cGMP-speci®c, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time-and concentration-dependent reversible activation in response to cGMP, with the highest activation (9-to 11-fold) observed at low substrate concentrations (0.1 mM cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildena®l than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.
Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG).The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle. Smooth muscle cells (SMCs)1 of the blood vessels, uterus, bladder, gastrointestinal tract, and respiratory tract relax or contract in response to hormonal or mechanical stimulation. Nitric oxide, nitrovasodilators, and natriuretic peptides act as relaxants, regulating smooth muscle tone by direct activation of guanylyl cyclase, leading to the elevation of cGMP and activation of cGMP-dependent protein kinase (PKG). Similarly cAMP (through -adrenergic stimulation) also induces relaxation of the same smooth muscle types. However, it has been reported recently that the cGMP/PKG pathway is independent from the cAMP/PKA pathway as a regulator of smooth muscle tone (1). Activation of PKG appears to mediate all of the NO/ cGMP relaxant effects in vascular smooth muscle as disruption of the PKG I gene totally abolishes NO/cGMP-dependent relaxation of smooth muscle in mouse aorta, whereas in the same cells cAMP-dependent relaxation remained intact. Defective responses also were found in intestinal smooth muscle, where pyloric stenosis developed as a result of PKG I disruption. Disruption of the PKG I gene also caused erectile dysfunction in mice (2). In male mice the ability to reproduce was greatly diminished, and no relaxation was induced by nerve...
The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides. ( J. Clin. Invest. 1997. 100:2611-2621.)
Abstract-Proliferation of arterial smooth muscle cells (SMCs) is a key event in the formation of advanced atherosclerotic lesions and restenosis after angioplasty. Cyclic nucleotides (cAMP and cGMP) inhibit arterial SMC proliferation, and elevation of cyclic nucleotides reduces neointimal formation after angioplasty in animal models. Degradation of cAMP and cGMP is catalyzed by cyclic nucleotide phosphodiesterases (PDEs). One of these, PDE1C, hydrolyzes cAMP and cGMP and is expressed in proliferating human SMCs but is absent in quiescent human aorta. Thus, PDE1C expression is low in cultured human SMCs made quiescent by attaching to fibrillar collagen type I. After release from the fibrillar collagen, PDE1C expression is induced and associated with traverse through S-phase of the cell cycle. Further, PDE1C is expressed in vivo in human fetal aorta containing proliferating SMCs, but not in newborn aorta in which SMC proliferation has ceased. Inhibition of PDE1C in SMCs isolated from normal aorta or from lesions of atherosclerosis using antisense oligonucleotides or a PDE1 inhibitor results in suppression of SMC proliferation.
The nitric oxide (NO)-cGMP pathway has been implicated as playing a crucial role in the induction of cerebellar long-term depression (LTD). The amplitude and duration of the cGMP signal is controlled by cyclic nucleotide phosphodiesterases (PDEs). Here we identify PDE5 and PDE1B as the two major cGMP-hydrolyzing PDEs specifically and differentially expressed in the Purkinje neurons of mouse cerebellum. PDE5 was found in all Purkinje neurons, whereas PDE1B was detected only in a subset of these cells, suggesting that individual Purkinje cells may differentially regulate cGMP, depending on the PDE isozymes expressed. Although expression of guanylate cyclase and/or cGMP-dependent protein kinase (PKG) in Purkinje cells have been reported, neither cGMP accumulation nor PKG activation in these cells in vivo has been demonstrated. To determine if changes in PKG activation and PDE5 regulation occur in vivo we have examined the phosphorylation of PDE5 in mouse cerebellar Purkinje cells by immunocytochemistry and Western blot analyses using a phosphospecific PDE5 antibody. Injection of sodium nitroprusside or selective PKG activators into the lateral ventricle of mouse brain induced PDE5 phosphorylation in vivo, but was completely missing in Purkinje cell-specific PKG I knock-out mice. In cerebellar slices, treatment with sildenafil or IBMX led to different levels of phospho-PDE5 accumulation and activation of PDE5. These results suggest that phosphorylation of PDE5 in Purkinje neurons after cGMP-PKG activation performs a critical role in the termination of the cGMP signal during LTD progression; moreover, PDE5 phosphorylation may be used as an in vivo indicator for PKG activation.
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