RESUMEN
Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Miometrio/metabolismo , Embarazo/fisiología , Neoplasias Uterinas/enzimología , Neoplasias Uterinas/patología , Útero/metabolismo , Citoesqueleto de Actina/ultraestructura , Adulto , Anciano , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Femenino , Humanos , Inmunohistoquímica , Proteínas de Microfilamentos , Persona de Mediana Edad , Miometrio/citología , Miometrio/patología , Grupos Raciales , Ratas , Valores de Referencia , Estados Unidos , Útero/citología , Útero/patología , CalponinasRESUMEN
Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Ciclo Menstrual/fisiología , Miometrio/enzimología , Adulto , Anciano , Western Blotting , Cuello del Útero/enzimología , Cuello del Útero/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Endometrio/irrigación sanguínea , Endometrio/enzimología , Endometrio/fisiología , Femenino , Humanos , Inmunohistoquímica , Menstruación/fisiología , Persona de Mediana Edad , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/fisiología , Miometrio/irrigación sanguínea , Miometrio/fisiología , Transducción de Señal/fisiología , Contracción Uterina/fisiologíaRESUMEN
Arterial smooth muscle cells undergo phenotypic and proliferative changes in response to balloon catheter injury. Nitric oxide (NO) and cGMP have been implicated in the inhibition of vascular smooth muscle cell proliferation and phenotypic modulation in cultured-cell studies. We have examined the expression of the major cGMP receptor protein in smooth muscle, cGMP-dependent protein kinase I (PKG), in response to balloon catheter injury in the swine coronary artery. On injury, there was a transient decrease in the expression of PKG in neointimal smooth muscle cells when compared with medial smooth muscle cells. The decrease in PKG expression was observed in the population of proliferating cells expressing the extracellular matrix protein osteopontin but not in cells present in the uninjured portion of the media. Coincident with the suppression of PKG expression in neointimal cells after injury, there was a marked increase in the expression of type II NO synthase (inducible NOS [iNOS], NOS-II) in the neointimal cells. These results suggest that PKG expression is transiently reduced in response to injury in the population of coronary arterial smooth muscle cells that are actively proliferating and producing extracellular matrix proteins. The reduction in PKG expression is also correlated temporally with increases in inflammatory activity in the injured vessels as assessed by iNOS expression. Coupled with our current knowledge regarding the role of PKG in the regulation of cultured cell phenotypes, these results imply that PKG may also regulate phenotypic modulation of vascular smooth muscle cells in vivo as well.
Asunto(s)
Vasos Coronarios/lesiones , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Angioplastia de Balón , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Autopsia , Western Blotting , Cateterismo , División Celular , Células Cultivadas , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Regulación hacia Abajo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Inmunohistoquímica , Modelos Animales , Músculo Liso Vascular/patología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Coloración y Etiquetado , Porcinos , Factores de Tiempo , Túnica Íntima/metabolismo , Túnica Íntima/patología , Cicatrización de HeridasRESUMEN
Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , GMP Cíclico/metabolismo , Neuronas Motoras/citología , Óxido Nítrico/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores Enzimáticos/farmacología , Feto/citología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/enzimología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/metabolismo , Oligopéptidos/farmacología , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Ratas , SolubilidadRESUMEN
Increases in guanosine 3',5'-cyclic monophosphate (cGMP) induced by nitric oxide (NO), nitrovasodilators, and atrial peptides correlate with relaxation of vascular smooth muscle. Relaxation of myometrial smooth muscle by increases in cGMP, however, has required unusually high concentrations of the cyclic nucleotide. We tested the hypothesis that the sensitivity of myometrium to relaxation by cGMP is increased during pregnancy. Aortic smooth muscle was more sensitive to relaxation by cGMP than myometrial tissues, and, contrary to our hypothesis, myometrium from pregnant rats was least sensitive. Although levels of cGMP were elevated after treatment with the NO donor, S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels of cGMP-dependent protein kinase (PKG) were significantly decreased in myometrium from pregnant rats compared with myometrium from nonpregnant cycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, and progesterone antagonized this response. We conclude that 1) myometrial tissues from pregnant rats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP is accompanied by progesterone-mediated decreases in the level of PKG expression.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relajación Muscular , Miometrio/enzimología , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , GMP Cíclico/análogos & derivados , Regulación hacia Abajo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Guanilato Ciclasa/metabolismo , Músculo Liso Vascular/enzimología , Óxido Nítrico/metabolismo , Ovariectomía , Penicilamina/análogos & derivados , Penicilamina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Embarazo , Ratas , Ratas Sprague-Dawley , S-Nitroso-N-AcetilpenicilaminaRESUMEN
The role of cGMP-dependent protein kinase (PKG) in the regulation of rat aortic vascular smooth muscle cells (VSMC) phenotype was examined using a transfected cell culture system. Repetitively passaged VSMC do not express PKG and exist in the synthetic phenotype. Transfection of PKG-l alpha cDNA, or the active catalytic domain of PKG-l alpha, resulted in the appearance of VSMC having a morphology consistent with the contractile phenotype. PKG-expressing cells also contained markers for the contractile phenotype (for example, smooth muscle specific myosin heavy chain, calponin, alpha-actin) and reduced levels of synthetic phenotype markers (osteopontin, thrombospondin). PKG-transfected VSMC have also reduced the levels of fibroblast growth factor receptors 1 and 2, consistent with the establishment of a more contractile phenotype. The regulation of PKG expression in VSMC is largely undefined; however, continuous exposure of cultured bovine aortic smooth muscle cells with nitric oxide (NO)-donor drugs or cyclic nucleotide analogues reduced the expression of PKG. These results suggest that PKG occupies a critical role in VSMC phenotype and that suppression of PKG expression during inflammation or injury promotes a more synthetic state of the VSMC.
Asunto(s)
AMP Cíclico/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiopatología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Enfermedades Vasculares/fisiopatología , Animales , Western Blotting , Células Cultivadas , Microscopía de Contraste de Fase , Fenotipo , Ratas , Ratas Sprague-Dawley , Transfección/fisiologíaRESUMEN
A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators.
Asunto(s)
AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/biosíntesis , GMP Cíclico/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Músculo Liso Vascular/enzimología , Transcripción Genética/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/enzimología , Bucladesina/farmacología , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Colforsina/farmacología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Isosorbida/farmacología , Cinética , Masculino , Músculo Liso Vascular/efectos de los fármacos , Nitroprusiato/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , ARN Mensajero/biosíntesis , Ratas , S-Nitroso-N-Acetilpenicilamina , Teofilina/farmacología , Tionucleótidos/farmacologíaRESUMEN
Nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) have been reported to prevent vascular smooth muscle cell (VSMC) proliferation and have beneficial effects to reduce intimal thickening in response to arterial injury. The purpose of this study was to determine whether the downstream effector molecule of NO-cGMP signaling, cyclic GMP-dependent protein kinase (PKG), regulates phenotypic modulation and proliferation in cultured rat aortic VSMC. PKG-expressing VSMC lines were created by transfection of PKG-deficient cell lines and characterized. All forms of PKG, i.e. PKG-I alpha and PKG-I beta, as well as the constitutively active catalytic domain of PKG-I, transformed dedifferentiated 'synthetic' VSMC to a more contractile-like morphology. PKG expression resulted in an increased production of the contractile phenotype marker proteins, smooth muscle myosin heavy chain-2, calponin and alpha-actin and restored the capacity of cAMP and cGMP analogues to inhibit platelet-derived growth factor (PDGF)-induced cell migration. On the other hand, PKG expression had no significant effects on PDGF-induced cell proliferation. These results suggest that PKG expression contributes to the regulation of a contractile-like phenotypic expression in cultured VSMC, and the suppression of PKG expression during cultured growth in vitro may permit the modulation of cells to a more synthetic, dedifferentiated phenotype.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Músculo Liso Vascular/citología , Animales , Western Blotting , División Celular , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Colforsina/farmacología , Músculo Liso Vascular/enzimología , Cadenas Pesadas de Miosina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Nitric oxide mediates diverse functions in development and physiology of vertebrate skeletal muscle. Neuronal type nitric oxide synthase-mu is enriched in fast-twitch fibers and binds to syntrophin, a component of the sarcolemmal dystrophin glycoprotein complex. Here, we show that cyclic GMP-dependent protein kinase type I, a primary effector for nitric oxide, occurs selectively at the neuromuscular junction, in mice and rats, and both neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I remain at skeletal muscle endplates at least two weeks following muscle denervation. Expression of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I are up-regulated following fusion of cultured primary myotubes. Interestingly, the highest levels of neuronal type nitric oxide synthase-mu in muscle are found complexed with dystrophin at the sarcolemma of intrafusal fibers in muscle spindles. Localization of neuronal type nitric oxide synthase-mu and cyclic GMP-dependent protein kinase type I at the neuromuscular junction suggests functions for nitric oxide and cyclic GMP in the regulation of synaptic actions of intra- and extrafusal muscle fibers.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Placa Motora/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Northern Blotting , Técnica del Anticuerpo Fluorescente Directa , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Microtúbulos/enzimología , Contracción Muscular/fisiología , Desnervación Muscular , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Ratas , Ratas Sprague-Dawley , Sarcolema/enzimologíaRESUMEN
Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.
Asunto(s)
Moléculas de Adhesión Celular/farmacología , Adhesión Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Glicoproteínas de Membrana/farmacología , Tenascina/farmacología , Animales , Aorta , Factor Natriurético Atrial/farmacología , Bovinos , Células Cultivadas , Colforsina/farmacología , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Endotelio Vascular , Inhibidores Enzimáticos/farmacología , Músculo Liso Vascular , Osteonectina/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , S-Nitroso-N-Acetilpenicilamina , Trombospondinas , TransfecciónRESUMEN
Nitric oxide and cGMP influence plasticity of nociceptive processing in spinal cord. However, effectors for cGMP have not been identified in sensory pathways. We now demonstrate that cGMP-dependent protein kinase I (cGKl) occurs in the DRGs at levels comparable to that in cerebellum, the richest source of cGKl in the body. Immunohistochemical studies reveal that cGKl is concentrated in a subpopulation of small- and medium-diameter DRG neurons that partially overlap with substance P and calcitonin gene-related polypeptide containing cells. During development, cGKl expression throughout the embryo is essentially restricted to sensory neurons and to the spinal floor and roof plates. Neuronal nitric oxide synthase (nNOS) is coexpressed with cGKl in sensory neurons during embryonic development and after peripheral nerve axotomy. The primary target for cGKl in cerebellum, G-substrate, is not present in developing, mature, or regenerating sensory neurons, indicating that other proteins serve as effectors for cGKl in sensory processing. These data establish sensory neurons as a primary locus for cGMP actions during development and suggest a role for cGKl in plasticity of nociception.
Asunto(s)
GMP Cíclico/metabolismo , Ganglios Espinales/enzimología , Óxido Nítrico Sintasa/metabolismo , Nociceptores/metabolismo , Proteínas Quinasas/metabolismo , Animales , Ratas , Médula Espinal/enzimología , Sustancia P/químicaRESUMEN
Recent studies indicate that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) may inhibit the proliferation of vascular smooth muscle cells (SMC) in vitro. The purpose of this study was to investigate the mechanism of NO- and cGMP-dependent inhibition of cultured rat aortic SMC. The cytokine interleukin-1 beta (IL-1 beta) inhibited serum- and platelet-derived growth factor-stimulated [3H]thymidine incorporation into DNA in subcultured rat aortic SMC. Incubation with IL-1 beta for 24 h markedly increased cGMP levels but not adenosine 3',5'-cyclic monophosphate (cAMP) levels. However, the IL-1 beta-induced increase in cGMP was correlated with an activation of the cAMP-dependent protein kinase (cAMP kinase) activity ratio. The activation of the cAMP kinase was prevented by treatments that blocked NO and cGMP production. The NO-generating vasodilator, S-nitroso-N-acetylpenicillamine (SNAP) also inhibited DNA synthesis and elevated cGMP levels. The inhibition of DNA synthesis by both IL-1 beta and SNAP was observed only when cGMP levels were elevated to high levels (10-fold or more). As was the case for IL-1 beta, SNAP increased the activity ratio of cAMP kinase. Selective inhibition of cAMP kinase using (R)-p-bromoadenosine 3',5'-cyclic monophosphorothioate prevented the inhibition of proliferation by IL-1 beta. By contrast, the inhibitor of the cGMP-dependent protein kinase, (R)-p-bromoguanosine 3',5'-cyclic monophosphorothioate, had no effect on IL-1 beta-induced inhibition of cellular proliferation. These studies suggest that cGMP-dependent activation of the cAMP kinase may be responsible in part at least for the NO-dependent inhibition of proliferation of subcultured rat aortic SMC.
Asunto(s)
GMP Cíclico/farmacología , Músculo Liso Vascular/citología , Óxido Nítrico/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Activación Enzimática , Interleucina-1/farmacología , Óxido Nítrico/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Cyclic GMP-dependent protein kinase (cGMP kinase) is the major receptor protein for cGMP in vascular smooth muscle. Vascular smooth muscle cells (VSMC) isolated from the rat aorta express type I cGMP kinase at high levels, but expression decreases markedly upon passage of the cells. In primary or early passage, the expression of cGMP kinase is lowest when cells are plated at low density as assessed by immunological and Northern analyses. Expression increases at confluence and is maintained in postconfluent cultures. With repeated passaging, however, the levels of cGMP kinase decrease even in confluent and postconfluent cultures so that after several passages enzyme levels are undetectable. The decrease in expression in passaged cells is not due to exposure to serum-derived growth factors, but rather on the repeated exposure of cells to conditions in which cell density is reduced (i.e., subculturing). These results indicate that aortic VSMC grown at low density or those repetitively passaged have reduced expression of cGMP kinase, and thus may not represent appropriate cultures with which to investigate the role of nitric oxide and cGMP in VSMC function.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Aorta/metabolismo , Secuencia de Bases , Western Blotting , Recuento de Células , Células Cultivadas , ADN Complementario , Datos de Secuencia Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
Nitric oxide (NO) down-regulates osteoclastic activity. The mechanism is unknown, although, in some cells NO acts by stimulating guanylate cyclase which activates cGMP-dependent proteins. We demonstrated cGMP-dependent protein kinase in osteoclasts by immunofluorescence microscopy. Specificity was confirmed by Western blot analysis showing a single 78 kDa band, the size of the Type I isoform, in isolated avian osteoclasts. Osteoclast function centers on HCl secretion at a specialized membrane organelle. We found that purified cGMP-dependent protein kinase inhibits ATP-dependent acid transport in reconstituted osteoclast membrane vesicles >90%, while cAMP-dependent kinase catalytic subunit, calmodulin kinase II, or cGMP alone were ineffective. This novel, direct modulation of acid transport by cGMP-dependent kinase and the occurrence of the enzyme in osteoclasts suggest that a mechanism of NO-regulation of bone turnover is via cGMP and cGMP-dependent protein kinase inhibition of HCl transport.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Ácido Clorhídrico/metabolismo , Osteoclastos/metabolismo , Animales , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Pollos , GMP Cíclico/metabolismo , Microscopía Fluorescente , Osteoclastos/enzimologíaRESUMEN
Cyclic GMP (cGMP) mediates vascular smooth muscle relaxation in response to nitric oxide and atrial natriuretic peptides. One mechanism by which cGMP decreases vascular tone is by lowering cytosolic Ca2+ levels in smooth muscle cells. Although mechanisms by which cGMP regulates cytosolic Ca2+ are unclear, an important role for the cGMP-dependent dependent protein kinase in regulating Ca2+ has been proposed. Cyclic GMP-dependent protein kinase has been shown to regulate several pathways that control cytosolic Ca2+ levels: inositol 1,4,5-trisphosphate production and action, Ca(2+)-ATPase ATPase activation, and activation of Ca(2+)-activated K+ channels. The pleiotropic action of cGMP-dependent protein kinase is proposed to occur through the phosphorylation of important proteins that control several signaling pathways in smooth muscle cells. One potential target for cGMP-dependent protein kinase is the class of okadaic acid-sensitive protein phosphatases that appears to regulate K+ channels among other potentially important events to reduce cytosolic Ca2+ and tone. In addition, cytoskeletal proteins are targets for cGMP-dependent protein phosphorylation, and it is now appreciated that the cytoskeleton may play a key role in signal transduction.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Tono Muscular , Músculo Liso Vascular/fisiología , Animales , Calcio/metabolismo , Calcio/fisiología , División Celular , GMP Cíclico/fisiología , Humanos , Membranas Intracelulares/metabolismo , Músculo Liso Vascular/citologíaRESUMEN
Cyclic GMP is recognized as an important intracellular mediator of extracellular signals such as nitric oxide and natriuretic peptides. Cyclic GMP interacts with three types of intracellular receptor proteins: cGMP-dependent protein kinases, cGMP-regulated ion channels, and cGMP-regulated cyclic nucleotide phosphodiesterases. This means that cGMP can alter cell function through protein phosphorylation or through mechanisms not directly related to protein phosphorylation. Cyclic GMP appears to regulate a number of intracellular processes, such as vascular smooth muscle relaxation and neutrophil activation, through these receptor proteins in the cell. It is also becoming clear that the localization of these cGMP receptor proteins in the cell is an important factor in the regulation of cell function by cGMP.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , GMP Cíclico/metabolismo , Canales Iónicos/metabolismo , Proteínas Quinasas/metabolismo , Animales , HumanosAsunto(s)
GMP Cíclico/metabolismo , Músculo Liso Vascular/enzimología , Neutrófilos/enzimología , Proteínas Quinasas/metabolismo , Animales , Aorta/citología , Compartimento Celular , Clonación Molecular , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Fragmentos de Péptidos/genética , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/aislamiento & purificación , Ratas , Proteínas Recombinantes/biosíntesisRESUMEN
The role of cGMP-dependent protein kinase in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells was examined by studying the effects of cGMP on the phosphorylation of the Ca(2+)-ATPase regulatory protein phospholamban. Cultured rat aortic smooth muscle cells incubated with atrial natriuretic peptide II or sodium nitroprusside responded with increased phosphorylation of the 6000-Da subunit of phospholamban. The identity of phospholamban was confirmed using immunoprecipitation methods. Phosphorylation was associated with an increase in the activation of membrane-associated ATPase by Ca2+. These results indicated that at least one site of action of cGMP in smooth muscle cells is the sarcoplasmic reticulum, where phosphorylation of proteins regulating Ca2+ fluxes occurs. Studies using confocal laser scanning microscopy to define the cellular distribution of cGMP-dependent protein kinase suggested that the enzyme was localized to the same cellular region(s) as was phospholamban. Phosphorylation of proteins by cGMP in broken cell fractions from rabbit aorta was also performed. Phospholamban and other proteins were phosphorylated in the presence of cGMP but not cAMP, suggesting that only cGMP-dependent protein kinase was associated with smooth muscle membrane fractions containing phospholamban. These results suggest that one mechanism of action of cGMP in the reduction of intracellular Ca2+ is the activation of sarcoplasmic reticulum Ca(2+)-ATPase via phosphorylation of phospholamban. The data also support the concept that compartmentalization of protein kinases with substrates in the intact cell is an important factor involved in protein phosphorylation.