RESUMEN
The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells.
Asunto(s)
Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Membranas Artificiales , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Plastoquinona/metabolismo , Animales , Transporte Biológico , Plaquetas/metabolismo , GMP Cíclico/metabolismo , Membrana Eritrocítica/metabolismo , Humanos , Liposomas/metabolismo , Compuestos Onio/química , Compuestos Organofosforados/química , Fosforilación , Plastoquinona/química , RatasRESUMEN
cGMP-specific phosphodiesterase (PDE5) has become a target for drug development for the treatment of a number of physiological dysfunctions, affected by changes in the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway. PDE5 has two highly homologous regulatory domains, GAF-A and GAF-B. We showed previously that PDE5 could be converted from a low-activity (nonactivated) state to a high-activity state upon cGMP binding to the GAF-A domain with higher sensitivities toward sildenafil (EMBO J 22:469-478, 2003). Here we investigated whether sildenafil sensitivity of PDE5 could be modified by cGMP-independent mechanisms. Individually expressed recombinant GAF-A and GAF-B proteins were tested for their ability to modulate full-length recombinant PDE5 affinity to sildenafil. The GAF-A domain protein had the most dramatic effect on the affinity of the nonactivated recombinant PDE5 for sildenafil, revealing much higher sensitivity to sildenafil inhibition. The apparent affinity for sildenafil increased from the nanomolar range to the picomolar range, providing evidence for the presence of a "super-high" sensitivity state of PDE5 for sildenafil inhibition. In human platelet, higher sensitivity of PDE5 for sildenafil inhibition has been detected after blocking cGMP-binding sites of the GAF-A domain. Thus, our data demonstrate that high sensitivity of PDE5 for sildenafil can be obtained not only through cGMP-induced activation of PDE, but also through cGMP-independent modulation of PDE5 in the nonactivated state, possibly through protein-protein interaction. Furthermore, data suggest that nonactivated PDE5 with "super-high" affinities for sildenafil inhibition may be responsible for therapeutic effects of long-term treatments with low doses of PDE5 inhibitors.
Asunto(s)
GMP Cíclico/fisiología , Inhibidores de Fosfodiesterasa 5 , Inhibidores de Fosfodiesterasa/farmacología , Piperazinas/farmacología , Sulfonas/farmacología , Animales , Plaquetas/enzimología , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Humanos , Ratones , Conformación Proteica , Estructura Terciaria de Proteína , Purinas/farmacología , Citrato de SildenafilRESUMEN
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.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Cerebelo/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Células de Purkinje/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-GMP Cíclico Fosfodiesterasas/química , Animales , Especificidad de Anticuerpos , Cerebelo/química , Cerebelo/citología , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Citosol/química , Citosol/enzimología , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos , Inmunohistoquímica , Inyecciones Intraventriculares , Depresión Sináptica a Largo Plazo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitroprusiato/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación/efectos de los fármacos , Células de Purkinje/enzimologíaRESUMEN
cGMP-specific, 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 micro M 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 sildenafil 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.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , GMP Cíclico/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/química , 3',5'-GMP Cíclico Fosfodiesterasas/genética , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Activación Enzimática , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Inhibidores de Fosfodiesterasa/metabolismo , Fosforilación , Piperazinas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Purinas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Citrato de Sildenafil , SulfonasRESUMEN
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. In conclusion, PDE1C expression is a marker of human SMC proliferation ex vivo and in vivo. Inhibition of PDE1C leads to inhibition of human SMC proliferation. Because PDE1C is absent in quiescent SMCs, PDE1C inhibitors may target proliferating SMCs in lesions of atherosclerosis or restenosis.
Asunto(s)
Músculo Liso Vascular/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Aorta/citología , Aorta/embriología , Aorta/enzimología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Colágeno Tipo I/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Recién Nacido , Isoenzimas/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacosRESUMEN
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.