磷酸二酯酶抑制剂与呼吸道疾病

磷酸二酯酶(PDE)存在于许多炎症细胞及结构细胞中,目前已发现11种.PDE抑制剂主要抑制体内环磷酸腺苷(cAMP)及环磷酸鸟苷(cGMP)水解,使细胞内cAMP及cGMP浓度增加,引起一系列生理功能,如平滑肌舒张、减轻细胞炎症及免疫反应等.PDE4特异性水解cAMP,选择性PDE4抑制剂具有广泛抗炎作用,如抑制细胞趋化,抑制中性粒细胞、嗜酸粒细胞、巨噬细胞及T细胞细胞因子及化学趋化物质释放.第二代PDE4抑制剂Cilomilast和Roflumilast已进入临床实验阶段,并已证实对支气管哮喘(简称哮喘)及慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)有效.由于胃肠道副作用,这类药物临床应用受到一定限制.PDE5可特异性水解cGMP,对缺氧性肺动脉高压和血管重塑有效.PDE3和PDE7特异性水解cAMP,PDE7参与T细胞激活.目前其他PDE抑制剂与PDE4抑制剂混合制剂正在研发中.PDE4-PDE7双重抑制剂可能对哮喘及COPD更有效.PDE3-PDE4双重抑制剂具有更强的支气管舒张作用及气道保护作用.     

Overview of cardiovascular physiologic and pharmacologic aspects of selective phosphodiesterase peak III inhibitors

In recent years several agents have been developed as selective inhibitors of the low Michaelis constant cyclic adenosine monophosphate (cAMP) phosphodiesterase (peak III), a fraction of the cyclic nucleotide phosphodiesterases that is specific for the metabolic breakdown of cAMP. These agents are often referred to as PDE III inhibitors and share similar pharmacologic profiles. The principal interest in these agents--the therapy of congestive heart failure--is based on the cardiovascular effects that result from sequential elevation of intracellular cAMP, cAMP-dependent protein kinase activation, phosphorylation of cellular proteins and change in cellular function. The selective PDE III inhibitors have a triad of cardiovascular activities that provide hemodynamic benefit to patients with congestive heart failure. As a representative drug from this class of compounds, milrinone increases myocardial contractility, increases the rate of ventricular relaxation, and unloads the heart by way of a peripheral vasodilator action. The selective PDE III inhibitors offer a new modality for oral therapy of congestive heart failure.     

Biochemical aspects of inhibition of cardiovascular low (Km) cyclic adenosine monophosphate phosphodiesterase

Multiple isozymes of cyclic nucleotide phosphodiesterase (PDE) exist in mammalian cells. At least 5 major types of PDE isozymes have been identified; they differ by substrate affinity, maximal activity, intracellular regulation or mechanism of pharmacologic inhibition. A low Michaelis constant (Km) cyclic adenosine monophosphate (cAMP) PDE, whose activity is inhibited by submicromolar concentrations of cyclic guanosine monophosphate and stimulated by cAMP-mediated phosphorylation, is present in both cardiac muscle and vascular smooth muscle. This PDE isozyme (referred to as peak IIIc PDE) is sensitive to selective inhibition by amrinone, milrinone, imazodan, CI-930, piroximone, and numerous other PDE inhibitors. The subcellular distribution of cardiac PDE IIIc varies according to species; it is found in the soluble fraction of guinea pig myocardium, in the particulate fraction of canine myocardium, and in both fractions of primate (simian and human) myocardium. Another PDE isozyme, which is sensitive to inhibition by rolipram and is less sensitive to inhibition by PDE IIIc inhibitors, is found in cardiac muscle of some species (i.e., soluble fractions of rat and canine myocardium) and is apparently not related to direct regulation of positive inotropy. Both positive inotropy and vasorelaxation by milrinone and other PDE IIIc inhibitors can be linked to inhibition of PDE IIIc and activation of the cAMP system. These significant relations are similar to those obtained for other cAMP-related positive inotrope/vasodilators (such as beta-adrenoreceptor agonists). Moreover, an increased rate of ventricular relaxation (lusitropy), which is apparent with PDE IIIc inhibitors, may also be attributable to activation of the cAMP system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages

Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE₂). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.     

Phosphodiesterase 4 inhibition synergizes with relaxin signaling to promote decidualization of human endometrial stromal cells

The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.     

Cyclic AMP phosphodiesterases in human lymphocytes

The function of lymphocytes, like platelets, has been shown to be inhibited by agents which increase intracellular cyclic AMP. Two high-affinity cAMP phosphodiesterases (PDEs), the cyclic GMP-inhibited cAMP phosphodiesterase, PDE3, and the cAMP-specific phosphodiesterase PDE4, are known to regulate cAMP concentration in haemopoietic cells by degrading cAMP to AMP. We characterized the relative contribution of the two PDEs to total lymphocyte PDE activity. We then determined which of the different gene products, PDE3A, typical of myocardium and platelets, or PDE3B, typical of adipocytes, were present in lymphocytes. The PDE3-specific inhibitor, milrinone, and the PDE4 inhibitor, rolipram, suppressed hydrolysis by 70% and 30% respectively, which indicated that both PDE4 and PDE3 were present, and that PDE3 was predominant. RT-PCR yields the expected size fragment for the primer pair PDE3B and not for PDE3A. The DNA sequence obtained had > 95% identity with PDE3B. PDE3B appears to be the major cAMP PDE in lymphocytes. In contrast to human platelets, human lymphocytes appear to contain the PDE3B subtype. Since PDE3B in adipocytes is subject to hormonal regulation, lymphocytes may be similarly modulated. Understanding the role of cAMP regulation and the involvement of cAMP in lymphocyte function may have important implications in drug development.     

Myocardial Phosphodiesterases and Regulation of Cardiac Contractility in Health and Cardiac Disease

Phosphodiesterase (PDE) inhibitors are potent cardiotonic agents used for parenteral inotropic support in heart failure. Contractile effects of these agents are mediated through cAMP-protein kinase A-induced stimulation of I _Ca2+ which ultimately results in increased Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. A number of additional effects such as increases in sarcoplasmic reticulum Ca²⁺ stores, stimulation of reverse mode Na⁺–Ca²⁺ exchange, direct or cAMP-mediated effects on sarcoplasmic reticulum ryanodine receptor, stimulation of the voltage-sensitive sarcoplasmic reticulum Ca²⁺ release mechanism, as well as A₁ adenosine receptor blockade could contribute to positive inotropic responses to PDE inhibitors. Moreover, some PDE inhibitors exhibit Ca²⁺ sensitizer properties as they could increase the affinity of troponin C Ca²⁺-binding sites as well as reduce Ca²⁺ threshold for thin myofilament sliding and facilitate cross-bridge cycling. Inotropic responses to PDE inhibitors are significantly reduced in cardiac disease, an effect largely attributed to downregulation of cAMP-mediated signalling due to sustained sympathetic activation. Four PDE isoenzymes (PDE1, PDE2, PDE3 and PDE4) are present in myocardial tissue of various mammalian species, of which PDE3 and PDE4 are particularly involved in regulation of cardiac myocyte contraction. PDE cAMP-hydrolysing activity is preserved in compensated cardiac hypertrophy but significantly reduced in animal models of heart failure. However, clinical studies have not revealed any changes in distribution profile as well as kinetic and regulatory properties of myocardial PDEs in failing human hearts. A reduction of PDE inhibitors-induced contractile responses in heart failure has therefore been ascribed to reduced cAMP synthesis due to uncoupling of adenylyl cyclase from β-adrenoreceptor. In cardiac myocytes, PDEs are targeted to distinct subcellular compartments by scaffolding proteins such as myomegalin, mAKAP and β-arrestins. Over subcellular microdomains, cAMP hydrolysis by PDE3 and PDE4 allows to control the activity of local pools of protein kinase A and therefore the extent of protein kinase A-mediated phosphorylation of cellular proteins.     

Role of cyclic nucleotide signaling in oocyte maturation

The development of the ovarian follicle, oocyte maturation, and ovulation require a complex set of endocrine, paracrine, and autocrine inputs that are translated into the regulation of cyclic nucleotide levels. Changes in intracellular cAMP mediate the gonadotropin regulation of granulosa and theca cell functions. Likewise, a decrease in cAMP concentration in the oocyte has been associated with the resumption of meiosis. Using pharmacological and molecular approaches, we determined that the expression of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, is compartmentalized in the ovarian follicle of all species studied, with PDE3 present in the oocytes and PDE4s in granulosa cells. The PDE3 expressed in the mouse oocyte was cloned, and the protein expressed in a heterologous system had properties similar to those of a PDE3A derived from somatic cells. Inhibition of the oocyte PDE3 completely blocked oocyte maturation in vitro and in vivo, demonstrating that the activity of this enzyme is essential for oocyte maturation. Heterologous expression of PDE3A in Xenopus oocyte causes morphological changes distinctive of resumption of meiosis (GVBD), as well as activation of mos translation and MAPK phosphorylation. Using mRNA and antibody microinjection in the Xenopus eggs, we have shown that PDE3 is downstream from the kinase PKB/Akt in the pathway that mediates IGF-1 but not progesterone-induced meiotic resumption. The presence of a similar regulatory module in mammalian oocytes is inferred by pharmacological studies with PDE3 inhibitors and measurement of PDE activity. Thus, PDE3 plays an essential role in the signaling pathway that controls resumption of meiosis in amphibians and mammals. Understanding the regulation of this enzyme may shed some light on the signals that trigger oocyte maturation.     

Interleukin-1beta induces phosphodiesterase 4B2 expression in human myometrial cells through a prostaglandin E2- and cyclic adenosine 3',5'-monophosphate-dependent pathway

Intrauterine infections are important etiological factors of preterm labor. They trigger an increase in proinflammatory cytokines, in particular IL-1beta, that induces a cascade of events resulting in the production of potent effectors of myometrial contractility, such as the prostaglandin E(2) (PGE(2)). Within the smooth muscle cells, contractility is under the control of cAMP content, partly regulated by cAMP-phosphodiesterase 4 (PDE4), the predominant family of PDEs expressed in human myometrium. In the present study, using a model of inflammation of human myometrial cells in culture, we demonstrated that exposing the cells to IL-1beta resulted in a significant up-regulation of PDE4 activity through an increase in PDE4B2 mRNA and protein levels. The IL-1beta-induced PDE4 activity occurs after an increase in PGE(2) production and subsequent cAMP augmentation. Pretreatment with indomethacin or NS 398 completely blocked this long-term effect of IL-1beta, revealing a PGE(2)-dependent pathway. Accordingly, our results demonstrated that the PDE4B2 variant can participate in the regulation of the inflammatory reaction that occurs at term or in preterm labor and leads to myometrial contractions. Knowing the myorelaxant effect of PDE4 inhibitors and the implication of the PDE4B2 in the inflammatory process, this isoform may be an appropriate target for discovering antiinflammatory drugs to manage infection-induced preterm deliveries.     

Platelet cyclic adenosine monophosphate phosphodiesterases: targets for regulating platelet-related thrombosis

Platelets contain two cyclic adenosine monophosphate (cAMP) phosphodiesterases (PDEs) that regulate the level of cAMP, the major inhibitor of platelet activation pathways. PDE3A hydrolyzes cAMP to 5' AMP with a low K (m). PDE3A is inhibited by cyclic guanosine monophosphate (cGMP), which provides a feedback control and controls basal levels of cAMP. In contrast, PDE2A hydrolyzes both cAMP and cGMP with a high K (m), is allosterically stimulated by cGMP at moderate levels, and may control the stimulated levels of cAMP. Using affinity labeling, chemical modification, and site-directed mutagenesis of highly conserved amino acids, the amino acids required for catalytic activity and/or metal binding are H752 and H756. The singular binding sites for cAMP include N845, E971, and F972, whereas the unique amino acids interacting with cGMP are Y751, H836, H849, and D950. Residues E866 and F1004 are present in both the overlapping cGMP and cAMP sites. Two inhibitors of PDE3A are used in clinical medicine: milrinone and cilostazol. Three amino acids, Y751, D950, and F1004, show decreased sensitivity to both inhibitors (increased K (i)). These inhibitors mimic cGMP as an inhibitor of PDE3A rather than compete for cAMP binding. New nonhydrolyzable affinity labels inactivate PDE3A and are protected by Sp-cAMPS, a nonhydrolyzable substrate of the enzyme. These compounds have the potential to identify amino acids that are unique for PDE3A. An inhibitor of platelet PDE2A increases cAMP more than inhibitors of PDE3A but has much less effect on platelet activation, suggesting that these enzymes are present in different compartments of the cell.     

Targeting Cyclic Nucleotide Phosphodiesterase in the Heart: Therapeutic Implications

The second messengers, cAMP and cGMP, regulate a number of physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression and cardiac structural remodeling. Emerging evidence suggests that multiple spatiotemporally distinct pools of cyclic nucleotides can discriminate specific cellular functions from a given cyclic nucleotide-mediated signal. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic AMP and/or cyclic GMP, control the amplitude, duration, and compartmentation of cyclic nucleotide signaling. To date, more than 60 different isoforms have been described and grouped into 11 broad families (PDE1–PDE11) based on differences in their structure, kinetic and regulatory properties, as well as sensitivity to chemical inhibitors. In the heart, PDE isozymes from at least six families have been investigated. Studies using selective PDE inhibitors and/or genetically manipulated animals have demonstrated that individual PDE isozymes play distinct roles in the heart by regulating unique cyclic nucleotide signaling microdomains. Alterations of PDE activity and/or expression have also been observed in various cardiac disease models, which may contribute to disease progression. Several family-selective PDE inhibitors have been used clinically or pre-clinically for the treatment of cardiac or vascular-related diseases. In this review, we will highlight both recent advances and discrepancies relevant to cardiovascular PDE expression, pathophysiological function, and regulation. In particular, we will emphasize how these properties influence current and future development of PDE inhibitors for the treatment of pathological cardiac remodeling and dysfunction.     

Absence of muscarinic cholinergic airway responses in mice deficient in the cyclic nucleotide phosphodiesterase PDE4D

Muscarinic cholinergic signaling plays an essential role in the control of the normal airway functions and in the development of pulmonary pathologies including asthma. In this paper we demonstrate that the airways of mice deficient in a cAMP-specific phosphodiesterase (PDE4D) are no longer responsive to cholinergic stimulation. Airway hyperreactivity that follows exposure to antigen was also abolished in PDE4D(-/-) mice, despite an apparently normal lung inflammatory infiltration. The loss of cholinergic responsiveness was specific to the airway, not observed in the heart, and was associated with a loss of signaling through muscarinic receptors with an inability to decrease cAMP accumulation. These findings demonstrate that the PDE4D gene plays an essential role in cAMP homeostasis and cholinergic stimulation of the airway, and in the development of hyperreactivity. In view of the therapeutic potentials of PDE4 inhibitors, our findings provide the rationale for novel strategies that target a single PDE isoenzyme.     

A biochemical and functional assessment of monocyte phosphodiesterase activity in healthy and asthmatic subjects

The aim of this study was to investigate whether cyclic adenosine 3'5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 +/- 7.0, n=9) compared to healthy individuals (46.3 +/- 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF alpha release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF alpha release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.     

Allergen challenge alters lymphocyte phosphodiesterase activity in horses with heaves

Heaves is an allergic airway disease in horses characterised by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation associated with a Th(2) response. Cyclic nucleotide-dependent signalling pathways can regulate lymphocyte function. In this study, we examined lymphocyte PDE activity comparing horses with heaves to healthy control animals. Total PDE activity and the effects of isoenzyme selective inhibitors were measured before, 5 and 24 h after the start of a 7 h allergen challenge. Allergen challenge had no effect on either total cAMP PDE activity or its inhibition by the PDE4 selective inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline. In contrast, the PDE3 selective inhibitor, quazinone, caused significantly greater inhibition of cAMP PDE activity before challenge in the heaves susceptible group. Additionally, total cGMP PDE activity was significantly lower 24 h after the start of challenge in the heaves affected group (11+/-2 and 21+/-3 pmol/min/mg for heaves and control animals, respectively) and the PDE5 selective inhibitor, zaprinast, caused significantly less inhibition in the heaves group at this time point. The functional significance of these findings was explored by examining the effect of PDE3, PDE4 and PDE5 selective inhibitors on mitogen-induced mononuclear cell proliferation before and 24 h after the start of allergen challenge. Proliferation decreased after challenge in the heaves group (stimulation index=328+/-110 and 200+/-72 before and after challenge, respectively) whilst remaining constant in the control group (stimulation index=161+/-13 and 183+/-45 before and after challenge, respectively). However, all three PDE inhibitors caused a similar amount of inhibition at each time point and the effect of a combination of a PDE3 and a PDE5 inhibitor was simply additive in both groups. These results suggest differences in the control of lymphocyte PDE activity in horses with heaves.     

Phosphodiesterase 4D is required for beta2 adrenoceptor subtype-specific signaling in cardiac myocytes

beta adrenoceptor (betaAR) signaling is finely regulated to mediate the sympathetic nervous system control of cardiovascular function. In neonatal cardiac myocytes, beta1AR activates the conventional Gs/cAMP pathway, whereas beta2AR sequentially activates both the Gs and Gi pathways to regulate the myocyte contraction rate. Here, we show that phosphodiesterase 4D (PDE4D) selectively impacts signaling by beta2AR in neonatal cardiac myocytes, while having little or no effect on beta1AR signaling. Although beta2AR activation leads to an increase in cAMP production, the cAMP generated does not have access to the protein kinase A-dependent signaling pathways by which the beta1AR regulates the contraction rate. However, this restricted access is lost in the presence of PDE4 inhibitors or after ablation of PDE4D. These results not only suggest that PDE4D is an integral component of the beta2AR signaling complex, but also underscore the critical role of subcellular cAMP regulation in the complex control of receptor signaling. They also illustrate a mechanism for fine-tuned betaAR subtype signaling specificity and intensity in the cardiac system.     

Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice

In eukaryotic cells, the inactivation of the cyclic nucleotide signal depends on a complex array of cyclic nucleotide phosphodiesterases (PDEs). Although it has been established that multiple PDE isoenzymes with distinct catalytic properties and regulations coexist in the same cell, the physiological significance of this remarkable complexity is poorly understood. To examine the role of a PDE in cAMP signaling in vivo, we have inactivated the type 4 cAMP-specific PDE (PDE4D) gene, a mammalian homologue of the Drosophila dunce. This isoenzyme is involved in feedback regulation of cAMP levels. Mice deficient in PDE4D exhibit delayed growth as well as reduced viability and female fertility. The decrease in fertility of the null female is caused by impaired ovulation and diminished sensitivity of the granulosa cells to gonadotropins. These pleiotropic phenotypes demonstrate that PDE4D plays a critical role in cAMP signaling and that the activity of this isoenzyme is required for the regulation of growth and fertility.     

Pseudohypoparathyroidism type Ib in 2015

The term pseudohypoparathryoidism (PHP) refers to a group of rare genetic and epigenetic disorders characterized by resistance to the action of parathyroid hormone (PTH) that activates cAMP signaling in target cells. Together with pseudohypoparathyroidism, Albright hereditary osteodystrophy (AHO) and progressive osseous heteroplasia (POH) represent rare, related and deeply impairing disorders encompassing heterogeneous features, such as brachydactyly, ectopic ossifications, short stature, mental retardation and endocrine deficiencies due to resistance to the action of different hormones. The two main subtypes, PHP-Ia and PHP-Ib, are caused by mutations in GNAS exons 1-13 and methylation defects in the imprinted GNAS cluster respectively, while mutations in the PRKAR1A and PDE4D genes (also involved in mediating cAMP signalling) have been demonstrated in patients with acrodysostosis, a disease of bone formation with characteristics similar to AHO. The molecular overlap among these disorders indicates the need for different classification models and seriously alters our understanding of the mechanisms through which GNAS defects, together with the new recently described defects involving other components of the cAMP signalling cascade, cause AHO-related disorders.     

Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells

Glucocorticoids are integral to successful treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. A large body of data indicates that in various model systems, elevation of cyclic adenosine monophosphate (cAMP) can potentiate glucocorticoid response, although this has not been well evaluated as a potential leukemia treatment. Although cAMP analogs have been studied, little data exist regarding the potential toxicity to leukemia cells of pharmacologic elevation of cAMP levels in leukemic blasts. Using MTT assays of cell proliferation on CEM ALL cells, we found that aminophylline and other nonspecific phosphodiesterase (PDE) inhibitors suppress cell growth. This effect is replicated by the PDE4-specific PDE inhibitor rolipram, but not by specific inhibitors of the PDE1 or PDE3 classes. We found that PDE inhibitors cause increased dexamethasone sensitivity and a synergistic effect with the adenylyl cyclase activator forskolin. We observed several important cellular characteristics associated with this treatment, including elevation of cAMP, induction of p53 and p21(WAF1/CIP1) proteins, G(1) and G(2)/M cell cycle arrest, and increased apoptosis. Sensitivity to forskolin and rolipram is shared by at least 2 pediatric ALL cell lines, CEM and Reh cells. Some cell lines derived from adult-type lymphoid malignancies also show sensitivity to this treatment. These findings suggest that PDE inhibitors have therapeutic potential in human ALL and characterize the molecular mechanisms that may be involved in this response.     

Role of phosphodiesterase and protein kinase G on nitric oxide-induced inhibition of prolactin release from the rat anterior pituitary

OBJECTIVE: In order to determine the mechanism by which nitric oxide (NO) inhibits prolactin release, we investigated the participation of cGMP-dependent cAMP-phosphodiesterases (PDEs) and protein kinase G (PKG) in this effect of NO. METHODS: Anterior pituitary glands of male rats were incubated with inhibitors of PDE and PKG with or without sodium nitroprusside (NP). Prolactin release, and cAMP and cGMP concentrations were determined by RIA. RESULTS AND CONCLUSIONS: The inhibitory effect of NP (0.5 mmol/l) on prolactin release and cAMP concentration was blocked by EHNA (10(-4)mol/l) and HL-725 (10(-4)mol/l), inhibitors of cGMP-stimulated cAMP-PDE (PDE2). 8-Br-cGMP (10(-4) and 10(-3)mol/l), which mimics cGMP as a mediator of NP effects on prolactin release, also decreased cAMP concentration. Zaprinast (10(-4)mol/l), a selective inhibitor of specific cGMP-PDE (PDE5), potentiated the NP effect on cAMP concentration. Rp-8-[(4-chlorophenyl)thio]-cGMP triethylamine (Rp-8-cGMP, 10(-7)-10(-6)mol/l), an inhibitor of PKG, reversed the effect of NP on prolactin release. The present study suggests that several mechanisms are involved in the inhibitory effect of NO on prolactin release. The activation of PDE2 by cGMP may mediate the inhibitory effect of NO on cAMP concentration and therefore on prolactin release. NO-activated PKG may also be participating in the inhibitory effect of NO on prolactin release.