选择性磷酸二酯酶抑制剂研究进展

磷酸二酯酶 (PDEs)迄今已报道有 9个基因家族 ,每个家族又包括多个亚家族。PDEs分布于多个组织中 ,其抑制剂具有广泛的生理作用。其中 ,PDE 4抑制剂被认为是作用于细胞内靶点的新型免疫调节、抗炎药物。新型PDE 5抑制剂sildenafil通过NO/cGMP通路舒张海绵体血管及平滑肌 ,应用于阳痿治疗取得了较好的临床效果。该文简要综述选择性PDEs抑制剂近年研究进展。     

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.     

Comparative actions of diazepam and other phosphodiesterase inhibitors on the effects of noradrenaline in rat myocardium

Diazepam inhibits different isoforms of the enzyme cyclic nucleotide phosphodiesterase and also potentiates the inotropic effect of endogenous catecholamines in rat heart. In the present study we have examined whether this late effect is the consequence of inhibition of a phosphodiesterase subtype or whether inhibition of several phosphodiesterase subtypes is involved. We compared the effect of diazepam with that of the selective inhibitors of phosphodiesterase1 (MIMX), phosphodiesterase2 (EHNA), phosphodiesterase3 (milrinone) and phosphodiesterase4 (rolipram) on the inotropic effect of noradrenaline in rat ventricle. Both rolipram or diazepam were equipotent and more effective than milrinone in potentiating the inotropic effect of noradrenaline whereas EHNA and MIMX had no effect. The results suggest that the diazepam induced potentiation of the contractile effect of noradrenaline is due principally to inhibition of phosphodiesterase4 isoenzyme activity.     

Endothelin receptors in adult human and swine isolated ventricular cardiomyocytes.

The present study aimed to investigate endothelin-1 (ET-1) receptors in human and swine cardiomyocytes with binding studies using ET(A) and ET(B) selective receptor antagonists (BMS-182874 and BQ-788, respectively). Cell distribution of mRNA expression for ET(A) and ET(B) subtypes was investigated by in situ hybridization using specific cDNA probes. The 1251-ET-1 binding, which reached equilibrium in about 120 min (Kobs = 0.051+/-0.003 min(-1)), was only partially displaceable by the addition of a large excess of ET-1 (about 15% with a half-life of 20 min). In equilibrium binding studies, 125I-ET-1 had a Kd of 0.43+/-0.08 nM and a maximum binding (Bmax) of 42.8+/-6.6 fmol/mg protein. ET(A) and ET(B) receptors are represented in human and swine cardiomyocytes with an 85:15 ratio as indicated by the biphasic pattern of competition of both BMS-182874 and BQ-788. In situ hybridization studies confirmed that myocytes mainly expressed mRNA for ET(A), whereas expression of mRNA for the ET(B) subtype was documented in non-myocyte cells. These results showed that ET-1 binds with high affinity and poor reversibility to specific receptors, in both human and swine isolated ventricular cardiomyocytes, without significant species differences.     

Relaxant effects of the selective phosphodiesterase inhibitors milrinone and OPC 3911 on isolated human mesenteric vessels

Several new positive inotropic drugs with vasodilating properties for which a major mechanism of action is believed to be inhibition of phosphodiesterase (PDE) have been introduced in the treatment of congestive heart failure. Hydrolysis of cyclic nucleotides is catalyzed by multiple forms of PDE, which may vary between organs and cell-types. These enzymes can be selectively inhibited by various agents, theoretically making it possible to produce tissue-selective responses. An enzyme, which belongs to a subclass of cGMP inhibited low Km cAMP PDE, was recently purified from rat adipose tissue. The enzyme was specifically and potently inhibited by the cilostamide derivative OPC 3911 (OPC) and milrinone (mil). We studied the relaxant effects of OPC and mil on isolated human mesenteric arteries and veins in vitro. In preparations contracted by noradrenaline (NA), both agents produced about 60% maximum relaxation; OPC was 3-4 times more potent than mil. Both OPC and mil caused rightward displacement of the NA concentration-response curve and depressed the maximum responses. Arteries, as compared to veins, were more sensitive to this inhibition of NA contraction. Both drugs relaxed arteries contracted by 30 mM K+, but not 127 mM K+; maximum relaxation was between 60 and 70%. OPC was 10 times more potent than mil. The interactions between mil/OPC and isoprenaline, forskolin and ouabain were also studied. In preparations pretreated with isoprenaline or forskolin, the relaxant effects of mil and OPC were additive to those of isoprenaline and forskolin. Ouabain pretreatment did not affect the actions of the phosphodiesterase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incomplete reversal of beta-adrenoceptor desensitization in human and guinea-pig cardiomyocytes by cyclic nucleotide phosphodiesterase inhibitors.

1. The decreased response to beta-adrenoceptor stimulation seen in heart failure may be related to a defect in cyclic AMP production. The inotropic effects of the selective phosphodiesterase (PDE) III inhibitors, SK&F 94120 and SK&F94836, and the non-selective PDE inhibitor, 3-isobutyl-l-methylxanthine (IBMX), alone and when combined synergistically with isoprenaline, were studied in control and beta-adrenoceptor-desensitized ventricular myocytes. 2. Myocytes isolated from noradrenaline-treated guinea-pigs had a reduced maximum response to isoprenaline compared with control animals (60.0 +/- 2.5%, n = 42 vs 79.5 +/- 1.7% maximum calcium: n = 46, P < 0.001). Together with an approximately 20 fold increase in the isoprenaline EC50, this is indicative of beta-adrenoceptor desensitization as a result with chronic infusion with noradrenaline. 3. The maximum inotropic response of IBMX was depressed following noradrenaline treatment, from 74.9 +/- 4.6% (n = 7) in control, to 61.7 +/- 2.70% (n = 6), as a percentage of maximum calcium in noradrenaline-treated guinea-pig ventricular myocytes (P < 0.02). The pD2 value for IBMX was also reduced (P < 0.02). No significant differences in the inotropic effects of SK&F94120 and SK&F94836 were seen between control and beta-adrenoceptor desensitized myocytes. 4. Threshold inotropic concentrations of SK&F94120 and SK&F94836 caused a five fold decrease in the EC50 of control myocytes for isoprenaline, and an 11 fold decrease in the noradrenaline-treated guinea-pig ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of non-selective and selective phosphodiesterase inhibitors on human eosinophil functions.

1. The effect of non-selective (3-isobutyl-1-methylxanthine, IBMX; theophylline) and type IV- or type III/IV-selective (rolipram, RP 73401; zardaverine, tolafentrine) phosphodiesterase (PDE) inhibitors on human eosinophil functions was investigated. 2. For this purpose human eosinophils were purified from blood of healthy donors by a magnetic cell separation (MACS) technique to a purity > or = 99%. From the stimuli investigated (complement C5a; N-formyl-methionyl-leucyl-phenylalanine, fMLP; platelet activating factor, PAF; opsonized zymosan) C5a was selected to test the influence of the above mentioned compounds on secretion of granule constituents (eosinophil cationic protein, ECP; eosinophil-derived neurotoxin, EDN) as well as on formation of reactive oxygen species measured by luminol-enhanced chemiluminescence in intact cells. For comparison, inhibition of PDE IV activity in the cytosol of disrupted cells, which contains about 75% of total PDE IV activity, was determined. 3. Both theophylline and IBMX inhibited the two cell responses with IC50 values which were in the range of their IC50 values obtained for inhibition of PDE IV activity in the cell-free system. The beta 2-adrenoceptor agonist, salbutamol (1 mumol l-1), which by itself did not substantially influence the two cell responses, only marginally improved the potency of theophylline and IBMX in inhibiting ECP/EDN secretion. Only the IC50 value of IBMX for inhibition of chemiluminescence was lowered by about one order of magnitude in the presence of salbutamol. 4. In contrast, none of the selective PDE inhibitors tested substantially inhibited the two cell responses at concentrations up to 10 mumol l-1. This was surprising because all of the compounds investigated inhibited PDE IV activity in the cell-free system with IC50 values which were at least 30 fold lower than the highest concentration of the compounds used with intact cells. In combination with salbutamol, however, both ECP/EDN secretion and chemiluminescence was inhibited by rolipram and zardaverine with IC50 values similar to the IC50 values for inhibition of PDE IV activity. Although RP 73401 and tolafentrine also inhibited both cell responses in the presence of salbutamol, the potency of these two compounds in inhibiting eosinophil function in intact cells was at least two orders of magnitude lower than would have been expected from the inhibition of PDE IV activity in the cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Opposing effects of endothelin-1 on C-type natriuretic peptide actions in rat cardiomyocytes.

C-type natriuretic peptide (CNP) and Endothelin-1 are paracrine peptides with opposing vascular and mitogenic actions. In cardiac myocytes, CNP reduced contractility and induced accumulation of cyclic guanosine monophosphate (cGMP). Endothelin-1 caused an increase in contractile amplitude, abolished the negative inotropic effect of CNP, reduced the negative inotropic effect of a membrane permeable cGMP, and inhibited cGMP accumulation induced by CNP. We conclude that endothelin-1 abolishes the negative inotropic effect of CNP. This effect may be mediated by inhibition of the negative inotropic actions of cGMP as well as by reduction of cGMP levels.     

Determination of beta-adrenoceptor subtype on rat isolated ventricular myocytes by use of highly selective beta-antagonists.

1. The relative proportions of beta 1- and beta 2-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2. Competition experiments using CGP 20712A or ICI 118,551 with [125I]-iodocyanopindolol ([125I]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta 1-adrenoceptor) and 50 nM for ICI 118,551 (beta 2-adrenoceptor). 3. The density of high-affinity (beta 1-adrenoceptor) and low-affinity (beta 2-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4. In myocytes, specific binding of [125I]-ICYP and [3H]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leucoisoprenochrome-o-semiquinone formation in freshly isolated adult rat cardiomyocytes

Sustained high levels of circulating catecholamines can lead to cardiotoxicity. There is increasing evidence that this process may result from metal-catalyzed catecholamine oxidation into semiquinones, quinones, and aminochromes. We have previously shown that Cu2+-induced oxidation of isoproterenol into isoprenochrome induces toxic effects in isolated cardiomyocytes. The aim of this study was to characterize the isoproterenol oxidation process and to locate the formation of semiquinone radicals in cardiomyocyte suspensions. Freshly isolated rat cardiomyocytes were incubated with 1 or 10 mM isoproterenol and 20 microM Cu2+ for 4 h. The formation of an isoproterenol oxidation radical was detected in the extracellular medium, cells, membranes, and heavy organelles by electron spin resonance spectroscopy. An electron spin resonance signal assigned to leucoisoprenochrome-o-semiquinone increased in a time-dependent manner in the extracellular medium. A second electron spin resonance signal, characteristic of an immobilized radical, was also found in the cardiomyocytes. The latter was attributed to leucoisoprenochrome-o-semiquinone immobilized on cellular components such as membranes, cytoskeleton, nucleus, and heavy organelles. In addition, the levels of leucoisoprenochrome-o-semiquinone decreased in the presence of glutathione. Computer simulations of the experimental spectra indicate the formation of two distinct isomeric leucoisoprenochrome-o-semiquinone radicals during isoproterenol oxidation. The present study shows that the isoproterenol oxidation in isolated rat cardiomyocytes correlates with the formation of leucoisoprenochrome-o-semiquinone in the cells and in the extracellular medium, suggesting that it might be involved in cardiotoxicity induced by the oxidation of catecholamines.     

Effects of phosphodiesterase inhibitors on normal and chemically-skinned isolated airway smooth muscle.

1. The effects of three phosphodiesterase inhibitors (papaverine, isobutyl methyl xanthine (IBMX) and SKF 94120) were examined on tension responses and cyclic nucleotide content (both cyclic AMP and cyclic GMP) of normal and Triton X-100 skinned isolated trachealis of the guinea-pig. 2. The three inhibitors were approximately equipotent in eliciting concentration-dependent relaxation of histamine-induced contractions of the trachealis. 3. Papaverine-induced relaxation was associated with concentration-related increases in the levels of both cyclic nucleotides. 4. IBMX at low concentrations (1 mumol l-1) produced significant relaxation (36%) of histamine-contracted trachealis without changing cyclic nucleotide levels. At a ten fold higher concentration IBMX-induced relaxation (95%) was associated with a selective increase in tissue cyclic GMP levels. Only at the highest concentration tested (100 mumol l-1) did IBMX increase cyclic AMP levels significantly. 5. SKF 94120 (1 mumol l-1) elicited a 23% relaxation of the contracted trachealis without altering the tissue content of either cyclic nucleotide. At the two higher concentrations tested (10 and 100 mumol l-1), SKF 94120-induced relaxation was accompanied by a selective increase in the levels of cyclic AMP. 6. In the skinned trachealis Ca2+ (10 and 20 mumol l-1)-induced contractions were significantly inhibited by the calmodulin antagonist calmidazolium (10 mumol l-1) and by cyclic AMP (10 mumol l-1), the catalytic subunit of cyclic AMP-dependent protein kinase (0.1 mumol l-1) and cyclic GMP (10 mumol l-1). 7. Papaverine (100 mumol l-1) significantly inhibited (31 +/- 6%) the Ca2+-induced contractions of the skinned trachealis. Both IBMX and SKF 94120 were without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of selective phosphodiesterase inhibitors on synaptic transmission in the guinea-pig ileum

The effect of selective phosphodiesterase (PDE) inhibitors was studied on neural transmission within the enteric nervous system employing a two-compartment bath (containing the oral and the anal end of a segment of guinea-pig ileum, respectively). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 45 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded. The partitioned bath enables PDE inhibitors and other drugs to be applied to enteric nerve pathways (in the anal compartment) without interfering with the recording of the smooth muscle contraction in the oral compartment. The PDE 4 inhibitors rolipram (0.01–10 μM) and Ro-20-1724 (0.01–10 μM) significantly (P<0.01) inhibited (10–91% and 9–83%, respectively) the nerve-mediated contractions. When both rolipram and Ro-20-1724 were tested after phentolamine (1 μM) or yohimbine (0.1 μM), they were significantly (P<0.01) less effective. By contrast prazosin (1 μM) was ineffective. Vinpocetine (50 μM), milrinone (30 μM) and zaprinast (100 μM), which inhibit PDE 1, 3 and 5, respectively, did not modify the nerve-mediated contractions. 8-Bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cyclic AMP) or N⁶,2’-O-dibutyryladenosine 3’,5’ cyclic monophosphate (dibutyryl cyclic AMP), two analogues of cyclic AMP, at lower concentrations (0.1–1 μM) significantly (P<0.01) inhibited (15–73% and 5–49%, respectively) the nerve-mediated contractions, while at higher concentrations (10–100 μM) they caused a significant (P<0.01) potentiating (48–68% and 77–78%, respectively) effect. These results indicate that inhibition of PDE 4 (but not PDE 1, PDE 3 or PDE 5) produces a depression of neural transmission within the enteric nervous system, possibly by releasing noradrenaline acting at α₂-adrenoceptors on enteric neurons. Received: 21 November 1997 / Accepted: 11 March 1998     

Alpha-adrenergic regulation of action potentials in isolated rat cardiomyocytes

Radioligand binding studies and studies in which inositol phosphate levels were assayed have provided substantial evidence for the existence of alpha 1-adrenoceptors in isolated rat ventricular myocytes, a pure cardiac preparation free of neuronal and vascular elements. Since correlational electrophysiological studies have not been carried out in this model, we investigated alpha-adrenergic effects on action potentials using intracellular microelectrodes to impale the myocytes. Epinephrine, in the presence of propranolol (10(-6) M), rapidly increased the action potential duration in a concentration-dependent manner (threshold concentration of 30 nM, EC50 of 100 nM). The maximal upstroke velocity and overshoot of Ca2+-mediated action potentials (15 mM K+) were also increased. Epinephrine did not significantly affect the resting membrane potential. The alpha 1-adrenoceptor antagonist, prazosin (10(-7) M) blocked epinephrine's effects. LiCl (10 mM), an inhibitor of inositol-phosphate phosphatases potentiated the effects of epinephrine (30 nM). The results suggest that the isolated rat cardiomyocyte is a suitable preparation for examining the ionic and molecular mechanisms of direct alpha-adrenergic effects on the cardiac membrane.     

Pentasubstituted quercetin analogues as selective inhibitors of particulate 3':5'-cyclic-AMP phosphodiesterase from rat brain

Some penta-O-substituted analogues of quercetin were synthesized and tested for the inhibition of cytosolic and particulate rat brain cyclic AMP and cyclic GMP phosphodiesterase activities. Ten of these compounds are potent and highly selective inhibitors of cAMP hydrolysis with respect to cGMP hydrolysis. They inhibit more potently the particulate enzyme than the cytosolic preparation. The highest selectivity was observed with penta-O-ethylquercetin and analogue 6d, which proved to be more selective and more potent inhibitors than the reference compound Ro 20-1724. Some structure-activity relationships are discussed.     

Molecular cardiopharmacology of selective inhibitors of cyclic nucleotide phosphodiesterase isozymes]

The existence of multiple isozymes of cyclic nucleotide phosphodiesterase (PDE) in many tissues including the heart has been demonstrated. Five isozyme families, each composed of several subtypes and having different tissue and subcellular distributions, have been characterized. Selective inhibitors of PDE III (cGMP-inhibited PDE) elevates the cAMP level which mediates positive inotropic actions with compartmentation of cAMP related to cardiac cell particulate structures. Both cardiac cytosolic and particulate PDE III were potently and selectively inhibited by the new cardiotonic agents competitively with respect to cAMP, except for vesnarinone. There might be at least two subtypes of PDE III, and vesnarinone may be a selective subtype inhibitor of PDE III in human heart. It was also reported that vesnarinone was beneficial in treating patients with congestive heart failure. Moreover, selective inhibitors of PDE III with ancillary properties such as calcium sensitization may prove to be more useful drugs for the treatment of heart failure.     

Inhibition of IgE-mediated histamine release from human peripheral leukocytes by selective phosphodiesterase inhibitors.

Several phosphodiesterase (PDE) inhibitors with the capacity to inhibit the PDE IV isoenzyme produce dose-dependent inhibition of IgE-mediated histamine release (HR) from human peripheral leukocytes in vitro. Inhibition reached a maximum after 20 min of preincubation (IC30: 6-30 microM, IC50: 30-80 microM). Motapizone--a potent inhibitor of the isoenzyme PDE III--was much less effective, thus giving indirect evidence that PDE IV plays a predominant role in the control of cAMP cleavage in human basophils. The inhibiting effect of PDE-III/IV-selective compounds on IgE-mediated HR did not exceed the action of PDE-IV-selective inhibitors. The inhibition of anti-IgE-induced HR by zardaverine (a PDE-III/IV-inhibiting compound) was synergistically enhanced in the combined presence of forskolin or the recently synthesized histamine H2-agonist FRA 19).     

Stimulation of fatty acid oxidation by phosphodiesterase III inhibitors in rat myocytes.

In order to study the regulation of fatty acid oxidation in the heart, the effects of some phosphodiesterase III inhibitors, such as enoximone and milrinone, on the oxidation of [1-14C]palmitic acid, [1-14C]octanoic acid, and [2-14C]pyruvate were studied in adult rat myocytes. Enoximone and milrinone, at a concentration of 0.25 mM, increased palmitate oxidation significantly, by 70 and 40%, respectively. Also, enoximone increased octanoate oxidation by 45%. In contrast, pyruvate oxidation was decreased by 60% by enoximone. To investigate the effects of enoximone or milrinone on the pathway of fatty acid oxidation, their effects on the oxidation of either [1-14C]palmitoyl-CoA or [1-14C]palmitoylcarnitine were studied with rat heart homogenates. Neither enoximone nor milrinone had any effects on the oxidation of these compounds. Compounds known to elevate intracellular [Ca2+] or cyclic AMP, such as the calcium ionophore A23187, ionomycin, dibutyryl cyclic AMP, or isoproterenol, had no effect on palmitate oxidation. Enoximone, at a concentration of 0.25 mM, increased palmitate uptake by 40% in rat myocytes. These results suggest that enoximone and milrinone increase fatty acid oxidation in myocytes by increasing their cellular transport, and they also show the usefulness of these compounds as a tool to study the regulation of this vital pathway in heart.