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1.
J Cereb Blood Flow Metab ; 37(12): 3759-3773, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28155571

RESUMO

Large conductance, Ca2+-activated K+ (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 µM) and sensitive to the BK channel inhibitor, penitrem-A (100 nM). Fostriecin (30 nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30 nM), a phosphodiesterase 5 (PDE5) inhibitor. Using proximity ligation assay (PLA)-based co-immunostaining, BK channels were observed to co-localize with PP2A, PDE5, and cGMP-dependent protein kinase (cGKI) (spatial restriction < 40 nm); cGKI co-localization increased following SNP exposure. SNP (10 µM) reversibly inhibited myogenic tone in cannulated cerebral arteries, which was augmented by either fostriecin or sildenafil and inhibited by penitrem-A. Collectively, these data suggest that (1) cGKI, PDE5, and PP2A are compartmentalized with cerebrovascular BK channels and determine the extent of BK current augmentation by NO/cGMP signaling, and (2) the dynamic regulation of BK activity by co-localized signaling enzymes modulates NO-evoked dilation of cerebral resistance arteries.


Assuntos
Artérias Cerebrais/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/análise , Masculino , Proteína Fosfatase 2/análise , Proteína Fosfatase 2/metabolismo , Ratos , Ratos Sprague-Dawley , Vasodilatação
2.
Int Urogynecol J ; 28(3): 431-436, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27686568

RESUMO

INTRODUCTION AND HYPOTHESIS: The objective was to investigate the expression of endothelial nitric oxide synthase (eNOS) and phosphodiesterase (PDE) 5 in vaginal tissue of premenopausal women experiencing stress urinary incontinence (SUI) with and without sexual dysfunction. METHODS: Women presenting for treatment of SUI were screened using the Female Sexual Function Index (FSFI) and 10 were selected who met the criteria for female sexual dysfunction (FSD) and 10 asymptomatic controls. Vaginal tissue specimens were obtained from those premenopausal women aged ≥40 years who had had sexual activity ≥2 times every month for the last 6 months and who were scheduled to undergo surgery for SUI. FSD criteria was FSFI scores <18 and arousal domain scores <3. The control group had FSFI scores ≥26 and individual domain scores ≥4. The expressions of eNOS and PDE 5 were compared in the two groups using immunofluorescence staining and western blotting. RESULTS: The mean total FSFI scores were 30.4 ± 2.6 and 15.3 ± 2.3 in the control and FSD groups respectively. In immunofluorescence staining, eNOS and PDE5 were localized in the vaginal epithelium. In western blotting, the expressions of eNOS and PDE5 were significantly lower in the FSD group than in the control group (p = 0.003 and p = 0.038 respectively). CONCLUSIONS: eNOS and PDE5 in the vagina may play important roles in the pathophysiology of FSD.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Epitélio/enzimologia , Óxido Nítrico Sintase/análise , Disfunções Sexuais Fisiológicas/enzimologia , Incontinência Urinária por Estresse/enzimologia , Vagina/enzimologia , Biomarcadores/análise , Western Blotting , Estudos de Casos e Controles , Feminino , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Pré-Menopausa , Disfunções Sexuais Fisiológicas/fisiopatologia , Incontinência Urinária por Estresse/fisiopatologia
3.
Sci Total Environ ; 565: 140-147, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27161135

RESUMO

A simple, fast and reliable analytical method for the determination of phosphodiesterase type V inhibitors in wastewater was developed and validated. The method was based on direct injection followed by liquid chromatography coupled to tandem mass spectrometry with triple quadrupole as mass analyzer. Transformation products and analogues were included in the target list besides the three active pharmaceutical ingredients (sildenafil, vardenafil and tadalafil). The method performance was thoroughly investigated, including the analyte stability in wastewater and matrix effect. All target compounds presented linear fits between their LOD and 500ng/L. The quantification limits ranged from 1.6 to 30ng/L for all compounds except for n-octylnortadalafil (LOQ: 100ng/L); precision calculated as intraday repeatability was lower than 30%; accuracy calculated as procedural recovery ranged successfully between 85 and 105% in all cases. The method was applied to samples collected during three week-long monitoring campaigns performed in 2013, 2014 and 2015 in three Dutch cities. Only sildenafil and its two metabolites, desmethyl- and desethylsildenafil, were present with normalized loads ranging from LOQ to 8.3, 11.8 and 21.6mg/day/1000 inh, respectively. Two additional week-long sets of samples were collected in Amsterdam at the time that a festival event took place, bringing around 350,000 visitors to the city. The difference in drug usage patterns was statistically studied: "weekday" versus "weekend", "normal" versus "atypical" week; and results discussed. The metabolite to parent drug concentration ratio evolution during consecutive years was discussed, leading to several possible explanations that should be further investigated. Finally, wastewater-based epidemiology approach was applied to back-calculate sildenafil consumption.


Assuntos
Cromatografia Líquida/métodos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Monitoramento Ambiental/métodos , Medicamentos sob Prescrição/análise , Águas Residuárias/análise , Poluentes da Água/análise , Países Baixos , Espectrometria de Massas em Tandem/métodos
4.
Urology ; 85(4): 964.e1-6, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25704994

RESUMO

OBJECTIVE: To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. METHODS: Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. RESULTS: Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. CONCLUSION: The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Músculo Liso/enzimologia , Uretra/enzimologia , Western Blotting , Peptídeo Relacionado com Gene de Calcitonina/análise , Humanos , Imuno-Histoquímica , Isoenzimas/análise , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Liso/inervação , Óxido Nítrico Sintase Tipo I/análise , Transdução de Sinais , Peptídeo Intestinal Vasoativo/análise
6.
J Urol ; 190(4): 1430-5, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23545097

RESUMO

PURPOSE: Phosphodiesterase type 5 inhibitors were recently introduced as a new treatment option for men with lower urinary tract symptoms. Safety and clinical effectiveness are well documented but the mode of action is still unclear. We determined and compared the expression of phosphodiesterase type 5 in the spinal cord of normal (sham operated) rats and rats with partial urethral obstruction induced bladder overactivity. We also assessed the urodynamic effects of intravenously and intrathecally administered sildenafil in the rats to determine whether phosphodiesterase type 5 inhibitors exert effects on the sacral spinal cord. MATERIALS AND METHODS: A total of 65 male Sprague-Dawley rats were used for molecular/morphological and functional experiments. Bladder overactivity was induced via surgical partial urethral obstruction in 39 of 65 rats. Spinal phosphodiesterase type 5 expression was assessed by histology and polymerase chain reaction. The effects of sildenafil administered intravenously or intrathecally were studied urodynamically. RESULTS: Phosphodiesterase type 5 was expressed in various regions of the lumbosacral spinal cord, including the sacral regions of micturition control. Expression was similar in normal rats and rats with partial urethral obstruction/bladder overactivity. In normal rats intravenous and intrathecal sildenafil had no urodynamic effect. When administered intravenously and intrathecally to rats with partial urethral obstruction/bladder overactivity, sildenafil decreased micturition frequency and bladder pressure. Doses tested intrathecally had no effect when given intravenously. CONCLUSIONS: Phosphodiesterase type 5 is expressed in the rat spinal cord. Intravenous sildenafil may exert part of its urodynamic effect in rats with partial urethral obstruction/bladder overactivity via an effect on the sacral spinal cord.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/fisiologia , Medula Espinal/metabolismo , Obstrução do Colo da Bexiga Urinária/complicações , Bexiga Urinária Hiperativa/etiologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Masculino , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Citrato de Sildenafila , Medula Espinal/química , Medula Espinal/efeitos dos fármacos , Sulfonas/farmacologia
7.
BJU Int ; 112(2): 246-57, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23452226

RESUMO

OBJECTIVE: To study PDE5 localisation by visualising the product of phosphodiesterase type 5 (PDE5) inhibition, namely cGMP, to determine the site of action of inhibitors in the urinary bladder. MATERIALS AND METHODS: Bladders of nine male guinea pigs were dissected and treated in wells containing 2 mL Krebs' solution and 1 µM of the specific PDE5 inhibitor vardenafil at 36 °C for 30 min. After stimulating tissues with 100 µM of the nitric oxide (NO) donor diethylamine-NONOate for 10 min, the tissues were snap-frozen and 9-10 µm sections were cut. Sections were examined for cGMP immunoreactivity and also stained for vimentin, a marker for interstitial cells and the neuromarkers protein gene product 9.5 (PGP9.5), synaptic vesicle protein 2 (SV2), neurofilament (NF) and calcitonin gene-related peptide (CGRP), using the two-step indirect immunohistochemistry technique. RESULTS: After PDE5 inhibition, cGMP was found to be present in the urothelium, suburothelial interstitial cells and endothelium of blood vessels. cGMP was not expressed in nerves positive for CGRP, NF and SV2, and was expressed only in very few efferent nerves positive for PGP9.5. CONCLUSION: Our data show that the possible sites of action of PDE5 inhibition in the bladder are the urothelium, suburothelial interstitial cells and blood vessels, rather than the bladder nerve fibres.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Bexiga Urinária/química , Bexiga Urinária/metabolismo , Animais , Cobaias , Masculino
8.
ACS Chem Neurosci ; 2(10): 600-7, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22860158

RESUMO

Eleven phosphodiesterase (PDE) families are known, each having several different isoforms and splice variants. Recent evidence indicates that expression of individual PDE family members is tissue-specific. Little is known concerning detailed PDE component expression in brain microvessels where the blood-brain-barrier and the local cerebral blood flow are thought to be regulated by PDEs. The present study attempted to identify PDE family members that are expressed in brain microvessels. Adult male F344 rats were sacrificed and blocks of the cerebral cortex and infratentorial areas were dissected. Microvessels were isolated using a filtration method, and total RNA was extracted. RNA quality and quantity were determined using an Agilent bioanalyzer. The isolated cortical and infratentorial microvessel total RNA amounts were 2720 ± 750 ng (n = 2) and 250 ± 40 ng (n = 2), respectively. Microarrays with 22 000 transcripts demonstrated that there were 16 PDE transcripts in the PDE superfamily, exhibiting quantifiable density in the microvessels. An additional immunofluorescent study verified that PDE4D (cAMP-specific) and PDE5A (cGMP-specific) were colocalized with RECA-1 (an endothelial marker) in the cerebral cortex using both F344 rats and Sprague-Dawley rats (n = 3-6/strain). In addition, PDE4D and PDE5A were found to be colocalized with alpha-smooth muscle actin which delineates cerebral arteries and arterioles as well as pericytes. In conclusion, a filtration method followed by microarray analyses allows PDE components to be identified in brain microvessels, and confirmed that PDE4D and PDE5A are the primary forms expressed in rat brain microvessels.


Assuntos
Encéfalo/enzimologia , Capilares/enzimologia , Diester Fosfórico Hidrolases/química , Actinas/metabolismo , Animais , Circulação Cerebrovascular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Imunofluorescência , Isoenzimas/química , Isoenzimas/genética , Masculino , Análise em Microsséries , Diester Fosfórico Hidrolases/genética , RNA/biossíntese , RNA/química , Ratos , Ratos Endogâmicos F344 , Recombinases Rec A/metabolismo
9.
J AOAC Int ; 94(6): 1770-7, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22320083

RESUMO

An analog of aildenafil, which is a potent and highly selective inhibitor of phosphodiesterase 5, was found in a dietary supplement marketed for enhancement of sexual function. The compound was isolated by silica gel column chromatography, and its structure was identified by means of 13C-NMR spectrometry, 1H-NMR spectrometry, high-resolution MS, and X-ray structure determination. The compound was identified to be sulfoaildenafil (other names: thioaildenafil, dimethyl sildenafil thione, and thiomethisosildenafil). Sulfoaildenafil is very similar to the compound thiohomosildenafil. As it is difficult to distinguish between them by LC-photodiode array detector analysis, ultra-performance LC (UPLC)/MS, ion trap LC/MS/MS (LC/IT-MS/MS), and GC/MS were performed. The mass spectra of thiohomosildenafil by UPLC/MS and LC/IT-MS/MS showed mass fragments of m/z 58, 72, and 355, and the mass spectrum by GC/MS showed mass fragments of m/z 56, 72, and 420. Some of these fragments had low intensities, but they were useful for distinguishing between the two compounds. The relationship between aildenafil (other names: dimethylsildenafil and methisosildenafil) and homosildenafil is similar to that between sulfoaildenafil and thiohomosildenafil. Therefore, these compounds were also examined.


Assuntos
Suplementos Nutricionais/análise , Piperazinas/análise , Sulfonas/análise , Cromatografia Líquida/métodos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Inibidores da Fosfodiesterase 5/análise , Espectrometria de Massas em Tandem/métodos
10.
Hypertens Res ; 33(9): 899-904, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20555333

RESUMO

Nitric oxide (NO) is a short-lived intercellular messenger that provides an efficient vascular regulatory mechanism to support homeostasis and prevent thrombosis. Endothelial dysfunction and reduced NO bioavailability have a central role in hypertension associated with pregnancy. The purpose of this study was to investigate the impact of pregnancy on the L-arginine-NO-cGMP pathway in platelets and its correlation to platelet function and blood pressure in normotensive rats and spontaneously hypertensive rats (SHRs). Platelets were obtained from blood on the 20th day of pregnancy from female SHRs (SHR-P) and normotensive controls (P) or age-matched nonpregnant rats (SHR-NP and NP). Intraplatelet NO synthase (NOS) activity was reduced in P compared to NP, despite unchanged L-arginine influx. The expression levels of endothelial NOS (eNOS) and inducible NOS (iNOS) were diminished during pregnancy in normotensive rats. Paradoxically, cyclic guanosine monophosphate (cGMP) levels were similar between NP and P, as were phosphodiesterase type 5 (PDE5) expression and platelet aggregation induced by adenosine diphosphate. In SHRs, L-arginine influx was reduced in SHR-P compared to SHR-NP. SHR-P exhibited impaired NOS activity and reduced iNOS expression compared with SHR-NP. Soluble guanylyl cyclase and PDE5 expression in platelets were lower in SHR-P than in SHR-NP, whereas no differences were noted between groups with respect to cGMP levels. However, increased levels of cGMP were observed in SHR-P compared to normotensive groups and platelet aggregability remained unaltered. In conclusion, these observations prompted the hypothesis that normal platelet aggregation in pregnant SHRs may be related to a reduction in PDE5 expression and consequently the maintenance of cGMP levels, independently of reduced platelet NO bioavailability.


Assuntos
Arginina/metabolismo , GMP Cíclico/metabolismo , Hipertensão/metabolismo , Óxido Nítrico/metabolismo , Animais , Arginina/fisiologia , Plaquetas/enzimologia , GMP Cíclico/análise , GMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Feminino , Guanilato Ciclase/análise , Hipertensão/fisiopatologia , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo III/análise , Agregação Plaquetária , Gravidez , Ratos , Ratos Endogâmicos SHR , Ratos Wistar
11.
Biochem J ; 414(3): 363-74, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18503409

RESUMO

Post-translational modification by isoprenylation is a pivotal process for the correct functioning of many signalling proteins. The Drosophila melanogaster cGMP-PDE (cGMP-specific phosphodiesterase) DmPDE5/6 possesses a CaaX-box prenylation signal motif, as do several novel cGMP-PDEs from insect and echinoid species (in CaaX, C is cysteine, a is an aliphatic amino acid and X is 'any' amino acid). DmPDE5/6 is prenylated in vivo at Cys(1128) and is localized to the plasma membrane when expressed in Drosophila S2 cells. Site-directed mutagenesis of the prenylated cysteine residue (C1128S-DmPDE5/6), pharmacological inhibition of prenylation or co-expression of DmPrBP (Drosophila prenyl-binding protein)/delta each alters the subcellular localization of DmPDE5/6. Thus prenylation constitutes a critical post-translational modification of DmPDE5/6 for membrane targeting. Co-immunoprecipitation and subcellular-fractionation experiments have shown that DmPDE5/6 interacts with DmPrBP/delta in Drosophila S2 cells. Transgenic lines allow targeted expression of tagged prenylation-deficient C1128S-DmPDE5/6 in Type I (principal) cells in Drosophila Malpighian tubules, an in vivo model for DmPDE5/6 function. In contrast with wild-type DmPDE5/6, which was exclusively associated with the apical membrane, the C1128S-DmPDE5/6 mutant form was located primarily in the cytosol, although some residual association occurred at the apical membrane. Despite the profound change in intracellular localization of C1128S-DmPDE5/6, active transport of cGMP is affected in the same way as it is by DmPDE5/6. This suggests that, in addition to prenylation and interaction with DmPrBP/delta, further functional membrane-targeting signals exist within DmPDE5/6.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prenilação de Proteína
12.
Cell Signal ; 20(8): 1423-31, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18467075

RESUMO

Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of PDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze PDE activity in living cells. It allows to sense cAMP gradients around particular PDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/análise , 3',5'-GMP Cíclico Fosfodiesterases/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Inibidores de Fosfodiesterase/farmacologia , Linhagem Celular , AMP Cíclico/análise , GMP Cíclico/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Humanos , Inibidores da Fosfodiesterase 3 , Inibidores da Fosfodiesterase 4 , Inibidores da Fosfodiesterase 5 , Proteínas Recombinantes de Fusão/análise
13.
Circ Res ; 102(2): 226-33, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17991881

RESUMO

In the pulmonary vasculature, cGMP concentrations are regulated in part by a cGMP-dependent phosphodiesterase (PDE), PDE5. Infants with persistent pulmonary hypertension of the newborn (PPHN) are often mechanically ventilated with high oxygen concentrations. The effects of hyperoxia on the developing pulmonary vasculature and PDE5 are largely unknown. Here, we demonstrate that exposure of fetal pulmonary artery smooth muscle cells (FPASMCs) to high levels of oxygen for 24 hours leads to decreased responsiveness to exogenous NO, as determined by a decreased intracellular cGMP response, increased PDE5 mRNA and protein expression, as well as increased PDE5 cGMP hydrolytic activity. We demonstrate that inhibition of PDE5 activity with sildenafil partially rescues cGMP responsiveness to exogenous NO. In FPASMCs, hyperoxia leads to increased oxidative stress without increasing cell death. Treatment of normoxic FPASMCs with H2O2 is sufficient to induce PDE5 expression and activity, suggesting that reactive oxygen species mediate the effects of hyperoxia in FPASMCs. In support of this mechanism, a chemical antioxidant, N-acetyl-cysteine, is sufficient to block the hyperoxia-mediated increase in PDE5 expression and activity and rescue cGMP responsiveness to exogenous NO. Finally, ventilation of healthy neonatal sheep with 100% O2 for 24 hours leads to increased PDE5 protein expression in the resistance pulmonary arteries and increased PDE5 activity in whole lung extracts. These data suggest that PDE5 expression and activity play a critical role in modulating neonatal pulmonary vascular tone in response to common clinical treatments for PPHN, such as oxygen and inhaled NO.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Regulação Enzimológica da Expressão Gênica , Hiperóxia/enzimologia , Miócitos de Músculo Liso/enzimologia , Artéria Pulmonar/embriologia , Animais , Animais Recém-Nascidos , Células Cultivadas , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Hipertensão Pulmonar , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/farmacologia , Artéria Pulmonar/citologia , RNA Mensageiro/análise , Carneiro Doméstico
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