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Abstract-In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated "contractile" phenotype to a dedifferentiated "synthetic" one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca 2ϩ -calmodulin-stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial "contractile" VSMCs but is nuclear in neointimal "synthetic" VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the "contractile" phenotype, whereas nuclear PDE1A is with the "synthetic" phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G 1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis. (Circ Res. 2006;98:777-784.) Key Words: PDE Ⅲ smooth muscle cell Ⅲ growth Ⅲ apoptosis Ⅲ vascular injury V ascular smooth muscle cells (VSMCs) in response to injury and hormonal stimuli exhibit phenotypic plasticity, changing from a differentiated (quiescent, contractile) phenotype to a dedifferentiated (active, synthetic) one. 1 This process was originally defined as "phenotypic modulation." 2 Under normal conditions, VSMCs residing in the media of vessels are quiescent with a very low turnover rate. 3,4 Quiescent VSMCs are fully differentiated cells that possess the "contractile" phenotype and function principally to maintain vascular tone. If the vessel is injured or cells are placed in tissue culture, VSMCs respond by changing from the "contractile" to the "synthetic" phenotype. 4 Synthetic VSMCs contribute to neointima formation by downregulating contractile proteins and acquiring the capacity to proliferate, migrate, and produce extracellular matrix proteins. 5 Therefore, phenotypic modulation of VSMCs plays a key role in the pathogenesis of cardiovascular disorders such as atherosclerosis, postangioplasty restenosis, bypass vein graft failure, and ca...

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K m values of ϳ1 M and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 M, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP-and Ca 2؉ -mediated signaling in these cells. PDE13 cyclic nucleotide phosphodiesterases are dual-specificity enzymes that bind and hydrolyze cAMP and cGMP in a mutually competitive manner (for review, see Ref. 1). Their activity is increased by their binding to Ca 2ϩ and calmodulin, a feature unique to the PDE1 family of cyclic nucleotide phosphodiesterases. Three PDE1 genes, PDE1A, PDE1B, and PDE1C, have been identified, and several protein isoforms are generated from these genes by alternative splicing. PDE1A and PDE1B isoforms have significantly higher affinities for cGMP than for cAMP, whereas PDE1C isoforms have similar affinities for both substrates (2-4). The modulation of the catalytic activity of PDE1 isoforms by Ca 2ϩ -dependent stimulation and their susceptibility to mutually competitive inhibition by cAMP and cGMP suggest that these enzymes may be points of interaction among several signaling pathways.Cyclic nucleotide phosphodiesterases are particularly important in cardiac muscle. In humans, alterations in cAMP metabolism and cAMP-mediated signaling leading to decreases in intracellular cAMP content and in the phosphorylation of some substrates of cAMP-dependent protein kinase are prominent features of the pathophysiology of heart failure, and inhibition of PDE3 cAMP hydrolytic activity has a major role in its treatment (5). Changes in cGMP-mediated signaling in cardiac disease have not been ch...

In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and cAMP hydrolysis at 1 M, at which it inhibits PDE1. In contrast, in preparations from the left ventricles of normal mice and mice with heart failure resulting from coronary artery ligation, the effects of sildenafil on cGMP hydrolysis were attributable to inhibition of both PDE5 and PDE1; PDE5 comprised ϳ22 and ϳ43% of the cytosolic cGMP-hydrolytic activity in preparations from normal and failing mouse hearts, respectively. These differences in PDE5 activities in human and mouse hearts call into question the extent to which the effects of sildenafil in mouse models are likely to be applicable in humans and raise the possibility of PDE1 as an alternative therapeutic target.The PDE5-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil is used in the treatment of erectile dysfunction and pulmonary hypertension. Its actions in these diseases result from its inhibition of cGMP-hydrolytic activity in vascular smooth muscle myocytes and the consequent increases in intracellular cGMP content and the potentiation of protein kinase G-mediated vasodilatory responses (Francis and Corbin, 2005;Kass et al., 2007;Movsesian et al., 2008).

This study assessed the effects of selective inhibitors of 3 ,5 -cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 ؎ 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 ؎ 25% and 235 ؎ 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 ؎ 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 ؎ 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 ؎ 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes. Adipose tissue functions as an energy storage organ in which excess calories are sequestered in the form of triglyceride (TG). TG contained in circulating chylomicrons and VLDL particles is hydrolyzed by extracellular lipoprotein lipase to yield glycerol and FFA. FFA is then taken up by adipocytes, converted to fatty acyl-CoA, and reesterified with glycerol-3-phosphate to form intracellular TG. The size of adipose TG stores is dynamically regulated by endocrine signals in response to energy intake and metabolic demands. Thus, anabolic hormones, such as insulin, stimulate adipocyte TG synthesis (lipogenesis), whereas catabolic hormones, such as epinephrine, glucagon, and corticotropin, stimulate hydrolysis of adipocyte TG to glycerol and FFA (lipolysis).Cyclic AMP is an important second messenger in the signaling pathways that mobilize fat stores (1). Catecholamines (epinephrine and norepinephrine) stimulate adipocyte lipolysis by binding to ␤ -adrenoceptors, which activate adenylyl cyclase (AC) via the stimulatory guanine nucleotide binding protein (G s ), leading to an increase in intracellular cAMP and activation of cAMP-dependent protein kinase (PKA). Initially, cAMP-mediated stimulation of lipolysis was thought to be attributable exclusively to PKA-dependent phosphorylation and activation of hormone-sensitive lipase (HSL), the primary neutral lipase in adipose tissue (2). However, adipocytes from HSL knockout mice retain considerable TG lipase activity, and lipolysis in these cells is partially responsive to the ␤ -adrenoceptor agonist isoproterenol (ISO), suggesting that other lipases besides HSL play a role in lipolysis (3-5). Another layer of regulation of lipolysis is revealed by the observation that lipolytic stimuli cau...

The aim of this study was to determine, in an animal model, the effects of tadalafil on myocardial infarct size (IS), hemodynamics and regional myocardial blood flow after myocardial ischemia and reperfusion. Patients with erectile dysfunction (ED) often have risk factors for coronary artery disease. Tadalafil, a long-acting inhibitor of the enzyme phosphodiesterase-5 (PDE5), is used for the treatment of ED; there are no previous data regarding tadalafil in the setting of coronary artery occlusion (CAO). Sprague-Dawley male rats were treated with tadalafil or vehicle (10 mg/kg, by gastric gavage), 2 h before a 30 min CAO. Heart rate was comparable between tadalafil and control groups. Tadalafil reduced mean arterial pressure (P ¼ 0.009), systolic (P ¼ 0.035) and diastolic (P ¼ 0.009) blood pressures during ischemia/reperfusion. Tadalafil significantly reduced IS (4272%) versus controls (5473%) (P ¼ 0.006). For the first time, we showed that the PDE5 inhibitor, tadalafil, was well tolerated and cardioprotective in the setting of an experimental myocardial infarction, by substantially reducing ischemic cell death.

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 4sCa2+ uptake into vesicles containing reconstituted calcium cnnes were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of asCa2+ uptake was linearly proportional to the extent of phosphorylation of the a, and 13 subunits of the calcium channel up to a stoichiometry of %1 mol of phosphate incorporated into each subunit. The calcium channeis activated by phosphorylation were determined to be incorporated into the reconstituted vescles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2 , Ni2+, and +. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation ofthe a, and 13 subunits ofthe purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.