoquinoline compounds antagonised the ability of calmodulin (CaM) to stimulate the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE). This ‘anti-CaM’ activity was related to the hydrophobicity of the non-polar terminal region of the antagonist molecule. Antagonistic potency increased with the increase of hydrophobicity; the anti-CaM activity did not change when the polar terminus was a tertiary amine or quarternary amine. The anti-CaM potency was greater for bisbenzylisoquinoline compounds than for monobenzylisoquinoline compounds. Among the bidbenzylisoquinoline compounds anti-CaM pathway was: D 3 > D 2 berbamine > daurisoline > dauriune. Compound D 3, which exhibited an IC 50 value of 2.8 μM, was one of the most potent calmodulin antagonists, among benzylisoquinoline compounds, so far reported.