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.
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