Expression, Structure/function And Regulation Of Phosphodiesterase 3 Isoforms

磷酸二酯酶 3 亚型的表达、结构/功能和调控

• 批准号: 7734977
• 负责人: VINCENT MANGANIELLO
• 金额: $ 164.15万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734977
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Phosphodiesterase type 3 (PDE3) is an important regulator of cAMP-mediated responses within the cardiovascular system. PDE3 exists as two subtypes: PDE3A and PDE3B, with distinct cellular and subcellular locations. Due to the lack of subtype-specific pharmacological tools, the definitive role of each subtype in regulating cardiovascular function has not been determined. We therefore have begun to examine the subcellualr localization of PDE3 isoforms and their contribution to functional regulation of cAMP signaling pathways in human myocardium.We attempted to determine if caveolae-enriched myocardial plasma membranes and internal membrane sarcoplasmic reticulum fractions represent specialized subdomains that concentrate and organize different isoforms of PDE3A and PDE3B in their regulation of cAMP-mediated signaling in human heart. Caveolae-enriched membrane fractions from failing heart myocardium are highly enriched in b2-ARs, b3-ARs, G(i), adenylyl cyclase 5/6 and caveolin-1, but not b1-ARs or G(s). Approximately 30% of the total membrane PDE3 activity is associated with PM/caveolae and 70% with internal membrane fractions. PDE3A3 is the dominant PDE3A isoform in PM/caveolae fractions, while all isoforms of PDE3A (A1-3) and PDE3B1 are present in internal membranes. Based on the histochemical staining with affinity purified antibodies, PDE3A is more highly expressed in cardiac myocytes than in endothelial cells or vascular smooth muscle myocytes, while PDE3B is more highly expressed in vascular smooth muscle myocytes. On gel filtration chromatography, PDE3 activity in solubilized membrane fractions from human ventricle was partitioned between distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. The HMW peaks likely contain multiprotein complexes, including PDE3A1, PDE3A2, and PDE3B as well as several proteins involved in b-adrenergic receptor-mediated signaling b1-AR, b2-AR, AC5/6 ,PKA-RII, caveolin-1, and most of the AKAPs that we tested. PDE3 activity in cytosolic fractions consisted primarily of PDE3A2 and PDE3A3, and eluted only in LMW fractions. These results indicate that PDE3 isoforms in myocardial membranes are likely to incorporate into multiprotein complexes. In collaborative studies, we also examined expression, activity and function of phosphodiesterases, including PDE3 isoforms, in the mature and immature ductus arteriosus. A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. Following PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE) mediated degradation. We used ductus from immature (n=25) and mature (n=21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activities that contribute to the decreased sensitivity of the late gestation ductus arteriosus to vasodilators like PGE2. Although mechanisms for acute activation of PDE3B and its potential physiological roles have been studied in isolated adipocytes and cultured cells, its' role(s)in human and animal physiology is not well understood. To evaluate these physiological functions, we introduced a targeted disruption in the murine PDE3B gene by homologous recombination. Obesity is a major risk factor for developing type 2 diabetes and cardiovascular disease. White adipose tissue (WAT) affects body fat and energy utilization via storage and turnover/hydrolysis of triglycerides. In addition, via production of endocrine factors, adipocytokines and lipids, WAT regulates and integrates important physiological pathways and homeostatic mechanisms, including energy utilization, glucose homeostasis, peripheral insulin sensitivity, and systemic inflammatory responses. WAT, unfortunately, can also contribute to metabolic dysregulation that characterizes insulin resistance and obesity-related metabolic and cardiovascular complications. Aquirement of BAT characteristics by WAT, with enhanced intra-adipocyte FAO (fatty and oxidation), represents a potential new strategy in treatment of obesity and diabetes. In PDE3B KO mice, epididymal WAT (EWAT) exhibits characteristics of BAT, including changes in morphology (increased mitochondria number and size in KO EWAT), increased vascularization, and increased expression of genes related to BAT differentiation, mitochondrial biogenesis and energy dissipation, including PGC1a, PPARa, uncoupling protein-1 (UCP-1), and B-oxidation enzymes. Mitochondria were isolated from wild-type and PDE3B KO mice using discontinuous sucrose gradients, and were studied by electron microscopic (EM) and proteomics techniques and functional assays. Sucrose gradient and EM data demonstrated two populations of mitochondria, with EWAT containing lighter and smaller mitochondria, and BAT, heavier and larger mitochondria. EWAT from KO contained both populations of mitochondria. In KO EWAT, isolated mitochondria are uncoupled, with dysfunction of mitochondrial complex I (NADH:ubiquinone oxidoreductase), consuming less oxygen and generating less ATP than WT EWAT mitochondria. Whereas mitochondrial membrane potential of EWAT mitochondria in WT and KO were not significantly different, EWAT mitochondria from KO mice were more sensitive to calcium in mitochondria swelling assays. 2-dimentional difference gel electrophoresis (DIGE) indicated that many FAO-related gene products were induced in KO. Alterations in cAMP/PKA- and AMP kinase-signaling pathways were reflected in alterations in lipolysis and FAO, in that the antilipolytic actions of insulin were inhibited and FAO was increased in isolated adipocytes from KO mice. Compared to WT mice, O2 consumption was increased in vitro in BAT and EWAT isolated from KO mice, and in intact KO mice treated with the B-3 adrenergic receptor agonist, CL316243 (CL). Taken together, these results suggested that PDE3B might function as a molecular switch determining white versus brown adipocyte differentiation, and thereby could play an important role in regulation of energy metabolism. Although, as we previously reported (J. Clin. Investig. 116:3240-3251, 2006), PDE3B seems to be important in regulating certain cAMP-signaling pathways, including lipolysis, insulin- induced anti-lipolysis, and cAMP-mediated insulin secretion, PDE3B KO mice also show signs of systemic insulin resistance, most likely due to dysregulation of hepatic glucose production. They are, however, lean and not diabetic, perhaps because, in PDE3B KO mice, the presence of good BAT in EWAT depots compensates for insulin resistance.

磷酸二酯酶3型（PDE3）是心血管系统中营地介导的反应的重要调节剂。 PDE3作为两个亚型存在：PDE3A和PDE3B，具有不同的细胞和亚细胞位置。由于缺乏亚型特异性药理学工具，尚未确定每个亚型在调节心血管功能中的明确作用。因此，我们已经开始研究PDE3同工型的子细胞定位及其对人心肌中cAMP信号通路的功能调节的贡献。我们试图确定是否可以确定卡韦洛伊 - 增强的心肌质膜和内部膜骨菌株的内部杂种和有限量的PELSIZE和OLINGERIASS的persize and Ornance and Organizize and Organsize of Persimize and Organsize of Off sectee and Organsize of Off pecs and Off insime and Organsize and Organsize and Organsize of and Organsize Off insime and Organsize。 PDE3B在调节人心脏中cAMP介导的信号传导中。富含Caveolae的心脏心肌富含Caveolae的膜级分在B2-ARS，B3-ARS，G（I），Adenylyl Cyclase 5/6和Caveolin-1中高度富集，但不具有B1-ARS或G（S）。总膜PDE3活性的大约30％与PM/Caveolae有关，而内膜内部分数为70％。 PDE3A3是PM/Caveolae级分中的主要PDE3A亚型，而PDE3A（A1-3）的所有同工型和PDE3B1都存在于内部膜中。基于与亲和力纯化抗体的组织化学染色相比，PDE3A在心肌细胞中比内皮细胞或血管平滑肌心肌细胞更高，而PDE3B在血管平滑肌肌细胞中的表达更高。在凝胶过滤色谱法上，将人心室溶解化膜级分的PDE3活性分为不同的高分子量（HMW）和低分子量（LMW）峰之间的PDE3活性。 HMW峰可能包含多蛋白络合物，包括PDE3A1，PDE3A2和PDE3B，以及几种参与B-肾上腺素能受体介导的信号B1-AR，B2-AR，AC5/6，PKA-RII，PKA-RII，CKA-RII，Caveolin-1，Caveolin-1，以及我们测试过的大多数AKAP的蛋白。胞质级分中的PDE3活性主要由PDE3A2和PDE3A3组成，仅在LMW级分中洗脱。这些结果表明，心肌膜中的PDE3同工型可能会掺入多蛋白络合物中。 在协作研究中，我们还研究了成熟和未成熟的动脉导管中磷酸二酯酶（包括PDE3同工型）的表达，活性和功能。专利导管动脉苏速于过早导管对PGE2的敏感性提高。 PGE2刺激后，未成熟的cAMP浓度高于成熟管道。 cAMP浓度取决于腺基环酶产生和磷酸二酯酶（PDE）介导的降解速率。我们使用未成熟（n = 25）和成熟（n = 21）胎羊的管道来研究PDE活性的发育增加是否可以解释成熟导管中PGE2刺激后的CAMP积累减少。随着妊娠的前进，导管中平滑肌PDE同工型（PDE1A，1B，1C，3A，3B，4D和5A）的mRNA表达和水解活性一样增加。 PDE1，PDE3和PDE4的选择性抑制剂在存在前列腺素和一氧化氮抑制剂的情况下放松了成熟和未成熟的管道。成熟的导管需要较高浓度的每种PDE抑制剂，以抑制其张力的程度与未成熟管道中相同的程度。胎儿主动脉的PDE表达没有发育变化。总之，我们观察到cAMP和CGMP PDE活动的发展增加，这有助于妊娠妊娠导管对PGE2（如PGE2）的血管舒张剂的敏感性降低。 尽管已经在孤立的脂肪细胞和培养的细胞中研究了PDE3B急性激活及其潜在生理作用的机制，但其在人类和动物生理学中的作用尚不清楚。 为了评估这些生理功能，我们通过同源重组引入了鼠PDE3B基因的靶向破坏。 肥胖是发展2型糖尿病和心血管疾病的主要危险因素。白脂肪组织（WAT）通过储存和周转/水解甘油三酸酯影响体内脂肪和能量利用。 此外，通过产生内分泌因子，脂肪细胞因子和脂质，WAT可以调节和整合重要的生理途径和稳态机制，包括能量利用，葡萄糖稳态，外周胰岛素敏感性和全身性炎症反应。 不幸的是，WAT还可以导致代谢失调，该代谢失调表征胰岛素抵抗以及与肥胖相关的代谢和心血管并发症。 WAT的蝙蝠特性含水，具有增强的脂肪细胞内粮农组织（脂肪和氧化），代表了治疗肥胖和糖尿病的潜在新策略。 In PDE3B KO mice, epididymal WAT (EWAT) exhibits characteristics of BAT, including changes in morphology (increased mitochondria number and size in KO EWAT), increased vascularization, and increased expression of genes related to BAT differentiation, mitochondrial biogenesis and energy dissipation, including PGC1a, PPARa, uncoupling protein-1 (UCP-1), and B-oxidation酶。 使用不连续的蔗糖梯度从野生型和PDE3B KO小鼠中分离线粒体，并通过电子显微镜（EM）以及蛋白质组学技术和功能测定进行研究。蔗糖梯度和EM数据显示了两个线粒体种群，含有较轻和较小的线粒体，蝙蝠，较重，更大的线粒体。来自KO的EWAT包含两个线粒体种群。在KO EWAT中，分离的线粒体与线粒体复合物I（NADH：泛氨基酮氧化还原酶）的功能障碍，消耗氧气较少，而与WT EWAT线粒体产生更少的ATP。尽管线粒体在WT和KO中的线粒体膜电位没有显着差异，但来自KO小鼠的EWAT线粒体对线粒体肿胀测定法对钙更敏感。二维差异凝胶电泳（DIGE）表明，KO中诱导了许多与FAO相关的基因产物。 CAMP/PKA和AMP激酶信号途径的改变反映在脂解和FAO的变化中，因为胰岛素的抗溶液作用被抑制，并且在KO小鼠的分离脂肪细胞中增加了FAO。 与WT小鼠相比，从KO小鼠中分离出的BAT和EWAT以及用B-3肾上腺素能受体激动剂CL316243（CL）处理的完整的KO小鼠中的O2消耗增加。 综上所述，这些结果表明，PDE3B可能是决定白色与棕色脂肪细胞分化的分子开关，因此可以在能量代谢的调节中发挥重要作用。尽管正如我们先前报道的那样（J. Clin。 生产。但是，它们是瘦而不是糖尿病患者，也许是因为在PDE3B KO小鼠中，EWAT仓库中存在良好的蝙蝠可以补偿胰岛素抵抗。

项目成果

Novel mechanisms of the regulation of protein kinase B in adipocytes; implications for protein kinase A, Epac, phosphodiesterases 3 and 4.

脂肪细胞中蛋白激酶B调节的新机制；

• DOI: 10.1016/j.cellsig.2006.05.024
• 发表时间: 2007
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: Zmuda-Trzebiatowska,Emilia;Manganiello,Vincent;Degerman,Eva
• 通讯作者: Degerman,Eva

Role of phosphodiesterase type 3A and 3B in regulating platelet and cardiac function using subtype-selective knockout mice.

• DOI: 10.1016/j.cellsig.2007.03.012
• 发表时间: 2007-08
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: Bing Sun;Haiquan Li;Y. Shakur;J. Hensley;S. Hockman;J. Kambayashi;V. Manganiello;Yongge Liu

Short-term regulation of PDE4 activity.

PDE4 活性的短期调节。

• DOI: 10.1038/sj.bjp.0704741
• 发表时间: 2002
• 期刊: British journal of pharmacology
• 影响因子: 7.3
• 作者: Manganiello,V

Systematic search for single nucleotide polymorphisms in the 5' flanking region of the human phosphodiesterase 3B gene: absence of evidence for major effects of identified polymorphisms on susceptibility to Japanese type 2 diabetes.

系统搜索人类磷酸二酯酶 3B 基因 5 侧翼区域的单核苷酸多态性：缺乏证据表明已识别的多态性对日本 2 型糖尿病易感性有重大影响。

• DOI: 10.1016/s1096-7192(03)00035-0
• 发表时间: 2003
• 期刊: Molecular genetics and metabolism
• 影响因子: 3.8
• 作者: Osawa,Haruhiko;Niiya,Toshiyuki;Onuma,Hiroshi;Murakami,Akiko;Ochi,Masaaki;Nishimiya,Tatsuya;Ogura,Takahiro;Kato,Kenichi;Shimizu,Ikki;Fujii,Yasuhisa;Ohashi,Jun;Yamada,Kazuya;Liang,Shu-Jian;Manganiello,VincentC;Fujita-Yamaguchi,
• 通讯作者: Fujita-Yamaguchi,

Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B.

腺病毒介导的鼠环核苷酸磷酸二酯酶 3B 的过度表达。

• DOI: 10.1385/1-59259-839-0:093
• 发表时间: 2005
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Ahmad,Faiyaz;Harndahl,Linda;Tang,Yan;Holst,LenaStenson;Manganiello,VincentC
• 通讯作者: Manganiello,VincentC