RESUMO
The acceleration of myocardial relaxation produced by ß-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.
Assuntos
Cálcio/metabolismo , Calpaína/farmacologia , Miócitos Cardíacos/fisiologia , Miofibrilas/fisiologia , Proteólise/efeitos dos fármacos , Serina/metabolismo , Troponina I/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Serina/químicaRESUMO
KEY POINTS: Transforming growth-factor-ß (TGF-ß) and RhoA/Rho-kinase are independently implicated in the airway hyper-responsiveness associated with asthma, but how these proteins interact is not fully understood. We examined the effects of pre-treatment with TGF-ß on expression and activity of RhoA, Rho-kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin-induced contraction, in airway smooth muscle. TGF-ß enhanced bradykinin-induced RhoA translocation, Rho-kinase-dependent phosphorylation and contraction, but partially suppressed bradykinin-induced RhoA activity (RhoA-GTP content). TGF-ß enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF-ß on RhoA and Rho-kinase activity and contraction, respectively. ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin-sensitized mice. ARHGEF1 is a key TGF-ß target gene, an important regulator of Rho-kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper-responsiveness. ABSTRACT: Transforming growth factor-ß (TGF-ß), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF-ß pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF-ß pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20 , MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20 , but induced de-phosphorylation of cofilin. TGF-ß pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF-ß on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-ß on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin-sensitized mice. Our data indicate that TGF-ß enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.
Assuntos
Asma/fisiopatologia , Contração Muscular , Músculo Liso/fisiologia , Sistema Respiratório/fisiopatologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Adulto , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Fosforilação , Ratos , Ratos Wistar , Sistema Respiratório/citologia , Sistema Respiratório/efeitos dos fármacos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Adulto Jovem , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Application of H2S ("sulfide") elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 µM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 µM) and by 50 µM ryanodine; the Ca2+ channel blocker nifedipine (1 µM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 µM), the NADPH oxidase (NOX) blocker VAS2870 (10 µM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 µM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 µM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.
Assuntos
Benzoquinonas/química , Sulfeto de Hidrogênio/farmacologia , Músculo Liso Vascular/fisiologia , Oxirredutases/metabolismo , Artéria Pulmonar/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sulfetos/química , Animais , Cálcio/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Músculo Liso Vascular/citologia , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
An increase in the H2 S (hydrogen sulphide, hereafter sulphide) concentration in pulmonary artery smooth muscle cells (PASMCs) has been proposed to mediate hypoxic pulmonary vasoconstriction (HPV). We evaluated this hypothesis in isolated rat intrapulmonary arteries (IPAs) by examining the effects of the sulphide precursor cysteine and sulphide-synthesis blockers on HPV and also on normoxic pulmonary vasoconstriction (NPV) stimulated by prostaglandin F2α (PGF2α ) and by the drug LY83583, which causes contraction in IPAs by increasing cellular reactive oxygen species levels. Experiments with several blockers of cystathionine γ-lyase (CSE), the enzyme responsible for sulphide synthesis in the vasculature, demonstrated that propargylglycine (PAG, 1 mm) had little or no effect on the NPV caused by PGF2α or LY83583. Conversely, other CSE antagonists tested, aminooxyacetic acid (AOAA, 100 µm), ß-cyanoalanine (BCA, 500 µm) and hydroxylamine (HA, 100 µm), altered the NPV to PGF2α (BCA increased, HA inhibited) and/or LY83583 (BCA increased, AOAA and HA inhibited). Preincubating IPAs in physiological saline solution (PSS) containing 1 mm cysteine increased the amplitude of the NPV to PGF2(α) by â¼50%, and had a similar effect on HPV elicited by hypoxic challenge with 0% O2 . The enhancement of both responses by cysteine was abolished by pretreatment with 1 mm PAG. Measurements carried out with an amperometric electrode demonstrated that incubation with 1 mm cysteine under anoxic conditions (to minimize sulphide oxidation) greatly potentiated the release of sulphide from pieces of rat liver and that this release was strongly antagonized by PAG, indicating that at this concentration PAG could enter cells intact and antagonize CSE. PAG at 1 mm had no effect on HPV recorded in control PSS, or in PSS supplemented with physiological concentrations of cysteine (10 µm), cystine (50 µm) and glutamate (100 µm) in order to prevent the possible depletion of intracellular cysteine during experiments. Application of a combination of 1 mm cysteine and 1 mm α-ketoglutarate to promote sulphide synthesis via the cysteine aminotransferase/mercaptopyruvate sulphurtransferase (CAT/MST) pathway caused an increase in HPV similar to that observed for cysteine. This was partially blocked by the CAT antagonist aspartate (1 mm) and also by PAG. However, HPV was not increased by 1 mm α-ketoglutarate alone, and HPV in the absence of α-ketoglutarate and cysteine was not attenuated by aspartate. Pretreatment of IPAs with dithiothreitol (DTT, 1 mm), proposed to promote the conversion of mitochondrial thiosulphate to sulphide, did not increase the release of sulphide from pieces of rat liver in either the presence or the absence of 1 mm cysteine, and virtually abolished HPV. The results provide evidence that the sulphide precursor cysteine can promote both NPV and HPV in rat IPA by generating sulphide via a PAG-sensitive pathway, presumably CSE. However, HPV evoked under control conditions was unaffected by the blockade of CSE. Moreover, HPV was not affected by the CAT antagonist aspartate and was blocked rather than enhanced by DTT. The data therefore indicate that sulphide generated by CSE or CAT/MST or from thiosulphate is unlikely to contribute to O2 sensing during HPV in these arteries.
Assuntos
Cistationina gama-Liase/antagonistas & inibidores , Sulfeto de Hidrogênio/metabolismo , Hipóxia/metabolismo , Artéria Pulmonar/metabolismo , Sulfurtransferases/antagonistas & inibidores , Vasoconstrição , Animais , Cisteína/farmacologia , Dinoprosta/farmacologia , Inibidores Enzimáticos/farmacologia , Hipóxia/fisiopatologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Ratos , Ratos WistarRESUMO
Asthma and chronic obstructive pulmonary disease (COPD) are highly prevalent respiratory diseases characterized by airway inflammation, airway obstruction and airway hyperresponsiveness. Whilst current therapies, such as ß-agonists and glucocorticoids, may be effective at reducing symptoms, they do not reduce disease progression. Thus, there is a need to identify new therapeutic targets. In this review, we summarize the potential of novel targets or tools, including anti-inflammatories, phosphodiesterase inhibitors, kinase inhibitors, transient receptor potential channels, vitamin D and protease inhibitors, for the treatment of asthma and COPD.
Assuntos
Asma/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Antiasmáticos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Humanos , Inibidores de Fosfodiesterase/uso terapêutico , Inibidores de Proteases/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Canais de Potencial de Receptor Transitório/uso terapêutico , Vitamina D/uso terapêuticoRESUMO
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
Assuntos
Regulação para Baixo , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axotomia , Comportamento Animal/fisiologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Fibras Nervosas Mielinizadas/metabolismo , Neuralgia/etiologia , Neuralgia/fisiopatologia , Medição da Dor , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/fisiopatologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Interferente Pequeno , Ratos , Ratos WistarRESUMO
Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.
Assuntos
Actinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Adenosina/farmacologia , Células Cultivadas , Citocalasina D/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Histamina/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Faloidina/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Trombina/farmacologiaRESUMO
AIMS: We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. CONCLUSIONS: srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.
Assuntos
Cálcio/metabolismo , Artéria Pulmonar/metabolismo , Quinases Associadas a rho/fisiologia , Quinases da Família src/fisiologia , Animais , Células Cultivadas , Dinoprosta/farmacologia , Masculino , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Ratos , Ratos Wistar , Tirosina/metabolismoRESUMO
Reactive oxygen species are implicated in pulmonary hypertension and hypoxic pulmonary vasoconstriction. We examined the effects of low concentrations of peroxide on intrapulmonary arteries (IPA). IPAs from Wistar rats were mounted on a myograph for recording tension and estimating intracellular Ca2+ using Fura-PE3. Ca2+ sensitization was examined in alpha-toxin-permeabilized IPAs, and phosphorylation of MYPT-1 and MLC(20) was assayed by Western blot. Peroxide (30 microM) induced a vasoconstriction with transient and sustained components and equivalent elevations of intracellular Ca2+. The transient constriction was strongly suppressed by indomethacin, the TP-receptor antagonist SQ-29584, and the Rho kinase inhibitor Y-27632, whereas sustained constriction was unaffected. Neither vasoconstriction nor elevation of intracellular Ca2+ was affected by removal of extracellular Ca2+, whereas dantrolene suppressed the former and ryanodine abolished the latter. Peroxide-induced constriction of permeabilized IPAs was unaffected by Y-27632 but abolished by PKC inhibitors; these also suppressed constriction in intact IPAs. Peroxide caused translocation of PKCalpha, but had no significant effect on MYPT-1 or MLC(20) phosphorylation. We conclude that in IPAs peroxide causes transient release of vasoconstrictor prostanoids, but sustained constriction is associated with release of Ca2+ from ryanodine-sensitive stores and a PKC-dependent but Rho kinase- and MLC(20)-independent constrictor mechanism.
Assuntos
Cálcio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteína Quinase C/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Vasoconstrição/efeitos dos fármacos , Animais , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.
Assuntos
Miócitos de Músculo Liso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rho de Ligação ao GTP/genética , Quinases da Família src/genética , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Aminoquinolinas/farmacologia , Animais , Regulação da Expressão Gênica , Pulmão/irrigação sanguínea , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miografia , Oxirredução , Fosforilação , Cultura Primária de Células , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Pirimidinas/farmacologia , Ratos , Ratos Wistar , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Vasoconstritores/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
AIMS: Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway. METHODS AND RESULTS: Rat mesenteric arteries and IPAs mounted on a myograph were challenged with â¼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by â¼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47(phox-/-), but not NOX2(-/-), mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583. CONCLUSION: Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , NADH NADPH Oxirredutases/fisiologia , Fosforilcolina/análogos & derivados , Artéria Pulmonar/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Vasoconstrição/efeitos dos fármacos , Animais , Canais de Cálcio/fisiologia , Óxidos N-Cíclicos/farmacologia , Relação Dose-Resposta a Droga , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Animais , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , NADPH Oxidases/farmacologia , NADPH Oxidases/fisiologia , Fosforilcolina/farmacologia , Proteína Quinase C-épsilon/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esfingosina/farmacologia , Marcadores de Spin , Fosfolipases Tipo C/farmacologia , Vasoconstrição/fisiologiaRESUMO
BACKGROUND AND PURPOSE: The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src-family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR-mediated ASM contraction and associated signalling events. EXPERIMENTAL APPROACH: Contraction was recorded in intact or α-toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light-chain-20 (MLC20 ) and myosin phosphatase targeting subunit-1 (MYPT-1) was evaluated in cultured human ASM cells (hASMC). [Ca(2+) ]i was evaluated in Fura-2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined. KEY RESULTS: Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho-kinase, while after α-toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho-kinase, but not FAK. CCh and BK increased phosphorylation of MYPT-1 and MLC20 and auto-phosphorylation of SrcFK and FAK. MYPT-1 phosphorylation was sensitive to inhibition of Rho-kinase and SrcFK, but not FAK. Contraction induced by SR Ca(2+) depletion and equivalent [Ca(2+) ]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization-induced contraction was sensitive to FAK inhibition only. SrcFK auto-phosphorylation was partially FAK-dependent, while FAK auto-phosphorylation was SrcFK-independent. CONCLUSIONS AND IMPLICATIONS: SrcFK mediates Ca(2+) -sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca(2+) influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease.
Assuntos
Bronquíolos/fisiologia , Cálcio/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Músculo Liso/fisiologia , Quinases Associadas a rho/fisiologia , Quinases da Família src/fisiologia , Adulto , Animais , Bradicinina/farmacologia , Bronquíolos/citologia , Broncoconstritores/farmacologia , Carbacol/farmacologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ratos Wistar , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Adulto JovemRESUMO
AIMS: ß-catenin has been shown to be regulated by inducible nitric oxide synthase (NOS) in endothelial cells. We investigated here whether ß-catenin interacts with and regulates endothelial NOS (eNOS) and whether eNOS activation promotes ß-catenin signalling. METHODS AND RESULTS: We identified ß-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass spectroscopy and western blot analyses of ß-catenin and eNOS immunoprecipitates. This was confirmed by in situ proximity ligation assay. eNOS activity, assessed by cGMP production and eNOS phosphorylation (Ser1177), was enhanced in ß-catenin(-/-) mouse pulmonary endothelial cells (MPECs) relative to wild-type MPECs. eNOS activation (using adenosine, salbutamol, thrombin, or histamine), or application of an NO donor (spermine NONOate) or cGMP-analogue (8-bromo-cGMP) caused nuclear translocation of ß-catenin in HUVEC as shown by western blotting of nuclear extracts. Exposure to spermine NONOate, 8-bromo-cGMP, or sildenafil (a phosphodiesterase type 5 inhibitor) also increased the expression of ß-catenin-dependent transcripts, IL-8, and cyclin D1. Stimulation of wild-type MPECs with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP, or sildenafil increased tube length relative to controls in an angiogenesis assay. These responses were abrogated in ß-catenin(-/-) MPECs, with the exception of that to bFGF which is NO-independent. In C57BL/6 mice, subcutaneous VEGF-supplemented Matrigel plugs containing ß-catenin(-/-) MPECs exhibited reduced angiogenesis compared with plugs containing wild-type MPECs. Angiogenesis was not altered in bFGF-supplemented Matrigel. CONCLUSION: These data reveal bidirectional cross-talk and regulation between the NO-cGMP and ß-catenin signalling pathways.
Assuntos
Células Endoteliais da Veia Umbilical Humana/enzimologia , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , beta Catenina/metabolismo , Animais , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Quinase 3 da Glicogênio Sintase , Glicogênio Sintase Quinase 3 beta , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Inibidores da Fosfodiesterase 5/farmacologia , Fosforilação , Ligação Proteica , Serina , Transdução de Sinais , beta Catenina/deficiência , beta Catenina/genéticaRESUMO
AIMS: To determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV). METHODS AND RESULTS: Studies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F2α (PGF2α, 3 µM) or 20 mM Kâº. The GJ inhibitors 18ß-glycyrrhetinic acid (18ß-GA, 30 µM), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 µM) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca²âº sensitization. The voltage-dependent Ca²âº channel blocker diltiazem (10 µM) had no effect on HPV, and did not alter the inhibitory action of 18ß-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca²âº sensitization, in the presence of high glucose 18ß-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca²âº using Fura PE-3 demonstrated that whilst 18ß-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca²âº. Consistent with this, 18ß-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18ß-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response. CONCLUSION: These results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca²âº sensitization during sustained HPV, but not elevation of intracellular Ca²âº, and may explain the dependence of the former on an intact endothelium.
Assuntos
Cálcio/metabolismo , Junções Comunicantes/metabolismo , Hipóxia/fisiopatologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Diltiazem/farmacologia , Junções Comunicantes/efeitos dos fármacos , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Hipóxia/metabolismo , Masculino , Fosforilação , Proteína Fosfatase 1/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca(2+) ([Ca(2+)](i)), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC(20)) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF(2alpha)-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF(2)alpha-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca(2+)](i), but caused a Y27632-sensitive constriction in alpha-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC(20) at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca(2+) sensitization and vasoconstriction independent of hydrogen peroxide.
Assuntos
Broncoconstrição/fisiologia , Hipertensão Pulmonar/enzimologia , Miócitos de Músculo Liso/fisiologia , Superóxidos/farmacologia , Quinases Associadas a rho/fisiologia , Quinases da Família src/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Amidas/farmacologia , Aminoquinolinas/farmacologia , Animais , Broncoconstrição/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Guanilato Ciclase/antagonistas & inibidores , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/prevenção & controle , Indóis/farmacologia , Masculino , Miócitos de Músculo Liso/patologia , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Artéria Pulmonar/patologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidoresRESUMO
AIMS: We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca(2+) sensitization and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC(20)) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca(2+)](i) was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC(20) phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca(2+)](i) that accompany HPV were also inhibited by PP2. CONCLUSION: Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca(2+) sensitization, and [Ca(2+)](i) responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.
Assuntos
Hipóxia/metabolismo , Artéria Pulmonar/metabolismo , Vasoconstrição/fisiologia , Quinases da Família src/metabolismo , Amidas/farmacologia , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Masculino , Modelos Animais , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC(50) is approximately 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (
Assuntos
Cálcio/metabolismo , Fosforilcolina/análogos & derivados , Proteína Quinase C-delta/fisiologia , Artéria Pulmonar/fisiologia , Esfingosina/análogos & derivados , Sistema Vasomotor/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Diltiazem/farmacologia , Dinoprosta/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Membranas Intracelulares/metabolismo , Masculino , Miócitos de Músculo Liso/enzimologia , Concentração Osmolar , Fosforilcolina/administração & dosagem , Fosforilcolina/farmacologia , Potássio/farmacologia , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Artéria Pulmonar/metabolismo , Ratos , Ratos Wistar , Esfingosina/administração & dosagem , Esfingosina/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
We have determined the subunit stoichiometry of the transient receptor potential C1 (TRPC1) channel by imaging isolated channels using atomic force microscopy (AFM). A frequency distribution of the molecular volumes of individual channel particles had two peaks, at 170 and 720 nm(3), corresponding with the expected sizes of TRPC1 monomers and tetramers, respectively. Complexes were formed between TRPC1 channels and antibodies against a V5 epitope tag present on each subunit. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 88 degrees and 178 degrees. This result again indicates that the channel assembles as a tetramer.