Forms Of Cyclic Nucleotide Phosphodiesterase Research Articles

Vascular activity of (-)-anonaine, (-)-roemerine and (-)-pukateine, three natural 6a(R)-1,2-methylenedioxyaporphines with different affinities for alpha1-adrenoceptor subtypes.

We have studied the mechanism of action of three 6a( R)-1,2-methylenedioxyaporphines as vasorelaxant compounds. The alkaloids assayed showed different affinities for the three human cloned alpha (1)-adrenoceptor (AR) subtypes stably expressed in rat-1 fibroblasts, showing lower affinity for alpha(1B)-AR with regard to the alpha(1A)- or alpha(1D)-subtypes. These three natural compounds are more potent inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. As all these alkaloids inhibited noradrenaline (NA)-induced [ (3)H]-inositol phosphate formation in cerebral cortex and rat tail artery, they may be safely viewed as alpha (1)-AR antagonists, as is demonstrated by the vasorelaxant responses observed in isolated rat tail artery and/or aorta precontracted with NA. The alkaloids also inhibited the contractile response evoked by KCl (80 mM) but with a lower potency than that shown against NA-induced contraction. We have also examined their ability to inhibit the different forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aortic smooth muscle and endothelial cells, with negative results. We conclude that N-methylation favours the interaction of (R)-aporphines with all alpha (1)-AR subtypes, and that the topography of the binding site recognizing the basic or protonated nitrogen atom is similar in all three alpha (1)-AR subtypes. The presence of a hydroxy group at C-11 has different effects on the affinity for each alpha (1)-AR subtype but decreases the affinity for Ca (2+) channels. These results confirm and extend the view that subtle changes in the hydroxylation patterns on the aromatic ring of the aporphine structure affect the interactions of these compounds with the three alpha (1)-AR subtypes in different ways, suggesting that the binding site recognizing the aporphine skeleton is different in each of the three subtypes.

Crystal Structures of the Semireduced and Inhibitor-bound Forms of Cyclic Nucleotide Phosphodiesterase from Arabidopsis thaliana

The crystal structure of the semireduced form of cyclic nucleotide phosphodiesterase (CPDase) from Arabidopsis thaliana has been solved by molecular replacement and refined at the resolution of 1.8 A. We have previously reported the crystal structure of the native form of this enzyme, whose main target is ADP-ribose 1",2"-cyclic phosphate, a product of the tRNA splicing reaction. CPDase possesses six cysteine residues, four of which are involved in forming two intra-molecular disulfide bridges. One of these bridges, between Cys-104 and Cys-110, is opened in the semireduced CPDase, whereas the other remains intact. This change of the redox state leads to a conformational rearrangement in the loop covering the active site of the protein. While the native structure shows this partially disordered loop in a coil conformation, in the semireduced enzyme the N-terminal lobe of this loop winds up and elongates the preceding alpha-helix. The semireduced state of CPDase also enabled co-crystallization with a putative inhibitor of its enzymatic activity, 2',3'-cyclic uridine vanadate. The ligand is bound within the active site, and the mode of binding is in agreement with the previously proposed enzymatic mechanism. Selected biophysical properties of the oxidized and the semireduced CPDase are also discussed.

Ipopamine treatment increases death rate in patients with severe heart failure

The goal of this study was to assess the relationship between the positive inotropic response to high concentrations of the vasodilators flosequinan and BTS 53 554 (the sulfone metabolite of flosequinan) and the effect of both compounds on different forms of cyclic nucleotide phosphodiesterase. In addition, the relationship between inotropic activity and phosphodiesterase inhibition for the cardiotonic milrinone was also evaluated. All three agents exerted a positive inotropic effect on human cardiac muscle fibers. The concentration of milrinone required to increase cardiac contractility was comparable to the concentration required to inhibit the milrinone-sensitive subclass of cyclic AMP-specific phosphodiesterase (type III phosphodiesterase). However, no such relationship was observed for flosequinan and BTS 53 554. These results suggest that the cardiac response to high concentrations of flosequinan and BTS 53 554 is not mediated by inhibition of type III phosphodiesterase.

Inhibition of calmodulin-dependent phosphodiesterase induces apoptosis in human leukemic cells.

Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca2+-calmodulin dependent PDE (PDE1) and cAMP-specific PDE (PDE4). In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity [Epstein, P. M., Moraski, S., Jr., and Hachisu, R. (1987) Biochem. J. 243, 533-539]. Using reverse transcription-polymerase chain reaction (RT-PCR), we show that the mRNA encoding the 63-kDa form of PDE1 (PDE1B1) is expressed in RPMI-8392 cells, but not in normal, resting HPBL. This mRNA is, however, induced in HPBL following mitogenic stimulation by phytohemagglutinin (PHA). Also using RT-PCR, the full open reading frame for human PDE1B1 cDNA was cloned from RPMI-8392 cells and it encodes a protein of 536 amino acids with 96% identity to bovine, rat, and mouse species. RT-PCR also identifies the presence of PDE1B1 in other human lymphoblastoid and leukemic cell lines of B- (RPMI-1788, Daudi) and T-(MOLT-4, NA, Jurkat) cell origin. Inhibition of PDE1 or PDE4 activity by selective inhibitors induced RPMI-8392 cells, as well as the other cell lines, to undergo apoptosis. Culture of RPMI-8392 cells with an 18-bp phosphorothioate antisense oligodeoxynucleotide, targeted against the translation initiation region of the RPMI-8392 mRNA, led to a specific reduction in the amount of PDE1B1 mRNA after 1 day, and its disappearance after 2 days, and induced apoptosis in these cells in a sequence specific manner. This suggests that PDEs, particularly PDE1B1, because its expression is selective, may be useful targets for inducing the death of leukemic cells.

Relationship between inotropic activity and phosphodiesterase inhibition for flosequinan and milrinone

The goal of this study was to assess the relationship between the positive inotropic response to high concentrations of the vasodilators flosequinan and BTS 53 554 (the sulfone metabolite of flosequinan) and the effect of both compounds on different forms of cyclic nucleotide phosphodiesterase. In addition, the relationship between inotropic activity and phosphodiesterase inhibition for the cardiotonic milrinone was also evaluated. All three agents exerted a positive inotropic effect on human cardiac muscle fibers. The concentration of milrinone required to increase cardiac contractility was comparable to the concentration required to inhibit the milrinone-sensitive subclass of cyclic AMP-specific phosphodiesterase (type III phosphodiesterase). However, no such relationship was observed for flosequinan and BTS 53 554. These results suggest that the cardiac response to high concentrations of flosequinan and BTS 53 554 is not mediated by inhibition of type III phosphodiesterase.

Investigations of the dual contractile/relaxant properties showed by antioquine in rat aorta

1. In the present study we assessed the activity of antioquine, a bisbenzyltetrahydroisoquinoline alkaloid isolated from Pseudoxandra sclerocarpa, by examining its effects on the contractile activity of rat isolated aorta, specific binding of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin to cerebral cortical membranes and the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta. 2. Contractions in rat aorta induced by high concentrations of KCl (80 mM) and noradrenaline (1 microM) were inhibited by antioquine in a concentration-dependent manner (0.1 microM- 300 microM). The alkaloid appeared more potent against KCl-induced contractions. This inhibitory effect was observed at both 37 degrees C and 25 degrees C. 3. Paradoxically, at the highest concentration tested (300 microM) antioquine induced a contractile response of similar magnitude in the presence and absence of extracellular calcium, at 37 degrees C. This activity was greatly attenuated at 25 degrees C. Antioquine-induced contractions were not inhibited by prazosin (0.1 microM), nifedipine (1 microM) or diltiazem (100 microM). On the contrary, prazosin and nifedipine slightly increased the contractions in the presence of extracellular calcium. Papaverine (100 microM) partially inhibited the contractile response to antioquine both in the presence and absence of extracellular calcium. 4. At 25 degrees C, in Ca(2+)-free solution, antioquine (300 microM) did not modify the contractile response (phasic and tonic) evoked by noradrenaline, but increased the phasic contraction induced by caffeine. At 37 degrees C, the contraction elicited by antioquine made it impossible to observe the noradrenaline-induced one. 5. Antioquine showed affinity for the [3H]-prazosin binding site and for the [3H]-(+)-cis-diltiazembinding site of the Ca2+-channel receptor complex, but had no effect at the dihydropyridine binding site in rat cerebral cortex.6. Antioquine weakly inhibited some PDE forms isolated from bovine aorta: a CaM-PDE (PDE I)which preferentially hydrolyzes cyclic GMP and is activated by calmodulin, and a rolipram-sensitive cyclic AMP-PDE (PDE IV) which hydrolyzed cyclic AMP. Antioquine did not exert any inhibitory effect on the other forms of PDE, a cyclic GMP selective form (PDE V) and a low Km cyclic AMP-PDEthat is inhibited by cyclic GMP (CGI-PDE, PDE III).7. The present work provides evidence that antioquine has properties both as a calcium entry blocker(possibly through the benzothiazepine recognition site in the calcium channel) and as a contractile agent.Its mechanism of action as a contractile agent is not related to Ca2+-entry and is hypothetically similar to that of calyculin-A or okadaic acid. The possible involvement of a-adrenoceptors in this paradoxical effect cannot be excluded. The rigidity of the molecule provides an interesting model for analyzing this contractile mechanism and the intracellular processes involved.

Chemotactic sorting to cAMP in the multicellular stages of Dictyostelium development.

Dictyostelium transformants that overproduce the extracellular form of cyclic nucleotide phosphodiesterase and so accumulate a reduced amount of cAMP are blocked in development after aggregation in the form of a tight mound, prior to formation of the apical tip. In such mounds, prespore cell differentiation is repressed, and the apical accumulation of prestalk cells is greatly retarded. When a source of cAMP is placed below the arrested mounds, prestalk cells that would normally migrate in an apical direction instead sort downwards to the substratum. Thus, by acting as the chemoattractant that draws prestalk cells to the apex, cAMP signaling directs the formation of a patterned structure.

Multiple actions of glaucine on cyclic nucleotide phosphodiesterases, alpha 1-adrenoceptor and benzothiazepine binding site at the calcium channel.

1. In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of alpha-adrenoceptors, by use of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. Contraction evoked by noradrenaline (1 microM) or depolarizing solution (60 mM KCl) were inhibited in a concentration-dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA-induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl-induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically. 3. In Ca(2+)-free solution, prazosin (0.1 microM) and glaucine (0.1 mM) inhibited the contraction evoked by NA; diltiazem (0.1 mM) diminished this contraction whereas nifedipine (1 microM) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mM) significantly inhibited the contractions evoked by NA or caffeine in Ca(2+)-free medium. 4. Glaucine and papaverine show affinity at the [3H]-prazosin binding site and at the benzothiazepine binding site of the Ca(2+)-channel receptor complex, but have no effect at the dihydropyridine binding site in rat cerebral cortex. Glaucine exerts some selectivity as an inhibitor of [3H]-prazosin binding as opposed to [3H]-(+ )-cis-diltiazem binding while papaverine appears to have approximately equal affinity in this respect.5. This study confirms the presence of four phosphodiesterase (PDE) activities in bovine aorta: a calmodulin-activated PDE (CaM-PDE type I) which hydrolyzed preferentially guanosine 3':5'-cyclic monophosphate (cyclic GMP); a cyclic GMP selective form (cGMP-PDE type V); and two low Km adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDEs that are insensitive to the stimulatory effect of CaM, one of which was inhibited by cyclic GMP (CGI-PDE, type III) and the other by rolipram (cAMP-PDE, type IV). Glaucine selectively inhibits one of the two forms of Ca2+-independent low Km cAMP-PDE, the type IV. In contrast, papaverine exerts a non-selective inhibitory effect upon all PDE forms.6. The present work provides evidence that glaucine, a benzyltetrahydroisoquinoline alkaloid, has interesting properties as an alpha l-adrenoceptor antagonist, calcium entry blocker (through the benzothiazepine recognition site in the calcium channel) and as a selective inhibitor of the rolipram-sensitive cAMP-PDE, type IV PDE.

Expression of early developmental genes in Dictyostelium discoideum is initiated during exponential growth by an autocrine-dependent mechanism

Throughout growth, Dictyostelium cells continuously produce an autocrine factor, PSF, that accumulates in proportion to cell density. Production of PSF declines rapidly when cells are shifted to starvation conditions, and the properties of PSF are distinct from those of regulatory factors produced by starving cells. During late exponential growth, PSF induces expression of several early developmental genes, including those for proteins important in cAMP signaling and cell aggregation. Examples are the aggregation stage cAMP receptor (cAR1), the aggregation-specific form of cyclic nucleotide phosphodiesterase, and gp24 (contact sites B). Through PSF, growing cells detect environmental conditions (cell number high, food approaching depletion) that are appropriate for production of the gene products needed to initiate aggregation and development.

Cellular distribution of phosphodiesterase isoforms in rat cardiac tissue.

We have resolved multiple forms of cyclic nucleotide phosphodiesterase (PDE) in whole rat ventricle and in isolated rat ventricular myocytes by use of anion-exchange high-performance liquid chromatography. One major form, the soluble calmodulin-stimulated PDE, is apparently absent from isolated myocytes. We discern four peaks of PDE activity (designated A-D in the order of their elution) in a soluble fraction obtained from whole rat ventricle. Peak A is stimulated twofold to threefold by the addition of calcium and calmodulin (Ca2+/CalM) and preferentially hydrolyzes cGMP over cAMP (in the presence of Ca2+/CalM, KmcGMP = 1.5 microM, KmcAMP = 17 microM). Peak B has similar affinities for both cAMP and cGMP (half-maximum velocities achieved at 30 microM substrate) and demonstrates positive cooperativity with cAMP but not with cGMP. The hydrolysis of cAMP by peak B is stimulated by cGMP at substrate concentrations up to 20 microM; the maximum effect is seen at 1 microM cAMP (25-fold stimulation by 3 microM cGMP). This pattern of stimulation by cGMP results from two kinetic changes: an increase in the enzyme's apparent affinity for cAMP (apparent KmcAMP decreases from 33 to 11 microM) and the abolition of positive cooperativity. Peaks C and D selectively hydrolyze cAMP, are not stimulated by Ca2+/CalM or cGMP, and differ in their affinities for substrate (peak C, apparent KmcAMP = 7.2 microM; peak D, 0.44 microM). In addition, peak D is much more sensitive than peak C to inhibition by cGMP, cilostamide, rolipram, and milrinone. Ro20-1724 is a slightly more potent inhibitor of peak D than of peak C. Peak D appears to consist of two different enzyme activities, one inhibited by cGMP, cilostamide, and cardiotonic drugs and the other potently inhibited by rolipram. In contrast to whole ventricle, the soluble fraction of isolated rat ventricular myocytes lacks peak A. Three major peaks in myocytes are entirely analogous to peaks B, C, and D of whole ventricle in terms of the NaCl concentration at which they elute, substrate affinities, and stimulation or inhibition by various drugs and effectors. We conclude that the soluble Ca2+/CalM-stimulated PDE in whole rat ventricle is present in nonmyocyte cells.

Differential inhibition of human neutrophil functions: Role of cyclic amp-specific, cyclic gmp-insensitive phosphodiesterase

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been characterized in various tissues and cells according to their substrate specificity, intracellular location, and calmodulin dependence. The purpose of this study was to evaluate the possible involvement of different molecular forms of phosphodiesterase in regulating the respiratory burst and lysosomal enzyme release responses of human neutrophils. Treatment with the selective cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase inhibitors Ro 20-1724 or rolipram, or the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), resulted in inhibition of respiratory burst stimulated by the chemoattractants formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) ( ic 50 values: 0.71−17 μM) and complement fragment C5a ( ic 50 values: 61–93 μM), but did not inhibit phagocytosis-stimulated respiratory burst (< 10% inhibition at 100 μM). Selective inhibitors of calmodulin-dependent phosphodiesterase (ICI 74,917), calmodulin-insensitive, cyclic GMP-specific phosphodiesterase (M&B 22,948), cyclic GMP-stimulated phosphodiesterase (AR-L 57), or cyclic AMP-specific, cyclic GMP-inhibited phosphodiesterase (amrinone and cilostamide) exhibited little or no inhibitory effect on FMLP- or phagocytosis-stimulated respiratory burst (0–42% inhibition at 100 μM). Regulation of neutrophil activation by phosphodiesterase was also response specific, as Ro 20-1724, rolipram and IBMX were less potent inhibitors of FMLP-induced lysosomal enzyme release (0–14% inhibition at 100 μM). Analysis of human neutrophil preparations confirmed the existence of a cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase, which was associated with the particulate fraction of the cell. These results demonstrate a role for the cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase in the regulation of human neutrophil functions, which appears to be both stimulus specific and response specific.

Effects of selective inhibitors on cyclic nucleotide phosphodiesterases of rabbit aorta

In this study three forms of cyclic nucleotide phosphodiesterase (PDE) isolated from rabbit aorta were pharmacologically characterized, and the consequence of selective inhibition of calmodulinstimulated PDE (CaM-PDE) and cGMP specific PDE (cG-PDE) was evaluated using PDE inhibitors. The cG-PDE (F1) was selectively inhibited by M&B 22948 ( IC 50 = 0.5 μ M) and dipyridamole ( IC 50 = 7 μ M) . The cAMP-PDE (cA-PDE, F3) was inhibited more effectively by the cA-PDE inhibitor milrinone than by other PDE inhibitors. The cA-PDE preparation appeared to contain both cG-inhibited PDE and cG-insensitive PDE based on an additive inhibition of the activity by milrinone and SQ 65442, respective inhibitors of these enzymes. Vinpocetine, 8-methoxymethyl isobutylmethylxanthine (8-MeOMeMIX) and M&B 22948 effectively inhibited CaM-PDE (F2). Vinpocetine was a more selective inhibitor of CaM-PDE than M&B 22948 or 8-MeOMeMIX. CaM-PDEs isolated from rabbit aorta and bovine brain exhibited a similar sensitivity to these inhibitors. Seventy-two percent of the cGMP-hydrolyzing activity of this rabbit aortic CaM-PDE preparation was immunoadsorbed to monoclonal antibody (ACC-1) against CaM bound to brain CaM-PDE. Vinpocetine, 8-MeOMeMIX and M&B 22948 at concentrations (30 and 100 μM) which inhibit CaM-PDE greater than 60% increased cGMP but not cAMP levels in l-norepinephrine (NE) preincubated rabbit aortic slices. At concentrations selectively inhibiting cG-PDE, dipyridamole and M&B 22948 increased cGMP levels in untreated slices but failed to increase cGMP levels significantly in NE-treated slices. By contrast, vinpocetine failed to increase cGMP significantly in untreated slices, although it increased cGMP levels in NE or KC1 preincubated slices. These data indicate that, in activated (precontracted) aorta, CaM-PDE is a major enzyme, whereas in untreated aorta cG-PDE is a predominant enzyme for the hydrolysis of cGMP. This study also shows a usefulness of selective inhibitors in identifying different forms of PDE and similar drug sensitivities and immunoadsorption of aortic and brain CaM-PDEs by a monoclonal antibody.

New mechanisms for positive inotropic agents: Focus on the discovery and development of imazodan

Intense efforts during the last decade to identify a useful positive inotropic agent to replace digitalis for the treatment of congestive heart failure have led to the discovery of several dozen potential substitutes, of which a number are currently undergoing clinical trials. In addition to producing a variety of new therapeutic entities, research in this area has also yielded valuable new information regarding the fundamental events that regulate calcium homeostasis and contractile function in the cardiac cell. For example, several of these new inotropic agents, including the calcium-channel stimulator BAY-k 8644, the sodium-channel stimulator DPI-201-186, and the sodium-calcium exchange inhibitor dichlorobenzamil, have provided considerable insight into the role of sodium and calcium in regulating contractility and the molecular events that mediate potential-dependent ion channels. Likewise, the discovery and development of agents like imazodan, amrinone, enoximone, and other selective type III phosphodiesterase inhibitors have provided new information regarding multiple molecular forms of cyclic nucleotide phosphodiesterase, compartmentation of cyclic AMP, and the importance of soluble vs. membrane-bound phosphodiesterases.

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta: Separation and inhibition by selective reference phosphodiesterase inhibitors

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.

61] High-affinity, calmodulin-dependent isoforms of cyclic nucleotide phosphodiesterase in rat testis

This chapter presents methods to examine changes in the pattern of isoforms during sexual development, and approaches to studying their localization within the various cellular compartments of the testis. To facilitate the latter, in addition to intact testes, methods for studies of Sertoli cell only (SCO) testes, Leydig cell tumors, and purified germ cell suspensions are also presented. The testis, like a wide range of other tissues, contains not one but several forms of cyclic nucleotide phosphodiesterase; three of these appear to be low-k m , calmodulin-dependent isoforms. That these isoforms are not simply artifacts of the chromatographic procedure but distinct isoenzymes is based on differences in substrate specificity, kinetics, thermal stability, and reactions to calmodulin and trifluoperazine. The relatively specific cGMP is prominent in both adult intact and SCO testes, especially in the latter, is the major isoform in the Leydig cell tumor, but is absent in round spermatids. These observations suggest that this phosphodiesterase is more characteristic of the endocrine tissue of the testis rather than the germinal elements, possibly the Leydig cells.

Cyclic AMP and cyclic GMP phosphodiesterases: Target for drug development

AbstractCyclic nucleotide phosphodiesterases catalyze the hydrolysis of 3′,5′ cyclic nucleotides to the corresponding nucleoside 5′ monophosphates. Multiple forms of cyclic nucleotide phosphodiesterase have been identified in both the cardiac muscle and vascular smooth muscle from various species. These isozymes differ in apparent molecular mass, chromatographic properties, substrate specificity, immunologic reactivity, sensitivity to inhibitors, and mode of regulation. The phosphodiesterases play important roles in the regulation of cyclic nucleotide concentrations, which in turn mediate cellular responses to a variety of biological stimuli. The development of phosphodiesterase inhibitors to increase intracellular cyclic nucleotide levels represents an important pharmacological approach to the treatment of various cardiovascular diseases.

Cardiotonic agents. 7. Inhibition of separated forms of cyclic nucleotide phosphodiesterase from guinea pig cardiac muscle by 4,5-dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-3(2H)-pyridazinones and related compounds. Structure-activity relationships and correlation with in vivo positive inotropic activity.

The structure-activity relationships of a series of 4,5-dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-3(2H)-pyridazinones and related compounds were investigated for the in vivo inhibition of different forms of cyclic nucleotide phosphodiesterase (PDE) isolated from guinea pig ventricular muscle. With few exceptions, these 4,5-dihydropyridazinones were potent inhibitors of cardiac type III phosphodiesterase, which is a low Km, cyclic AMP specific form of the enzyme. The inhibitory effects on cardiac type I and type II phosphodiesterase, both of which hydrolyze cyclic AMP as well as cyclic GMP, were minimal. The most selective PDE III inhibitor was CI-930 (10), the 5-methyl analogue of imazodan (CI-914, 1), with an IC50 of 0.6 microM. The most potent inhibitor of PDE III was the 4,5,6,7-tetrahydrobenzimidazole analogue of 10 (31), with an IC50 of 0.15 microM. This paper describes the structural features that impart both selectivity for inhibiting type III phosphodiesterase and potency of inhibition. In addition, correlations between in vitro PDE inhibitory potency, in vivo positive inotropic potency, and physicochemical properties are discussed.

ChemInform Abstract: Inhibition of Cyclic Nucleotide Phosphodiesterases from Pig Coronary Artery by Benzo‐Separated Analogues of 3‐Isobutyl‐1‐methylxanthine.

AbstractDie im Titel genannten Verbindungen (I) und (II) werden, wie im Formelschema skizziert, hergestellt.

Inhibition of cyclic nucleotide phosphodiesterases from pig coronary artery by benzo-separated analogs of 3-isobutyl-1-methylxanthine

The linear and proximal benzo-separated analogues of 7-benzyl-3-isobutyl-1-methylxanthine, 3-isobutyl-1,8-dimethylxanthine, 3-isobutyl-8-tert-butyl-1-methylxanthine, 3-isobutyl-8-(methoxymethyl)-1-methylxanthine , and 1-isoamyl-3-isobutylxanthine have been prepared and assayed as inhibitors of the peak I and peak II forms of cyclic nucleotide phosphodiesterase from pig coronary artery. Most of the benzo analogues were less effective inhibitors of these phosphodiesterases when compared to 3-isobutyl-1-methylxanthine (IBMX) even though the active sites of both enzyme forms tolerated the stretched-out xanthines. Indeed, the linear benzo-separated analogue of 7-benzyl-IBMX was a more potent inhibitor of peak I activity than was IBMX.

Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets: Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been identified previously in several tissues and cell types using a variety of different isolation methods. In the present study, the different molecular forms of phosphodiesterase (PDE) were isolated from cardiac muscle (guinea pig left ventricle), vascular smooth muscle (bovine coronary arteries) and human platelets using the same isolation procedure in each instance. These enzymes were then characterized kinetically, and the effects of various reference PDE inhibitors and cardiotonic agents on each form were examined. A low K m, low V max form of phosphodiesterase (PDE I) was found in all three tissue/cell types. PDE I activity was stimulated by calmodulin in cardiac and smooth muscle, but not in platelets. In smooth muscle and platelets, PDE I preferentially hydrolyzed cyclic GMP, whereas cardiac muscle PDE I hydrolyzed cyclic AMP and cyclic GMP equally. A high K m , high V max form of phosphodiesterase (PDE II) was found in cardiac muscle and platelets, but not in smooth muscle. PDE II activity was not stimulated by calmodulin and there was no substrate specificity. A low K m , low V max cyclic AMP-specific form of phosphodiesterase (PDE III) was found in all three tissue/cell types. The activity of PDE III was not stimulated by calmodulin. The reference inhibitors theophylline and papaverine exerted nonspecific inhibitory effects on all forms of phosphodiesterase. Other reference inhibitors (M & B 22,948 and dipyridamole) and several cardiotonic agents (AR-L 57, CI-914, CI-930, amrinone, and MDL 17,043) exerted selective inhibitory effects on only one molecular form of phosphodiesterase. The degree of selectivity was often dependent upon the tissue or cell from which the molecular form of phosphodiesterase was isolated. These studies demonstrate that (i) there is heterogeneity regarding the number of phosphodiesterases present in various tissue/cell types, as well as their substrate specificity and their ability to be stimulated by calmodulin, and (ii) these different molecular forms of phosphodiesterase can be selectively inhibited by different pharmacological agents. The possibility exists that such selective inhibitors may produce discrete changes in cyclic AMP or cyclic GMP levels, and that these changes may be produced in specific tissues and/or cells.