Cyclic guanosine monophosphate-dependent protein kinase I promotes adhesion of primary vascular smooth muscle cells.
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ABSTRACT:
SUBMITTER: Weinmeister P
PROVIDER: S-EPMC2555919 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We previously reported that hypoxia attenuates nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated fetal pulmonary vessel relaxation by inhibiting cGMP-dependent protein kinase 1 (PKG1) activity, but not all the mechanisms by which acute hypoxia inhibits PKG1 activity have been delineated. Here we demonstrate for the first time, to the best of our knowledge, that acute hypoxia induces an accumulation of ubiquitinated PKG1 in ovine fetal and newborn pulmonary artery smooth muscle cells. Such a modification was not evident in ovine fetal systemic (cerebral) artery smooth muscle cells. The accumulation of polyubiquitinated PKG1 observed after 4 hours of hypoxia was affected neither by the activation of PKG1 kinase activity with the cell-permeable cGMP analogue 8-bromo-cGMP, nor by its inhibition with DT-3 in fetal pulmonary artery smooth muscle cells. Ubiquitinated PKG1? was unable to bind the cGMP analogue 8-(2-aminoethyl)thioguanosine-3',5' (AET)-cGMP, a ligand for the unmodified protein. Inhibition of the proteasomal complex with MG132 led to the accumulation of polyubiquitinated PKG1 in normoxia, indicating the involvement of the ubiquitin-26S proteasomal system in degradation and clearance of this protein under normoxic conditions. The ubiquitinated PKG1 under hypoxic conditions, however, was not predominantly targeted for proteasomal degradation. Importantly, reoxygenation reversed the acute hypoxia-induced accumulation of ubiquitinated PKG1. Our results suggest that the PKG1 ubiquitination induced by acute hypoxia plays a unique role in the regulation of the pulmonary vascular smooth muscle cell vasoreactivity and relaxation mediated by the NO-cGMP-PKG1 pathway.
| S-EPMC3326432 | biostudies-literature
Project description:Previous studies have shown that progesterone could inhibit muscle contraction in various sites of the gastrointestinal tract. The underlying mechanisms responsible for these inhibitory effects of progesterone are not fully known. The aim of the current study was to investigate the effect of progesterone on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway and muscle contraction in the stomach. Single gastric smooth muscle cells from female Sprague-Dawley rats were used. The expression of progesterone receptor (PR) mRNA was analyzed by reverse transcription polymerase chain reaction. NO and cGMP levels were measured via specific ELISAs. Acetylcholine (ACh)-induced contraction of single gastric muscle cells preincubated with progesterone was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, Nω-Nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and expressed as percent shortening from resting cell length. PR expression was detected in the stomach muscle cells. Progesterone inhibited ACh-induced gastric muscle cell contraction. Furthermore, progesterone increased NO and cGMP levels in single gastric muscle cells. Most notably, pre-incubation of muscle cells with either L-NNA or ODQ abolished the inhibitory action of progesterone on muscle contraction. These present observations suggest that progesterone promotes muscle cell relaxation in the stomach potentially via the NO/cGMP pathway.
| S-EPMC6256236 | biostudies-literature
Project description:The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.
| S-EPMC3496052 | biostudies-literature
Project description:Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.
| S-EPMC5376330 | biostudies-literature
Project description:BACKGROUND: Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process. METHODS AND RESULTS: Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit and recording of the associated cGMP-gated current (ICNG). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of ICNG, whereas NO donors (S-nitroso-N-acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 micromol/L) had little effect. The ICNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 micromol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 micromol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I(CNG) activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide. CONCLUSIONS: These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function.
| S-EPMC1877795 | biostudies-literature
Project description:OBJECTIVE:Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the progression of several vasculopathologies. Adenosine monophosphate-activated protein kinase (AMPK) has been shown to play a pivotal role in cellular proliferation and migration. However, the roles of AMPK in VSMC migration and its underlying molecular mechanisms remain elusive. APPROACH AND RESULTS:VSMC migration and the neointima formation were studied in cultured mouse VSMCs or in carotid artery ligation of wild-type C57BL/6J mice, AMPK?2, AMPK?1 homozygous-deficient (AMPK?2(-/-), AMPK?1(-/-)) mice. Deletion of AMPK?2, but not AMPK?1, led to increased phosphorylation of both I?B kinase ? and its downstream target nuclear factor ?B2/p100 at serine 866/870. Consequently, phosphor-p100 at S866/870 bound with E3 ubiquitin ligase ?-transducin repeat-containing protein resulting in the proteolytic processing of the p100 precursor and nuclear factor ?B2/p52 induction. Interestingly, acetylation of histone H3 at lysine 56 mediated by histone deacetylase-3 reduction was enhanced significantly in AMPK?2(-/-) VSMCs compared with wild-type or AMPK?1(-/-) VSMCs. Moreover, the augmented association of p52/acetylation of histone H3 at lysine 56 with the promoter of ubiquitin E3 ligase, S-phase kinase-associated protein 2, was shown in AMPK?2(-/-) VSMCs by chromatin immunoprecipitation assay. Furthermore, AMPK?2 deletion caused S-phase kinase-associated protein 2-mediated E-cadherin downregulation. S-Phase kinase-associated protein 2 siRNA abolished the increased migration of AMPK?2(-/-) VSMCs via E-cadherin upregulation. Finally, neointima formation after ligation of carotid artery was increased in AMPK?2(-/-), but not AMPK?1(-/-), mice. CONCLUSIONS:We conclude that deletion of AMPK?2 causes aberrant VSMC migration with accelerated neointima formation in vivo.
| S-EPMC3910301 | biostudies-literature
Project description:In mammals, the well-organized activation of quiescent primordial follicles is pivotal for female reproductive reserve. In the present study, we examined the mechanisms underlying primordial follicle activation in mice. We found that endothelial nitric oxide synthase (eNOS) and its downstream effectors, cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG), were expressed in pre-granulosa cells and promoted primordial follicle activation, oocyte growth and granulosa cell proliferation in neonatal ovaries. Mammalian target of rapamycin (mTOR) colocalized with PKG in pre-granulosa cells and was essential for eNOS/cGMP/PKG pathway-induced primordial follicle activation. The eNOS/cGMP/PKG pathway was found to stabilize mTOR protein. The mRNA levels of F-box and WD repeat domain containing 7 (FBXW7), an E3 ubiquitin ligase, correlated negatively with mTOR protein levels in neonatal ovaries. FBXW7 bound to and destabilized mTOR protein in pre-granulosa cells in a ubiquitin/proteasome-dependent manner. However, agonists of the eNOS/cGMP/PKG pathway reduced FBXW7 mRNA levels. FBXW7 overexpression suppressed primordial follicle activation and prevented the eNOS/cGMP/PKG pathway from activating primordial follicles and stabilizing mTOR protein. These findings demonstrate that the eNOS/cGMP/PKG pathway activates primordial follicles by suppressing FBXW7-induced ubiquitination of mTOR in mice.
| S-EPMC7835019 | biostudies-literature
Project description:The cGMP-dependent protein kinase G-1? (PKG-1?) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1? activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1? is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1?, were both less susceptible to nitration than WT PKG-1?, but only Y247F-PKG-1? exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1? activity. The overexpression of WT- or Y247F-PKG-1? decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1?. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1? in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1?. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1? for cGMP, which we confirmed using a [(3)H]cGMP binding assay. Our study shows that the nitration of Tyr(247) and the attenuation of cGMP binding is an important mechanism regulating in PKG-1? activity and SMC proliferation/differentiation.
| S-EPMC3953305 | biostudies-literature
Project description:The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.
| S-EPMC5971447 | biostudies-literature
Project description:Gene-targeted deletion of the immediate early responsive gene X-1 (IEX-1) results in a significant increase in systemic arterial blood pressure, but the underlying mechanism is not understood. Studies of arterial reactivity in isolated aortas revealed normal endothelium-dependent and -independent vasorelaxation and vasoconstriction but reduced cAMP-dependent vasorelaxation in the absence of IEX-1. This defect in cAMP signaling was also evident in endothelium-denuded aortic rings, consistent with the enhancement of mitochondrial O2·- production only in IEX-1-deficient vascular smooth muscle cells, not in endothelial cells. Excessive production of reactive oxygen species at mitochondria augmented the expression of G?(i2), suppressing cAMP production in vascular smooth muscle cells. The role of mitochondrial reactive oxygen species in the upregulation of G?(i2) leading to the development of hypertension was supported by the ability of antioxidant or pertussis toxin to restore the cAMP-dependent vasorelaxation to a normal level and reverse established hypertension in IEX-1 homozygous knockout mice. Our results suggest that hypertension in IEX-1 knockout mice may arise primarily from impaired cAMP signaling induced by overproduction of mitochondrial reactive oxygen species in vascular smooth muscle cells and demonstrate a causal relationship between mitochondrial dysfunction and cAMP-dependent vasorelaxation.
| S-EPMC3157252 | biostudies-literature